18 4

PHARMEUROPA 18.
4
CONTENTS
October 2006
Pharmeuropa, Pharmeuropa Bio and

Pharmeuropa Scientific Notes Online
555
Publication of Supplements 5.7 and 5.8
522
New: HELPDESK
524
General Information
525
Electronic version of the 5 Edition of the European

Pharmacopoeia
CRS: conditions of sale
List of CRS adopted at the June 2006 session
List of texts adopted at the June 2006 session
List of texts published in Supplement 5.7
Comments concerning some revised/corrected texts
published in Supplement 5.7
Updated work programme of the European
Pharmacopoeia (June 2006)
List of Standard Terms: 5th Edition (2004)
List of new Standard Terms
Standard Terms: introduction and guidance for use
Knowledge database
List of codes of groups of experts
Elaboration / Revision of a monograph (Procedure 1)
Technical Guide: 4th Edition (2005)
Proceedings of conferences of the EDQM
Pharmeuropa Scientific Notes
Pharmeuropa Bio
Press releases
11th Annual Meeting of the European Network of
Official Medicines Control Laboratories (OMCLs)
(Limassol, Cyprus, 9-12 May 2006)
Pharmacopoeial Discussion Group (PDG)
(Yokohama, Japan, 5-8 June 2006)
The EDQM continues to collaborate closely with its
Chinese partners
Russian Federation and Republic of Kazakhstan: new
observers to the European Pharmacopoeia
5th Edition of the European Pharmacopoeia now
available in Spanish
th
International Conferences
Requirements for Production and Control of Avian
Influenza Vaccines
19-20 October 2006, Strasbourg, France
New Frontiers in the Quality of Medicines
13-15 June 2007, Strasbourg, France
Training Sessions on the 5th Edition of the European
Pharmacopoeia: Chemicals
13-14 November 2006, Dublin, Ireland
13-14 September 2007, Poland
6-7 December 2007, London, United Kindom
Readers Tribune
Herbal Reference Standards
Scientific Notes
A Liquid Chromatographic Method using a Reversedphase Hybrid Stationary Phase to Control Potential
Impurities of Imipramine Hydrochloride
Draft Monographs and General Texts

for Comment
N-Acetyltyrosine
PHARMEUROPA Vol. 18, No. 4, October 2006
525
526
531
532
533
535
540
541
541
542
540
547
548
549
551
552
553
554
555
546
546
544
557
558
569
564
557
557
577
577
579
579
585
585
Adrenaline
Assay of 2-antiplasmin (2.7.25)
Assay of human -1-antitrypsin (2.7.32)
Assay of oxygen in gases (2.5.27)
Assay of protein C (2.7.30)
Assay of protein S (2.7.31)
Bilberry fruit dry extract, rened and standardised
Calcium dobesilate monohydrate
Clodronate disodium tetrahydrate
Coccidiosis vaccine (live) for chickens
Cod-liver oil
Cod-liver oil, farmed
Crospovidone
Equine inuenza vaccine (inactivated)
Esomeprazole magnesium trihydrate
Etamsylate
Flucloxacillin magnesium octahydrate
Fludeoxyglucose (18F) (prepared by nucleophilic
substitution) injection
Human -1-antitrypsin
Human normal immunoglobulin
Human plasma (pooled and treated for virus inactivation)
Imipramine hydrochloride
Lauromacrogol 400
Levodropropizine
Measurement of consistency by penetrometry (2.9.9)
Methylphenidate hydrochloride
Methyltestosterone
Mianserin hydrochloride
Noroxacin
Omeprazole magnesium
Orciprenaline sulphate
Oxacillin sodium monohydrate
Pesticide residues (2.8.13)
Racecadotril
Semi-solid preparations for cutaneous application
Sevourane
Spanish sage oil
Sulfacetamide sodium
Swine-fever vaccine (live, prepared in cell cultures),
classical
Teicoplanin
Test for anti-D antibodies in human immunoglobulin for
intravenous administration (2.6.26)
Tinidazole
Illustrations of powdered drugs in herbal monographs:
Birch leaf
Capsicum
Cinchona bark
Devils claw root
Ginger
International Harmonisation
Crospovidone
PDG state of work (June 2006)
Projected timetable for publication and implementation
of texts signed off by the PDG (July 2006)
Contents of the JP Forum (Vol. 15, No. 2)
Contents of the USP Forum (Vol. 32, No. 4)
587
588
589
590
590
591
592
594
596
597
601
605
609
612
614
617
618
621
625
626
628
630
632
635
637
639
640
642
645
647
650
652
656
660
662
664
666
668
670
673
676
677
679
679
680
680
681
683
683
686
688
690
691
521
THE EUROPEAN PHARMACOPOEIA

5th Edition: initial volume 5.0 (2 volumes) + 8 Supplements (5.1 - 5.8)
Supplements 5.6, 5.7, 5.8 published in 2006
The 5th Edition 5.0 (2 volumes) has been available since June 2004 (for prices and ordering
information please consult http://book.pheur.org). It is comprised of texts that were implemented
on 1st January 2005 and a cumulative list of reagents.
Supplements
The supplements are not cumulative and are to be kept for the duration of the 5th Edition.
Modifications (revisions/corrections) to texts are indicated by a line in the margin.
Supplement 5.1 has been available since September 2004; it is comprised of texts that were
implemented on 1st April 2005.
Supplement 5.2 has been available since December 2004; it is comprised of texts that were
implemented on 1st July 2005.
Supplement 5.3 has been available since June 2005; it is comprised of texts that were
implemented on 1st January 2006.
Supplement 5.4 has been available since September 2005; it is comprised of texts that were
implemented on 1st April 2006 and a cumulative list of reagents.
Supplement 5.5 has been available since December 2005; it is comprised of texts that were
Supplement 5.6 has been available since June 2006; it is comprised of texts that will be
implemented on 1st January 2007.
Supplement 5.7 has been available since September 2006; it is comprised of texts that will
be implemented on 1st April 2007 and a cumulative list of reagents.
Supplement 5.8 will be available in December 2006; it is comprised of texts that will be
_____________________________________________________________________________
Online access is available to the database giving names of reagents,
especially chromatographic columns. The address is:
http://www.pheur.org/knowledge.htm
522
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523
The EDQM HELPDESK

for rapid user support
available at www.pheur.org
IF YOU HAVE A QUESTION

OR YOU WOULD LIKE
TO CONTACT THE EDQM
USE THE NEW EDQM HELPDESK
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HOW CAN I CONTACT AND/OR SEND

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Enter your account details. If you do not have an account,
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Complete the electronic form with details of your question(s)
and click on validate. Please ensure that all the elds are lled in as
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All questions received will be considered in our continuous
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nd out more about this new service, visit the HELPDESK today:
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524
General Information
General Information
5th EDITION OF THE EUROPEAN PHARMACOPOEIA
ELECTRONIC VERSION
1970 New and Revised Monographs and 304 General Texts
With the electronic version of the 5th Edition of the European Pharmacopoeia you can view 1970 monographs,
304 general texts (including general monographs and methods of analysis), and 2350 reagents, and also have a
direct internet link to the most recent catalogue of reference substances, which contains 1834 references.
The electronic format has the following convenient features:
hierarchical table of contents, subject index and keyword search;
hyperlinks in the text of a monograph giving access to information on general methods, reagents and reference
substances used in the monograph;
changed (inserted and deleted) texts indicated in both the HTML version and the Acrobat version;
direct access from a monograph or a general method to the CRS database on the internet;
use of a standard internet browser to access the data;
direct printing of an Acrobat version for each individual monograph;
internet and intranet versions available;
direct links to the general notices and the list of general monographs from each text.
NEW: in addition to the official English and French online versions, a Spanish online version is also available for the
convenience of users.
PRICES: SEE THE CATALOGUE ON OUR WEBSITE http://book.pheur.org

Our prices are indicated in EUR but we accept payments in national currencies.
________________________________________________________________________________
FREQUENTLY ASKED QUESTIONS:

WHAT IS THE ROLE OF
THE EUROPEAN PHARMACOPOEIA?
The European Pharmacopoeia is the original source of harmonised quality standards for medicines; all of its
published texts have undergone European harmonisation. These texts are mandatory in 35 European countries* and
in the European Union, and replace any pre-existing national texts on the same subject.
As laid down in their national legislation, certain member states** may continue to issue a national pharmacopoeia
that republishes all or some of the harmonised European texts (most often General Chapters), translated if necessary
into the national language. In all cases it is the European text that is implemented and made legally binding in all of
the member states.
*Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Montenegro, Netherlands, Norway, Portugal, Romania, Serbia, Slovak
Republic, Slovenia, Spain, Sweden, Switzerland, the former Yugoslav Republic of Macedonia, Turkey, United Kingdom.
**Austria, Bulgaria, Czech Republic, Germany, Greece, Hungary, Portugal, Romania, Spain, Switzerland, United Kingdom.
525
General Information
REFERENCE STANDARDS
OF THE EUROPEAN PHARMACOPOEIA
The reference substances and preparations are selected
and verified batches suitable for use as prescribed in the
European Pharmacopoeia. The European Pharmacopoeia
Commission does not guarantee their use for purposes
other than those prescribed. Each vial supplied contains a
quantity sufficient for the prescribed use.
It is recommended that the products be used as soon as
possible.
The stability of the contents of opened vials or ampoules
cannot be guaranteed.
It should be noted that no certificates of analysis,
expiry dates, nor any data not relevant to the use of
the products as defined by the Ph. Eur. monograph
are provided with the reference material. The products
comply with the requirements of the monograph and are
monitored regularly.
NEW: the reference standards database is now updated
daily with information on availability (see the EDQM
website http://www.pheur.org). Other information
available includes details on the origin, assigned value
and batch validity.
NEW: the EDQM has launched a new system for
submitting questions through the HELPDESK, which is
accessible from the EDQM website
(http://www.pheur.org).
1834 reference substances, reference preparations

and reference spectra are supplied by the Technical
Secretariat of the European Pharmacopoeia Commission.
Prices
PRICE LIST
Prices are identified for each product in the CRS

catalogue.
However, please note that prices and package sizes are
subject to change without notice.
The European Directorate for the Quality of Medicines
(EDQM) does not operate a discount policy. The sale
prices are exclusive of duties and taxes and are given
in Euros. It is the responsibility of the buyer (or the
recipient of the delivery if different from the buyer) to
contact the national fiscal or customs authorities to pay
the duties and taxes. In no event shall the said duties and
taxes be paid by the Council of Europe (EDQM).
In the European Union (EU), there is no VAT identification number for organisations with diplomatic status.
The Council of Europe (EDQM) therefore has no VAT
identification number and is not subject to duties and
taxes.
526
The goods remain the property of the Council of Europe

(EDQM) until the invoice has been paid in full. Catalogue
items are not returnable for exchange or refund.
DELIVERY AND RELATED COSTS
Unless otherwise stated below or specifically agreed

with the customer, the goods are shipped to the buyer
on a DDU (Incoterms 2000) basis, namely, delivered
duty unpaid insurance included. Where the shipment
is identified below as airport consignment (see section
Delivery charges), the goods are shipped to the buyer
on a CIP (Incoterms 2000) basis, namely carriage and
insurance included.
The Council of Europe (EDQM) delivers the goods to
the buyer not cleared for import and not unloaded by
any means of transport.
The Council of Europe (EDQM) bears the cost and risks
of packing, transport to the delivery site and insurance.
In no event shall the Council of Europe (EDQM) be
held responsible for any deterioration of the goods due
to their delayed delivery by the carrier.
The buyer is responsible for the cost of import
customs clearance, for paying the duties and taxes
required in the country of import and for unloading
the goods.
Where the shipping costs are paid by the customer,
the goods are shipped to the buyer on an EX Works
(Incoterms 2000) basis, with neither carriage nor
insurance included. Therefore, the Council of
Europe (EDQM) takes no responsibility in any case of
deterioration or loss of goods.
The buyer shall be entirely responsible if the goods
are held up at customs at the time of import into
the buyers country. In no event shall the Council of
Europe (EDQM) be able to provide any assistance.
Delivery charges
The extra charges are applied per shipment. A shipment
comprises only the reference standards that can be
shipped under the same conditions. Consequently, goods
requiring specific packaging (eg ice, dry-ice), dangerous
goods or controlled substances will be invoiced separately
from the rest of the order and extra charges will be
incurred. As one order could include several shipments,
the Council of Europe (EDQM) advises its customers
to re-group their orders by type of shipment so the
customers can better track the progress of a complete
order and to save money in shipping charges.
Where the buyer requests shipping conditions other than
those recommended in our catalogue, or another carrier,
the Council of Europe (EDQM) takes no responsibility in
any case of deterioration of the goods or loss of parcel.
Extra charges (postage and packaging) will be applied in
the following cases. Please note that prices are subject to
change without notice:
General Information
a) Shipment at ambient temperature

France: no extra charge, price is inclusive of
packaging and postage. At the clients request, express
courier delivery is charged at 18 EUR per shipment
EU: 18 EUR per shipment
Other European countries: 80 EUR per shipment
Outside Europe: 120 EUR per shipment
(Note: for India, South America and Africa, our
shipment is by airport consignment only)
Shipping costs paid by the customer: 10 EUR per
shipment
b) Shipment under ice (+ 5 C): sent in cooled freight
containers, either by express courier or by airfreight
(Note: for all countries inside the EU (with the
exception of Cyprus, Estonia, Malta, Poland) our
shipment is on a door to door basis. For all other
countries (and the exceptions above), our shipment is
by airport consignment only)
shipment
c) Shipment under ice (- 20 C): sent in cooled freight
containers, either by express courier or by airfreight
shipment
d) Shipment under dry-ice: sent in cooled freight
containers (dry-ice), either by express courier or by
airfreight
shipment
e) Hepatitis C virus BRP, B19 virus DNA for NAT: dry
ice + dangerous goods from 5 to 100 vials (from 1
to 20 sales units), sent by carrier chosen by EDQM.
For orders of over 100 vials (20 sales units): prices on
request
(Note: for countries outside France, our shipment is

f) Dangerous goods: sent by airfreight chosen by EDQM
(Note: for countries outside France, our shipment is
EU: 150 EUR per item
Other European countries: 180 EUR per item
Outside Europe: 250 EUR per item
g) Dangerous goods in excepted quantities: sent by
carrier chosen by EDQM
(Note: for countries inside the EU (with the exception
of Cyprus and Malta), our shipment is on a door to
door basis. For all other countries (and the exceptions
above), our shipment is by airport consignment only)
h) Dangerous goods sent by road: carrier chosen by
EDQM
EU: 150 EUR per item (door to door)
Other European countries: 180 EUR per item (door
to door)
Outside Europe: cannot be sent
i) Precursors (controlled drugs: sent by carrier chosen
by EDQM)
(Note for countries outside the EU, our shipment is by
airport consignment only)
NB: these extra charges include packaging, shipping
and management of permits
j) Psychotropic substances (controlled drugs: sent by
carrier chosen by EDQM)
(Note for countries outside France, our shipment is by
EU (except France): 110 EUR per shipment
Outside EU: 160 EUR per shipment
527
General Information
k) Narcotics (controlled drugs: sent by carrier chosen by

EDQM)
(Note for countries outside France, our shipment is by
France: 50 EUR per shipment
EU (except France): 110 EUR per shipment
Outside EU: 160 EUR per shipment
details of the Delivery/Dispatch address (if different)

including name of company, post code, town,
country (please note STREET ADDRESS ONLY, no
P.O. Boxes)
contact name, telephone number, fax number and
e-mail address: an e-mail address is required for order
confirmation and shipping notification purposes
VAT number (mandatory within the European
Union)
Your order reference/purchase order reference
l) Reference spectra
Item order code
France: France: no extra charge, price is inclusive of

Official name of the Reference Standard as set out in

this catalogue

Other countries: 50 EUR per shipment
shipment.
How do I order?
The reference standards are supplied by the EDQM.
ORDER FORM
Please send your order using the CRS order form (see
the following page or page xv of the CRS catalogue) or by
sending an official purchase order on company letterhead
to the EDQM. The order form may be downloaded from
www.pheur.org under Reference Standards (Ph. Eur.
Reference Standards).
Fax: +33 (0)3 88 41 27 71 for the attention of CRS Sales
E-mail: orders@pheur.org
Letter: Council of Europe, European Directorate for
the Quality of Medicines, FAO CRS Sales Team,
BP907, F-67029 Strasbourg Cedex, France
Sales/unit quantity
Name and account number of the carrier (if you wish
to use your own)
If orders are received without the official name of the
Reference Standard and the full item order code (as set
out in the catalogue) the EDQM takes no responsibility
for an incorrect item being dispatched.
Unfortunately, we will not be able to process any orders
received without the above information.
Payment
Payment can be made by cheque made payable to the
Council of Europe/EDQM and be sent to the above
address (see 3.1) or by bank transfer.
Socit Gnrale, 255, route de Mittelhausbergen, 67200
Strasbourg, France
IBAN Account Number for International Transfers:
(FR 76) 30003 02360 00550034256 76
National transfers: 30003 02360 00550034256 76
SWIFT:
SOGEFRPP
Customers are financially responsible for duplicate orders You can also pay by credit card (Visa, Eurocard,
in the following cases:
Mastercard or American Express) by writing down the
card number, the expiry date, the card holders name and
confirmation orders that are not clearly marked as
not forgetting the card holders signature. Please note
being a confirmation of an order that has already
that we do not accept credit card numbers by telephone.
been sent to the Council of Europe (EDQM)
In all cases, the payment should be net of charge for
submission of the same order multiple times (i.e., via the Council of Europe and invoices should be paid
fax, e-mail, mail or any combination thereof)
within 30 days from the date of invoice. Any other fees,
such as customs duties, taxes, or tariffs are also the
Please note that we do not accept orders by telephone.
responsibility of the customer.
If you are using any other documentation other than the
For certain countries, especially those having strict
official CRS order form please ensure you have included:
monetary regulations, new clients and large orders,
we reserve the right to require pre-payment. In case of
details of the Invoicing/Billing address including
doubt, please contact us at orders@pheur.org. Payment
name of company, post code, town, country and
by letter of credit is not accepted.
telephone number
528
General Information
Council of Europe
European Directorate for the Quality of Medicines
CRS Order Form

Tel: +33 (0)3 88 41 30 30
BP 907, 67029 Strasbourg Cedex 1 (France)

website: http://www.pheur.org
Helpdesk: http://www.pheur.org/site/page_521.php
SIRET: 778860080010
APE Code APE:990Z
VAT N: Not applicable to Council of Europe - diplomatic privilege.
Fax: + 33 (0)3 88 41 27 71
BILLING ADDRESS
Your Client Code
Company Name*
Invoice Address*
DELIVERY ADDRESS
(Please complete if different from invoicing address)
Company Name*
Delivery Address*
City*
PostCode*
Country*
City*
PostCode*
Country*
Contact Name*
VAT N(* in Europe)
Tel*
E-mail
Contact Name*
VAT N(* in Europe)
Tel*
Email
Fax
Fax
All items marked with an asterisk (*) are mandatory
Your Order Reference* [reference]

Reference*
Date*
Item*
Unit Price
(1) A CRS/BRP may include several individual vials (see sale unit in catalogue), in such instances do not order in terms of total number
of vials.
Quantity(1)*
Total
Total Goods/
DELIVERY CHARGES and PRICES

The price should not be regarded as representing the selling price of a commercial product. The prices quoted in our catalogue are
exclusive of duties and taxes. Extra handling charges may be applied. Please see our catalogue for details.
CONDITIONS OF SALE
We sell on our standard terms of business. For details please see our catalogue.
Payment
I would like to pay now. I will automatically receive an invoice/receipt
I enclose a cheque made payable to Council of Europe/EDQM
I wish to pay by credit card
Visa N
Euro/Mastercard N
Expiry Date
Name
American Express N
Signature
I will pay on receipt on a invoice
529
General Information
ORDER FORM
CATALOGUE OF
- CHEMICAL REFERENCE SUBSTANCES - BIOLOGICAL REFERENCE PREPARATIONS - INFRARED REFERENCE SPECTRA
The catalogue of reference standards of the European Directorate for the Quality of Medicines is
a publication of the Council of Europe, issued three times a year to include the latest substances
adopted by the European Pharmacopoeia Commission.
This catalogue is free; if you would like to receive subsequent updated versions, please complete
and return this form.
RECIPIENT:
Please check the appropriate box:
Prof
Dr
Mr
Ms
Surname...................................................................First name...........................................................
Company/Laboratory ..........................................................................................................................
Industrial Laboratory
Private Control Laboratory
Public Control Laboratory
Department:
Invoicing/Purchasing
Analytical Laboratory
Address.................................................................................................................................................
..............................................................................................................................................................
City.............................................................Postal Code .........................Country.................................
Tel.............................................................................Fax......................................................................
ADDITIONAL RECIPIENT IN YOUR COMPANY/LABORATORY:

Please check the appropriate box:
Prof
Dr
Mr
Ms
Surname...................................................................First name...........................................................
Department: Invoicing/Purchasing
Analytical Laboratory
Other service (please describe)...................................................................................
EUROPEAN DIRECTORATE FOR THE QUALITY OF MEDICINES

226, avenue de Colmar - BP 907 - F 67029 Strasbourg Cedex 1, France
Fax +33(0)3 88 41 27 71
530
General Information
LIST OF REFERENCE STANDARDS ADOPTED

AT THE JUNE 2006 SESSION OF
THE EUROPEAN PHARMACOPOEIA COMMISSION,
AND NOW AVAILABLE
NEW REFERENCE STANDARDS
Name
Code
Name
Code
E-Acetyldigoxin CRS
E-Acetyldigoxin for peak identification CRS
Altizide CRS
Azithromycin for peak identification CRS
Bendroflumethiazide impurity A CRS
Bisacodyl for peak identification CRS
Cefepime dihydrochloride monohydrate
for system suitability CRS
Chlorogenic acid CRS
Dembrexine hydrochloride monohydrate CRS
Digoxigenin CRS
Dopexamine hydrochloride CRS
Dopexamine impurity B CRS
Dopexamine impurity F CRS
Doxazosin mesilate CRS
Enalaprilat for system suitability CRS
Ethyl indole-3-carboxylate CRS
Febantel CRS
Febantel for system suitability CRS
Fluconazole CRS
Y0000565
Y0000642
Y0000606
Y0000637
Y0000564
Y0000608
Fluconazole for peak identification CRS

Fluconazole impurity B CRS
Fluconazole impurity C CRS
Gemcitabine hydrochloride CRS
Gemcitabine impurity A CRS
Iohexol for peak identification CRS
Letrozole CRS
Loratadine for system suitability CRS
Loratadine impurity F CRS
Loratadine impurity H CRS
Methotrexate for peak identification CRS
Modafinil CRS
Modafinil for system suitability CRS
Paroxetine for system suitability CRS
Prednisolone acetate for peak
identification CRS
Sodium laurilsulfate CRS
Tropisetron hydrochloride CRS
Tropisetron impurity B CRS
Verbenalin CRS
Y0000558
Y0000573
Y0000574
Y0000675
Y0000676
Y0000672
Y0000685
Y0000603
Y0000604
Y0000605
Y0000602
Y0000635
Y0000636
Y0000630
Y0000631
Y0000569
Y0000611
Y0000640
Y0000612
Y0000613
Y0000614
Y0000553
Y0000629
Y0000617
Y0000556
Y0000660
Y0000557
Y0000596
Y0000620
Y0000616
Y0000618
Y0000661
REPLACEMENT BATCHES
Name
Code
Name
Code
Arginine hydrochloride CRS 2

Clomifene citrate CRS 2
Digoxin CRS 7
4-Epioxytetracycline CRS 6
Flumequine impurity B CRS 2
Heparin sodium CRS 3
Hydrochlorothiazide CRS 4
Itraconazole CRS 2
A1271000
C2320000
D1900000
E0530000
F0189020
H0200000
H1200000
I7000000
Kanamycin B sulphate CRS 2

Lysine hydrochloride CRS 2
Maltitol CRS 2
Morantel hydrogen tartrate CRS 2
Orciprenaline sulphate CRS 2
Proguanil impurity D CRS 2
Sumatriptan impurity mixture CRS 3
Zolpidem impurity A CRS 3
K0100000
L0900000
M0160000
Y0000028
O0180000
Y0000182
Y0000026
Z2500010
531
General Information
LIST OF TEXTS ADOPTED

AT THE JUNE 2006 SESSION
OF THE EUROPEAN PHARMACOPOEIA COMMISSION
Unless otherwise indicated, these texts will be published in Supplement 5.8 of the European Pharmacopoeia. Individual copies of texts will not be supplied.
NEW TEXTS
GENERAL CHAPTERS
Homoeopathic preparations
2.4.32. Total cholesterol in oils rich in omega-3-acids

5.1.7. Viral safety
Methods of preparation of homoeopathic stocks and

potentisation (2371)
MONOGRAPHS
Monographs
General monographs
Castor oil, refined (2367)

Dwarf pine oil (2377)
Lemon verbena leaf (1834)
Metolazone (1757)
Mirtazapine (2338)
Moxifloxacin hydrochloride (2254)
Paclitaxel (1794)
Poly(vinyl acetate) dispersion 30 per cent (2152)
Ritonavir (2136)
Testosterone decanoate (1736)
Testosterone isocaproate (1737)
Triglycerol diisostearate (2032)
Essential oils (2098)

Vaccines for veterinary use
Salmonella Enteritidis vaccine (inactivated) for
chickens (1947)
Salmonella Typhimurium vaccine (inactivated) for
chickens (2361)
Radiopharmaceutical preparations
Iobenguane sulphate for radiopharmaceutical
preparations (2351)
REVISED TEXTS
GENERAL CHAPTERS
2.6.7. Mycoplasmas
MONOGRAPHS
General monographs
Allergen products (1063)
Extracts (0765)
Homoeopathic preparations (1038)
Immunosera for human use, animal (0084)
Monoclonal antibodies for human use (2031)
Recombinant DNA technology, products of (0784)
Substances for pharmaceutical use (2034)
Vaccines for human use (0153)
Vegetable fatty oils (1579)
Dosage forms
Tablets (0478)
Vaccines for human use
Rabies vaccine for human use prepared in cell
cultures (0216)
Monographs
Adrenaline tartrate (0254)
Ampicillin trihydrate (0168)
Bisacodyl (0595)
Bromocriptine mesilate (0596)
Calcium lactate, anhydrous (2118)
Calcium lactate monohydrate (2117)
532
Calcium lactate pentahydrate (0468)

Calcium lactate trihydrate (0469)
Calcium phosphate (1052)
Capsicum (1859)
Capsicum oleoresin, refined and quantified (2336)
Capsicum tincture, standardised (2337)
Castor oil, hydrogenated (1497)
Cilastatin sodium (1408)
Desmopressin (0712)
Dihydrostreptomycin sulphate for veterinary use (0485)
Dopexamine dihydrochloride (1748)
Enalaprilat dihydrate (1749)
Enoxaparin sodium (1097)
Fluphenazine dihydrochloride (0904)
Hypromellose phthalate (0347)
myo-Inositol (1805)
Oxytocin (0780)
Oxytocin concentrated solution (0779)
Poloxamers (1464)
Pravastatin sodium (2059)
Sesame oil, refined (0433)
Sodium aurothiomalate (1994)
Somatostatin (0949)
Somatropin (0951)
Somatropin concentrated solution (0950)
Somatropin for injection (0952)
Sulbactam sodium (2209)
Terazosin hydrochloride dihydrate (2021)
Thioridazine (2005)
Tranexamic acid (0875)
General Information
LIST OF TEXTS PUBLISHED IN SUPPLEMENT 5.7

A vertical line in the margin indicates where part of a text has been revised or corrected. A horizontal line in the
margin indicates where part of a text has been deleted. It is to be emphasised that these indications, which are not
necessarily exhaustive, are given for information and do not form an official part of the texts. Editorial changes are
not indicated.
Individual copies of texts will not be supplied.
NEW TEXTS
GENERAL CHAPTERS
2.4.31. Nickel in hydrogenated vegetable oils
2.8.18. Determination of aatoxin B1 in herbal drugs
MONOGRAPHS
The monographs below appear for the first time in the
European Pharmacopoeia. They will be implemented on
1 April 2007 at the latest.
Sodium molybdate (99Mo) solution (ssion) (1923)
Monographs
Ferrous sulphate, dried (2340)
Helium (2155)
Hydrocodone hydrogen tartrate 2.5-hydrate (1784)
Indian frankincense (2310)
Leunomide (2330)
Opium dry extract, standardised (1839)
Opium tincture, standardised (1841)
Pelargonium root (2264)
Spectinomycin sulphate tetrahydrate for veterinary
use (1658)
Tamsulosin hydrochloride (2131)
Tibolone (1739)
Valerian dry hydroalcoholic extract (1898)
Valerian tincture (1899)
Yohimbine hydrochloride (2172)
REVISED TEXTS
GENERAL CHAPTERS
2.2.1. Clarity and degree of opalescence of liquids
2.5.12. Water: semi-micro determination
2.7.6. Assay of diphtheria vaccine (adsorbed)
2.7.8. Assay of tetanus vaccine (adsorbed)
4.
Reagents (cumulative list)
MONOGRAPHS
The monographs below have been technically revised
since their last publication. They will be implemented on
1 April 2007.
General monographs
Feline viral rhinotracheitis vaccine (inactivated) (1207)
Feline viral rhinotracheitis vaccine (live) (1206)
Monographs
Amiodarone hydrochloride (0803)
Artichoke leaf (1866)
Calcium hydrogen phosphate, anhydrous (0981)
Calcium hydrogen phosphate dihydrate (0116)
Cefazolin sodium (0988)
Ceftriaxone sodium (0991)
Cefuroxime axetil (1300)
Cellulose, microcrystalline (0316)
Cellulose, powdered (0315)
Cladribine (2174)
Cloxacillin sodium (0661)
Croscarmellose sodium (0985)
Erythromycin lactobionate (1098)
Ethyl parahydroxybenzoate sodium (2134)
Glutathione (1670)
Glycerol distearate (1428)
Glycerol monostearate 40-55 (0495)
Glycine (0614)
Hypromellose (0348)
Interferon alfa-2 concentrated solution (1110)
Iotrolan (1754)
Lactose, anhydrous (1061)
Lactose monohydrate (0187)
Levodropropizine (1535)
Mepyramine maleate (0278)
Methacrylic acid - ethyl acrylate copolymer (1:1) (1128)
Methylcellulose (0345)
Minocycline hydrochloride dihydrate (1030)
Morantel hydrogen tartrate for veterinary use (1546)
Naloxone hydrochloride dihydrate (0729)
Nimesulide (1548)
Nitrous oxide (0416)
Noradrenaline hydrochloride (0732)
Noradrenaline tartrate (0285)
Paroxetine hydrochloride, anhydrous (2283)
Paroxetine hydrochloride hemihydrate (2018)
Phenylephrine (1035)
Phenylephrine hydrochloride (0632)
533
General Information
Sodium calcium edetate (0231)

Sodium stearate (2058)
Spectinomycin dihydrochloride pentahydrate (1152)
Sultamicillin (2211)
Sultamicillin tosilate dihydrate (2212)
Timolol maleate (0572)
Trimethoprim (0060)
Tuberculin puried protein derivative, avian (0535)
Tuberculin puried protein derivative, bovine (0536)
Valerian root (0453)
Vecuronium bromide (1769)
CORRECTED TEXTS
The texts below have been corrected and are republished in their entirety. These corrections are to be taken into
account from the publication date of Supplement 5.7.
GENERAL CHAPTERS
2.9.3. Dissolution test for solid dosage forms
MONOGRAPHS
Iobenguane (123I) injection (1113)
Iobenguane (131I) injection for diagnostic use (1111)
Iobenguane (131I) injection for therapeutic use (1112)
Monographs
Bromazepam (0879)
Brotizolam (2197)
Cefuroxime sodium (0992)
Ciclosporin (0994)
Colistimethate sodium (0319)
Colistin sulphate (0320)
Ispaghula husk (1334)

Magnesium lactate dihydrate (2160)
Metoclopramide (1348)
Milk-thistle fruit (1860)
Mometasone furoate (1449)
Narrow-leaved coneower root (1821)
Olsalazine sodium (1457)
Oxybutynin hydrochloride (1354)
Pale coneower root (1822)
Polymyxin B sulphate (0203)
Povidone (0685)
Purple coneower herb (1823)
Purple coneower root (1824)
Pyrrolidone (2180)
Temazepam (0954)
Ursodeoxycholic acid (1275)
Yarrow (1382)
TEXTS WHOSE TITLE HAS CHANGED

The title of the following texts has been changed in Supplement 5.7.
MONOGRAPHS
Feline viral rhinotracheitis vaccine (live) (1206) (previously Feline viral rhinotracheitis vaccine (live), freeze-dried)
Monographs
Minocycline hydrochloride dihydrate (1030) (previously Minocycline hydrochloride)
SUPPRESSION OF TEXTS
The following text is deleted on 1 January 2007.
GENERAL CHAPTERS
2.9.13. Limit test of particle size by microscopy
The following text was deleted on 1 April 2006.
MONOGRAPHS
Monographs
Glucagon (0612)
534
General Information
COMMENTS CONCERNING SOME REVISED/

CORRECTED TEXTS PUBLISHED IN SUPPLEMENT 5.7
Here follows information concerning certain technical modifications to some revised/corrected texts adopted by the
European Pharmacopoeia Commission at the March 2006 session. This information completes the modifications
indicated by lines in the margin in the supplement. Therefore, the information below is not necessarily exhaustive.
ANALYTICAL METHODS
2.2.1. Clarity and degree of opalescence of liquids
Instrumental determination of opalescence: the required
accuracy of the instrument has been modied, since at
low measurement ranges it is necessary to take account
of the instrument resolution, which becomes comparable
with other sources of incertitude.
2.5.12. Water: semi-micro determination
The general method has been revised to replace
iodosulphurous reagent R (which contains pyridine
and is no longer used in practice) by commercial Karl
Fischer reagents. The suitability of these reagents has to
be demonstrated for each substance to be examined. The
description of the method has been adapted to modern
apparatus.
2.7.6. Assay of diphtheria vaccine (adsorbed)
A serological model in guinea-pigs has been added to the
present methods using intradermal or lethal challenge
with diphtheria toxin in guinea-pigs. The serological
model is intended for use as an alternative to challenge
methods in the circumstances described in the preamble
and is, because of animal welfare, a preferred method
potentially applicable to all combinations of diphtheria
vaccine (adsorbed) for routine analyses.
The development of the serological model has been
carried out as an extensive project in the Biological
Standardisation Programme of the EDQM and was
supported by the Council of Europe and the European
Commission. One of the reports of this project is

available in Pharmeuropa Bio 2003-2, and the other in
Pharmeuropa Bio 2006-1.
The serological model has advantages for animal welfare,
since it avoids challenge with toxin. It can also lead to a
reduction in the number of animals needed, particularly
where a simplied model such as a single-dilution model
is used.
In addition, the serological assay of diphtheria vaccine
(adsorbed) can now be performed with the same group
of animals (guinea-pigs) used for the serological assay of
tetanus vaccine (adsorbed), further reducing the number
of animals needed.
2.7.8. Assay of tetanus vaccine (adsorbed)
The serological assay of tetanus vaccine (adsorbed)
can now be performed with the same group of animals
(guinea-pigs) used for the serological assay of diphtheria
vaccine (adsorbed), reducing the number of animals
needed.
The development of the serological model has been
carried out as an extensive project in the Biological
Standardisation Programme of the EDQM and was
supported by the Council of Europe, the European
Commission and the European Centre for the Validation
of Alternative Methods of the European Commission.
The report of the project is available in Pharmeuropa
Bio 2001-2.
GENERAL MONOGRAPHS
Protein content: the monograph has been revised in
order to allow, where justied and authorised, a higher
limit of content, in accordance with certain licensed
preparations.
Denition: addition of a statement that the monograph
does not apply to herbal drugs, herbal drug preparations
or extracts. This revision stems from a request for

clarication from the Herbal Medicinal Products
Committee of the EMEA. The recently adopted revision
of the general chapter 5.1.4. Microbiological quality of
pharmaceutical preparations has general acceptance
criteria for the category substances for pharmaceutical
use. Since the chapter also has provisions for herbal
drugs and herbal drug preparations, it has been decided
to indicate that they are not within the category
substances for pharmaceutical use.
VACCINES FOR VETERINARY USE

Feline viral rhinotracheitis vaccine (inactivated) (1207)
Feline viral rhinotracheitis vaccine (live) (1206)
Potency: this test, which is based on a scoring system
of signs observed for vaccinated and control cats, has
been harmonised with what is done in practice by the
manufacturers and also with similar potency tests, such
as the test described in the feline calicivirosis vaccine

monographs: the statement that if signs of disease are
observed on more than one day they are only recorded
once has been deleted, because in practice, signs of
disease are recorded each time they are observed and
scores are added.
535
General Information
MONOGRAPHS
Amiodarone hydrochloride (0803)
Due to the toxicity of impurity H, its limit has been
reduced from 0.2 per cent to 0.02 per cent.
Artichoke leaf (1866)
An illustration of the powdered drug has been added.
Calcium hydrogen phosphate, anhydrous (0981)
The monograph has been signed-off by the USP, JP
and Ph. Eur. To implement the harmonised version
of the monograph, it has been necessary to revise the
monograph by making the following changes.
Identication: reaction (b) of phosphates and (b) of
calcium (2.3.1) have been replaced.
Tests:
test for acid-insoluble substances has been added;
test for chlorides (2.4.4) has been replaced;
test for uorides (2.4.5) has been replaced by
Potentiometry (2.2.36, Method II);
test for sulphates (2.4.13) has been replaced;
test for barium the has been revised;
test for loss on drying has been replaced by test for
loss on ignition.
Assay: the method has been revised.
Calcium hydrogen phosphate dihydrate (0116)
The monograph has been signed-off by the USP, JP
and Ph. Eur. To implement the harmonised version
of the monograph, it has been necessary to revise the
monograph by making the following changes.
Identication: reaction (b) of phosphates and (b) of
calcium (2.3.1) have been replaced.
Tests:
test for acid-insoluble substances has been added;
test for chlorides (2.4.4) has been replaced;
Labelling: this section has been deleted as it contained

information covered by the general monograph
Substances for pharmaceutical use (2034).
Ceftriaxone sodium (0991)
Denition: precisions introduced on the products origin.
Related substances: limits expressed to 1 decimal place.
Bacterial endotoxins: limit decreased to 0.08 IU/mg
to take account of the maximum parenteral dose
administered (4 g).
Labelling: section deleted as it contained information
covered by the general monograph Substances for
pharmaceutical use (2034).
Cefuroxime axetil (1300)
Related substances and Impurities: new impurity
introduced (impurity E).
Cellulose, microcrystalline (0316)
Cellulose, powdered (0315)
The European Pharmacopoeia Commission has decided
that more attention will be paid to functionality-related
characteristics (FRCs) in monographs on excipients.
While these characteristics are of importance for the
intended use of many excipients, they are essentially a
matter for agreement between the excipient supplier and
the manufacturer of a medicinal product. For this reason,
FRCs are to be dealt with in a specic non-mandatory
section of the monograph and the particular use or uses
for which a given FRC is relevant are to be specied.
Cladribine (2174)
Cladribine is rather costly and the quantity of the
substance that could be obtained is not sufcient to
establish a CRS for the monograph as it stands. The
monograph has therefore been revised to reduce the
amount of cladribine CRS needed.
Identication by IR spectrophotometry: no differences
in the IR spectra of cladribine CRS before and after
re-crystallisation were noticeable, therefore the recrystallisation procedure can be restricted to the
substance to be examined.
test for uorides (2.4.5) has been replaced by

Potentiometry (2.2.36, Method II);
TLC for impurity E: it is no longer necessary to use a CRS

in reference solution (a) as the test is no longer used for
identication.
test for sulphates (2.4.13) has been replaced;
Cloxacillin sodium (0661)
test for barium has been revised;
Denition: upper limit for content increased to

102.0 per cent according to general chapter 2.2.46.
Chromatoraphic separation techniques; precisions
introduced on the products origin.
test for loss on ignition has been added.

Assay: the method has been revised
Cefazolin sodium (0988)
Related substances and Impurities: impurity F has been
deleted as this synthesis impurity does not appear in
commercial batches, thus detection at 210 nm is no
longer necessary. Impurity L has been added to the
transparency list (previously misidentied as impurity I
in the chromatogram).
536
Related substances: limits expressed to 1 decimal place.

Bacterial endotoxins: limit decreased to 0.20 IU/mg to
take account of the dose and route of administration, as
in the monograph Oxacillin sodium monohydrate (2260).
Labelling: section deleted as it contained information
covered by the general monograph Substances for
pharmaceutical use (2034).
General Information
Croscarmellose sodium (0985)
Glycine (0614)

Identication A: the preparation method has been deleted

as it has been shown that a higher concentration than the
one previously prescribed gives a better spectrum.
Erythromycin lactobionate (1098)

Denition: upper limit for content increased to
102.0 per cent according to general chapter 2.2.46.
Chromatoraphic separation techniques; precisions
introduced on the products origin.
Related substances: 2-8 C range now given for the
storage temperature of the solutions instead of 5 C,
which placed unnecessary constraints on the user;
reference solutions (e) and (f) introduced to allow the
identication of impurities D, E and F; further precisions
given on the zone where the 70 C temperature must
be attained; relative retentions added for all impurities;
limits expressed according to current policy and
correction factors given for impurities E and F.
Pyrogens: replaced by a test for bacterial endotoxins
according to the general policy for reducing the number
of animals used.
Labelling: section deleted as a consequence of the
deletion of the test for pyrogens.
Ethyl parahydroxybenzoate sodium (2134)
Hypromellose (0348)
Interferon alfa-2 concentrated solution (1110)
Potency: the FS-71 cell line given as an example in the
assay is not commercially available, and has therefore
been replaced by the A-549 cell line, which is also suitable
and sensitive to infection by the encephalomyocarditis
virus, and is available to users.
Iotrolan (1754)
Related substances: a spray reagent has been introduced
to improve the sensitivity of the method and allow correct
detection of the spots at 0.10 per cent. A pretreatment of
the plate has also been introduced.
Lactose, anhydrous (1061)
Lactose monohydrate (0187)
Sulphated ash: reference to the method harmonised with
the USP and JP.
Particle size distribution: reference to the general

Heavy metals: problems with method G have been
methods.
reported and a variation of method A has been introduced
Levodropropizine (1535)
in its place.
Glutathione (1670)
Related substances: based on batch results, the limit for
impurity C has been increased to 1.0 per cent and the
limit for unspecied impurities has been replaced by a
limit for any other impurities, which has been increased
to 0.2 per cent as glutathione is a fermentation product.
Glycerol distearate (1428)
Composition of fatty acids: glycerol distearate type III has
been unavailable owing to its non-compliance with the
requirements for stearic acid content, so its lower limit
for stearic acid content has been reduced from 90.0 per
cent to 80.0 per cent.
Nickel: method 2.4.27 has been replaced by method
2.4.31.
Glycerol monostearate 40-55 (0495)
Composition of fatty acids: glycerol monostearate 40-55
type III has been unavailable owing to its non-compliance
with the requirements for stearic acid content and
melting point, so its lower limit for stearic acid content
has been reduced from 90.0 per cent to 80.0 per cent.
Nickel: method 2.4.27 has been replaced by method
2.4.31.
Impurity B and related substances: the preparation

of reference solution (b) has been modied, because
the peak due to impurity B was not detectable in the
chromatogram obtained with this solution.
Enantiomeric purity: it was difcult to meet the
resolution criterion prescribed, so the value has
been changed and is now determined with a solution
containing impurity A at its maximum authorised limit.
Mepyramine maleate (0278)
Identication: IR identication is sufcient, so the
melting point has been deleted from the 1st series; the
TLC test formerly used for both Identication and
Related substances is now described under Identication;
chloroform has been replaced by methylene chloride.
Related substances: the TLC has been replaced by an LC.
Methacrylic acid - ethyl acrylate copolymer (1:1) (1128)
The previous version of the monograph described only
one type of product, the acid form. Another grade of
methacrylic acid-ethyl acrylate copolymer (1:1) is now
available and is partially neutralised using sodium
hydroxide. This revised monograph denes specications
for this 2nd type (type B) and ways to differentiate both
types.
537
General Information
A section on functionality-related characteristics has

been added.
Methylcellulose (0345)
Minocycline hydrochloride dihydrate (1030)
Noradrenaline tartrate (0285)

Identication A: ether has been replaced by methylene
chloride.
Related substances: an LC method is prescribed in place
of the test for noradrenalone by absorbance and the test
for adrenaline by TLC.
Pale coneower root (1822)
Characters: it has been decided generally to delete
the cross-reference to identication tests A and B
(macroscopic and microscopic description), since the
Identication section is mandatory while the Characters
section is only informative. As a consequence, this section
has been deleted.
Paroxetine hydrochloride, anhydrous (2283)

Title and Denition: hydration status has been introduced Identication by IR: the spectra can be recorded on
to avoid reported risks of confusion.
KBr discs without problems and the description of the
preparation has been deleted.
Test for impurity D:
the composition of the mobile phase has been adapted
Morantel hydrogen tartrate for veterinary use (1546)
to obtain a better separation;
Test for pH: the limits have been revised based on batch
data.
Naloxone hydrochloride dihydrate (0729)
the resolution criterion has been replaced by a

peak-to-valley ratio determined on a solution
corresponding to the substance to be examined spiked
with about 1 per cent of impurity D; this gives a better
idea of the separation at the concentration of the test
solution;
Related substances: specied impurity F has been added

and the expression of limits has been modied; since
impurity F is not present in the chromatogram shown for the symmetry of the peak due to the main substance
information on the EDQM internet site, a CRS for peak
does not meet the requirement of chapter 2.2.46.
identication has been added.
Chromatoraphic separation techniques; this has no
consequence on the quantication of impurity D and
Narrow-leaved coneower root (1821)
it has been indicated that the general requirement is
not applicable.
Test for impurities H and I: the peak due to paroxetine in
the chromatogram obtained with reference solution (b) is
section is only informative. As a consequence, this section not visible at 263 nm; the signal-to-noise ratio has to be
checked on the peak due to impurity H.
has been deleted.
Paroxetine hydrochloride hemihydrate (2018)
Nimesulide (1548)
Related substances: due to difculties in obtaining
Related substances: the preparation of reference
enough of impurity C to establish a replacement
solution (a) has been revised to take into account the new batch, a revision has been adopted to include the use
presentation of nimesulide impurity D CRS. Impurity C is of paroxetine for system suitability CRS (containing
commercially available and is now described as a reagent. impurity C) in place of impurity C CRS. The preparation
of the reference solutions has been modied accordingly.
Nitrous oxide (0416)
Identication: the assay and identication test A
are based on the same distinctive characteristic of
nitrous oxide; the assay is performed using an infrared
analyser and identication test A is done using infrared
spectrophotometry.
By analogy to the monograph Oxygen (0417),
identication test A has been replaced by a reference to
the assay.
Noradrenaline hydrochloride (0732)
Identication B: ether has been replaced by methylene
chloride.
Related substances: an LC method is prescribed in place
of the test for noradrenalone by absorbance and the test
for adrenaline by TLC.
538
Phenylephrine (1035)
Absorbance: this test has been deleted because
impurities C and E are now covered by the test for related
substances.
Related substances: the TLC has been replaced by an
LC test; limits are based on current batch data.
Impurities: this section has been updated to indicate only
those impurities that are detected by the test for related
substances.
Phenylephrine hydrochloride (0632)
Related substances: the TLC has been replaced by an
LC test; limits are based on current batch data.
Ketones: this test has been deleted because impurities C
and E are now covered by the test for related substances.
General Information
Impurities: this section, harmonised with the Impurities

section in the monograph for Phenylephrine (1035), has
been added.
Purple coneower herb (1823)
Purple coneower root (1824)
Trimethoprim (0060)
Related substances (part A): due to difculties in
obtaining a sufcient quantity to prepare a replacement
batch of trimethoprim impurity E CRS, the preparation
of reference solution (b) has been revised to describe
the use of trimethoprim for system suitability CRS;
this new CRS consists of a mixture of trimethoprim and
impurity E.

Tuberculin puried protein derivative, avian (0535)
section is only informative. As a consequence, this section Tuberculin puried protein derivative, bovine (0536)
has been deleted.
Potency: as the test prescribes 8 injections per guinea-pig
Pyrrolidone (2180)
(4 per ank), 8 is the appropriate minimum number of
Identication B: the limits have been revised to dene
guinea-pigs that should be used in this test with a Latin
the relative density taking the correction factor given in
square design; the potency test has been modied, not
general chapter 2.2.5. Relative density into account.
only to indicate a suitable minimum number of guinea
pigs when using a Latin square design but also to allow
Sodium calcium edetate (0231)
the use of designs other than the Latin square.
The monograph has been signed-off by the USP, JP and
Valerian root (0453)
Ph. Eur.
Identication D, Chlorides, Water, Assay: these tests have
been revised to implement the harmonised version of the
monograph.
Sodium stearate (2058)
Denition:
the limit for essential-oil content in the whole or
fragmented drug has been reduced;
Test for acidity or alkalinity: it is necessary to heat the

substance under reux to allow complete dissolution.
since the whole or fragmented drug is no longer

available in sufcient quantities and losses are
unavoidable during processing, an additional section
with lower limits for the content of essential oil
and of sesquiterpenic acids in the cut drug has been
introduced.
Spectinomycin dihydrochloride pentahydrate (1152)
Characters:
Related substances: the relative retention for impurity B

has been deleted as it is not a specied impurity;
identication of impurities C, F and G is no longer
required as impurity F is not in the CRS and impurities C
and G, though specied, are limited to the same level as
other detectable impurities; the reference to peaks due to
the anomers has been deleted under the disregard limit
as precautions are taken to avoid their formation in this
test, as opposed to the assay.
the reference to the characteristic odour has been

deleted;
Test for chlorides: the limit of 0.1 per cent was too strict a maximum value of 0.2 per cent better reects the actual
levels in sodium stearate.
Water: the test sample has been halved to 0.100 g as this

quantity is sufcient.
it has been decided generally to delete the crossreference to identication tests A and B (macroscopic
and microscopic description), since the Identication
section is mandatory while the Characters section is
only informative.
Identication:
the microscopic description has been updated;

the TLC method has been revised, replacing

uoroscein by acetoxyvalerenic acid in the reference
solution.
Sultamicillin (2211)
Sultamicillin tosilate dihydrate (2212)
Assay: dantron has been replaced by valerian

standardised dry extract CRS in the reference solution,
since the reproducibility of the correction factor for
dantron was not satisfactory.
Related substances: system suitability requirements for

resolution have been relaxed as experience has shown
they were too strict.
Timolol maleate (0572)
Enantiomeric purity: (R)-timolol maleate CRS has been
replaced by (R)-timolol CRS.
Vecuronium bromide (1769)

Test for impurity B: the concentration of reference
solution (a) has been increased to give a better idea of the
separation at the concentration of the test solution.
539
General Information
UPDATED WORK PROGRAMME OF

THE EUROPEAN PHARMACOPOEIA
(June 2006)
The following items have recently been added to the work programme of the European Pharmacopoeia. Interested
parties are invited to contact the EDQM (lynn.kelso-eleuterio@pheur.org) with a view to participating in the work on
items of interest.
TEXTS TO BE REVISED
5.2.3. Cell substrates for the production of vaccines for
human use: Residual host-cell DNA limits
Allergen products (1063): Terminology, water content,
abnormal toxicity
Butyl parahydroxybenzoate (0881): Related substances,
assay
Cefoxitin sodium (0990): Related substances
Ceftazidime (1405): Related substances
Ethyl parahydroxybenzoate (0900): Related substances,
assay
Fish oil rich in omega-3-acids (1912): Oligomers
Folic acid (0067): Identication
Human normal immunoglobulin (0338): Tests for anti-A
and anti-B haemagglutinins and anti-D antibodies for
products administered subcutaneously
(1646): Test for hepatitis A virus by NAT
Methyl parahydroxybenzoate (0409): Related substances,

assay
Omega-3-acid ethyl esters 60 (2063): Oligomers
Omega-3-acid ethyl esters 90 (1250): Oligomers
Omega-3-acid triglycerides (1352): Oligomers
Propyl parahydroxybenzoate (0431): Related substances,
assay
Ribavirin (2109): Related substances
Sodium calcium edetate (0231): Identications C and D
Sodium fusidate (0848): Related substances
Sodium methyl parahydroxybenzoate (1262): Related
substances, assay
Sodium propyl parahydroxybenzoate (1263): Related
substances, assay
Spiramycin (0293): Related substances
Tablets (0478): Addition of a test for uniformity of
content of multi-sectional tablets under Production
_______________________________________________________________________________________________
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540
General Information
LIST OF STANDARD TERMS

5th EDITION
(27 European languages)
The present list of Standard Terms is a revised list that was drawn up in response to a request from the European
Commission. It covers medicines for both human and veterinary use. These Standard Terms are to be used in
answering the questions in Module 1 (items 1.2 and 1.3) of the EU application form.
The list of Standard Terms is composed of:
an introduction:
a section of general principles and instructions for the use of Standard Terms,
the summary of the changes (amendments, additions, deletions) performed since the last publication
(December 2002),
the procedure for the addition, deletion or modification of terms in the list of Standard Terms (requests
restricted to licensing authorities);
3 lists of standard terms:
list of pharmaceutical forms,
list of routes and/or methods of administration,
list of containers, closures and administration devices.
The 5th Edition contains translations in 27 European languages: Bulgarian, Croatian, Czech, Danish, Dutch, English,
Finnish, French, German, Greek, Hungarian, Icelandic, Italian, Macedonian, Norwegian, Polish, Portuguese, Slovak,
Slovenian, Spanish, Swedish and Turkish. 5 new languages have been added compared to the printed version
published in December 2002: Estonian, Latvian, Lithuanian, Maltese and Romanian.
The corresponding online version is available only to those who ordered the printed version of the 5th Edition
(December 2004).
Price: see the catalogue on our website (http://book.pheur.org).
________________________________________________________________________________
LIST OF NEW STANDARD TERMS

The following new standard terms were adopted in May 2006 and have been included in the Standard Terms
database, accessible on our website (www.pheur.org). The translations have been included by the national
pharmacopoeia authorities.
Those who have already bought the printed version of the Standard Terms list (5th Edition, December 2004) can find
on the inside front cover the instructions to access the database.
NEW TERMS
DEFINITIONS
Transdermal system
Assembly of components intended for transdermal delivery driven by external forces

(e.g. electric current, chemical reaction). Transdermal patch is excluded.
Solution for skin-prick test
Monograph No. 1063. Allergen product for diagnostic use
Solution for skin-scratch test
Monograph No. 1063. Allergen product for diagnostic use
Solution for provocation test
Monograph No. 1063. Allergen product for provocation test by the nasal, ocular or
bronchial route
Plaster for provocation test
Monograph No. 1063. Allergen product for provocation test
Medicinal gas, compressed
Gas packaged under pressure, which is entirely gaseous at - 50 C.
Medicinal gas, cryogenic
Gas that liquefies at 1.013 bar at a temperature below - 150 C
Medicinal gas, liquefied
Gas packaged under pressure, which is partially liquid (gas over a liquid) at - 50 C
Fixed cryogenic vessel
Static, thermally insulated container designed to maintain the contents in the liquid
state
Mobile cryogenic vessel
Mobile, thermally insulated container designed to maintain the contents in the liquid
state
541
General Information
STANDARD TERMS
INTRODUCTION AND GUIDANCE FOR USE
General principles and instructions
for the use of the lists
The lists of Standard Terms were drawn up by the
European Pharmacopoeia Commission further to
the request of the EU Commission for the use in the
marketing authorisation application, SPC, labelling and
electronic communications. It has the double purpose
of bringing information to the patient/user/prescriber
and distinguishing medicinal products having the same
trade-name. Because of the SPC and labelling purposes
its is imperative that any Standard Term and combination
of Standard Terms is constructed with a view to the
1.5.
patient. It should convey essential information on the
properties and uses of the particular medicinal products.
However, information on the container and the route
of administration need not always be included in the
Standard Term but may appear elsewhere in the labelling,
package leaflet and SPC.
1. Pharmaceutical forms
1.1.
The essential information to be conveyed

for all products is the pharmaceutical form
defined as follows: the pharmaceutical form
is the combination of the form in which a
pharmaceutical product is presented by the
manufacturer (form of presentation) and the form
in which it is administered including the physical
form (form of administration).
1.2.
The list of Standard Terms for pharmaceutical

1.6.
forms presents the basic terms needed to
characterise the pharmaceutical form. The
Standard Terms for pharmaceutical forms may
be used for products intended for human as well
as veterinary use. Additional terms specific for
preparations for veterinary use are identified by
insertion of the abbreviation Vet. before the terms
in the table.
1.3.
The list of Standard Terms for pharmaceutical

forms is based on the following guiding principles
and assumptions:
terminology is to be used consistently
throughout the list;
each term should be as short as possible,
commensurate with providing the necessary
information to inform the patient or the user;
each term needs to convey several elements of
information; the number of elements will vary
from one product type to another.
1.4.
542
In certain cases, a complete characterisation

of the pharmaceutical form requires additional
information about the immediate container.
This applies in any case to pre-filled syringes,
pressurised preparations and single-dose
preparations; it also applies in cases where
the administration of the same physical form
differs due to different design of the container/

administration device. For example, the term
cutaneous spray, solution is acceptable as such
if only one form is licensed. If it is supplied in a
pressurised container as well as a preparation with
a spray pump, this presents two pharmaceutical
forms: cutaneous spray, solution, pressurised
container and cutaneous spray, solution, spray
pump.
The same principle applies to the construction,
when needed, of a product-specific term
by combination of a Standard Term for the
pharmaceutical form and a Standard Term for the
route of administration, e.g. Solution for injection
for intrathecal use. The common parenteral
routes for intravenous use and for intramuscular
use need not be added to a Standard Term unless
they are needed to distinguish medicinal products
having the same trade name.
In particular, the complete characterisation
of a pharmaceutical form of a preparation for
veterinary use may require a combination of
Standard Terms or elements thereof due to the
specificity and multiplicity of veterinary routes
of administration and associated administration
devices.
If the physical form in which the product is
supplied by the manufacturer is different from that
in which it is to be administered to/used by the
patient, that is, if transformation of the product is
required before it can be administered/used, both
these elements of information need to be conveyed
within the term. However, this does not mean that
in the case of a powder that is dissolved in a small
amount of solvent before it is diluted in a larger
volume to be infused, the Standard Term should
contain the word concentrate. The appropriate
Standard Term is Powder for solution for infusion
or analogous variants thereof (policy valid as of
June 2006).
1.7.
If the product has certain special characteristics

that are relevant to its use, these need to be
included in the term.
1.8.
In some cases, the level of information

required for the purpose of Part 1A and 1B of the
application dossier (and leaflets) may be higher
than required for inclusion on the product label.
Therefore, in addition to the Standard Terms given
in the Pharmaceutical dosage forms section, short
terms that may be used for labelling only are given
in the Short terms section. Where such a short
term is given in the Short terms section, this is
indicated in the Pharmaceutical dosage forms
General Information
section by insertion of the corresponding short

term in the relevant box.
1.9.
Definitions of Standard Terms and requirements

applicable to the different categories are given in
the general monographs of the latest edition of the 5.2
European Pharmacopoeia.
1.10. In some cases, established usage allows one word

within a term to convey more than one element of
information. For example, the term Tablet, unless
otherwise qualified, denotes a product for oral use,
i.e. to be swallowed.
11.11 The term modified-release is not sufficiently
precise for describing a particular product. A more
specific term such as prolonged-release, delayedrelease or gastro-resistant should be used,
wherever applicable.
2. Routes of administration
In some cases, a product may be intended for more

than one route and/or method of administration.
In these cases, the two (or more) terms may be
included, for example solution for injection/
infusion.
5.4
In other cases, where no existing Standard Term

applies, characterisation of the pharmaceutical
form may be obtained by combination of elements
of Standard Terms for pharmaceutical forms,
or combination of such elements with Standard
Terms, or elements thereof, for the route of
administration. For example, when the form of
presentation (e.g. solution/suspension/emulsion
or ointment/cream/gel) differs from that of the
existing Standard Term, replace the form in the
Standard Term. Where necessary, combine the
term with the appropriate term for the route of
administration.
In some cases, a product may be intended for more

than one route and/or method of administration.
In these cases, both terms may be included, for
example solution for injection/infusion.
3.1.
3.2.
The pharmaceutical form is supplied in an

immediate packaging, which is the container or
other form of packaging immediately in contact
with the product. A closure is a means to close
an immediate container for the purpose of the
correct storage and use of the product. In some
cases a special administration device is needed
for the correct administration of the product. The
administration device may be an integral part of
the immediate container or closure.
Definitions, requirements and recommendations
for containers are provided in chapter 3.2 of the
latest edition of the European Pharmacopoeia.
Combination of existing Standard Terms, e.g.

combination of the pharmaceutical form and
the immediate container (see item 1.4) and
combination of the pharmaceutical form and
the route of administration (see item 1.5), may
be necessary for safety reasons or to distinguish
marketed products.
5.3
The route of administration indicates the part of

the body on which, through which or into which
the product is to be introduced. Mostly in the
veterinary field, the method of administration
indicates the way the product is to be supplied to
the animals.
3. Containers, closures and administration devices
be included in the list of combinations of existing

Standard Terms. The list is available online at
www.pheur.org and in the electronic version of the
Standard Terms (http://st.pheur.org).
PROCEDURE FOR THE ADDITION, DELETION OR

MODIFICATION OF TERMS IN THE LIST OF STANDARD
TERMS
Before submitting any request for the addition, deletion
or modification of a Standard Term, please read the
Introduction to Standard Terms publication, which
provides information on the general principles on which
Standard Terms are established. In particular, attention
is drawn to the expectation that a request for a new
Standard Term will only be made when the nature of
the product is such that no existing Standard Term or
combination of Standard Terms accurately describes it.
1.
The member state, the European Commission or the

EMEA sends the request form, duly filled in, to the
EDQM.
2.
A request for a new Standard Term will only be
made to the European Directorate for the Quality
of Medicines (EDQM) when the nature of the
3.
product is such that no existing Standard Term
or combination of Standard Terms accurately
describes it. Such requests will be made in
accordance with the procedure described hereafter. 4.
5. Combinations of existing Standard Terms
The EDQM immediately (within one week) sends the

proposal to the Working Party on Standard Terms of
the European Pharmacopoeia Commission.
4. New Standard Terms
5.1
In the case of a novel product, the member state

or the European Medicines Agency (EMEA) may
consider that a suitable product-specific term
can be constructed by a combination of existing
Standard Terms or elements thereof. The member
state or the EMEA is requested to notify the EDQM
of such cases so that the suitability of the proposed
combination of terms may be confirmed by the
EDQM. Where appropriate, the combination will
The Working Party examines the proposal by

correspondence or in a meeting, if required, and if
necessary designates a rapporteur.
Within 6 weeks, unless a meeting is required, the
Working Party gives an opinion together, if necessary
for a new term, with a proposal for a provisional
definition.
The European Commission, the member states
competent authorities and the EMEA are informed
about this opinion and are asked to comment within
1 month. Any disagreement should be justified.
The opinion is submitted to Group of Experts No. 12
of the European Pharmacopoeia Commission so
that, where necessary, it can make a proposal on the
543
General Information
authorities, each authority translating it into its own

language.
revision of the corresponding monograph or on the

elaboration of a new monograph.
5.
Taking into account any comment received and

the advice of the Working Party, the European
Pharmacopoeia adopts the Standard Term or
adopts the deletion or modification of a Standard
Term so that the change can be introduced in the
list of Standard Terms. It authorises, if necessary,
the revision of the corresponding monograph or
elaboration of a new monograph.
A new or modified Standard Term is introduced
in the updated list of Standard Terms and is then
translated into the various languages by the national
6.
In case the request for a new Standard Term is made

for a licence application, the request mentions the
date of the receipt of the valid application so that an
approved Standard Term is provided within 120 days.
In such cases, to avoid any potential delay,
delegations to the Commission are requested, if
necessary, to adopt the new term by correspondence.
The EDQM is duly informed of the result of the
assessment of the licence application before
publishing the new Standard Term.
________________________________________________________________________________
5th EDITION OF THE EUROPEAN PHARMACOPOEIA

NOW AVAILABLE IN SPANISH
A new version of the European Pharmacopoeia is now
available: the Spanish online version. The rst two
volumes 5.0 were made available in July, followed by
supplements 5.1 to 5.3 in August.
dedicated team within the EDQM and used the latest IT

tools available to translate, format and validate all of the
texts accurately. The features of this version are the same
as the English and French online versions.
The EDQM is committed to keeping pace with its users

needs and to utilising the most current technologies
to allow access to the European Pharmacopoeia, the
reference work for the quality control of medicines and
substances for pharmaceutical use.
If you already hold a subscription to the 5th Edition

European Pharmacopoeia online version, you will
automatically be granted free access to the Spanish
online version, as well as access to our technical support
service. If not, but you are interested in ordering, you
can do so directly from the EDQM or through your local
distributor.
This, the rst Spanish online version of the European

Pharmacopoeia, will make it easier for Spanish-speaking
users to use, understand and meet the requirements of
the European Pharmacopoeia, and will serve to improve
and promote public health worldwide.
The work, which began at the end of 2005 in liaison
with the Spanish Medicines Agency, was managed by a
Additional information on our publications and pricing is

available on our website (http://www.pheur.org/site/page_
581.php and http://book.pheur.org).
See also the page opposite.
________________________________________________________________________________
COUNTERFEIT MEDICINES - SURVEY REPORT (2006)

Counterfeit medicines pose an ever-greater threat to public health in Europe today.
In an effort to adequately measure the scope of the phenomenon and reduce the
inherent risks, the Council of Europe has commissioned a survey on issues related
to this particularly disquieting form of fraud. This report covers, among others:
the current and estimated market and trade matters; the status of pharmaceutical
regulation; national and international co-operation between authorities, the industry
and wholesalers; detection systems and procedures; the adequacy of legal, judicial
and administrative systems; and professional training in the matter. It also sets out to
dene counterfeit medicine and pharmaceutical crime.
Authors: Jonathan Harper, Bertrand Gellie
ISBN: 92-871-5863-0
Price: 25 EUR (Europe) / US$ 38 (outside Europe), + 10 % postage
Available in English only
Council of Europe Publishing - Sales Unit
Ms Sophie Lobey, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 25 81 - Fax: +33 (0)3 88 41 39 10
E-mail: publishing@coe.int - Website: http://book.coe.int
544
General Information
NEW! FOR THE FIRST TIME
European Pharmacopoeia
5th Edition 2006

I N SPA NISH
(online version only)
NOVEDAD! POR PRIMERA VEZ

a
La 5 Edicin 2006
de la Farmacopea Europea
EN ESPA OL
(nicamente versin internet)
HOW TO ORDER
You can order online at https://book.pheur.org
and choose your preferred method of payment
HOW TO CONTACT US
Use the HELPDESK on the EDQM website at
COMO EFECTUAR UN PEDIDO

Usted puede realizar un pedido va on-line en la direccin
de internet https://book.pheur.org y elegir la modalidad
de pago que le resulte ms cmoda
COMO CONTACTARNOS
Utilice el HelpDesk de la pgina web de la DEQM en
EUROPEAN DIRECTORATE
FOR THE QUALITY OF MEDICINES
545
General Information
THE EDQM CONTINUES TO COLLABORATE

CLOSELY WITH ITS CHINESE PARTNERS
A multidisciplinary team from the EDQM (Publications, Public Relations and Certification) took part in an official
journey on 17-29 June 2006 with the following missions:
to inspect production sites of manufacturers that have been granted certificates of suitability of monographs
of the European Pharmacopoeia (CEP/COS) for their raw materials to be used in the manufacture of medicines
for the European market; the purpose of these inspections was to check compliance with good manufacturing
practice (GMP) and with the information provided by the manufacturer in the application for certification.
to hold meetings with the Chinese authorities in charge of quality control of medicines at the national and
provincial levels as laid down in the mutual agreements;
to participate in an international conference in Shanghai;
to organise, with the support of the Chinese Pharmaceutical Association (CPA), a training session in Shanghai
for Chinese users of the European Pharmacopoeia;
to participate via a stand and a conference in the 6th CPhI (specialised fair-exhibition for Chinese and other
Asian producers of raw materials); over 1000 visitors were received at the stand and during the conference; the
documents of the EDQM had been translated into Chinese for this occasion.
The EDQM/European Pharmacopoeia is pleased with the quality of the various exchanges that took place during
this mission; they will help the EDQM/Council of Europe become known in one of the two leading countries in
terms of world production of pharmaceutical raw materials.
________________________________________________________________________________
RUSSIAN FEDERATION AND REPUBLIC OF

KAZAKHSTAN: TWO NEW OBSERVERS TO THE
EUROPEAN PHARMACOPOEIA COMMISSION
The Russian Federation and the Republic of Kazakhstan
requested and obtained observer status to the European
Pharmacopoeia Commission. The decision was voted
for unanimously by the delegates from the member
countries during the June 2006 session, and allows new
collaborations to be developed with the authorities of
these two countries.
The Russian Federation and the Republic of Kazakhstan
are indeed keen to participate in the work of the
European Pharmacopoeia and to share information and
experiences, to ensure the quality control of medicines
and protect public health in their countries.
The Convention on the Elaboration of a European
Pharmacopoeia is an international convention drafted
by the Council of Europe in 1964. It has the objective
of progressively elaborating in Europe a pharmacopoeia
that is common to all the member states, and which
defines a single set of specifications that will become the
official standards applicable within these countries for
active substances and excipients used in medicines; the
European Pharmacopoeia also describes test methods to
ensure the quality of these medicines.
546
The EDQM has as its mission the protection and

promotion of public and animal health, through the
elaboration of quality standards for medicines for human
and veterinary use. Medicines need to be safe, effective
and of good quality in order to produce the expected
therapeutic effect. The EDQM works closely with its
international and European partners to strengthen
measures to ensure that substandard or counterfeit
medicines do not reach the marketplace.
There are currently 36 members* of the European
Pharmacopoeia Commission and 20 observers**.
* Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Montenegro, Netherlands, Norway, Portugal, Romania, Serbia, Slovak
Republic, Slovenia, Spain, Sweden, Switzerland, the former Yugoslav
Republic of Macedonia, Turkey, United Kingdom and the European Union.
* * The World Health Organisation (WHO); 6 countries in Europe:
Albania, Georgia, Poland, Republic of Kazakhstan, Russian Federation
and Ukraine; 13 other countries in the world: Algeria, Australia, Brazil,
Canada, China, Israel, Madagascar, Malaysia, Morocco, Senegal, Syria,
Tunisia and United States of America.
General Information
LIST OF CODES OF GROUPS OF EXPERTS

(July 2006)
GROUPS OF EXPERTS
1
6
6B
7
9G
10A
10B
10C
10D
Microbiology
Biological substances
Human blood and blood products
Antibiotics
Medicinal gases
Organic chemistry - synthetic products
Organic chemistry - synthetic products
Organic chemistry - synthetic Products
Organic chemistry - synthetic Products
11
Organic chemistry - natural products
12
Galenical products
13A
Phytochemistry
13B
Phytochemistry
13H
Fatty oils and derivatives
14
Radioactive compounds
15
Sera and vaccines
15V
Veterinary sera and vaccines
WORKING PARTIES
BOT
Botulinum toxin
LEC
BSR
Bovine serum
MAB Monoclonal antibodies
CEL
Cellulose derivatives
MAT
CLP
Cloud point
MMM Alternative microbiological methods
Lecithins for pharmaceutical purposes

Monocyte activation test
CND Conductivity
MYC Mycoplasmas
CRB
Carbohydrates
NIR
CST
Chromagraphic separation techniques
NMR Nuclear magnetic resonance spectrometry
CTP
Cell therapy products
P4
FRC
Functionality-related characteristics
POW Powder characterisation techniques
GEL
Gelatin
PST
GTP
Gene therapy products
RGN Reagents
HFA
Propellants
SRP
Near-infrared spectrophotmetry
Procedure 4
Pesticides in herbal drugs
Special revision programme
HMM Homoeopathic manufacturing methods
ST
Standard terms
HOM Homeopathy
STA
Statistics
ICP
Inductively coupled plasma spectrometry
VIT
Vitamins
INC
Inorganic chemistry
WAT
Water
INH
Inhalations
WXT Water for the preparation of extracts
547
General Information
ELABORATION / REVISION OF A MONOGRAPH

(Procedure 1)
The European Pharmacopoeia Commission decides to
elaborate/revise a monograph
j
Group of experts:
a rapporteur prepares a draft monograph, which is
evaluated by the experts
j
j
Pharmeuropa (4 issues per year):

the draft monograph is published for public enquiry,
which lasts 3 months
j
The national pharmacopoeia authorities process the
comments received on the draft
The EDQM-Division 1 compiles the comments sent by

the national authorities
The revised draft is

published for further
enquiry, if necessary
j
j
The group of experts examines the comments and

revises the draft monograph accordingly
j
The draft is proposed to the
Commission
j
European Pharmacopoeia Commission
j
- adopts the monograph, if necessary with slight
modications
- adopts the implementation date (about 1 year
after the adoption of the monograph)
j
does not adopt the monograph
j
EUROPEAN PHARMACOPOEIA (3 supplements per year):
the monograph is published about 6 months after adoption
548
General Information
ETHICAL EYE - BIOMEDICAL RESEARCH (2004)

What are the rules and underlying values governing biomedical research in Europe? What form do these values take
and where do they originate? Does biomedical research pose a threat to individuals and their rights? What balance
should be struck between freedom of research and protection of the individual? All these questions are examined in
this book from a pan-European perspective.
The authors look at various international and European standards, including the Helsinki Declaration of the World
Medical Association, EU Directive 2001/20 on pharmaceutical research and the Council of Europes Convention on
Human Rights and Biomedicine. The last named was signed in Oviedo in 1997 and is the rst binding international
treaty on the subject, with a special chapter on scientic research on human beings. The Convention establishes a
common minimum level of protection of fundamental rights throughout Europe. It will soon be supplemented by an
additional protocol specically concerned with biomedical research.
The book contains a glossary and a list of relevant international conventions and treaties, websites and publications.
It is aimed at both specialists and a wider public interested in this subject.
Contents
BIOMEDICAL RESEARCH IN EUROPE
Preface
Introduction by Claude Huriet (France)
History and denitions by Povl Riis (Denmark)
Germany: current legislation by Jochen Taupitz (Germany)

Central and eastern Europe: research-related problems for
transition countries by Eugenijus Gefenas (Lithuania)
Italy: some shortcomings of biomedical research by Stphane
Bauzon (Italy)
United Kingdom: data protection and condentiality by
Michel Coleman and Vivienne Harpwood (United Kingdom)
ETHICAL DILEMMAS IN RESEARCH

Uses and abuses of biomedical research
by Jan Helge Solbakk (Norway)
Selection and recruitment of participants: European standards
by Herman Nys (Belgium)
Placebo: its action and place in health research
by Andrzej Grski (Poland)
Cancer clinical trials by Maxime Seligmann (France)
Some ethical considerations in industry-sponsored clinical
trials by Tom Gallacher and N. Sreeharan (United Kingdom)
Women in biomedical research by Outi Leena L. Hovatta
(Sweden)
EUROPE AND BIOMEDICAL RESEARCH

European law and biomedical research by Peteris Zilgalvis
(Council of Europe)
APPENDICES
Selected websites
Draft protocol on biomedical research, Council of Europe
The Helsinki Declaration, World Medical Association
ISBN 92-871-5462-7. Price: 15 EUR (Europe) / US$ 23 (outside Europe), + 10 % postage.

This book is available in French and English from:
Ms Sophie Lobey, F- 67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 25 81 - Fax: +33 (0)3 88 41 39 10
E-mail: publishing@coe.int - Website http://book.coe.int
________________________________________________________________________________
TECHNICAL GUIDE FOR THE

ELABORATION OF MONOGRAPHS - 4th Edition (2005)
now available as a free download on the EDQM website
The Technical Guide for the Elaboration of Monographs describes the scientific approach used for the elaboration of
monographs and the establishment of specifications of the European Pharmacopoeia. The guide also describes how
to scientifically elaborate the various sections that must be included in each monograph, for example, definition,
characters, the physical and chemical reactions constituting the identification section, purity tests, assay methods
and storage conditions. It is continually being updated.
Available in English and French as a free download from the EDQM website (http://www.pheur.org).
549
General Information
GUIDE TO SAFETY AND QUALITY ASSURANCE

FOR ORGANS, TISSUES AND CELLS
2nd Edition (2004)
The purpose of this guide is to provide guidance for all those involved in transplantation to maximise the quality,
and thereby the success rate, of transplants, and to minimise the risks to all involved in this complex procedure. It
includes safety and quality standards for the procurement, preservation, processing and distribution of organs, tissues
and cells of human origin (allogeneic and autologous) used for transplantation purposes. This guide will be regularly
updated, in line with the latest technical advances.
As the European Union Directive on Tissues and Cells (2004/23/EC) was recently adopted, the European Commission
will build on the Council of Europes guide when establishing technical standards under the directive. This
co-operation will ensure that the same standards are applied throughout Europe.
The 2nd Edition of the Guide can be obtained in English and French from:
Tel: +33 (0)3 88 41 25 81 - Fax : +33 (0)3 88 41 39 10
Questions and comments on the content should be sent directly to the division in charge:
Council of Europe, Health Division
Mr Karl-Friedrich Bopp, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 22 14 - Fax: +33 (0)3 88 41 27 26
E-mail: karl-friedrich.bopp@coe.int
Council of Europe website: www.coe.int
Health and Ethics: www.coe.int/T/E/Social_Cohesion/Health
________________________________________________________________________________
VISIT OUR WEBSITE http://www.pheur.org

and get the news as it happens at the
European Directorate for the Quality of Medicines (EDQM)
and the
(site is in English only)
access the latest news on official publications

consult the list of adopted monographs and CRSs after each session of the Commission
download about 1600 safety datasheets and about 190 leaflets
find out about the new developments in the procedure for certification of suitability of monographs of the
access the work programme database of the European Pharmacopoeia (KNOWLEDGE DATABASE), the
certification and the reference substance databases
NEW: the reference substances database is now updated daily with information on availability
other accessible information includes details on the origin, assigned value and batch validity
find official surveys in progress, announcements of conferences and international seminars, etc.
keep up-to-date on the latest technical, scientific and regulatory developments
find out about career opportunities at the EDQM
access the EDQMs online publications ordering service at http://book.pheur.org
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through the EDQM website at http://www.pheur.org/site/page_521.php
Please send us your comments and suggestions to help us develop this site!
550
General Information
EDQM CONFERENCE PROCEEDINGS

The following free Conference Proceedings are available to download from the EDQM website
(http://www.pheur.org/site/page_601.php).
Certicates of Suitability of Monographs of the
European Pharmacopoeia: Implementation of the
5th Edition New Procedures for Revision and
Renewal of Certicates
Quality on the Move: Dynamics of the European

Pharmacopoeia
4-6 October 2004, Budapest, Hungary
Process Analytical Technologies International Symposium
3-4 May 2004, Cannes, France
27-28 October 2005, Istanbul, Turkey
OMCL Information Day: Place and Role of the European Microbiological Control Methods in the European
Pharmacopoeia: Present and Future
OMCL Network within the Regulatory Framework in
Europe
5-6 May 2003, Copenhagen, Denmark
27 May 2005, Rome, Italy
Foot and Mouth Disease Vaccines: Current Situation

17-18 March 2003, Strasbourg, France
Alternatives to Whole Cell Pertussis Vaccine Potency

Assay
Standardisation and Quality Control Cell and Gene

Therapy Products
24-25 February 2003, Strasbourg, France
16 March 2005, Geneva, Switzerland
Quality of Homoeopathic Products in the New European

Replacement, Reduction and Renement of the Use of
Legislative Framework
Animals in the Quality Control of Vaccines
15 February 2005, Strasbourg, France
7-8 November 2002, Strasbourg, France
Serological Potency Tests for Diphtheria and Other
Excipients: Classical Requirements and Functionality
Vaccines
Related Testing
4-5 April 2002, Brussels, Belgium
6-7 October 2004, Budapest, Hungary
******
The following Conference Proceedings can be ordered from the EDQM. For prices and ordering please consult the
catalogue on our website (http://book.pheur.org).
Certication of Suitability of Monographs of the
European Pharmacopoeia (CEP) - New Developments
of the Procedure, How to Apply for a CEP
8-9 November 2001, Athens, Greece
The Future Face of the European Pharmacopoeia Current Concerns in Pharmaceutical Analysis
8-9 February 2001, Cannes, France
Herbal Medicinal Products: Quality Evaluation Contribution of the European Pharmacopoeia
16-17 November 2000, Nice, France
Tetanus Vaccine for Human Use
Mycoplasma Testing: The Potentialities and Role
of PCR Tests
13-14 March 2000, Paris, France
Biologicals beyond 2000: Challenge for Quality

Standards in an Evolving Field
27-29 September 1999, Strasbourg, France
General Monographs on Dosage Forms and PharmacoTechnological Test Methods
26-27 October 1998, Seville, Spain
The Vision of the European Pharmacopoeia in the
21st Century - The Dynamics of Quality of Medicines in
Europe
4-7 December 1996, Prague, Czech Republic
Sterility Tests and Efcacy of Antimicrobial
Preservation
5-6 February 1996, Barcelona, Spain
All above proceedings are available in English only and are not included in the subscription to Pharmeuropa.
551
General Information
PHARMEUROPA SCIENTIFIC NOTES

As from Pharmeuropa 17.4, Scientic Notes are principally published in a new publication called Pharmeuropa
Scientic Notes.
Articles published in Pharmeuropa Scientic Notes are indexed in the PubMed database of the National Library of
Medicine, available online (www.ncbi.nlm.nih.gov).
These issues are included in the subscription to Pharmeuropa.
SCIENTIFIC NOTES 2006-1
SCIENTIFIC NOTES 2005-1
Pyrogen Testing of Lipidic Parenterals with a Novel

in vitro Test - Application of the IPT Based on
Cryopreserved Human Whole Blood
Few Bicyclic Acetals at Reducing End of LowMolecular-Weight Heparins: Might they Restrict
Specication of Pharmacopoeia?
Evaporative Light Scattering Detection (ELSD): a Tool

for Improved Quality Control of Drug Substances
The Control of Impurities in Chlortalidone

Using a Reversed-Phase Stationary Phase
In vitro Pyrogenicity of Gram-Positive Bacteria

Validation of the Kit using Fresh Human Whole Blood
Factor VIII Test in Reference Preparations:

Compensation for Different Dilutions
Possible Alternative to European Pharmacopoeias

Method of Analysis Test for Fc Function of
Immunoglobulin (2.7.9) by using Tetanus Toxoid as
Antigen
The Control of Impurities in Amitriptyline

Hydrochloride Using a Reversed-Phase
Hybrid Stationary Phase
Large Variation in the Ovalbumin Content in Six

European Inuenza Vaccines
A Precise Colour Determination Method for Tablets an Application of Instrumental Colour Measurement
in the Pharmaceutical Development
Effect of Temperature on Plasma Freezing under

Industrial Conditions
Development of an in vivo Test Procedure for the Ease

of Breaking of Scored Tablets
Isoform Number I - a New Tool to Evaluate the Quality

of Erythropoeitin
Chromogenic Assay of Human Coagulation Factor VIII:

Statistical Comparison of 2 Working Dilution
Procedures
Viral Safety Evaluation of Biopharmaceuticals and

Homoeopathic Preparations of Human or Animal Impurity Profile of Amino Acids?
Origin
Batch Variability of Bacitracin: HPLC versus MEKC
The Use of a Two-way Analysis of Variance to Compare
Quality Criteria of Homoeopathic Mother Tinctures:
the Methods for the Assay of Anhydrous Dibasic
Considerations Regarding Suitable Tests for
Calcium Phosphate in the European, Japanese and
Homoeopathic Monographs
United States Pharmacopoeias
Instructions for Authors
Instructions for Authors
Available (English only)
Available (English only)
For prices and ordering information please consult the catalogue on our website http://book.pheur.org
552
General Information
PHARMEUROPA BIO
These issues are included in the subscription to Pharmeuropa.
BIOLOGICALS 2006-1
Validation of in vitro Potency Assays for Tetanus
Immunoglobulin
Validation of a New ELISA Method for in vitro Potency
Assay of Hepatitis B-containing Vaccines
Establishment of the Human Coagulation Factor VII
Concentrate European Pharmacopoeia Biological
Reference Preparation Batch 1
Collaborative Study for the Establishment of
Replacement Batches for Somatropin CRS Batch 1
Collaborative Study to Establish Immunoglobulin BRP
Batch 3 and Human Immunoglobulin (Molecular Size)
BRP Batch 1
International Collaborative Study to Establish
Immunoglobulin (Anti-D Test) BRP Batch 1
Establishment of European Pharmacopoeia
Mycoplasma Reference Strains
Collaborative Study for the Validation of Serological
Methods for Potency Testing of Diphtheria Toxoid
Vaccine (Part 2)
Available soon (English only)
BIOLOGICALS 2005-1
Capillary Electrophoresis for the Control of Impurities

of rDNA Somatropin
Collaborative Study to Establish the Low-MolecularMass Heparin for Assay European Pharmacopoeia
Biological Reference Preparation (BSP060)
Available now (English only)
BIOLOGICALS 2003-2
Collaborative Studies for the Establishment of
Reference Substances for the Microbiological Assay of
Antibiotics
Collaborative Study for Establishment of a Global
Standard for the Potency Assay of Human Anti-D
Immunoglobulin
Collaborative Study for Establishment of a European
Pharmacopoeia Biological Reference Preparation (BRP)
For B19 Virus DNA Testing of Plasma Pools by Nucleic
Acid Amplication Technique
Vaccines: Part 1
Vaccines: Extended study: Correlation of Serology with
In Vivo Toxin Neutralisation
Establishment of European Pharmacopoeia Biological
Reference Preparations Batch 2 for rDNA Hepatitis B
vaccine (Method A and B)
Control of Clostridium Perfringens Vaccines by Means
of an Indirect Competitive ELISA for the Epsilon
Toxin Component Examination of the Assay by a
Collaborative Study
Collaborative Study to Establish a New Biological

Reference Preparation for Prekallikrein Activator
Collaborative Study for the Establishment of the Ph.
Eur. BRP Batch 1 for Anti-Vaccinia Immunoglobulin
Feasibility Study to Develop a Common in vitro
D-Antigen Assay for Inactivated Poliomyelitis Vaccines
Allergy Vaccines: a Need for Standardisation in Mass
Units of Major Allergen
Efficacy Demonstration of Tetanus Vaccines by Double
Antigen ELISA
International Symposium on Alternatives to Whole Cell
BIOLOGICALS 2003-1
Pertussis Vaccine Potency Assay
Collaborative Study for the Establishment of The
European Pharmacopoeia BRP Batch 1 for
BIOLOGICALS 2004-1
Diphtheria Toxin
Validation Study to Evaluate the Reproducibility of a
Candidate In Vitro Potency Assay of Newcastle Disease
European Pharmacopoeia BRP Batch 2 for Inactivated
Vaccines and to Establish the Suitability of a Candidate
Poliomyelitis Vaccine for In Vitro D Antigen Assay
Biological Reference Preparation
Collaborative Study for the Establishment of A Global
Establishment of Batch 4 of the Biological Reference
(WHO International / US/Ph. Eur.) Standard for the
Preparation (BRP) for Rabies Vaccine (Inactivated) for
Potency Assay of Human Anti-D Immunoglobulin
Veterinary Use
Feasibility Study to Evaluate the Correlation Between
Results of a Candidate In Vitro Assay and Established
Erythropoietin BRP Batch 2
In Vivo Assays for Potency Determination of Newcastle
Disease Vaccines
Somatropin and its Variants: Structural
Characterization and Methods of Analysis
For prices and ordering information please consult the catalogue on our website (http://book.pheur.org).
553
General Information
11th ANNUAL MEETING OF THE EUROPEAN

NETWORK OF OFFICIAL MEDICINES CONTROL
LABORATORIES (OMCLs)
Limassol, Cyprus, 9-12 May 2006
At the invitation of the European Directorate for the

Quality of Medicines (EDQM) of the Council of Europe,
almost 180 representatives from 32 countries* attended
the annual meeting of the European Network of OMCLs.
The meeting was hosted in Limassol by the State General
Laboratory and was opened by the Minister of Health of
the Republic of Cyprus, Dr. Andreas Th. Gavrielides. It
was organised to review the activities of the Network in
2005 and to discuss the work programme for the year to
come.
This meeting was the opportunity to bring together the
OMCLs of the General European Network in different
areas of interest (physico-chemical, pharmaceutical,
biological) to discuss and exchange viewpoints of
common interest in the field of controlling and testing
medicines by independent OMCLs.
The following matters were discussed.
The annual reports of activities of each laboratory,
highlighting key issues and results; in this context, a
preliminary discussion on the risk analysis approach
in defining the testing programme of the OMCLs was
held.
Results of tests on marketed medicines for human use
and veterinary use (MSS studies); discussion of the
current programme and adoption of the action plan
for 2006; these collaborative studies organised by the
EDQM serve as reliable indicators of the quality of
products on the European pharmaceutical market.
The progress report on the implementation of a
harmonised programme on Quality Assurance in the
different OMCLs including a review of the actions
in assistance and maintenance of QA throughout
the Network; the proficiency testing studies (PTS)
programme for the coming year in both physicochemical and biological fields, as well as the key
outcomes of the 2005 programme; the Network agreed
to consider making publicly available their relevant
guidelines and procedures as well as key outcomes.
The status of the OMCL inventory database project:
the future database for internal use within the OMCL
Network will host information about competences
available throughout the network and is planned
to become productive in 2007; the new computer
application intends to improve communication and

quick information exchange between the members of
the Network and aims to help to establish first contact
in case there is a need to exchange expertise within the
Network, initiate work sharing and build up further
mutual recognition of data.
Guidelines on general policies, such as the definition
of an OMCL, terms of reference of the Advisory Group
of the GEON, etc., and issues concerning the specific
activities of OMCLs, such as the further development
of qualification of equipment (for example, gas
chromatography); this is to update the established set
of specific guidelines.
Concerning the role of OMCLs in combating
counterfeiting, a procedure of key issues to take into
consideration as well as an information exchange
system with the Network; some OMCLs presented their
recent results and the strategies developed in this field
of activity; in particular, the wish to intensify the cooperation with industry was endorsed.
In the field of Official Control Authorities Batch Release
(OCABR), 1 new specific guideline for influenza vaccine
and 10 revised guidelines were all adopted. All these
guidelines will be published by the end of 2006, for
implementation by 1 January 2007, and will also be
electronically available on the EDQM website, as in
the past. Several internal documents and procedures
were also adopted. Additionally, 2 new and 2 revised
product-specific guidelines for vaccines were approved for
external consultation within the months to come.
A breakout session concerning MRP product testing
was held, in which the final report of the 2005 testing
program was presented. In total, 14 OMCLs actively
participated in the program and approximately 280
projects could be finalised (180 in 2004). The testing
group adopted a general document describing the
principle of the co-operation in post-marketing
surveillance of MRP products. A status report of the
MRP-product testing database project, which will be
finalised in 2007, was provided.
The testing of traditional Chinese medicines was also
discussed in a workshop of specialists to examine the
possible contribution of the OMCLs in this field of
activity.
* Austria, Belgium, Bosnia-Herzegovina, Bulgaria, Canada, Croatia, Czech Republic, Cyprus, Denmark, Estonia, Finland, France, Germany,
Greece, Hungary, Ireland, Italy, Latvia, Lithuania, Luxembourg, Netherlands, Norway, Poland, Portugal, Romania, Serbia-Montenegro, Slovakia,
Slovenia, Spain, Sweden, Switzerland, and United Kingdom.
554
General Information
PHARMACOPOEIAL DISCUSSION GROUP (PDG)

Yokohama, Japan, 5-8 June, 2006
The Pharmacopoeial Discussion Group [European
Pharmacopoeia (EP), Japanese Pharmacopoeia (JP),
United States Pharmacopeia (USP)] met in association
with the Expert Working Groups of the International
Conference on Harmonisation (ICH).
Hypromellose phthalate: this harmonised monograph
was signed off.
Methyl paraben, Ethyl paraben, Propyl paraben and
Butyl paraben: it was agreed to initiate the revision of
these monographs and the EP was nominated as the
coordinating pharmacopoeia.
Uniformity of delivered dose of inhalations: this general
chapter was added as a new topic and the EP was
nominated as the coordinating pharmacopoeia.
Revision of PDG Working Procedures: the PDG has
elaborated a revision proposal of the PDG Working
Procedures, last revised in July 2003, in order to reflect
the interaction with the ICH Q4B EWG. This proposal
changes the interpretation of Stages 6 & 7 and adds a
Stage 6C (Indication of harmonisation). The former
Stage 7 (Inter-regional implementation) was redefined
to be Inter-regional acceptance. The date of Stage 7
will be common to all 3 pharmacopoeias and will be
assigned after receiving formal notification of regulatory
acceptance from Q4B. The PDG Working Procedures will
be officially revised at the next PDG meeting.
These efforts will be beneficial for users of the
pharmacopoeias and facilitate the work of the Q4B EWG.
Interaction with ICH Q4B: in order to achieve common
regulatory acceptance of harmonised monographs and
general chapters in the ICH regions, 5 documentation
packages for harmonised general chapters have been

submitted by the PDG to the Q4B EWG for their
evaluation. These packages detail the harmonised
methods for dissolution, extractable volume, particulate
matter in parenterals, residue on ignition/sulphated
ash and the sterility test. The methods for residue on
ignition/sulphated ash and extractable volume have been
recognised to be interchangeable by the Q4B group; the
PDG is awaiting feedback on dissolution and particulate
matter in parenterals. A number of issues remain to
be resolved for the sterility test in order to achieve
regulatory acceptance. Additionally, packages for the PDG
harmonised texts on disintegration, uniformity of dosage
units and microbiological quality are in preparation for
submission to Q4B. On 7 June, 2006, the PDG held a
joint meeting with the Q4B EWG.
Industry Associations: a meeting with industry
associations from the 3 ICH regions was held on 6 June,
2006, to exchange information on PDG updates, progress
with the current work program and future harmonisation
needs. Industry associations were encouraged to play an
active role in the harmonisation process as important
stakeholders.
Excipients Councils: a meeting was held on 8 June, 2006,
with Tri-PEC (IPEC Americas, IPEC Europe, Japanese
Pharmaceutical Excipients Council) to discuss the work
program on harmonisation of excipient monographs.
Current issues include the policy for functionalityrelated characteristics, use of additives and processing
aids in excipients, co-processed excipients, impurities in
excipients, and the future of harmonisation.
The PDG will hold its next meeting on 23-26 October,
2006, in Chicago, USA.
________________________________________________________________________________
PHARMEUROPA, PHARMEUROPA BIO AND

PHARMEUROPA SCIENTIFIC NOTES ONLINE
Pharmeuropa, Pharmeuropa Bio and Pharmeuropa Scientic Notes Online are now available as a complementary
service for subscribers to the printed edition of Pharmeuropa, and will be offered for a trial period without an
additional fee for those who ordered the printed version of Pharmeuropa Vol. 18 (2006). All issues stretching back to
volume 10 (1998) are stored as Acrobat PDF les, and can be searched with a search engine identical to the one used
for the online versions of the European Pharmacopoeia and the Standard Terms.
A username and a password are required to access Pharmeuropa Online, and instructions for creating these using the
EDQM Certicate of Authenticity can be found on the inside-front cover of Pharmeuropa 18.1.
555
General Information
GUIDE TO THE PREPARATION, USE AND QUALITY

ASSURANCE OF BLOOD COMPONENTS - 12th Edition (2006)
In the absence of substitutes, the use of blood components remains essential in therapy. This guide contains a
compendium of measures designed to ensure the safety, efcacy and quality of blood components and is particularly
intended for all those working in blood transfusion services. In accordance with the approach recommended by
the Council of Europe in this eld, it is based on the premise of voluntary, non-remunerated blood donation. It
describes the different blood components and gives information on their clinical indications and possible side effects.
This guide continues to be the gold standard for blood transfusion services and forms the basis for many national
guidelines in Europe and around the world. For example, in 2000 Australia mandated the guide in its standard for
blood components. During the elaboration of this 12th Edition, the Council of Europe and the European Commission
have worked closely together to ensure that the requirements set under Article 29 of the European Union Directive
2002/98/EC are compatible with those of this guide. Where necessary, chapters have been revised to take into account
the new technological advances. The Guide to the preparation, use and quality assurance of blood components will
be of interest to blood transfusion centres, legislators, health personnel and to all those working in the eld of blood
transfusion.
The European Pharmacopoeia monograph on human plasma for fractionation refers inter alia to the
recommendations made in this Guide.
The 12th Edition of the Guide can be obtained in English and French from:
Tel: +33 (0)3 88 41 25 81 - Fax: +33 (0)3 88 41 39 10
Questions and comments on the content should be sent directly to the division in charge:
Council of Europe, Health Division
Mr Karl-Friedrich Bopp, F-67075 Strasbourg Cedex, France.
Tel: +33 (0)3 88 41 22 14 - Fax: +33 (0)3 88 41 27 26
E-mail: karl-friedrich.bopp@coe.int
Council of Europe website: www.coe.int
Health and Ethics: www.coe.int/T/E/Social_Cohesion/Health
NEWS
EUROPEAN PHARMACOPOEIA
5th Edition of the list of Standard Terms (Dec. 2004)

Printed and online versions available
The present list of Standard Terms is a revised list that was drawn up
in response to a request from the European Commission. It covers
medicines for both human and veterinary use. These Standard Terms
are to be used in answering the questions in Module 1 (item 1.2 and
1.3) of the EU application form.
The publication contains 3 lists of Standard Terms:
the list of pharmaceutical forms,
the list of routes and/or methods of administration,
the list of containers, closures and administration devices.
The 5th Edition contains translations in 27 European languages. New
languages that have been added since 2002 are: Estonian, Latvian,
Lithuanian, Maltese and Romanian.
With every printed publication of the 5th Edition ordered, access is
granted FREE to the online version.
How to purchase
this publication
All EDQM publications may be
ordered quickly and easily using
our online bookshop:
http://book.pheur.org
Ordering online is entirely secure
and helps us to process your order
more efciently.
Other ways to order include:
by fax: 00 33 (0) 3 88 41 27 71
by telephone: 00 33 (0) 3 88 41 30 30
by e-mail: orders@pheur.org
by surface mail:
Council of Europe
226 avenue de Colmar BP 907
F- 67029 STRASBOURG Cedex 1, FRANCE
For more information, visit our internet site:

556
AGENDA 2006-2007
INTERNATIONAL CONFERENCES & SYMPOSIA

Requirements for Production and Control of Avian Inuenza Vaccines
19-20 October 2006, Strasbourg, France
***
TRAINING SESSIONS ON THE 5th EDITION
OF THE EUROPEAN PHARMACOPOEIA
CHEMICALS PRODUCTS
13-14 November 2006, Dublin, Ireland
***
NEW ANNOUNCEMENT!
New Frontiers in the Quality of Medicines

Early-bird registration deadline: 15/01/2007
***
2007 TRAINING SESSIONS
13-14 September 2007, Poland **

6-7 December 2007, London, United Kingdom
The EDQM is pleased to announce a new training programme on the European Pharmacopoeia 5th Edition. The
new programme aims to provide professionals with an in-depth and up-to-date knowledge of the most important
and practical aspects of the European Pharmacopoeia. New additions for 2006 include practical examples and case
studies, question and answer sessions giving you the opportunity to clarify any issues that may arise and the chance
to meet one-to-one with the speakers who work in a certain area, thus providing you with more meaningful and
worthwhile interactions.
Do not miss these opportunities to meet the EDQM/European Pharmacopoeia.
More information will be available on the EDQM website www.pheur.org and in the next issues of Pharmeuropa.
GENERAL CONDITIONS FOR REGISTRATION AT THE EDQM CONFERENCES

AND TRAINING SESSIONS
HOW TO REGISTER
Register as soon as possible: places are limited.
We recommend using a separate form for each participant. Please ll in the registration form* and return it to
the EDQM Public Relations Unit, by post, by fax or via the HelpDesk duly completed with the selected method of
payment.
NEW Online Registration: Online conference registration is now possible. To use the online registration form, just
click on the icon EDQM Events - Register online and follow the steps described.
REGISTRATION FEE
A special rate is given to permanent staff of national authorities, university, R&D public centres.
The registration fee is not subject to VAT and covers attendance at the lectures, working documents, lunches,
coffee breaks and the ofcial dinner.
The registration fee does not include your hotel accommodation costs (see hotel reservation form) and travel
expenses.
METHOD OF PAYMENT
You can make your payment: by bank transfer, by enclosed cheque or by credit card.
The relevant bank transfer information is given on the invoice.
CANCELLATION POLICY
One month before the event 80% of the registration fee will be refunded; no refunds will be given after this date.
However, registration may be transferred to another person at any time. In case of no show, the registration fee is
due.
* All information requested on the registration form is necessary and used only for the organisation of the seminar.
** Date to be conrmed
557
558
2006 EDQM SYMPOSIUM: STRASBOURG

REQUIREMENTS FOR PRODUCTION AND CONTROL OF
AVIAN INFLUENZA VACCINES
19-20 OCTOBER 2006
Duration: 1,5 days, Working language: English
***
PROGRAMME
Strasbourg, 19-20 October 2006, Holiday Inn Hotel
REQUIREMENTS FOR PRODUCTION AND CONTROL OF AVIAN INFLUENZA VACCINES
THURSDAY 19 OCTOBER 2006

8:00-9:00
Registration
9:00-9:10
OPENING SESSION
Opening remarks
Dr Michael Morris, Chair of the European Pharmacopoeia Commission
9:10-9:30
Expectations regarding requirements for avian influenza vaccines, in

particular for immunogenicity and potency testing
SESSION I: GENERAL OVERVIEWS

Moderator: Dr Maria Tollis,
Director of Research - Istituto Superiore di Sanita, Roma (I)
9:30-10:00
Overview on avian influenza infections

Dr Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezie (I)
10:00-10:30
Avian influenza vaccine technologies and laboratory methods for assessing

protection
Dr David Swayne, US Department of Agriculture (USA)
10:30-11:00
Coffee break
11:00-11:20
Rapid molecular diagnostic tools for avian influenza

Dr David Suarez, US Department of Agriculture (USA)
11:20-12:00
The control of avian influenza in poultry and the DIVA (Differentiating Infected
from Vaccinated Animals) system
Dr Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezie (I)
12:00-12:20
Control of avian influenza in Italy: From stamping out to emergency and to

prophylactic vaccination
Dr Stefano Marangon, Istituto Zooprofilattico Sperimentale delle Venezie (I)
12:20-12:40
Preventive vaccination campaign in France

Dr Vronique Jestin, Afssa (F)
12:40-14:00
Lunch Break
SESSION II: SPECIFIC TOPICS ON AVIAN INFLUENZA VACCINES

Moderator: Dr Lukas Bruckner
Chairman of Group of Experts N 15V on Veterinary vaccines of the European Pharmacopoeia
Strasbourg, 19-20 October 2006, Holiday Inn Hotel
REQUIREMENTS FOR PRODUCTION AND CONTROL OF AVIAN INFLUENZA VACCINES
14:00-14:20
Avian influenza: The international veterinary response

Dr Christianne Bruschke, OIE (F)
14:20-14:40
Measures to promote the availability of vaccines against avian influenza in the

European Union
Dr David MacKay, EMEA (GB)
14:40-15:00
CVMP Guideline on avian influenza vaccines

Dr Ralph Woodland, Veterinary Medicines Directorate (GB)
15:00-15:20
Quality control and standardisation

Dr Hok Oei, CIDC-Lelystad (NL)
15:20-15:40
Questions & Answers
15:40-16:10
Coffee break
16:10-16:30
Effect of vaccination on avian influenza virus transmission

Dr Jeanet van der Goot, CICD-Lelystad (NL)
16:30-17:30
16:30
General overview of the production of avian influenza vaccines

. Avian influenza vaccines - Fort Dodge Animal Health initiatives
Dr Mahesh Kumar, Fort Dodge Animal Health (USA)
. Production of avian influenza vaccines world-wide
Dr Hans de Smit, Intervet International (NL)
. Avian influenza vaccines: a panel of solutions
Dr Franois-Xavier Le Gros, Mrial (F)
16:50
17:10
17:30-18:00
Round table discussion

With the participation of industry associations, EMEA, regulatory authorities
FRIDAY 20 OCTOBER 2006
SESSION III: MONOGRAPH SPECIFICATIONS

Moderator: Mr Peter Castle
Secretary of the European Pharmacopoeia Commission, EDQM/Council of Europe
9:00-9:20
What specifications are needed? Requirements for immunogenicity and potency

tests?
Dr Riks Maas, CIDC-Lelystad (NL)
9:20-9:40
Regulatory assessment and expectations

Dr Carmen Jungbaeck, PEI (D)
9:40-10:00
Questions & Answers
10:00-10:30
Coffee break
10:30-11:30
10:30
Manufacturers approach to specifications

. Special considerations for an emergency (temporary?) problem
Dr Mahesh Kumar, Fort Dodge Animal Health (USA)
. The release specifications of vaccines against avian influenza worldwide
Dr Hans de Smit, Intervet (NL)
. The complexity of specifications of avian influenza vaccines
Dr Franois-Xavier Le Gros, Merial (F)
10:50
11:10
11:30-12:00
Round-table discussion
With the participation of industry associations, EMEA, regulatory authorities
12:00
Final addresses and Closure of the meeting
13:30-16:00
DEBRIEFING MEETING
This meeting is open to national authority participants only.
559
19-20 October 2006 Location: Holiday Inn Hotel Strasbourg, France
REGISTRATION FORM:
SYMPOSIUM AVIAN INFLUENZA
Please complete and send this form to the Public Relations Unit: By fax: +33 3 88 41 27 71, or via the EDQM
HELPDESK http://www.pheur.org/site/page_521.php: Go to the topic Events, Public Relations & Library and
read the question May I send my registration form via the EDQMs internet site for further instructions, or
alternatively register ONLINE, click on Events Register Online and select the name of the event.
REGISTRATION DETAILS
DATE OF REGISTRATION (DD/MM/YY) :
_ __ __ _
REGISTRATION FEE (*See general conditions)

650 *
or
450 *
REGISTRATION FEE ONLINE: If you register online, you benefit from a reduced registration fee of
600 * for industry and 400 * for National Authorities, University. (*See general conditions)
MEETING FOR NATIONAL AUTHORITIES DELEGATES ONLY
Yes, I shall be attending No, I shall not
LUNCH ON 20 OCT
Yes, I shall be dining in the hotel
No, I shall not be dining
PARTICIPANT DETAILS Please complete one form per participant

Title (Dr., Mr, Mrs, Ms, )
First Name
Family Name
Company/Institution
Address for
Correspondence
Postcode
Town
Country
Telephone
Fax
E-mail
AREA OF ACTIVITY/
OCCUPATION:
Regulatory authority:
University
Manufacturer of raw material

Manufacturer of medicines for human use for veterinary use
QA
QC
R&D
Regulatory affairs
Licensing
Pharmacopoeias
Inspection
OMCL
Other (please specify)__________________________________________________________________
PAYMENT (NEW)
Following receipt of your registration form, we will send you an invoice. Please note that we must receive payment
before the conference takes place. Details of payment methods will be outlined on the invoice. However, you will
be able to settle your invoice by:
1. PERSONAL OR COMPANY CHEQUE made payable to Council of Europe/EDQM
2. BANK TRANSFER
3. CREDIT CARD
DETAILS FOR INVOICING PURPOSES (if different from participant details)
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Address
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Country
VAT Number (EU only)
Contact Name
Telephone
Fax
E-mail
PO Number/ Reference
CANCELLATION CHARGES: I have read and accept the cancellation terms as stated on our website.
Date
Signature
FOR MORE INFORMATION ABOUT CONFERENCES ORGANISED BY THE EDQM
PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
560
50 single rooms for Wednesday 18 and Thursday 19 October 2006,

15 single rooms for Friday 20 and Saturday 21 October 2006
Contact reservations: HOLIDAY INN, 20 Place de Bordeaux, 67000 Strasbourg, France,
Contact: Fabrice Beyer, Tel +33 (0)3 88 37 80 95, Fax +33 (0)3 88 25 61 64,
E-mail: fabrice.beyer@alliance-hospitality.com
General information: The quotas of rooms reserved will be available until 5 September 2006.
After this date the availability and the negotiated prices will not be guaranteed. The
rooms should be reserved individually by each participant on a first come, first
served basis. Cancellation policy: before 20 September 2006 at no further cost.
Cancellation after 18:00 pm on 20 September 2006 and in case of no show, the
full accommodation amount will be charged to the credit card provided.
Location: Strasbourg, France
Global reservation:
19-20 October 2006
HOTEL REGISTRATION FORM:
AVIAN INFLUENZA SYMPOSIUM

19-20 OCTOBER 2006, STRASBOURG
HOLIDAY INN HOTEL RESERVATION FORM
FAX: +33 (0) 3 88 25 61 64
To be filled in and sent by fax before the 5 September 2006
Participant:
Forename
Company/ employer
Address
City
Postal Code
Country
E-mail
Tel N
Fax N
Arrival day/hour /..... Departure day/hour /..

Please indicate the selected nights by a circle
Hotel reservation:
HOLIDAY INN
Surname
NIGHT
18/10/2006
YES
150 *
168 *
* Taxes and breakfast inclusive
SINGLE
Method of payment:
Credit card number:
DBLE
Credit card:
Visa
NIGHT
19/10/2006
YES
NIGHT
20/10/2006
YES
NIGHT
21/10/2006
YES
EuroCard / MasterCard Amex

Expiry date
Cardholder: ______________________________________________________
I authorise the HOLIDAY INN HOTEL to charge against my credit card the amount equivalent to
one night in order to block my reservation for the duration of the seminar.
Date: _____________________ Signature: ________________________________________
HOTEL CONFIRMATION RESERVATION N:___________ Signature:________________
561
30 single rooms have been reserved for Wednesday 18 October, 50 single rooms for
Thursday 19 and Friday 20 October 2006 and 10 single rooms for Saturday 21 October
Contact reservations: BEST WESTERN - MONOPOLE METROPOLE HOTEL: 16 rue Kuhn,
67000 Strasbourg, France; Contact: Vronique Tel +33 (0)3 88 14 39 14,
Fax +33 (0) 3 88 32 82 55, E-mail: infos@bw-monopole.com.
General information: The quotas of rooms reserved will be available until 25 September 2006.
rooms should be reserved individually by each participant on a first come, first served
basis. Cancellation policy: before 25 September 2006 at no further cost, after this date
and in case of no show one night will be charged on your credit card.
Global reservation:
19-20 October 2006

MONOPOLE METROPOLE HOTEL RESERVATION FORM
FAX: +33 (0) 3 88 32 82 55
Participant:
Forename
Company/ employer
Address
City
Postal Code
Country
E-mail
Tel N
Fax N

Hotel reservation:
BEST
WESTERN
MONOPOLE
METROPOLE
Surname
SINGLE
DBLE
80 *
95 *
NIGHT
18/10/2006
YES / NO
NIGHT
19/10/2006
YES / NO
NIGHT
20/10/2006
YES / NO
NIGHT
21/10/2006
YES / NO
Method of payment:
Credit card number:
Credit card:
Visa

Expiry date
Cardholder: ______________________________________________________
I authorise the BEST WESTERN MONOPOLE METROPOLE HOTEL to charge against my
credit card the amount equivalent to one night in order to block my reservation for the duration of the
seminar.
Date: _____________________ Signature: ________________________________________
562
Global reservation:
19-20 October 2006

MERCURE ST JEAN HOTEL RESERVATION FORM
FAX: +33 (0) 3 88 23 05 39
Contact reservations:
General information:
Participant:
Surname
Forename
Company/ employer
Address
City
Postal Code
Country
E-mail
Tel N
Fax N

Hotel reservation:
MERCURE
ST JEAN
20 single rooms have been reserved for Wednesday 18 October, 40 single rooms for
Thursday 19 October and 20 single rooms for Friday 20 October and Saturday 21
October 2006
MERCURE ST JEAN HOTEL: 3 rue du Maire Kuss, 67000 Strasbourg, France
Contact: Tel +33 (0)3 88 32 80 80, Fax +33 (0)3 88 23 05 39;
E-mail: h1813@accor.com
The quotas of rooms reserved will be available until 20 September 2006.
rooms should be reserved individually by each participant on a first come, first
served basis. Cancellation policy: before 20 September 2006 at no further cost, after
this date and in case of no show one night will be charged on your credit card.
NIGHT
18/10/2006
YES / NO
102 *
92 *
SINGLE
Method of payment:
Credit card number:
DBLE
Credit card:
Visa
NIGHT
NIGHT
NIGHT
19/10/2006 20/10/2006 21/10/2006
YES / NO YES / NO YES / NO

Expiry date
Cardholder: ______________________________________________________
I authorise the MERCURE ST JEAN HOTEL to charge against my credit card the amount
equivalent to one night in order to block my reservation for the duration of the seminar.
Date: _____________________ Signature: ________________________________________
563
2006 TRAINING SESSION

HOW TO USE THE EUROPEAN PHARMACOPOEIA 5TH EDITION
CHEMICAL PRODUCTS
13-14 NOVEMBER 2006
Duration: 1,5 days, Location: Clontarf Castle, Dublin, Ireland
Working language: English
Location: Clontarf Castle, Dublin, Ireland
13-14 November 2006
PROGRAMME: TRAINING SESSION 5TH EDITION
***
PROGRAMME
MONDAY 13 NOVEMBER 2006
8:30 - 9:15 Registration
Opening remarks and general introduction
9:15 - 9:30 Dr Michael Morris, Chair of the European Pharmacopoeia Commission
The European Regulatory System: Interactive and complementary relationship between EU/European
Commission/European Council/EMEA/EDQM/Council of Europe
The place and role of the European Pharmacopoeia
9:30 - 9:50 Dr Michael Morris, Chair of the European Pharmacopoeia Commission
General organisation of the EDQM. How the European Pharmacopoeia is elaborated
The relationship between the European Pharmacopoeia Commission and the EDQM
9:50 - 10:10 Mr Peter Castle, Secretary to the European Pharmacopoeia Commission, EDQM, Council of
Europe
How to use the European Pharmacopoeia: Understanding the general notices, general chapters,
general monographs and monographs on dosage forms. How to use them in practice.
10:10 - 10:35 Mr Peter Castle
10:35 - 10:45 Discussion with the speakers panel
10:45 - 11:15 Coffee break
How to use the general monograph Substances for pharmaceutical use to control impurities and decision
trees for impurities. How to interpret chromatograms and list of impurities.
11:15 - 11:35 Dr Michael Wierer, Dept of the European Pharmacopoeia, EDQM, Council of Europe
Cases studies of specific monographs (active substances and excipients), use of reference standards
11:35 - 11:55 Dr Michael Wierer
12:30 - 13:45 Lunch break
Current progress in the field of international harmonisation
Regulatory interchangeability between FDA (US) / EU / *MHLW (J)
13:45 - 14:10 Dr Michael Morris
14:10 - 14:25 Discussion
Identification of the need and uses of a reference standard. Overview of the policy and process used to
establish of a reference standard
*Ministry of Health, Labour and Welfare
564
How we build a monograph

13-14 November 2006
PROGRAMME: TRAINING SESSION 5TH EDITION
How to successfully submit a monograph?

ONE TO ONE CONSULTATIONS
16:30 - 17:30 Topics under which a consultation can be arranged: General questions on the EDQM,
the European regulatory framework and harmonisation; Technical questions on PhEur monographs and
texts; Reference standards.
TUESDAY 14 NOVEMBER 2006 (MORNING ONLY)
How to use the European Pharmacopoeia in your daily laboratory activity. Case study. Verification
and/or validation of test procedures. System suitability requirements. Reagent and reference
standards. Alternative methods.
The European Pharmacopoeia Publications (printed and electronic publications)
9:30 - 10:00 Dr Hans-Joachim Bigalke, Publications & Multimedia Dept, EDQM, Council of Europe
EDQM Internet sites: Features which will help you conduct your regulatory surveillance. How to
make the best use of the online services, specialised databases and the new users support: the
HELPDESK.
10:00 - 10:30 Ms Fiona Gilchrist, Public Relations Unit, EDQM, Council of Europe
10:30 - 10:45 Discussion with the panel of speakers
General considerations on the Certification procedure and Inspections
Origin, scope, how it works, who is involved
11:15 11:35 Mr Peter Castle
How to apply for a Certificate
Content of the dossier for chemical substances
Comments on the main deficiencies found in dossiers
Revisions and renewals
11:35 - 12:45 Ms Fiona McLeod, Scientific Officer, Certification Division, EDQM, Council of Europe
13:00 Final addresses and Closure of the meeting
13:00 - 14:15 Lunch break
ONE TO ONE CONSULTATIONS
14:15 - 15:15
Topics covered: General questions on the EDQM, the European regulatory framework and harmonisation;
Technical questions on PhEur monographs and texts; Certification procedure; Publications and services;
Reference standards; Electronic version of the European Pharmacopoeia
During coffee breaks, the electronic/online version of the European Pharmacopoeia will be set up and
demonstrated to participants interested in learning more about this version.
FOR MORE INFORMATION PLEASE VISIT THE WEBSITE : http://www.pheur.org
565
13-14 November 2006 Location: Clontarf Castle, Dublin, Ireland
REGISTRATION FORM:
TRAINING SESSION 5th EDITION
REGISTRATION DETAILS
DATE OF REGISTRATION (DD/MM/YY) :
_ __ __ _

650 *
or
300 *
REGISTRATION FEE ONLINE: If you register online, you benefit from a reduced registration fee of
600 * for industry and 250 * for National Authorities, University. (*See general conditions)
ONE TO ONE MEETING
Yes, I am interested in reserving a meeting
No, I am not interested
Which topic(s)? EU Regulations Monographs, revisions Publications Certification
Internet
LUNCH ON 14 NOV
Yes, I shall be dining in the hotel
No, I shall not be dining

First Name
Family Name
Company/Institution
Address for
Correspondence
Postcode
Town
Country
Telephone
Fax
E-mail
AREA OF ACTIVITY/ Manufacturer of raw material
Manufacturer of medicines
OCCUPATION:
QA
QC
Licensing
Inspection
OMCL
Other (please specify)
for human use for veterinary use

R&D
Regulatory affairs
Pharmacopoeias
University
PAYMENT (NEW)
2. BANK TRANSFER
3. CREDIT CARD
Company/Institution
Address
Postcode
Town
Country
Contact Name
Telephone
Fax
E-mail
Date
Signature
FOR MORE INFORMATION PLEASE VISIT REGULARLY THE WEBSITE : http://www.pheur.org
566
HOTEL RESERVATION FORM

2006 TRAINING COURSE DUBLIN: 13-14 NOVEMBER 2006
Location: Clontarf Castle Hotel, Dublin, Ireland
13-14 November 2006

CLONTARF CASTLE HOTEL RESERVATION FORM
Fax: +353 1 833 0418
To be filled in and sent by fax before the 13 August 2006
Global reservation:
25 rooms reserved from Sunday 12th November to Monday 13th included.
Contact reservations:
CLONTARF CASTLE HOTEL, Castle Avenue, Clontarf, Dublin 3, Ireland
Contact: Ms Sabrina McKenna; Tel: + 353 1 883 2321; E-mail: info@clontarfcastle.ie
The quotas of rooms reserved will be available until 13 August 2006.
General information:
After this date the availability and the negotiated prices will not be guaranteed. The rooms should be reserved
individually by each participant and are available on a first come, first served basis.
Cancellation policy: One week before (before 5th November), at no further cost, after this date and in the case
of no show the full amount will be charged on the credit card provided.
Participant:
First name:
Surname:
Address:
Company / Employer:
Postcode / County:
Town / City:
Country:
Tel:
Fax:
Email:
Hotel reservation:
Arrival day/hour
Departure day/hour
Please indicate the room and nights required by a circle.
CLONTARF
NIGHT
NIGHT
SINGLE
DOUBLE/ TWIN
CASTLE
12/11/2006
13/11/2006
OCCUPANCY
OCCUPANCY
HOTEL
145 EUR*
165 EUR*
YES
YES
* Taxes, service charge and breakfast inclusive
Method of payment: Credit card: Visa EuroCard/ MasterCard Amex Diners Card
Credit card number:
Expiry date
Cardholder: ______________________________________________________
I authorise the CLONTARF CASTLE HOTEL to charge against my credit card the amount
equivalent to one night in order to block my reservation for the duration of the training session.
Date:
Signature:
HOTEL CONFIRMATION RESERVATION N: ____________ Signature: ______________________
567
ONE TO ONE CONSULTATION QUESTION FORM

13-14 November 2006 Location: Dublin, Ireland
ONE-TO-ONE RESERVATION FORM:
2006 TRAINING SESSION: DUBLIN

EUROPEAN PHARMACOPOEIA 5TH EDITION CHEMICALS
13-14 NOVEMBER 2006
If you would like to register for a one-to-one consultation (15 minutes) with a member of the EDQM
scientific team during the training course, please complete this consultation request form and send it
to the EDQM by fax to +33 3 88 41 27 71 or e-mail via the EDQM Helpdesk on the website:
Priority will be given to those who book in advance.
PARTICIPANT DETAILS
Title (Dr., Mr, Mrs, Ms)
First Name
Family Name
Company/Institution
E-mail
The time slots available for one-to-one consultations will be limited. The EDQM shall do all it can to
accommodate all interested participants.
If it is on a specific application, please mention the reference number
Your question:
Date
Signature
FOR MORE INFORMATION ABOUT TRAINING SESSIONS AND CONFERENCES ORGANISED BY THE EDQM
568
NEW FRONTIERS IN THE QUALITY OF MEDICINES

13-15 June 2007 - Location: Council of Europe, Strasbourg, France
DRAFT PROGRAMME: New Frontiers in the Quality of Medicines
International Conference organised by the

European Directorate for the Quality of Medicines (EDQM), Council of Europe

***
Duration: 2,5 days, Working language: English
DRAFT PROGRAMME
Registration
WEDNESDAY 13TH JUNE 2007

13h45-18h30
OPENING SESSION
Welcome addresses:
The Secretary General of the Council of Europe
PLENARY SESSION
International Co-operation and Synergy in Quality Standards
Vision from Europe
Commission of the European Communities
European Agency for the Evaluation of Medicinal Products (EMEA)
The Heads of Agencies (HMA)
The European Pharmacopoeia Dynamics: the 6th Edition - New concepts
Coffee break
Perspectives for co-operation
Partnership with the Regulatory Authorities and the PDG Pharmacopoeias
Food and Drug Administration (FDA), United States of America
Health Canada, Canada
State Food and Drug Administration (SFDA), Peoples Republic of China
Central Drug Authority, India
ANVISA/Brazilian Pharmacopoeia, Brazil
Russian Health and Pharmaceutical Authorities, The Russian Federation
The United States Pharmacopoeia (USP), United States of America
The Japanese Pharmacopoeia (JP), Japan
Partnership with Industry
European Federation of Pharmaceutical Industries and Associations (EFPIA)
Discussion
Closure of meeting
569
WORKSHOPS & ONE-TO-ONE SESSIONS
Thursday 14th June 2007

8h30-12h15 & 14h15-18h00
Workshops
Certification
Anti-counterfeiting of medicines
European Biological Standardisation Programme (BSP): Focus on new
vaccines
Excipients
Herbals (1): Implementation of the new legislative framework
Herbals (2): Traditional Chinese Medicines
Homoeopathy
International harmonisation: Microbiological techniques
New concepts in quality and new methods (2 workshops)
Official Medicines Control Laboratories (OMCL)
Pharmacopoeial Discussion Group (PDG) and the ICH Q4B
Reference Standards
Process Analytical Technology (PAT)
Batch Release of Human Biologicals: Impact on Safety
Biosimilar Products: EDQM contribution for setting standards
Radiopharmaceuticals (To be confirmed)
One-to-One Sessions
Certification
Reference standards and preparations (CRS/BRP)
Publications
International harmonisation ICH
Herbals
Biotechnology
Closure
Conference Dinner
570
Friday 15th June 2007

8h30-12h45
Recommendations from the workshops
PLENARY SESSION
Action Plan for the Future:

Recommendations from the Industry
European Federation of Pharmaceutical Industries and Associations (EFPIA)
European Generic Medicines Association (EGA)
Association Europenne des Spcialits Grand Public (AESGP)
European Chemical Industry Council, Active Pharmaceutical Ingredients Committee
(CEFIC/APIC)
International Pharmaceutical Excipients Council (IPEC-Europe)
The Pharmaceutical Research and Manufacturers of America (PhRMA)
Coffee break
Working towards regulatory acceptability (RAAPAC): Pharmacopoeial
Discussion Group (PDG) & ICH
Final Round-table discussion with the Regulatory Authorities, the Global

Co-operation Group and the World Health Organisation (WHO)
Final Conclusions
571
REGISTRATION DETAILS DATE OF REGISTRATION (DD/MM/YY) : _

__ __ _
850 * or 450 *
REGISTRATION FEE ONLINE: If you register online and/or before 15 January 2007, you benefit from a reduced
registration fee
13-15 June 2007 Location: Strasbourg, France
REGISTRATION FORM: CONFERENCE ON NEW FRONTIERS IN THE QUALITY OF MEDICINES
WORKSHOPS (Choose 4 workshops or 3 workshops and 1 one-to-one meeting)

8:30-10:00
New concepts (1)
BSP
Herbal (1)
Batch Release
10:45-12:15
New concepts (2)
PAT
Herbals (2)
Certification
14:15-15:45
Microbio. techniques Excipients
Homoeopathy
OMCL
16:30-18:00
PDG/ICH
Biosimilar prod Ref. Standards Anti-Counterfeiting
ONE TO ONE MEETINGS: Certification CRS/BRP Publications ICH Herbals Biotechnology

First Name
Family Name
Company/Institution
Address for
Correspondence
Postcode
Town
Country
Telephone
Fax
E-mail
AREA OF ACTIVITY/
OCCUPATION:
University
Manufacturer of raw material

Manufacturer of medicines for human use for veterinary use
QA
QC
R&D
Regulatory affairs
Licensing
Pharmacopoeias
Inspection
OMCL
Other (please specify)__________________________________________________________________
PAYMENT
2. BANK TRANSFER
3. CREDIT CARD
Company/Institution
Address
Postcode
Town
Country
Contact Name
Telephone
Fax
E-mail
Date
Signature
FOR MORE INFORMATION ABOUT CONFERENCES ORGANISED BY THE EDQM
572
10 single rooms have been reserved for Tuesday 12 June 2007, 100 single
rooms for Wednesday 13 and Thursday 14 June 2007, 20 single rooms for
Friday 15 June 2007 and 10 single rooms for Saturday 16 June 2007
Contact reservations: HILTON STRASBOURG, Avenue Herrenschmidt, 67000 Strasbourg, France,
Tel +33 (0)3 88 37 10 10, Fax +33 (0)3 88 25 55 03,
E-mail: infoseminaire@hilton-strasbourg.com
General information: The quotas of rooms reserved will be available until 10 April 2007. After this
date the availability and the negotiated prices will not be guaranteed. The rooms
should be reserved individually by each participant on a first come, first served
basis.
Cancellation policy: Before 1st June at no further cost. In the event of a cancellation after this date or
a no-show, one night will be charged to the credit card provided. Any
cancellation of a reservation of three or more nights after this date or a no
show, the full accommodation amount will be charged.
Global reservation:
13-15 JUNE 2007 Location: Strasbourg, France
INTERNATIONAL CONFERENCE
13-15 JUNE 2007, STRASBOURG
HILTON STRASBOURG HOTEL RESERVATION FORM
FAX: +33 (0) 3 88 25 55 03
To be filled in and sent by fax before the 10 April 2007
Surname
Participant:
Forename
Company/ employer
Address
City
Postal Code
Country
E-mail
Tel N
Fax N
Hotel reservation:

HILTON
STRASBOURG
NIGHT
NIGHT
NIGHT
NIGHT
NIGHT
12/06/2007 13/06/2007 14/06/2007 15/06/2007 16/06/2007
YES
YES
YES
YES
YES
160 *
170 *
SINGLE
Method of payment:
DBLE
Credit card:
Credit card number:
Visa
EuroCard / MasterCard
Amex
Expiry date
Cardholder: ______________________________________________________
I authorise the HILTON STRASBOURG HOTEL to charge against my credit card the amount
equivalent to one night in order to block my reservation for the duration of the seminar.
Date: _____________________ Signature: ___________________________________________
573
80 single rooms for Wednesday 13 and Thursday 14 June 2007,

20 single rooms for Friday 15 June and Saturday 16 June 2007
Contact reservations: HOLIDAY INN, 20 Place de Bordeaux, 67000 Strasbourg, France,
Contact: Fabrice Beyer, Tel +33 (0)3 88 37 80 29, Fax +33 (0)3 88 25 61 64,
E-mail: fabrice.beyer@alliance-hospitality.com
General information: The quotas of rooms reserved will be available until 13 April 2007. After this
date the availability and the negotiated prices will not be guaranteed. The rooms
should be reserved individually by each participant on a first come, first served
basis.
Cancellation policy: Before 13 May 2007 at no further cost. Cancellation after 18:00 pm on 13 May
and in case of no show, the full accommodation amount will be charged to the
credit card provided.
Global reservation:
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Readers Tribune
Readers Tribune
The Secretariat of the European Pharmacopoeia recalls that articles reect the authors opinions. In order that all
opinions are taken into account, concerned users and readers of Pharmeuropa are invited to send their comments
to the Secretariat. Readers are reminded that details regarding contributions made to Pharmeuropa are cited on the
rear cover of this issue.
After we published Herbal reference standards by Dr K. Helliwell in Pharmeuropa 18.2 (April 2006), we received
comments from readers. We have decided to publish the following letter.
Comments from Dr F. Lang, Dr F. Waimer*

From our viewpoint as a phytopharmaceuticals
manufacturer, Dr. K. Helliwells article is indeed welcome.
The classication into
herbal drug reference standards
herbal drug preparation reference standards and
chemical reference standards
provides clarity and transparency with regard to
nomenclature and systematics.
The recent trend to introduce chemical reference
standards in conjunction with monographs on medicinal
plants and their preparations appears to be expedient
from all points of view. Dened reference substances
are just as essential as analytical procedures for
establishing uniform standards in phytoanalysis. They
enable the quality of herbal medicinal substances to be
determined in a comparable and unequivocal manner.
For this reason, it is appropriate that the European
Pharmacopoeia should give uniform reference substances
in addition to uniform methods and specications.
In the following, we would like to make a number of
comments, particularly with regard to herbal drug
preparation reference standards and chemical reference
standards.
1. BASIC REMARKS
Primary standards and working standards
In pharmaceutical practice in general, and in
phytoanalysis, so-called primary reference standards are
normally used. If necessary, secondary working standards
were derived from those.
Primary reference standards for chemically-dened
drugs are usually pure substances which are produced
synthetically and which are of high purity (usually
> 99 %). Natural substances, in contrast, are mostly
obtained from the respective plant material by means
of complex enrichment procedures (isolation) and only
rarely via synthesis.
Following elaborate concentration stages, content and
degree of purity are determined by a combination of
analytical procedures.
A particular problem regarding reference substances
obtained by isolation is that the chemical structure of
potential contamination products may be unknown. A
specic quantitative assay of potential impurities (as with

synthetic chemical substances) is therefore not possible.
Usually, attempts are made to produce high-purity
primary reference standards with contents of over 99 %.
The primary reference standards are extremely expensive
and should only be used sparingly. The majority of
primary reference standards for natural substances are
made available commercially by a number of specialised
manufacturers, in some cases with data on extraction
and characterisation of the substances as required for
registration procedures.
In practice, secondary working standards are often
derived in the respective laboratories using the
analytical methods in operation in those laboratories.
These standards are then available in larger quantities.
Normally, extracts from the own production are used as
working standards. During calibration with such working
standards, the assay values obtained must be identical to
those found during calibration using the original primary
reference substances. This is not a simple task, as the
determination of the quantity of a component (e.g. in an
extract) depends on the rigour of the analytical method.
As far as possible, the pattern of peaks for the reference
extract should correspond to that of the sample, as
otherwise peak distortions may result.
The reference extract should also be prepared using
the same extraction agent and the same extraction
procedure as those employed for the extract to be
analysed. Even slight changes in peak resolution within
a single laboratory, or between laboratories, may lead
to discrepancies despite the consistency of the methods
used.
Furthermore, the fact that the reference substance in
the working standard is only present in small amounts
and that the content may change due to instability of the
reference substance itself or of other substances in the
multi-component extract should be taken into account.
The derived content of reference substance in the extract
should therefore be checked regularly against a primary
standard.
Moreover, the fact that many extracts are highly
hygroscopic must also be taken into consideration:
water content or loss on drying should therefore also be
checked frequently.
* Schwabe Phytopharmaceuticals
577
Readers Tribune
Consequently, the assay and content specications for a

working standard/extract will depend on the respective
analytical laboratory, even when the same analytical
procedure is used.
Secondary working standards are therefore usually
derived by each laboratory itself and not supplied by
commercial manufacturers.
The introduction of HDPRS into the European
Pharmacopoeia would result in reference extracts, which
have so far only been used as internal laboratory working
standards, being used quasi as primary standards. In view
of the problems cited above, the use of such standards
would lead to limitations with regard to the accuracy of
the results obtained and their comparability between the
different test institutions using these extracts.
We therefore propose that chemical reference substances
or herbal drug preparation reference standards should be
used in conjunction with the extract categories given in
the Ph. Eur. general monograph Extracts (0765).
assaying a typical representative of this group. Only

this substance is characterised as a reference substance
(e.g. avonoids calculated as rutin). In exceptional
cases, reference substances of non-related structure but
similar spectroscopic properties may also be used. The
calculation is then performed using response factors.
This should be continued in future, so that synthetic
substances, which are inexpensive and of reproducible
high purity, can still be used.
4. POTENTIAL PROBLEMS DUE TO THE
INTRODUCTION OF HDPRS AND CRS
The possibility that different values may occur during
practical analysis when the new pharmacopoeial
standards are used cannot be excluded.
Signicant differences are mainly to be expected for
HDPRS, for the reasons already given above.
The general monograph Extracts (0765) distinguishes

three types:
In order to avoid coming into conict with registered

values and specications, the newly available
pharmacopoeial standards should be comprehensively
checked with regard to user acceptance prior to their
introduction. The derivation of the content of HPDRS
from synthetic primary standards, as well as the origin,
the extraction agent and possibly other relevant data
(residual solvents, hygroscopic characteristics, etc.)
should be made available.
standardised, quantied and other extracts.
5. CONCLUSION
For extracts of the standardised or quantied

type, ranges with clear specications regarding the
quantity of constituents which determine or codetermine therapeutic activity are needed. Chemical
reference standards of dened content, which allow the
constituents to be standardised within a pre-dened
range in a reproducible manner, are therefore absolutely
indispensable.
The recent trend to introduce chemical reference

standards in conjunction with monographs for medicinal
plants and their preparations appears appropriate from all
viewpoints.
2. REFERENCE SUBSTANCES IN CONJUNCTION

WITH THE EXTRACT CATEGORIES GIVEN
IN THE PH. EUR. GENERAL MONOGRAPH
EXTRACTS (0765)
This is not necessary for the type other extracts. The

range of content for marker substances is not normally
required in this case. However, marker substances are
used for quality assurance and for subsequent assays
of these other extracts in nished preparations. The
latter enable batch-related assays of the extract used
for each respective batch of nished pharmaceutical to
be made, for example. The marker substances may be
selected separately (with supporting arguments) by each
individual manufacturer.
For this type of relative assay, absolute reference
standards are not entirely necessary. In extreme cases,
reference standards with no precisely dened, but
constant content of the marker substance in question
may be used. Herbal drug preparation reference standards
are suitable for this extract group.
3. USE OF NON-AUTHENTIC REFERENCE
STANDARDS FOR ASSAYS
So far, the quantitative analysis of a group of
structurally similar constituents has been based on
578
For plants with constituents which (co-)dene efcacy

and extracts of the standardised or quantied
type, absolute specications for the content of those
constituents are required. For this purpose, uniform
reference standards of dened content are necessary,
whereby synthetic CRS substances are particularly well
suited as primary standards. This also applies for purity
tests with a corresponding quantitative upper limit.
The herbal drug preparation reference standards appear
to be less suited. Corresponding working standards
should be directly derived and established in the
laboratories concerned.
However, HDPRS would be quite suitable for batchrelated control of other extracts, if an appropriate
marker substance is used.
In addition, the possibility of using non-authenticated
standards and response factors should also be maintained
in the future.
In order to avoid coming into conict with registered
values and specications, the newly available
pharmacopoeial standards should be checked
carefully with regard to user acceptance prior to their
introduction.
The Control of Impurities of Imipramine Hydrochloride
Scientific Notes
The Secretariat of the European Pharmacopoeia recalls that articles reflect the authors opinions. In order that all
opinions are taken into account, concerned users and readers of Pharmeuropa are invited to send their comments
to the Secretariat. Readers are reminded that details regarding contributions made to Pharmeuropa are cited on
the rear cover of this issue.
$ /LTXLG &KURPDWRJUDSKLF 0HWKRG XVLQJ D

5HYHUVHGSKDVH +\EULG 6WDWLRQDU\ 3KDVH WR
&RQWURO 3RWHQWLDO ,PSXULWLHV RI ,PLSUDPLQH
+\GURFKORULGH
S.P. Dixon, F. La Torre, A. Lodi, J.H.McB. Miller, G.G. Skellern
$%675$&7
A reversed-phase liquid chromatographic method has been developed for the detection and control of impurities
of imipramine hydrochloride at a level of 0.2 g/ml (corresponding to 0.02 per cent in the procedure described).
This method is included in a proposed revision of the monograph (Pharmeuropa 18.4) and replaces the thin-layer
chromatographic method currently employed.
.(<:25'6
Reversed-phase, liquid chromatography, impurities, imipramine.
pharmaceuticals [12 & 13]. These tricyclic anti-depressants
present the same problem of poor symmetry as do other
The monograph for imipramine hydrochloride [1] of
substances of the same family, when chromatographed on
the European Pharmacopoeia contains a thin-layer
silica based reversed-phase liquid chromatographic columns.
chromatographic test to limit impurities, whereas the
A relatively new hybrid organic-inorganic stationary phase
current policy requires the use of quantitative methods,
[14] has been developed which decreases reactive residual
compatible with the general monograph on Substances for silanol groups, therefore reducing association of these
Pharmaceutical Use (2034) [2] and general chapter 5.10
groups with highly basic substances such as tricyclic
Control of impurities in substances for pharmaceutical use anti-depressants, resulting in improved symmetry of the
[3]. It is, therefore, necessary to revise the tests for related
peaks. The use of this stationary phase has been successfully
substances in these monographs to replace the thin-layer
applied to the separation of specified impurities of the
chromatographic method, by a liquid chromatographic
tricyclic anti-depressant amitriptyline hydrochloride [15].
method in order to include limits for specified impurities,
This paper describes a liquid chromatographic procedure
unspecified impurities, the sum of impurities and to give
using a hybrid reversed-phase column for the control of the
the chemical structures of the specified impurities. There
specified impurities of imipramine hydrochloride.
is currently only one specified impurity, iminodibenzyl,
(;3(5,0(17$/
which is controlled at the level of 0.2 per cent, as is any
other impurity detected in the monograph of imipramine
The following reagents were employed: acetonitrile, HPLC
hydrochloride. The specified impurities controlled by
gradient grade (Riedel de Hahn, Germany), dipotassium
manufacturers of imipramine are, desipramine (impurity
hydrogen phosphate, hydrochloric acid, glacial acetic acid,
A), N-(3-dimethylaminopropyl)-iminostibene (impurity
sulphuric acid, potassium dichromate, ethylacetate (Merck,
B), dimethylaminopropylacridone (impurity C) and
Germany) and phosphoric acid (Fluka, Germany). 7 samples
iminodibenzyl (impurity D). The first two impurities are not of imipramine hydrochloride from two different origins were
only synthetic impurities but also decomposition products.
tested. The impurities were supplied by a manufacturer.
Chromatographic system. The liquid chromatographic
A number of liquid chromatographic methods have been
system consisted of an Alliance 2690 Separation Module
reported for the determination of tricylic anti-depressants
and a Waters 996 Photodiode Array Detector (Waters
[4-11] usually in biological fluids but there are also
Corporation, USA).
some concerning the analysis of these substances in
,1752'8&7,21
S.P. Dixon. Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK

F. La Torre. Institute Superiore di Sanita, Viala Regina Elena, Rome, Italy
A. Lodi. Division III, European Directorate for the Quality of Medicines, Council of Europe, Strasbourg, France
J.H.McB. Miller. (corresponding author: e-mail: John.Miller@pheur.org), Division III, European Directorate for the Quality of Medicines, Council
of Europe, , Strasbourg, France
G.G. Skellern. Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK
Pharmeuropa Vol 18, No. 4, October 2006
579
Stationary phase (liquid chromatography): X-Terra RP-18,

150 x 4.6 mm (5 m) (Waters, USA) maintained at 40 C.
Mobile phase (liquid chromatography): 0.03M solution of
dipotassium hydrogen phosphate, adjusted to pH 7.0 with
phosphoric acid and acetonitrile (60:40 V/V). The flow rate
was 1.0 ml.min-1, the detection wavelength was 220 nm and
the injection volume was 10 l.
Test solution: 50.0 mg of the sample was dissolved in 50ml
of the mobile phase and diluted to 100.0 ml with the mobile
phase.
Reference solution. 1.0 ml of the test solution was diluted to
100.0 ml with the mobile phase.
Reference solutions (for linearity and precision). 50.0 mg of
imipramine hydrochloride was dissolved in the mobile phase
and diluted to 100.0 ml with the mobile phase. The solution
was further diluted with the mobile phase to give solutions
of 0.25 g.ml-1, 0.5 g.ml-1, 1.0 g.ml-1 and 2.5 g.ml-1.
Stationary phase (thin-layer chromatography): Silica gel
G (Alltech, USA).
Mobile phase (thin-layer chromatography): A mixture of
hydrochloric acid : water: glacial acetic acid:ethylacetate
(5:5:35:35 V/V/V/V).
Visualisation reagent: 0.5 per cent m/v solution of
potassium dichromate in a mixture of sulphuric acid:water
(1:4 V/V).
5(68/76 $1' ',6&866,21
Presently the monograph for imipramine hydrochloride
describes a thin-layer chromatographic method to control
NH
Iminodibenzyl (Impurity D)
CH3
CH3
one named impurity (iminodibenzyl) and any other unknown

impurities each at the level of 0.2 per cent.
The presently described thin-layer chromatographic
method, however, suffers from a number of disadvantages
which include: the spot due to iminodibenzyl migrates
with the solvent front; the colours of the impurity spots
differ from that of the reference spot (imipramine); and
N-(3-dimethylaminopropyl)iminostilbene cannot be detected
at the 0.2 per cent level.
Since it is now the policy of the European Pharmacopoeia to
apply the concepts of the ICH Guideline on impurities to all
monographs of synthetically produced medicinal substances,
a preliminary study has been conducted to replace the TLC
test by a liquid chromatographic test.
Since it is well known that the peaks of tricyclic
anti-depressants in reversed-phase chromatography exhibit
poor symmetry, a hybrid reversed-phase stationary phase
has been employed. It had already been demonstrated
[15] that adequate symmetry of the peak of amitriptyline
could be achieved using this type of stationary phase. The
principal synthetic route for the preparation of imipramine
is the reaction of iminodibenzyl (impurity D) with lithium
hydride and dimethylaminopropyl chloride [16]. Imipramine
has been reported to be stable both as a solid and in
aqueous solution, but on heating in acid conditions at pH
4 the major decomposition product is iminodibenzyl [16].
Desipramine has also been reported as a decomposition
product. A manufacturer of imipramine tests not only for the
presence of impurity D, but also for impurities A, B and C.
An isocratic reversed-phase chromatographic system using
a hybrid stationary phase and a simple mixture of solvents
Dimethylamino-propylacridone (Impurity C)
CH3
N-(3-dimethylaminopropyl)-iminostilbene (Impurity B)
CH3
N
H
CH3
Desipramine (Impurity A)
Figure 1 - Specified impurities of imipramine hydrochloride

580
has been developed which separates all of these impurities.

This method has been verified for sensitivity, selectivity,
linearity and precision.
6HOHFWLYLW\
It was shown that the symmetry of the imipramine peak

using these chromatographic conditions was good and
that the actual known impurities of imipramine could
be separated from each other and from imipramine in a
reasonable time (Figure 2). Using isocratic conditions,
chromatography can be completed in 35 minutes. A
selectivity requirement, the resolution between impurity B
and imipramine, could be proposed as a system suitability
criterion with a minimum resolution of 5.0.
5HVSRQVH )DFWRUV
The optimal wavelength for the detection of impurities

was at 220 nm where the responses of impurities A and
D, and imipramine were considered to be equivalent. The
response factor of impurity B was about 1.5 (Table 1).
Since it is proposed to use impurity B in the test for
resolution, its presence in the test solution can be compared
to the corresponding peak of impurity B in the reference
solution. The other impurities can be compared to the peak
corresponding to imipramine.
6HQVLWLYLW\
The limit of quantification of the peak due to imipramine

corresponds to a concentration of 0.20 g/ml, which is
equivalent to 0.02 per cent of the test solution. This is
sufficiently sensitive for the current limits of 0.2 per cent
and even for the proposed limits of 0.1 per cent for each
impurity, and thus a disregard limit of 0.05 per cent for the
summation of impurities.
A linear relationship has been demonstrated for area

response at various concentrations in the range of
0.25 g/ml to 2.5 g/ml (Figure 3) equivalent to 0.025
to 0.25 per cent (limiting concentration). Six replicate
injections were performed at each concentration. Areas
recorded, mean areas and relative standard deviations are
given in Table 2.
$QDO\VLV RI EDWFKHV RI LPLSUDPLQH K\GURFKORULGH
Impurity profiles of the batches are given in Table 3. All

batches examined contained desipramine (impurity A) and
N-(3-dimethylaminopropyl)iminostilbene (impurity B), close
to the limit of quantification but mostly well below the
disregard limit (0.05 per cent). All batches also contained
traces of impurities with unadjusted relative retention values
of 1.6 and 1.7 but not exceeding the disregard level. Only one
sample showed a minute trace of iminodibenzyl (impurity D),
whilst dimethylaminopropylacridone (impurity C) was not
detected at all in the samples examined.
&21&/86,21
This liquid chromatographic procedure has been shown
to separate all the specified impurities of imipramine
hydrochloride as well as some unknown impurities, all below
0.1 per cent. In fact, the batches examined are relatively
pure and no impurity was present at levels greater than
0.1 per cent, therefore, consideration should be given as to
appropriate impurity limits. It is proposed, for continuity,
to limit impurities A and B to 0.2 per cent and any other
impurities to 0.1 per cent, the total of all impurities being
limited to 0.3 per cent. This method is proposed as a
replacement for the existing TLC procedure, which is less
sensitive and less selective.
Imipramine
ne HCl
H
0.03
0.
030
/LQHDULW\ DQG 3UHFLVLRQ
0.02
0.
028
0.02
0.
026
0.02
0.
024
0.02
0.
022
0.02
0.
020
0.01
0.
018
AU
0.01
0.
016
0.01
0.
014
0.01
0.
012
0.01
0.
010
0.00
0.
008
0.00
0.
006
0.00
0.
004
0.00
0.
002
0.00
0.
000
0.00
0.
00
5.00
5.
10.00
15.00
20.00
25.00
Minutes
30.00
35.00
40.00
45.00
50.00
Figure 2 - Chromatogram of a spiked imipramine solution. Stationary phase: X-Terra RP18 150 x 4.6 mm x 5 m;
Column temperature: 40 C; Mobile phase: Dipotassium hydrogen phosphate (pH 7.0): Acetontrile 60:40 (V/V); UV
detection at 220 nm; Flow rate: 1.0 ml/min. 1.0 mg/ml imipramine solution with impurities at 0.1 %
581
Table 1 - Retention times, unadjusted retention times and response factors of impurities relative to imipramine
hydrochloride. (Detection wavelength: 220 nm)
Rt (mins)
Unadjusted relative retention
Rf
Dimethylaminopropylacridone (impurity C)
3.0
0.44
Desipramine (impurity A)
3.9
0.59
1.06
N-(3-dimethylaminopropyl)iminostilbene (impurity B)
4.9
0.74
1.53
Imipramine
6.6
1.0
1.00
Iminodibenzyl (impurity D)
31.0
4.7
0.85
Compound
Table 2 - Precision of replicate injection of reference substances of imipramine hydrochloride

Concentration (g/ml)
0.25
1.25
2.50
Peak Area
3839
4751
5287
5115
5341
4072
19909
20354
18911
17924
20471
20011
37311
37767
37470
37457
36821
38289
Mean Area
4734
19597
37519
SD
642
987
488
RSD (%)
13.55
5.04
1.30
Table 3 - Impurity profiles of batches of imipramine hydrochloride from different manufacturers

Identification No.
Per cent impurities by normalisation

Unadjusted Relative Retention
0.4
0.6
0.7
0.9
1.6
1.7
2.3
14.7
D
Manufacturer A
19279
19280
19281
0.04
0.02
0.05
0.02
0.02
0.02
0.05
0.04
0.05
0.02
0.03
0.02
0.02
Manufacturer B
19407
19408
19409
0.01
0.01
0.01
0.01
0.01
0.01
0.03
0.03
0.03
0.01
0.01
0.02
0.01
0.01
0.01
Manufacturer C
24940
0.05
0.02
0.02
0.02
0.01
0.07
0.01
45000
40000
35000
Peak Area
30000
25000
20000
y = 1.456E+04x + 1.201E+03
R 2 = 9.974E-01
15000
10000
5000
0
0.00
0.50
1.00
1.50
2.00
2.50
3.00
[Im ipr amin e] g/ml
Figure 3 - Linear relationship between peak area and

concentration of imipramine hydrochloride in the range
of 0.25 g/ml to 2.5 g/ml
582
5()(5(1&(6
[1] Imipramine hydrochloride, monograph 0029. Ph. Eur.
5th edition. Council of Europe; Strasbourg, France:
2005;(vol. 2).
[2] Substances for pharmaceutical use, monograph 2034.
Ph. Eur. 5th edition. Strasbourg, France: Council of
Europe; 2005.
[3] Control of impurities in substances for pharmaceutical
use, general chapter 5.10. Ph. Eur. 5th edition. Council
of Europe; Strasbourg, France: 2005.
[4] Wahlund K-G, Sokolowski A. Reversed-phase ion-pair
chromatography of antidepressive and neuroleptic
amines and related quaternary ammonium compounds.
J Chromatogr 1977;151:299-310.
[5] Hung C T, Taylor R B, Paterson N. The analysis
of tricyclic anti-depressants at therapeutic blood
levels by reversed-phase high-performance liquid
chromatography using pairing and organic counter-ions.
J Pharm Biomed Anal 1983:73-82.
[6] Oztunc A, Onal A, Ertunk S. 7,7,8,8-tetracyanoquinodimethane as a new derivatisation reagent for high
performance liquid chromatography and thin-layer chromatography: rapid screening for some anti-depressants.
J Chromatogr B 2002;774:149-155.
[7] Chen A G, Wing Y K, Chiu H et al. Simultaneous

determination of imipramine, desipramine and their
2- and 10-hydroxylated metabolites in human plasma
urine by high performance liquid chromatography. J
Chromatogr B 1997;693:153-158.
[8] McIntrye J M, King C V. et al. Dual ultra-violet
wavelength high performance liquid chromatography
method for the forensic or chemical analysis of 17
anti-depressants and some selected metabolites. J
Chromatogr 1993;621:215-223.
[9] Queiroz R H C, Lanchote V L, Bonato P S et al.
Simultaneous HPLC analysis of tricyclic anti-depressants
and metabolites in plasma samples. Pharm Acta Helv
1995;70:181-186.
[10] Kollroser M, Schober C. Simultaneous determination
of seven tricyclic antidepressant drugs in human
plasma by direct-injection HPLC-APCI-MS-MS with
an ion trap detector. Therapeutic Drug Monitoring
2002;24:537-544.
[11] Karpinska J, Starczewska B. Simultaneous LC
determination of some antidepressants combined with
neuroleptics. J Pharm Biomed Anal 2002;29:519-525.
[12] Bermudez-Saldana J M, Quinones-Torrelo C, Sagrado

S et al. A micellar liquid chromatographic method
for quality control of pharmaceutical preparations
containing tricyclic antidepressants. Chromatographia
2002;56:299-306.
[13] Ruiz-Angel M J, Carda-Broch S, Sim-Alfonso E F. et
al. Optimised procedures for the reversed phase liquid
chromatographic analysis of formulations containing
tricyclic antidepressants. J Pharm Biomed Anal
2003;32:71-84.
[14] Cheng Y-F, Walter T H, Lu Z et al. Hybrid
organic-inorganic particle technology: breaking through
traditional barriers of HPLC separations. LCNGC North
America 2000;18:1162-1172.
[15] Dixon S P, Lodi A, Miller J H McB et al. The control of
impurities in Amitriptyline HCl using a reversed-phase
hybrid stationary phase. Pharmeuropa Scientific Notes
2005;1:15-20.
[16] Kender D N, Schiesswohl R E. Imipramine hydrochloride
in Analytical Profiles of Drug Substances. Academic
Press Ed. K. Florey; 1985;14:37-76.
583
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584
N-Acetyltyrosine
Draft monographs and

general texts for comment
IMPORTANT NOTICE
This section contains proposals for new and revised monographs and general texts, intended for inclusion in the
European Pharmacopoeia and submitted for public comment. You can comment through the appropriate national
authority at the address listed on the inside-back cover of this issue. Readers from countries that are not members of the
European Pharmacopoeia Commission should send their comments directly to the European Directorate for the Quality
of Medicines. In order to facilitate the work of the Secretariats of the National Authorities and the European Directorate
for the Quality of Medicines who collect the comments, please mention in any correspondence the PA/PH reference
number indicated at the beginning of each text. Comments that propose modifications of limits should be supported by
analytical data obtained on a significant number of batches. Proposed changes of methodology should be supported by
experimental results of a comparative trial of the method published in Pharmeuropa for comment and the proposed
alternative. Only comments sent before 31 December 2006 will be considered before the final version is prepared.
It is stressed that these proposals have not been adopted by the European Pharmacopoeia Commission and must not
be regarded as official standards.
In the case of proposals for revision, text to be deleted is crossed out and replacements or additions are underlined.
In certain cases, draft monographs require, for appropriate checking, the use of a reference material that is not yet
commercially available; in exceptional circumstances, we will try to make the necessary substance available; please
enquire to the European Directorate for the Quality of Medicines.
Reference: PA/PH/Exp. 11/T (06) 58 ANP
B. Infrared absorption spectrophotometry (2.2.24).

Comparison : N-acetyltyrosine CRS.
NOTE ON THE MONOGRAPH
C. Thin-layer chromatography (2.2.27).
Identification : the TLC test formerly used for both
Test solution. Dissolve 80 mg of the substance to be
identification and related substances is now described
examined in 6 ml of a mixture of equal volumes of glacial
under Identification.
acetic acid R and water R, and dilute to 10 ml with
Impurity A : TLC test for related substances has been
anhydrous ethanol R.
replaced by an LC in accordance with current policy as
Reference solution. Dissolve 80 mg of
part of a special revision programme. The proposed LC is
N-acetyltyrosine CRS in a mixture of 3 volumes
not suitable to cover impurity B mentioned in the current
of water R, 3 volumes of glacial acetic acid R and
monograph. However, there are no impurities other
94 volumes of anhydrous ethanol R, and dilute to 10 ml
than A in the production and therefore no need for other
with the same mixture of solvents.
requirements.
Plate : TLC silica gel F254 plate R.
XXXX:1384
Mobile phase : water R, glacial acetic acid R, ethyl
acetate R (10:15:75 V/V/V).
N-ACETYLTYROSINE
Application : 5 l.
N-Acetyltyrosinum
Development : over a path of 10 cm.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
Results : the principal spot in the chromatogram obtained
with the test solution is similar in position and size to
the principal spot in the chromatogram obtained with
the reference solution.
C11H13NO4
Mr 223.2 D. Solution S (see Tests) is strongly acid (2.2.4).
DEFINITION
(2S)-2-(Acetylamino)-3-(4-hydroxyphenyl)propanoic acid.
Content : 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or
colourless crystals.
Solubility : freely soluble in water, practically insoluble in
cyclohexane.
IDENTIFICATION
First identification : A, B.
Second identification : A, C, D.
A. Specific optical rotation (see Tests).
TESTS
Solution S. Dissolve 2.50 g in water R and dilute to 100.0 ml
with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and
colourless (2.2.2, Method II).
Specific optical rotation (2.2.7) : + 46 to + 49 (dried
substance).
Dilute 10.0 ml of solution S to 25.0 ml with water R.
Related substances. Examine by thin-layer chromatography
(2.2.27), using a TLC silica gel F254 plate R.
Test solution (a). Dissolve 0.80 g of the substance to be
examined in 6 ml of a mixture of equal volumes of glacial
acetic acid R and water R and dilute to 10 ml with ethanol R.
585
N-Acetyltyrosine
in the lower watch-glass and dissolve in 0.5 ml of water R.

To the solution add 0.30 g of heavy magnesium oxide R.
Briefly triturate with a glass rod. Immediately close the cell
Reference solution (a). Dissolve 80 mg of
by putting the 2 watch-glasses together. Heat at 40 C for
N-acetyltyrosine CRS in a mixture of 3 volumes of
15 min. The litmus paper is not more intensely coloured
water R, 3 volumes of glacial acetic acid R and 94 volumes
blue than the litmus paper positioned in the same manner
of ethanol R and dilute to 10 ml with the same mixture of
above a standard prepared at the same time using 0.1 ml of
solvents.
ammonium standard solution (100 ppm NH4) R, 0.5 ml of
water R and 0.30 g of heavy magnesium oxide R.
Reference solution (b). Dilute 0.5 ml of test solution (b) to
10 ml with ethanol R.
Iron (2.4.9) : maximum 20 ppm.
Reference solution (c). Dissolve 40 mg of tyrosine CRS in
In a separating funnel, dissolve 0.5 g in 10 ml of dilute
20 ml of a mixture of equal volumes of water R and glacial hydrochloric acid R. Shake with 3 quantities, each of 10 ml,
acetic acid R and dilute to 50 ml with ethanol R.
of methyl isobutyl ketone R1, shaking for 3 min each time.
Apply separately to the plate 5 l of each solution. Develop To the combined organic layers add 10 ml of water R and
shake for 3 min. The aqueous layer complies with the test
over a path of 10 cm using a mixture of 10 volumes of
water R, 15 volumes of glacial acetic acid R and 75 volumes for iron.
of ethyl acetate R. Allow the plate to dry in air. Examine in Heavy metals (2.4.8) : maximum 10 ppm.
ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with test solution (a), apart from the principal spot, Dissolve 2.0 g in water R and dilute to 20 ml with the same
solvent. 12 ml of the solution complies with test A. Prepare
is not more intense than the spot in the chromatogram
the reference solution using lead standard solution (1 ppm
obtained with reference solution (b) (0.5 per cent). Spray
Pb) R.
with ninhydrin solution R and heat at 100 C to 105 C
Loss on drying (2.2.32) : maximum 0.5 per cent, determined
for 10 min. Examine in daylight. Any spot corresponding
on 1.000 g by drying in an oven at 100-105 C.
to tyrosine is not more intense that the spot in the
chromatogram obtained with reference solution (c) (1 per
Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
cent).
on 1.0 g.
Impurity A. Liquid chromatography (2.2.29).
Pyrogens (2.6.8). If intended for use in the manufacture
of parenteral dosage forms without a further appropriate
Test solution. Dissolve 50.0 mg of the substance to be
procedure for the removal of pyrogens, it complies with the
examined in water R and dilute to 100.0 ml with the same
test for pyrogens. Inject, per kilogram of the rabbits mass,
solvent.
1.0 ml of a freshly prepared solution in water for injections R
Reference solution. Dissolve 10.0 mg of tyrosine CRS in
containing, per millilitre, 10.0 mg of the substance to be
water R and dilute to 100.0 ml with the same solvent. Dilute examined and 9.0 mg of pyrogen-free sodium chloride R.
1.0 ml of this solution to 20.0 ml with water R.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml
with ethanol R.
Column :
size : l = 0.15 m, = 4.6 mm ;
stationary phase : octadecylsilyl silica gel for
chromatography R (5 m)(1) ;
mobile phase : mix 3 volumes of methanol R and
197 volumes of a 5.44 g/l solution of potassium
dihydrogen phosphate R adjusted to pH 4.35 ;
flow rate : 1.0 ml/min.
Detection : spectrophotometer at 214 nm.
Injection : 100 l.
ASSAY
Dissolve 0.180 g in 50 ml of carbon dioxide-free water R.
Titrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2.2.20).
1 ml of 0.1 M sodium hydroxide is equivalent to 22.32 mg
of C11H13NO4.
STORAGE
Store protected from light. If the substance is sterile, store
in a sterile, airtight, tamper-proof container.
Limits :
LABELLING
The label states, where applicable, that the substance is
apyrogenic.
impurity A : not more than the area of the corresponding

peak in the chromatogram obtained with the reference
solution (1 per cent).
IMPURITIES
Specified impurities : A.
Run time : 1.5 times the retention time of N-acetyltyrosine.
Chlorides (2.4.4) : maximum 200 ppm.

Dilute 10 ml of solution S to 15 ml with water R.
A. tyrosine,
Sulphates (2.4.13) : maximum 200 ppm.

Dissolve 1.0 g in distilled water R and dilute to 20 ml with
the same solvent.
Ammonium : maximum 200 ppm. Prepare a cell consisting of
2 watch-glasses 60 mm in diameter placed edge to edge. To
the inner wall of the upper watch-glass stick a 5 mm 5 mm
piece of red litmus paper R with a few drops of water R.
B. (2S)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic acid
Finely powder the substance to be examined, place 50 mg
(diacetyltyrosine).
(1) Inertsil ODS 2 is suitable.
586
Adrenaline
Reference: PA/PH/Exp. 10A/T (06) 57 ANP
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : adrenaline CRS.
This monograph has been adapted from the BP. A proposal B. Specific optical rotation (see Tests).
was published previously in Pharmeuropa (16.2). Based on
TESTS
the comments received, the proposal has been modified.
Appearance of solution. The solution is clear (2.2.1) and
The major changes are the following:
Identification B : replacement of a colour reaction using not more intensely coloured than reference solution BY5
(2.2.2, Method II).
diethoxytetrahydrofuran by a reference to the test for
Dissolve 1.25 g in dilute hydrochloric acid R and dilute
specific optical rotation.
to 25 ml with the same solvent. Examine the solution
Appearance of solution : introduction of this test for
immediately.
giving a general indication on the stability of the
Specific optical rotation (2.2.7) : 50.0 to 53.0 (dried
substance.
substance).
Related substances : peak identification of the specified Dissolve 1.00 g in 1 M hydrochloric acid and dilute to
impurities by injection of CRS and introduction of a
25.0 ml with the same acid.
limit for the total.
Related substances. Liquid chromatography (2.2.29).
Impurities : the chemical structure of impurity A has
Prepare the solutions protected from light.
been elucidated.
Solvent mixture A. Dissolve 5.0 g of potassium dihydrogen
XXXX:2303 phosphate R and 2.6 g of sodium octanesulphonate R in
water for chromatography R and dilute to 1000 ml with
the same solvent. It is usually necessary to stir for at least
ADRENALINE
30 min to have the samples dissolved. Adjust to pH 2.8 with
phosphoric acid R.
Adrenalinum
Solvent mixture B : acetonitrile R1, solvent mixture A
(130:870 V/V).
examined in 5.0 ml of 0.1 M hydrochloric acid and dilute to
50.0 ml with solvent mixture B.
Reference solution (a). Dilute 1.0 ml of the test solution
to 100.0 ml with solvent mixture B. Dilute 1.0 ml of this
C9H13NO3
Mr 183.2 solution to 10.0 ml with solvent mixture B.
Reference solution (b). Dissolve 1.5 mg of noradrenaline
tartrate CRS (impurity B) and 1.5 mg of adrenalone
DEFINITION
hydrochloride R (impurity C) in solvent mixture B, add
(1R)-1-(3,4-Dihydroxyphenyl)-2-(methylamino)ethanol.
1.0 ml of the test solution and dilute to 100.0 ml with solvent
mixture B.
Synthetic product.
Reference solution (c). Dissolve 4 mg of adrenaline for
peak identification CRS (containing impurities D and E) in
0.5 ml of 0.1 M hydrochloric acid and dilute to 5.0 ml with
CHARACTERS
solvent mixture B.
Appearance : white or almost white crystalline powder. It
Reference solution (d). Dissolve 4 mg of adrenaline with
becomes coloured on exposure to air and light.
impurity A CRS in 0.5 ml of 0.1 M hydrochloric acid and
dilute to 5.0 ml with solvent mixture B.
Solubility : practically insoluble in water, practically
insoluble in ethanol (96 per cent) and in methylene chloride. Blank solution : 0.1 M hydrochloric acid, solvent mixture B
It is soluble in hydrochloric acid.
(1:9 V/V).
The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.
Figure 2303-1. Chromatogram for the test for related substances of adrenaline
587
2.7.25. Assay of 2-antiplasmin
Column :
size : l = 0.10 m, = 4.6 mm ;
stationary phase: end-capped octadecylsilyl silica gel
for chromatography R (3 m)(2) ;
temperature : 50 C.
Mobile phase :
mobile phase A : acetonitrile R1, solvent mixture A
(5:95 V/V) ;
mobile phase B : acetonitrile R1, solvent mixture A
(45:55 V/V) ;
Time
(min)
0 - 15
Mobile phase A
(per cent V/V)
92 50
Mobile phase B
(per cent V/V)
8 50
15 - 20
50 92
50 8
20 - 25
92
Flow rate : 2.0 ml/min.

Injection: 20 l.
Identification of impurities : use the chromatogram supplied
with adrenaline for peak identification CRS and the
chromatogram obtained with reference solution (c) to identify
the peaks due to impurities D and E ; use the chromatogram
supplied with adrenaline with impurity A CRS and the
chromatogram obtained with reference solution (d) to
identify the peak due to impurity A.
Relative retention with reference to adrenaline
(retention time = about 4 min) : impurity A = about
0.2 ; impurity B = about 0.8 ; impurity C = about 1.3 ;
impurity D = about 3.3 ; impurity E = about 3.7.
System suitability : reference solution (b) :
resolution : minimum 3.0 between the peaks due to
impurity B and adrenaline.
Limits :
correction factors : for the calculation of content,
multiply the peak areas of the following impurities by
the corresponding correction factor : impurity D = 0.7 ;
impurity E = 0.6 ;
impurities A, B, C : for each impurity, not more than
twice the area of the principal peak in the chromatogram
obtained with reference solution (a) (0.2 per cent) ;
impurities D, E : for each impurity, not more than the
area of the principal peak in the chromatogram obtained
with reference solution (a) (0.1 per cent) ;
unspecified impurities : for each impurity, not more
than the area of the principal peak in the chromatogram
total: not more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (a)
(0.5 per cent) ;
disregard limit : 0.5 times the area of the principal peak
(0.05 per cent).
on 1.000 g by drying over diphosphorus pentoxide R at a
pressure not exceeding 0.7 kPa for 18 h.
on 1.0 g.
ASSAY
Dissolve 0.150 g in 50 ml of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 18.32 mg
of C9H13NO3.
STORAGE
Under nitrogen, protected from light.
IMPURITIES
Specified impurities : A, B, C, D, E.
A. (1R)-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanesulfonic acid,
B. noradrenaline,
C. R = H : 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone,
E. R = CH2-C6H5 : 2-(benzylmethylamino)-1-(3,4dihydroxyphenyl)ethanone,
D. (1R)-2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl)ethanol.
Reference: PA/PH/Exp. 6B/T (06) 30 ANP

XXXX:20725
2.7.25. ASSAY OF 2-ANTIPLASMIN

Plasma protein 2-antiplasmin is an inhibitor of the serine
protease plasmin pathway of fibrinolysis by rapidly forming
a complex with free plasmin. Furthermore, upon blood
coagulation, 2-antiplasmin is cross-linked to fibrin strands
by factor XIII, and interferes with binding of the proenzyme
plasminogen to fibrin.
The potency of 2-antiplasmin is estimated by comparing
the ability of the preparation to be examined to inhibit the
cleavage of a specific chromogenic substrate by plasmin with
the same ability of a reference preparation of 2-antiplasmin.
Plasmin cleavage of the chromogenic substrate yields a
chromophore that can be quantified spectrophotometrically.
The individual reagents for the assay may be obtained
separately or in commercial kits. Both end-point and kinetic
methods are available. Procedures and reagents may vary
between different kits and the manufacturers instructions
(2) Inertsil ODS 3 and Kromasil are suitable.
588
2.7.32. Assay of human -1-antitrypsin
are followed. The essential features of the procedure are

described in the following example of a microtitre plate
kinetic method.
REAGENTS
Dilution buffer pH 7.5. According to the manufacturers
instructions, a suitable buffer is used. Adjust the pH (2.2.3)
if necessary.
Plasmin. A preparation of human plasmin which should
not contain significant amounts of other proteases is
preferably used. Reconstitute and store according to the
manufacturers instructions.
Plasmin chromogenic substrate. A suitable specific
chromogenic substrate for plasmin is used such
as D-Novaryl-cyclohexylalanyl-lysis-pNA.HCl, or
pyroGlu-Phe-Lys-pNA.HCl. Reconstitute in water R to give
a suitable concentration according to the manufacturers
instructions.
METHOD
Varying quantities of the preparation to be examined are
mixed with a given quantity of plasmin and the remaining
plasmin activity is determined using a suitable chromogenic
substrate.
Reconstitute or thaw the preparation to be examined
according to the manufacturers instructions. Dilute with
dilution buffer pH 7.5 and prepare at least 2 independent
series of 3 or 4 dilutions for both the preparation to be
examined and the reference preparation. Dilutions may
need to be adjusted to ensure that they fall on the steepest,
most linear part of the dose-response curve and also provide
maximum overlap between the reference preparation and the
preparation to be examined.
or human neutrophil elastase) with the same ability of a

reference preparation of human -1-antitrypsin calibrated
in milligrams of active (functional) -1-antitrypsin. Varying
quantities of the preparation to be examined are mixed with a
given quantity of elastase and the remaining elastase activity
is determined using a suitable chromogenic substrate. The
method described below is given as an example.
REAGENTS
Tris-BSA buffer solution. Dissolve 24.23 g of trometamol R
in water R, adjust to pH 8.0 0.3 using hydrochloric
acid R1, and dilute to 1000 ml with water R. To 100 ml of
this solution add 0.5 ml of a 20 per cent solution of human
albumin R or bovine albumin R.
Buffer solution containing human or bovine albumin must
be prepared fresh on the day of its use ; otherwise, it can be
conserved by sterile filtration (0.2 m) and stored at 2-8 C
for up to 2 weeks.
METHOD
Prepare 2 series of 4 or 5 dilutions in an appropriate human
-1-antitrypsin concentration range, for both the preparation
to be examined and the reference preparation, using the trisBSA buffer solution.
Transfer 50 l of the reference solution dilutions into the

wells of a microtitre plate and add 150 l of a porcine
pancreatic elastase solution diluted to an appropriate
concentration with the tris-BSA buffer solution to each
well. Incubate for a defined time in the range of 3-10 min
at room temperature. Since the activity of the solutions
Mix 0.020 ml of each dilution with 0.020 ml of dilution
of the different porcine pancreatic elastases may vary, the
buffer pH 7.5 and warm to 37 C. Add 0.040 ml of a plasmin concentration of elastase can be adjusted by evaluation
solution (test concentration in the range of 0.2 nkat/ml to
of blank values containing elastase but no human
1.6 nkat/ml) previously heated to 37 C and leave for 1 min -1-antitrypsin, to exhibit a suitable change of absorbance
at 37 C. Add 0.020 ml of chromogenic substrate solution,
at 405 nm under the actual assay conditions. The greatest
previously heated to 37 C, to each mixture and measure the linearity of the standard curve is typically observed using
optical density at a wavelength of 405 nm. Substract the
dilutions of human -1-antitrypsin that, for a given porcine
optical density of the blank (prepared with dilution buffer
elastase concentration, result in 85-50 per cent residual
pH 7.5) from the optical density of the preparation to be
elastase activity.
examined with the test sample. Check the validity of the
assay and calculate the potency of the preparation to be
Add to each well 100 l of a solution of a suitable
examined by the usual statistical methods (5.3).
chromogenic substrate (for example, N-succinyl-tri-L-alanyl
4-nitroanilide (Suc-Ala-Ala-Ala-pNA)), reconstituted in
dimethyl sulphoxide R to give a solution containing
4.5 mg/ml and further diluted with the tris-BSA buffer
solution to a concentration suitable for the assay (for
example 0.45 mg/ml). Immediately start measurement
of the change in absorbance at 405 nm (2.2.25) using a
microtitre plate reader, continuing the measurement for
at least 5 min. Calculate the rate of change of absorbance
(A/min). Alternatively, an end-point assay may be used by
stopping the reaction with acetic acid and measuring the
absorbance at 405 nm. In case of performing the assay in
XXXX:20732 test tubes and using spectrophotometers for monitoring the
change in absorbance at 405 nm, the volumes of reagent
solutions are changed proportionally.
2.7.32. ASSAY OF HUMAN

-1-ANTITRYPSIN
Human -1-antitrypsin content is determined by comparing

the ability of the preparation to be examined to inactivate
the serine protease elastase (porcine pancreatic elastase
The rate of change of absorbance (A/min) is inversely

proportional to -1-antitrypsin activity.
Check the validity of the assay and calculate the potency of
the test preparation by the usual statistical methods (5.3).
589
2.5.27. Assay of oxygen in gases
Reference: PA/PH/Exp. 9G/T (06) 17 ANP

Method 2.5.27 is used to assay oxygen in gases, in
particular in Medicinal air (1238), Synthetic medicinal air
(1684) and Oxygen (0417).
According to the current operating conditions, the
calibration of the apparatus is carried out by passing air
through that contains 20.9 per cent V/V O2.
Using oxygen of a defined purity gives a better accuracy,
therefore it is proposed to use oxygen R instead of air as
the reference standard gas.
XXXX:20527
2.5.27. ASSAY OF OXYGEN IN GASES

Oxygen in gases is determined using a paramagnetic
analyser.
The principle of the method is based on the high
paramagnetic sensitivity of the oxygen molecule. Oxygen
exerts a strong interaction on magnetic fields, which is
measured electronically, amplified and converted to a
reading of oxygen concentration. The measurement of
oxygen concentration is dependent upon the pressure
and temperature and, if the analyser is not automatically
compensated for variations in temperature and pressure,
it must be calibrated immediately prior to use. As the
paramagnetic effect of oxygen is linear, the instrument must
have a suitable range with a readability of 0.1 per cent or
better.
Calibration of the instrument. Make the setting in the
following manner :
set the zero by passing nitrogen R1 through the
instrument at a suitable flow rate until a constant reading
is obtained. It should be set to zero according to the
manufacturers instructions ;
set the appropriate limit by passing air (20.9 per
cent V/V O2) pass oxygen R through the instrument at a
suitable flow rate until a constant reading is obtained.
The limit should be set to 20.9 per cent V/V in accordance
with the manufacturers instructions.
Assay. Pass the gas to be examined through the instrument
at a constant flow rate until a suitable stable reading is
obtained. Calculate the concentration of oxygen in the gas
to be examined.
The potency of protein C is estimated by comparing the

ability of the preparation to be examined to cleave a
chromogenic substrate with the same ability of a reference
preparation of protein C calibrated in International Units.
The International Unit is the activity of a stated amount of
the International Standard for protein C. The equivalence in
International Units of the International Standard is stated
by the World Health Organisation.
The individual reagents for the assay may be obtained
separately or in commercial kits. Both end-point and kinetic
methods are available. Procedures and reagents may vary
described in the following example of a microtitre plate
end-point method.
REAGENTS
Dilution buffer pH 8.4. Dissolve 6.055 g of
tris(hydroxymethyl)aminomethane R and 16.84 g
of caesium chloride R in water R and adjust the pH (2.2.3)
if necessary. Dilute to 1000.0 ml with water R.
Protein C activator. A protein isolated from the venom of
the viper Agkistrodon contortrix contortrix that specifically
activates protein C. Reconstitute and store according to
the manufacturers instructions. Dilute to 0.25 U/ml with
water R before use in the assay.
Activated protein C chromogenic substrate. A suitable
specific chromogenic substrate for APC, such as
pyroGlu-Pro-Arg-pNA.HCl. Reconstitute with water R to give
a concentration of 4.5 mmol/l. Dilute further with dilution
buffer pH 8.4 to give a concentration of 1.1 mmol/l before
use in the assay.
METHOD
water R to produce at least 3 separate dilutions for each
preparation in the range 0.050-0.200 IU/ml, preferably in
duplicate. Dilutions may need to be adjusted to ensure
that they fall on the steepest, most linear part of the
dose-response curve and also provide maximum overlap
between the reference preparation and the preparation to
be examined.
Step 1. Mix 0.025 ml of each dilution with 0.050 ml of
protein C activator, both pre-warmed to 37 C, and leave for
XXXX:20730 exactly 10 min at 37 C. For each dilution, prepare a blank
in the same manner, using water R instead of the protein C
activator.
2.7.30. ASSAY OF PROTEIN C
Step 2. Add 0.150 ml of diluted chromogenic substrate,
1. CHROMOGENIC ASSAY
previously warmed to 37 C, to each mixture and leave
Protein C is a vitamin K-dependent plasma protein that, upon for exactly 10 min at 37 C. The incubation time must be
adjusted, if necessary, to ensure a linear development of
activation to activated protein C (APC), can inhibit blood
chromophore with time. Terminate the reaction by adding
coagulation through the proteolytic cleavage of factors Va
0.050 ml of a 50 per cent V/V solution of glacial acetic
and VIIIa. Protein C activity is estimated using a two-step
acid R.
method ; in the first step protein C in the preparation is
Cleavage of the chromogenic substrate by APC causes
activated by a specific activator from snake venom ; in
the release of chromophore pNA, in proportion to the
the second step the activated protein C cleaves a specific
concentration of protein C in the preparation. Measure the
chromogenic substrate to form a chromophore that can be
optical density at a wavelength of 405 nm. Subtract the
quantified spectrophotometrically.
590
2.7.31. Assay of protein S
optical density of the blank from the optical density of the

test sample. Check the validity of the assay and calculate
the potency of the preparation to be examined by the usual
statistical methods (5.3.).

XXXX:20731
2.7.31. ASSAY OF PROTEIN S

2. CLOTTING ASSAY
Protein C activity is estimated following cleavage to APC
by a specific activator extracted from the venom of the
viper Agkistron contortrix contortrix. The resulting APC
inhibits the factors V and VIII, and thus prolongs the
APTT (activated partial thromboplastin time) of a system
in which all the coagulation factors are present, constant
and in excess, except for protein C, which is derived from
the preparation being testing. Prolongation of the clotting
time is proportional to the concentration of protein C in the
preparation.
The potency of protein C is estimated by comparing the
ability of the preparation to be examined to prolong
the clotting time with the same ability of a reference
preparation of protein C calibrated in International Units.
the International Standard for protein C. The equivalence in
Individual reagents may be obtained separately or in
commercial kits. Procedures and reagents may vary
described in the following example.
REAGENTS
Dilution buffer pH 7.4. An isotonic non-chelating buffer.
Protein C-deficient plasma. Citrated human plasma with
no measurable protein C content. Reconstitute and store
according to the manufacturers instructions.
Protein S is a vitamin K-dependent plasma protein that acts

as a cofactor for the anticoagulant functions of activated
protein C (APC). Protein S activity may be determined
by the clotting assay described below, which is sensitive
to the ability of protein S to accelerate the inactivation
of factor Va by APC. In practice, the assay involves the
addition of protein S to a reagent mixture containing APC,
factor Va and protein S-deficient plasma. Prolongation
of the clotting time is proportional to the concentration
of protein S in the preparation. Methods in which APC
is added directly as a reagent are preferred over those in
which APC is generated during the assay by the addition of
a specific protein C activator purified from snake venom.
Activation of coagulation is initiated by the addition of
an activating reagent such as thromboplastin, or activated
factor X, together with phospholipids and calcium chloride.
During the assay, factor Va is generated from factor V in
the protein S-deficient plasma following the activation of
coagulation. The assay procedure must ensure that protein S
is the only limiting factor.
The potency of protein S is estimated by comparing the
ability of the preparation to be examined to prolong
the clotting time with the same ability of a reference
preparation of protein S calibrated in International Units.
the International Standard for protein S. The equivalence in
Individual reagents may be obtained separately or in
commercial kits. Procedures and reagents may vary
described in the following example.
REAGENTS
Dilution buffer pH 7.4. Isotonic non-chelating buffer, for
example : dissolve 6.08 g of tris(hydroxymethyl)aminomethane R and 8.77 g of sodium chloride R in water R
and adjust the pH (2.2.3) if necessary ; add 10 g of bovine
albumin R or human albumin R and dilute to 1000.0 ml
with water R.
METHOD
Protein S-deficient plasma. Citrated human plasma with
no measurable protein S content and, preferably, also free
of C4b-binding protein.
dilution buffer pH 7.4 to produce at least 3 separate dilutions Coagulation activator. This reagent is used to initiate
for each preparation in the range 0.010-0.150 IU/ml,
coagulation in the protein S-deficient plasma and in so
preferably in duplicate. Dilutions may need to be adjusted
doing it also provides a source of activated factor V. The
to ensure that they fall on the steepest, most linear part of
activator may consist of tissue factor, activated factor X, or
the dose-response curve and also provide maximum overlap an agent capable of directly activating factor X that may be
purified from snake venom (Vipera russelli). The reagent
be examined.
also contains APC, phospholipids and calcium chloride R,
or, alternatively, calcium chloride may be added separately
Mix 1 volume of each dilution with 1 volume of
after a timed activation period.
protein C-deficient plasma and 1 volume of protein C
activator, all pre-warmed to 37 C. Add 1 volume of calcium METHOD
chloride R previously warmed to 37 C, and record the
clotting time.
The clotting time is proportional to the concentration of
dilution buffer pH 7.4 to produce at least 3 separate dilutions
protein C in each dilution. Check the validity of the assay
for each preparation in the range 0.020-0.100 IU/ml,
and calculate the potency of the preparation to be examined preferably in duplicate. Dilutions may need to be adjusted
by the usual statistical methods (5.3).
to ensure that they fall on the steepest, most linear part of
Protein C activator. A protein isolated from the venom of

the viper Agkistrodon contortrix contortrix that specifically
activates protein C. Reconstitute and store according to the
manufacturers instructions.
591
Bilberry fruit dry extract, refined and standardised
the dose-response curve and also provide maximum overlap

be examined.
Mix 1 volume of each dilution with 1 volume of
protein S-deficient plasma, both previously heated to 37 C.
Add 2 volumes of coagulation activator, previously heated
to 37 C, and record the clotting time.
Alternative procedures may use a coagulation activator
without calcium chloride, and require a precisely timed
activation period before the addition of calcium chloride and
the measurement of clotting time.
The clotting time is proportional to the concentration of
protein S in each dilution. Check the validity of the assay
and calculate the potency of the preparation to be examined
by the usual statistical methods (5.3).
mobile phase B : water R, acetic acid R (40:60 V/V).

Application : 10 l [or 2 l], as bands.
Development : over a path of 10 cm [or 6 cm].
Drying : in air.
Detection : examine in daylight.
Procedure : after application of the reference solution
and the test solution, elute with mobile phase A, dry the
plate using warm air and elute using mobile phase B.
Results : see below the sequence of zones present in the
chromatograms obtained with the reference solution
and the test solution. Furthermore, other zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
A faint violet-red zone
_______

XXXX:2394
BILBERRY FRUIT DRY EXTRACT,

REFINED AND STANDARDISED
Myrtilli fructus extractum siccum
raffinatum et normatum
DEFINITION
Refined and standardised dry extract produced from Bilberry
fruit, fresh (1602).
Content : 32.4 per cent to 39.6 per cent of anthocyanins
(dried extract).
PRODUCTION
The extract is produced by an appropriate procedure, using
ethanol (96 per cent) or methanol. Purification may be
performed by chromatography.
CHARACTERS
Appearance : dark reddish-violet, amorphous powder.
IDENTIFICATION
First identification : B.
Second identification : A.
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 0.10 g of the extract to be
examined in 25 ml of methanol R. Stir for 15 min and
filter.
Reference solution. Dissolve 2 mg of chrysanthemin R
and 2 mg of myrtillin R in 5 ml of methanol R.
Plate : TLC plate coated with cellulose for
chromatography R (5-40 m) [or TLC plate coated with
cellulose for chromatography R (2-10 m)].
Mobile phase :
mobile phase A : hydrochloric acid R, acetic acid R,
water R (3:15:82 V/V/V) ;
_______
A violet-red zone
Chrysanthemin : a violet-red
zone
Myrtillin : a violet-red zone
A violet-red zone
(chrysanthemin)
A violet-red zone (myrtillin)
_______
Reference solution
_______
Test solution
B. Liquid chromatography (2.2.29) as described in the test

for total free anthocyanidins.
The characteristic anthocyanin peaks (peaks 1-8, 10-15
and 17) in the chromatogram obtained with the test
solution are similar in their retention times to those in
the chromatogram obtained with reference solution (b).
TESTS
Total free anthocyanidins. Liquid chromatography (2.2.29).
Maintain the solutions at 4 C.
Test solution. Dissolve 125.0 mg of the extract to be
examined in a mixture of 2 volumes of hydrochloric acid R
and 98 volumes of methanol R and dilute to 25.0 ml with
the same mixture of solvents. Dilute 5.0 ml of this solution
to 20 ml with dilute phosphoric acid R.
Reference solution (a). Dissolve 10.0 mg of cyanidin
chloride CRS in a mixture of 2 volumes of hydrochloric
acid R and 98 volumes of methanol R and dilute to 25.0 ml
with the same mixture of solvents. Dilute 2.0 ml of this
solution to 100 ml with dilute phosphoric acid R.
Reference solution (b). Dissolve 125.0 mg of bilberry dry
extract CRS in a mixture of 2 volumes of hydrochloric acid R
and 98 volumes of methanol R and dilute to 25.0 ml with
the same mixture of solvents. Dilute 5.0 ml of this solution
to 20 ml with dilute phosphoric acid R.
Column :
size : l = 0.250 m, = 4.6 mm ;
temperature : 30 C.
(3) Agilent Zorbax Extend-C18, 5 m particle size, 80 pore size is suitable.
592
Bilberry fruit dry extract, refined and standardised
Mobile phase :
mobile phase A : anhydrous formic acid R, water R

(10:90 V/V) ;
mobile phase B : anhydrous formic acid R, acetonitrile R, Injection : 10 l.

methanol R, water R, (10:22.5:22.5:40 V/V/V/V) ;
Identification of peaks : use the chromatogram obtained
with reference solution (a) and the chromatogram supplied
Time
Mobile phase A
Mobile phase B
with bilberry dry extract CRS to identify the peaks.
(min)
(per cent V/V)
(per cent V/V)
0 - 35
93 75
7 25
35 - 45
75 35
25 65
45 - 46
35 0
65 100
46 - 50
100
50 - 51
0 93
100 7
51 - 60
93

peak-to-valley ratio : minimum 2.0, where Hp = height
above the baseline of the peak due to cyanidin
3-O-galactoside (peak 3) and Hv = height above the
baseline of the lowest point of the curve separating this
peak from the peak due to delphinidin 3-O-arabinoside
(peak 4).
1. delphinidin 3-O-galactoside chloride
11. petunidin 3-O-arabinoside chloride
2. delphinidin 3-O-glucoside chloride
12. peonidin 3-O-glucoside chloride
3. cyanidin 3-O-galactoside chloride
13. malvidin 3-O-galactoside chloride
4. delphinidin 3-O-arabinoside chloride
14. peonidin 3-O-arabinoside chloride
5. cyanidin 3-O-glucoside chloride (chrysanthemin)
15. malvidin 3-O-glucoside chloride
6. petunidin 3-O-galactoside chloride
16. cyanidin chloride
7. cyanidin 3-O-arabinoside chloride
17. malvidin 3-O-arabinoside chloride
8. petunidin 3-O-glucoside chloride
18. petunidin chloride
9. delphinidin chloride
19. peonidin chloride
10. peonidin 3-O-galactoside chloride
20. malvidin chloride
Figure 2394.-1. Chromatogram for the assay of refined and standardised bilberry fruit dry extract
593
Calculate the percentage of total free anthocyanidins using

the following expression :
A1
m1
sum of the areas of the peaks due to the

anthocyanidins (peaks 9, 16, 18 and 20) in the
chromatogram obtained with the test solution ;
area of the peak due to cyanidin chloride in
the chromatogram obtained with reference
solution (a) ;
mass of the extract to be examined, in milligrams ;
mass of cyanidin chloride CRS in reference
solution (a), in milligrams ;
percentage content of cyanidin chloride in
cyanidin chloride CRS.
Limits : not more than 1.0 per cent of total free

anthocyanidins.
ASSAY

Related substances : TLC test for Hydroquinone has been
replaced by an LC for related substances in accordance
with current policy as part of a special revision programme.
The limits proposed are based on batches results.
XXXX:1183
CALCIUM DOBESILATE
MONOHYDRATE
Calcii dobesilas monohydricum
C12H10CaO10S2,H2O
Mr 436.4
DEFINITION
Calcium di(2,5-dihydroxybenzenesulphonate).
Content : 99.0 per cent to 102.0 per cent (anhydrous
substance).
Liquid chromatography (2.2.29) as described in the test for

total free anthocyanidins with the following modification.
CHARACTERS
Appearance : white or almost white, hygroscopic powder.
Injection: test solution and reference solution (b).
Solubility : very soluble in water, freely soluble in anhydrous
ethanol, very slightly soluble in 2-propanol, practically
Calculate the percentage content of total anthocyanins using insoluble in methylene chloride.
the following expression :
IDENTIFICATION
A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 0.100 g in water R and dilute
to 200.0 ml with the same solvent. Dilute 5.0 ml of the
solution to 100.0 ml with water R.
= sum of the areas of the peaks due to the
A
Spectral range : 210-350 nm.
anthocyanins (peaks 1-8, 10-15 and 17) in the
Absorption maxima : at 221 nm and 301 nm.
Specific absorbance at the absorption maximum at
A1 = area of the peak due to chrysanthemin (peak 5)
301 nm : 174 to 181.
in the chromatogram obtained with reference
B.
Mix 1 ml of ferric chloride solution R2, 1 ml of a freshly
solution (b) ;
prepared
10 g/l solution of potassium ferricyanide R
m
= mass of the extract to be examined, in milligrams ;
and 0.1 ml of nitric acid R. To this mixture add 5 ml of
m1 = mass of bilberry dry extract CRS in reference
freshly prepared solution S (see Tests) : a blue colour and
solution (b), in milligrams ;
a precipitate are immediately produced.
p
= percentage content of chrysanthemin in bilberry
C. 2 ml of freshly prepared solution S gives reaction (b) of
calcium (2.3.1).
dry extract CRS.
Loss on drying (2.8.17) : maximum 4.5 per cent.
Total ash (2.4.16) : maximum 2.0 per cent.
STORAGE
In an airtight container, protected from light.
Reagents
Chrysanthemin. C21H21ClO11. (Mr 484.8). XXXXXXX.
[7084-24-4]. Cyanidin 3-O-glucoside chloride.
Myrtillin. C21H21ClO12. (Mr 500.8). XXXXXXX. [6906-38-3].
Delphinidin 3-O-glucoside chloride.
594
TESTS
Solution S. Dissolve 10.0 g in carbon dioxide-free water R
and dilute to 100 ml with the same solvent.
Appearance of solution. Solution S, when freshly prepared,
is clear (2.2.1) and colourless (2.2.2, Method II).
pH (2.2.3) : 4.5 to 6.0 for solution S.
Hydroquinone. Examine by thin-layer chromatography
(2.2.27), using as the coating substance a suitable silica gel
with a fluorescent indicator having an optimal intensity at
254 nm.
Test solution. Dissolve 2.0 g of the substance to be examined
in water R and dilute to 10 ml with the same solvent.
Reference solution. Dissolve 10 mg of hydroquinone R in
water R and dilute to 50 ml with the same solvent.
Apply to the plate 10 l of each solution and dry the starting

points in a current of cool air. Develop over a path of 15 cm
using a mixture of 20 volumes of methylene chloride R,
30 volumes of methyl acetate R and 50 volumes of ethyl
acetate R. Dry the plate in a current of hot air and examine
in ultraviolet light at 254 nm. Any spot corresponding to
hydroquinone in the chromatogram obtained with the test
solution is not more intense than the principal spot in the
chromatogram obtained with the reference solution (0.1 per
cent).
Related substances. Liquid chromatography (2.2.29). Use
freshly prepared solutions. Maintain them at 6 C.
Buffer solution. Dissolve 1.2 g of sodium dihydrogen
phosphate anhydrous R in 900 ml of water for
chromatography R. Adjust the pH to 6.5 with disodium
hydrogen phosphate solution R and dilute to 1000 ml with
water for chromatography R.
solvent.
to 100.0 ml with water R. Dilute 1.0 ml of this solution to
10.0 ml with water R.
Reference solution (b). Dissolve 5.0 mg of
hydroquinone CRS (impurity A) in water R and
dilute to 25.0 ml with the same solvent.
Reference solution (c). Mix 1 ml of reference solution (b)
with 4 ml of the test solution.
Reference solution (d). Dilute 1.0 ml of reference solution (b)
to 20.0 ml with water R.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 m)(4).
Mobile phase : acetonitrile R1, buffer solution (10:90 V/V).
Injection : 10 l of the test solution and of reference

solutions (a), (c) and (d).
Run time : 2.5 times the retention time of dobesilate.
Relative retention with reference to dobesilate (retention
time = about 6 min) : impurity A = about 1.7.
System suitability : reference solution (c) :
resolution : minimum 5 between the peaks due to
dobesilate and impurity A.
Limits :
impurity A : not more than the area of the principal peak
in the chromatogram obtained with reference solution (d)
(0.1 per cent) ;
total : not more than 0.2 per cent ;
(0.05 per cent).
Heavy metals (2.4.8) : maximum 15 ppm.
1.0 g complies with test C. Prepare the reference solution
using 1.5 ml of lead standard solution (10 ppm Pb) R.
Iron (2.4.9) : maximum 10 ppm, determined on 10 ml of
solution S.
Water (2.5.12) : 4.0 per cent to 6.0 per cent, determined on
0.500 g.
ASSAY
Dissolve 0.200 g in a mixture of 10 ml of water R and 40 ml of
dilute sulphuric acid R. Titrate with 0.1 M cerium sulphate,
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M cerium sulphate is equivalent to 10.45 mg of
C12H10CaO10S2.
STORAGE
Figure 1183.-1. Chromatogram for the test for related substances of calcium dobesilate monohydrate : solution of
calcium dobesilate monohydrate spiked with impurity A
(4) Atlantis dC18 and Hypersil C18 are suitable.
595
Clodronate disodium tetrahydrate
Reference solution (b). Dissolve 1 mg of clodronate

impurity D CRS in 10 ml of water R, sonicate for 10 min and
dilute to 20.0 ml with water R. Mix 2.0 ml of this solution
with 10.0 ml of the test stock solution and dilute to 20.0 ml
with water R.
Apparatus : ion-exchange chromatograph (HPIC) with
suppression(5).
A. benzene-1,4-diol (hydroquinone).
Precolumn :
size : l = 0.05 m, = 4 mm ;
stationary phase : anion exchange resin R(6) ;
bead size : 9 m.
Column :
XXXX:1777 size : l = 0.25 m, = 4 mm ;
stationary phase : anion exchange resin R(7) ;
CLODRONATE DISODIUM
bead size : 9 m.
TETRAHYDRATE
Mobile phase :
mobile phase A : 0.21 g/l solution of sodium hydroxide R
in carbon dioxide-free water R ; close immediately, mix
Dinatrii clodronas tetrahydricum
and use under helium pressure ;
mobile phase B : 4.2 g/l solution of sodium hydroxide R
in carbon dioxide-free water R ; close immediately, mix
and use under helium pressure ;
IMPURITIES
CH2Cl2Na2O6P2,4H2O
Mr 360.9
DEFINITION
Disodium (dichloromethylene)bis[hydrogen (phosphonate)]
tetrahydrate.
substance).
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water, practically insoluble in
ethanol (96 per cent), slightly soluble in methanol.
Time
(min)
0
Mobile phase A
(per cent V/V)
90
Mobile phase B
(per cent V/V)
10
0 - 10
90 60
10 40
10 - 22
60 50
40 50
22 - 23
50 20
50 80
23 - 25
20
80
25 - 26
20 90
80 10
26 - 31
90
10
Flow rate : 1 ml/min.

Detection : conductivity detector.
IDENTIFICATION
Injection : 20 l.
Relative retention with reference to clodronate (retention
Comparison : clodronate disodium tetrahydrate CRS.
time = about 12.8 min) : impurities A and B = about 0.7.
B. Dissolve 0.5 g in 10 ml of water R. The solution gives
reaction (a) of sodium (2.3.1).
peak-to-valley ratio : minimum 3, where Hp= height above
TESTS
the baseline of the peak due to impurity D and Hv = height
above the baseline of the lowest point of the curve
Appearance of solution. The solution is clear (2.2.1) and
separating this peak from the peak due to clodronate.
Dissolve 0.50 g in carbon dioxide-free water R and dilute to Limits :
10 ml with the same solvent.
sum of impurities A and B : not more than the area of
the principal peak in the chromatogram obtained with
pH (2.2.3) : 3.0 to 4.5.
reference solution (a) (0.2 per cent) ;
Dissolve 0.500 g in carbon dioxide-free water R and dilute
to 10 ml with the same solvent.
than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (a)
plastic vials.
(0.10 per cent) ;
Test solution. Dissolve 0.125 g of the substance to be
disregard
limit : 0.25 times the area of the principal peak
examined in 30 ml of water R, sonicate for 10 min and dilute
in
the
chromatogram
obtained with reference solution (a)
to 50.0 ml with water R (test stock solution). Dilute 10.0 ml
(0.05
per
cent).
of the test stock solution to 20.0 ml of water R.
2.0 g complies with test G. Prepare the reference solution
using lead standard solution (2 ppm Pb) R.
(5) Dionex DX-500 HPIC & Dionex ASRS are suitable.
(6) IonPac AG11-HC Dionex is suitable.
(7) IonPac AS11-HC Dionex is suitable.
596
1. impurities A + B
2. clodronate
3. impurity D
Figure 1777.-1. Chromatogram for the test for related substances of clodronate disodium tetrahydrate spiked with
impurities A, B and D
Water (2.5.12) : 18.5 per cent to 21.0 per cent, determined
on 0.100 g.
ASSAY
Dissolve 0.140 g in 10 ml of water R. Add 10 ml of
concentrated sodium hydroxide solution R and some glass
beads. Boil for 10 min. Cool in an ice-bath and add 30 ml
of water R and 10 ml of nitric acid R. Titrate with 0.1 M
silver nitrate, determining the end-point potentiometrically
(2.2.20).
1 ml of 0.1 M silver nitrate is equivalent to 14.44 mg of
CH2Cl2Na2O6P2.
IMPURITIES
Specified impurities : A, B.
Other detectable impurities (the following substances
would, if present at a sufficient level, be detected by one
or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
impurities and/or by the general monograph Substances for
pharmaceutical use (2034). It is therefore not necessary to
identify these impurities for demonstration of compliance.
See also 5.10. Control of impurities in substances for
pharmaceutical use) : C, D.
A. [dichloro[hydroxy(isopropoxy)phosphoryl]methyl]phosphonic acid,
B. phosphoric acid,
C. carbonylbis(phosphonic acid),
D. (chloromethylene)bis(phosphonic acid).
Reference: PA/PH/Exp. 15V/T (04) 47 ANP 2R

A new draft proposal has been prepared in order to take
into account :
comments received after the enquiry in
Pharmeuropa 17.3 and also comments discussed
at the hearing with coccidiosis vaccine manufacturers
in January 2006 ;
the new proposal for the immunogenicity test, which
links signs that may be observed post-challenge
specifically to the relevant Eimeria challenges ;
the reply from the producers of SPF eggs on the status
of these eggs with respect to coccidiosis.
Furthermore, it is proposed that the weight gain parameter
is deleted from the specific test for the safety of the vaccine
composition (section 2-3-1), whereas this parameter is kept
in the immunogenicity test (section 2-3-4).
XXXX:2326
COCCIDIOSIS VACCINE (LIVE) FOR

CHICKENS
Vaccinum coccidiosidis vivum ad pullum
1. DEFINITION
Coccidiosis vaccine (live) for chickens is a preparation of
sporulated oocysts of a suitable line or lines of species of
coccidial parasites (Eimeria species). This monograph
applies to vaccines intended for administration to chickens
for active immunisation.
597
2. PRODUCTION
2-1. PREPARATION OF THE VACCINE
Oocysts are grown in chickens from a flock free from
specified pathogens (SPF) (5.2.2) or in embryonated hens
eggs from an SPF flock (5.2.2). The eggs must be subject to
disinfection and incubation conditions validated to ensure
the inactivation of any Eimeria that may be on the shells.
The hatched chickens must then be reared in disinfected
premises, in isolation conditions to ensure no infection
with Eimeria. The chickens must not have been treated
with coccidiostats. Oocysts are collected from faeces or
contents of the intestinal tract of infected chickens during
the patent period. Oocysts of different Eimeria lines are
grown separately. Oocysts are isolated, purified, disinfected,
sporulated and counted. The vaccine is produced by
blending defined numbers of sporulated oocysts of each line
in a suitable medium.
2-2. SEED LOTS
2-2-1. Identification. The identity of each Eimeria master
seed is established from the characteristics of the coccidia
produced from it, based on an appropriate selection of the
following : size and shape of the oocyst ; localisation of the
developmental stages in the chicken gut ; pathognomic
lesions (E. tenella, E. acervulina, E. necatrix, E. maxima
and E. brunetti) and lack of macroscopic lesions (E. praecox
and E. mitis) ; size of schizonts in intestinal mucosa ; size of
gametocytes in the mucosa ; differences in the electrophoretic
mobilities of certain isoenzymes, e.g. lactate dehydrogenase
and glucose phosphate isomerase ; and by the use of
molecular biology techniques. Artificially attenuated lines
may be distinguished from the parent strains by studying
parameters appropriate to the method of attenuation.
2-2-2. Extraneous agents. Carry out tests 1-6 of chapter
2.6.24. Avian viral vaccines : tests for extraneous agents
in seed lots. General provisions a-d, f and h and section 7
of chapter 2.6.24 are also applicable. In these tests on the
master seed lot, use organisms that are not more than
5 passages from the master seed lot at the start of the tests.
Each master seed lot complies with the requirements of each
test.
2-3. CHOICE OF VACCINE COMPOSITION
Only coccidial lines shown to be satisfactory with respect to
residual pathogenicity and increase in virulence may be used
in the preparation of the vaccine, and the tests described
below (sections 2-3-2 and 2-3-3) may be used to demonstrate
this. The vaccine shall be shown to be satisfactory with
respect to safety (5.2.6) and efficacy (5.2.7) for the chickens
for which it is intended. The following tests under Specific
test for the safety of the vaccine composition (section 2-3-1)
and Immunogenicity (section 2-3-4) may be used during the
demonstration of safety and efficacy.
2-3-1. Specific test for the safety of the vaccine composition.
Carry out the test with a preparation containing oocysts of
each species at the least attenuated passage level that will be
present in a batch of vaccine. Use not fewer than 20 chickens,
from an SPF flock (5.2.2). The chickens must be hatched
and reared as described in section 2-1 and must not have
been treated with coccidiostats. Use 14-day-old chickens.
During the test, chickens are housed in suitable conditions
with the use of floor pens to favour reinfection with oocysts.
Administer by gavage or another suitable route to each
chicken a quantity of vaccinal oocysts consisting of the
equivalent of not less than 10 times the maximum quantity
of oocysts of each coccidial species likely to be contained in
1 dose of the vaccine. Observe the chickens at least daily for
598
21 days. The test is not valid if more than 10 per cent of the
vaccinated chickens die from causes not attributable to the
vaccinal oocysts. The vaccine complies with the test if no
vaccinated chicken shows notable clinical signs of coccidiosis
or dies from causes attributable to the vaccine.
2-3-2. Test for residual pathogenicity. Carry out a separate
test with each coccidial species and line to be included in the
vaccine. Use in each case a preparation containing oocysts
at the least attenuated passage level that will be present
between the master seed lot and a batch of the vaccine. For
each test use not fewer than 20 chickens not less than the
minimum age to be recommended for vaccination and not
more than 14 days of age, from an SPF flock (5.2.2). The
chickens must be hatched and reared as described in section
2-1 and must not have been treated with coccidiostats.
During the test, the chickens are placed in cages. Administer
by gavage or another suitable route to each chicken the
equivalent of not less than 10 times the maximum quantity
of the vaccinal oocysts likely to be contained in 1 dose of
the vaccine. Observe the chickens at least daily for 14 days.
The test is not valid if more than 10 per cent of chickens die
from causes not attributable to the vaccinal oocysts. Collect
faeces and determine oocyst production daily from day 3
until day 14. On one day between day 4 and day 8, depending
on the length of the pre-patent period, when lesions are
expected to be maximal, and on day 14, euthanise not fewer
than 9 chickens and examine the intestinal tract for specific
lesions indicative of infection with the coccidial species or,
for species known not to induce macroscopic lesions (e.g.
E. mitis and E. praecox), microscopic evidence of infection
such as demonstration of oocysts or developing oocysts in
the gut content or scrapings of the gut wall. For species
that have the potential to produce relevant macroscopic
pathological changes if not attenuated, a scoring system
with a scale from 0 to 4 is used to record the species-specific
lesions visible in the intestine as follows.
Eimeria acervulina
0
No gross lesions.
Scattered, white plaque-like lesions containing

developing oocysts are confined to the duodenum.
These lesions are elongated with the longer axis
transversely oriented on the intestinal walls like the
rungs of a ladder. They may be seen on either the
serosal or mucosal intestinal surfaces. There may be
up to a maximum of 5 lesions per square centimetre.
Lesions are much closer together, but not coalescent ;
lesions may extend as far posterior as 20 cm below
the duodenum in 3-week-old birds. The intestinal
walls show no thickening. Digestive tract contents
are normal.
Lesions are numerous enough to cause coalescence
with reduction in lesion size and give the intestine a
coated appearance. The intestinal wall is thickened
and the contents are watery. Lesions may extend as
far posterior as the yolk sac diverticulum.
The mucosal wall is greyish with completely
coalescent colonies. Congestion may be confined to
small petechiae or, in extremely heavy infections,
the entire mucosa may be bright red in colour.
Individual lesions may be indistinguishable in the
upper intestine. Typical ladder-like lesions appear in
the middle part of the intestine. The intestinal wall is
very much thickened and the intestine is filled with
a creamy exudate, which may bear large numbers of
oocysts. Birds dying of coccidiosis are scored as 4.
Eimeria brunetti
0
No gross lesions.
No gross lesions. In the absence of distinct lesions,

presence of parasites may go undetected unless
scrapings from suspicious areas are examined
microscopically.
Intestinal wall may appear grey in colour. The lower
portion may be thickened and flecks of pink material
sloughed from the intestine are present.
Intestinal wall thickened and a blood-tinged catarrhal
exudate present. Transverse red streaks may be
present in the lower rectum and lesions occur in the
caecal tonsils. Soft mucous plugs may be present in
this latter area.
Extensive coagulation necrosis of the mucosal surface
of the lower intestine may be present. In some birds
a dry necrotic membrane may line the intestine and
caseous cores may plug the entrance to the caeca.
Lesions may extend into the middle or upper intestine.
Dead birds are scored as 4.
2
3
Eimeria maxima
0
No gross lesions.
Small red petechiae may appear on the serosal side of

the mid-intestine. There is no ballooning or thickening
of the intestine, though small amounts of orange
mucous may be present.
Serosal surface may be speckled with numerous red
petechiae. The intestine may be filled with orange
mucous, but there is little or no ballooning of the
intestine. There is thickening of the wall.
Intestinal wall is ballooned and thickened. The
mucosal surface is roughened. Intestinal contents are
filled with pinpoint blood clots and mucous.
The intestinal wall may be ballooned for most of its
length. It contains numerous blood clots and digested
red blood cells giving a characteristic colour and
putrid odour. The wall is greatly thickened. Dead
birds are scored as 4.
3
4
Eimeria necatrix
0
No gross lesions.
Small scattered petechiae and white spots are easily

seen on the serosal surface. There is little, if any,
damage apparent on the mucosal surface.
Numerous petechiae are seen on the serosal surface.
Slight ballooning confined to the midgut area may
be present.
There is extensive haemorrhage into the lumen of
the intestine. The serosal surface is covered with red
petechiae and/or white plaques, and is rough and
thickened with many pinpoint haemorrhages. Normal
intestinal contents are lacking. Ballooning extends
over the lower half of the small intestine.
Extensive haemorrhage gives the intestine a dark
colour, and the intestinal contents consist of red or
brown mucous. Ballooning may extend throughout
much of the length of the intestine. Dead birds are
scored as 4.
2
3
Eimeria tenella
0
No gross lesions.
Very few scattered petechiae are seen on the caecal

wall, and there is no thickening of the caecal walls.
Normal caecal contents are present.
Lesions are more numerous with noticeable blood in
the caecal contents, and the caecal wall is somewhat
thickened. Normal caecal contents are present.
Large amounts of blood or caecal cores are present,
and the caecal walls are greatly thickened. There is
little if any normal faecal contents in the caeca.
Caecal wall is greatly distended with blood or large
caseous cores. Faecal debris is lacking or included in
the cores. Dead birds are scored as 4.
2
3
4
The species and line comply with the test for attenuation
if no more than mild coccidial lesions or limited signs of
infection are observed ; where the scoring system described
above is appropriate, the average lesion score on each
of days 5 or 6 and 14 is not greater than 1.5 points and no
individual score is greater than 3 points. The quantity and
time of oocyst production is determined.
2-3-3. Increase in virulence. Carry out a separate test with
each coccidial species and line to be included in the vaccine.
Use a preparation containing oocysts at the least attenuated
passage level that will be present between the master seed
lot and a batch of the vaccine. For each test use chickens
7-14 days of age, from an SPF flock (5.2.2). The chickens
must be hatched and reared as described in section 2-1 and
must not have been treated with coccidiostats. During the
test, chickens are placed in cages. Administer by gavage
or another suitable route to each of 5 chickens a quantity
of oocysts that will allow recovery of the oocysts for the
passages described below. Collect faeces daily from day 2
to day 14 after infection and prepare a pooled suspension
of sporulated oocysts from the 5 chickens. Administer a
suitable quantity by gavage or another suitable route to each
of 5 further chickens. Carry out this passage operation not
fewer than 5 times, verifying the presence of oocysts at each
passage. Repeat the test for residual pathogenicity (section
2-3-2), using the maximally passaged oocysts that have been
recovered and administering a similar quantity of sporulated
oocysts per bird to that used in the test with unpassaged
oocysts. Compare the results obtained for signs of lesions
or infection in the intestinal tract and oocyst output from
administration of passaged and unpassaged oocysts. The
line complies with the test if no indication of an increase
in virulence of the maximally passaged oocysts compared
with the unpassaged oocysts is observed. The test is invalid
if oocysts are not recovered at any passage level.
2-3-4. Immunogenicity. The efficacy of each coccidial
species and line included in the vaccine has to be determined
in a separate study with an appropriate challenge strain.
For each component, a test is carried out with vaccine
administered by each route and method of administration
to be recommended, using in each case chickens not older
than the youngest age to be recommended for vaccination.
The quantity of each of the components in the batch of
vaccine administered to each chicken is not greater than the
minimum number of oocysts to be stated on the label and
the oocysts are at the most attenuated passage level that will
be present in a batch of vaccine. Use for the test not fewer
than 40 chickens from an SPF flock (5.2.2). The chickens
must be hatched and reared as described in section 2-1 and
must not have been treated with coccidiostats. Vaccinate
not fewer than 20 chickens and maintain not fewer than
20 chickens as controls. For the evaluation of weight gain
599
with Eimeria strains showing a low pathogenicity, the

number of chickens used may be higher. After vaccination,
the chickens are housed in suitable conditions with the use
of floor pens to favour reinfection with oocysts. On a suitable
day between days 14 and 21 after vaccination, weigh each
chicken, move them to cages and challenge each chicken by
gavage or another suitable route with a sufficient quantity of
virulent coccidia to induce in the unvaccinated controls signs
of disease characteristic of the Eimeria challenge species.
Observe the birds at least daily until the end of the test.
Record deaths and the number of surviving chickens that
show clinical signs of disease. Collect faeces and determine
oocyst production from day 3 after challenge until the end of
the test. On an appropriate day between days 4 and 8 after
challenge, depending on the length of the pre-patent period
of the challenge species, weigh each chicken. Euthanise
10 birds from each group and examine them for lesions in
the intestinal tract. Where appropriate, record the specific
lesions indicative of the coccidial challenge species (using
the scoring system described in section 2-3-2). For species
known not to induce macroscopic lesions (E. mitis and
E. praecox), examine the birds for microscopic evidence of
infection such as demonstration of oocysts or developing
oocysts in the intestinal contents or scraping of the intestinal
wall. On day 14 after challenge, weigh each of the remaining
birds.
the percentage of sporulated oocysts and the oocyst count.

The values obtained are within the limits shown to allow
preparation of a satisfactory vaccine.
2-4-2. Batch potency test for each Eimeria species in
the vaccine. It is not necessary to carry out the potency
test (section 3-7) for each batch of the vaccine if it has
been carried out using a batch or batches of vaccine with
minimum potency and sporulated oocyst content. Where
the test is not carried out, an alternative validated method
is used, the criteria for acceptance for each component
being set with reference to a batch of vaccine that has given
satisfactory results in the test described under Potency.
3. BATCH TESTS
3-1. Identification
3-1-1. Microscopical examination is used to confirm the
presence of coccidial oocysts in the batch of vaccine.
3-1-2. The potency test (or batch potency test) is used to
confirm the presence of oocysts of each of the Eimeria
species stated on the label.
3-2. Bacteria and fungi. The vaccine and where applicable
the liquid supplied with it comply with the test for sterility
prescribed in the monograph Vaccines for veterinary use
(0062) and comply with the test with a medium selective
for Campylobacter spp.
The test is invalid if :
3-3. Mycoplasmas. The vaccine complies with the test for
mycoplasmas (2.6.7).
during the period between vaccination and challenge,
more than 10 per cent of the vaccinated or control
3-4. Extraneous agents. Carry out tests 1-6 of chapter
chickens show abnormal clinical signs or die from causes 2.6.25. Avian live virus vaccines : tests for extraneous
not attributable to the vaccine ;
agents in batches of finished product. General provisions
a-d, g and h are also applicable. The vaccine complies with
and/or for challenges with E. tenella, E. acervulina,
E. necatrix, E. maxima or E. brunetti, fewer than 80 per the requirements of each test.
cent of control chickens euthanised between days 4
3-5. Safety. Use not fewer than 10 chickens from an SPF
and 8 have marked characteristic lesions of the challenge flock (5.2.2), and of the minimum age recommended for
infection in the intestine at post mortem examination (e.g. vaccination. The chickens must be hatched and reared as
lesions scores not less than 2) ;
described in section 2-1 and must not have been treated
with coccidiostats. Administer by a recommended route and
and/or for challenges with E. mitis or E. praecox, fewer method to each chicken preferably 10 doses of vaccine. If
than 80 per cent of the controls chickens euthanised
the volume of 10 doses is too large, administer the largest
between days 4 and 8 are infected.
possible volume. After vaccination, the chickens are housed
in suitable conditions with the use of floor pens to favour
The vaccine complies with the test if:
reinfection with oocysts. Observe the chickens at least daily
for all the Eimeria challenge species, the production
for 21 days. The test is not valid if more than 20 per cent of
of the oocysts is significantly decreased in vaccinates
the chickens show abnormal clinical signs or die from causes
compared with controls ;
not attributable to the vaccine. The vaccine complies with
the test if no chicken shows notable clinical signs of disease
for all the Eimeria challenge species, no vaccinated
or dies from causes attributable to the vaccine.
chicken dies due to the challenge infection ;
for challenge with E. tenella, E. acervulina, E. necatrix, 3-6. Sporulated oocyst count. The sporulated oocysts
content per dose is determined by counting the sporulated
E. maxima or E. brunetti, at least 80 per cent of the
oocysts in a suitable counting chamber, under the
vaccinates show no more than mild signs of disease and
microscope. The contents are not less than the minimum
these are less marked than those in the controls ;
and not more than the maximum content of sporulated
for challenges with E. tenella, E. acervulina, E. necatrix, oocysts stated on the label.
E. maxima or E. brunetti, at least 80 per cent of the
3-7. Potency. The vaccine complies with the requirements
vaccinates have no or minimal lesions in the intestine
of the test prescribed under Immunogenicity (section 2-3-4)
(e.g. mean lesions scores not greater than 1) and no bird using 1 dose of the vaccine administered by a recommended
has a lesion score of 4 ;
route.
for challenges with E. tenella, E. acervulina, E. necatrix,
4. LABELLING
E. maxima, E. brunetti, E. mitis, or E. praecox, the
growth rate in the vaccinates is significantly greater than The label states :
in the controls.
the Eimeria species included in the vaccine ;
2-4. MANUFACTURERS TESTS
the minimum and maximum number of sporulated
oocysts per dose ;
2-4-1. In-process test for sporulation rate and oocyst count.
A sample of each oocyst bulk is examined microscopically
the extent to which the vaccine causes a reduction in
after the sporulation step and before blending to determine
weight-gain, if any, after vaccination.
600
Cod-liver oil
Reference: PA/PH/Exp. 13H/T (06) 45 ANP
Peroxide value (2.5.5, Method B) : maximum 10.0.

Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
determined on 2.0 g, and extracting with 3 quantities, each
The Ph. Eur. contains 2 monographs on cod-liver oil : one of 50 ml, of peroxide-free ether R.
on type A (including a test for anisidine value) and one on
type B (no test for anisidine value). It is proposed to merge Stearin. 10 ml remains clear after cooling in iced water for
3 h. Heat 10 ml to 60-130 C then allow to cool in a bath of
these monographs.
iced water or a thermostatically controlled bath at 0 0.5 C
Vitamin A and Vitamin D3 : the system suitability test
for 3 h. If necessary, to eliminate insoluble matter, filter the
criteria of both assays have been reviewed and modified ;
sample after heating. The sample remains clear.
nd
in particular, the 2 criterion has been deleted because it
Composition of fatty acids. Gas chromatography (2.2.28).
did not give a true indication of the validity of the assay.
XXXX:1192 Trivial name of
NomenclaLower limit
Upper limit
COD-LIVER OIL (TYPE A)

Iecoris aselli oleum
DEFINITION
Purified fatty oil obtained from the fresh livers of Gadus
morhua L. and other species of Gadidae, solid substances
being removed by cooling and filtering. A suitable
antioxidant may be added.
The monograph covers types A and B defined by the
anisidine value.
Content : 600 IU (180 g) to 2500 IU (750 g) of vitamin A
per gram and 60 IU (1.5 g) to 250 IU (6.25 g) of vitamin D3
per gram.
CHARACTERS
Appearance : clear, yellowish, viscous liquid.
Solubility : practically insoluble in water, slightly soluble in
ethanol (96 per cent), miscible with light petroleum.
IDENTIFICATION
First identification : A, B, C.
Second identification : C, D.
A. In the assay for vitamin A using method A, the test
solution shows an absorption maximum (2.2.25) at
325 2 nm. In the assay for vitamin A using method B,
the chromatogram obtained with the test solution shows
a peak corresponding to the peak of all-trans-retinol in
the chromatogram obtained with the reference solution.
B. In the assay for vitamin D3, the chromatogram obtained
with test solution (a) shows a peak corresponding to the
peak of cholecalciferol in the chromatogram obtained
with reference solution (b).
C. Composition of fatty acids (see Tests).
D. To 0.1 g add 0.5 ml of methylene chloride R and 1 ml of
antimony trichloride solution R. Mix. A deep blue colour
develops in about 10 s.
TESTS
Colour : not more intensely coloured than a reference
solution prepared as follows : to 3.0 ml of red primary
solution add 25.0 ml of yellow primary solution and dilute
to 50.0 ml with a 10 g/l solution of hydrochloric acid R
(2.2.2, Method II).
Relative density (2.2.5) : 0.917 to 0.930.
Refractive index (2.2.6) : 1.477 to 1.484.
Acid value (2.5.1) : maximum 2.0.
Anisidine value (2.5.36) : maximum 30.0.
type A : maximum 30.0 ;
type B : more than 30.0.
Iodine value (2.5.4, Method B) : 150 to 180.
Use starch solution R2.
fatty acid
ture
area
(per cent)
area
(per cent)
14:0
Myristic acid
16:0
Palmitic acid
18:0
Stearic acid
Mono-unsaturated fatty acids :
2.0
7.0
1.0
6.0
14.0
4.0
16:1 n-7
Palmitoleic acid
18:1 n-7
cis-Vaccenic acid
18:1 n-9
Oleic acid
20:1 n-11
Gadoleic acid
20:1 n-9
Gondoic acid
22:1 n-9
Erucic acid
22:1 n-11+13
Cetoleic acid
(22:1 n-11)
Poly-unsaturated fatty acids :
4.5
2.0
12.0
1.0
5.0
0
5.0
11.5
7.0
21.0
5.5
17.0
1.5
12.0
18:2 n-6
18:3 n-3
18:4 n-3
20:5 n-3
0.5
0
0.5
7.0
3.0
2.0
4.5
16.0
22:6 n-3
6.0
18.0
Saturated fatty acids :
Linoleic acid
-Linolenic acid
Moroctic acid
Timnodonic
(eicosapentaenoic)
acid (EPA)
Cervonic
(docosahexaenoic)
acid (DHA)
Test solution. Introduce about 0.45 g of the substance to be

examined into a 10 ml volumetric flask, dissolve in hexane R
containing 50 mg of butylhydroxytoluene R per litre and
dilute to 10.0 ml with the same solvent. Transfer 2.0 ml of
this solution into a quartz tube and evaporate the solvent
with a gentle current of nitrogen R. Add 1.5 ml of a 20 g/l
solution of sodium hydroxide R in methanol R, cover with
nitrogen R, cap tightly with a polytetrafluoroethylene-lined
cap, mix and heat in a water-bath for 7 min. Cool, add
2 ml of boron trichloride-methanol solution R, cover with
nitrogen R, cap tightly, mix and heat in a water-bath for
30 min. Cool to 40-50 C, add 1 ml of trimethylpentane R,
cap and vortex or shake vigorously for at least 30 s.
Immediately add 5 ml of saturated sodium chloride
solution R, cover with nitrogen R, cap and vortex or shake
vigorously for at least 15 s. Allow the upper layer to become
clear and transfer it to a separate tube. Shake the methanol
layer once more with 1 ml of trimethylpentane R and
combine the trimethylpentane extracts. Wash the combined
extracts with 2 quantities, each of 1 ml, of water R and dry
over anhydrous sodium sulphate R. Prepare 2 solutions for
each sample.
Column :
material : fused silica ;
size : l = 30 m, = 0.25 mm ;
stationary phase : macrogol 20 000 R (film thickness
0.25 m).
Carrier gas : hydrogen for chromatography R or helium for
chromatography R, where oxygen scrubber is applied.
Split ratio : 1:200.
601
Cod-liver oil
Temperature :
Column
Time
(min)
0 - 55
Temperature
(C)
170 225
55 - 75
225
Injection port
250
Detector
280
Detection : flame ionisation.

Injection: 1 l, twice.
System suitability :
the 15 fatty acids to be tested are satisfactorily identified
from the chromatogram shown in Figure 1192.-1 ;
injection of a mixture of equal amounts of methyl
palmitate R, methyl stearate R, methyl arachidate R and
methyl behenate R gives area percentages of 24.4, 24.8,
25.2 and 25.6 ( 0.5 per cent), respectively ;
methyl oleate and methyl cis-vaccenate ; the resolution
between the pair due to methyl gadoleate and methyl
gondoate is sufficient for purposes of identification and
area measurement.
Calculate the area per cent for each fatty acid methyl ester
using the following expression :
Ax
peak area of fatty acid x ;
At
sum of the peak areas (up to C22:6 n-3).
The calculation is not valid unless :

the total area is based only on peaks due solely to fatty
acid methyl esters ;
the number of fatty acid methyl ester peaks exceeding
0.05 per cent of the total area is at least 24 ;
the 24 largest peaks of the methyl esters account for more
than 90 per cent of the total area. (These correspond
to, in common elution order : 14:0, 15:0, 16:0, 16:1 n-7,
16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3,
18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6,
20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3,
22:5 n-3, 22:6 n-3).
ASSAY
Vitamin A. Carry out the test as rapidly as possible,
avoiding exposure to actinic light and air, oxidising agents,
oxidation catalysts (for example, copper and iron) and
acids.
Use method A. If method A is found not to be valid, use
method B.
METHOD A
Ultraviolet absorption spectrophotometry (2.2.25).
Test solution. To 1.00 g in a round-bottomed flask, add
3 ml of a freshly prepared 50 per cent m/m solution of
potassium hydroxide R and 30 ml of anhydrous ethanol R.
Boil under reflux in a current of nitrogen R for 30 min.
Cool rapidly and add 30 ml of water R. Extract with 50 ml
of ether R. Repeat the extraction 3 times and discard the
lower layer after complete separation. Wash the combined
upper layers with 4 quantities, each of 50 ml, of water R, and
evaporate to dryness under a gentle current of nitrogen R
at a temperature not exceeding 30 C or in a rotary
evaporator at a temperature not exceeding 30 C under
reduced pressure (water ejector). Dissolve the residue in
sufficient 2-propanol R1 to give an expected concentration
of vitamin A equivalent to 10-15 IU/ml.
Measure the absorbances of the solution at 300 nm,
310 nm, 325 nm and 334 nm and at the wavelength of
maximum absorption with a suitable spectrophotometer in
1 cm specially matched cells, using 2-propanol R1 as the
compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in
International Units per gram, using the following expression :
A325 =
m
=
V
=
1830 =
1821
absorbance at 325 nm ;
mass of the substance to be examined, in grams ;
total volume of solution containing 10-15 IU of
vitamin A per millilitre ;
conversion factor for the specific absorbance of
all-trans-retinol, in International Units.
Figure 1192.-1. Chromatogram for the test for composition of fatty acids of cod-liver oil (type A)
602
Cod-liver oil
The above expression can be used only if A325 has a value of Calculate the content of vitamin A in International Units
not greater than A325,corr/0.970, where A325,corr is the corrected per millilitre of reference solution (a) using the following
absorbance at 325 nm and is given by the following equation : expression, taking into account the assigned content of
retinol acetate CRS :
A designates the absorbance at the wavelength indicated

by the subscript.
If A325 has a value greater than A325,corr/0.970, calculate the
content of vitamin A using the following expression :
A326 =
V1
volume of reference solution (a) used ;
V2
volume of the diluted solution ;
1900 =

retinol acetate CRS, in International Units.
Reference solution (b). Proceed as described for the test

solution but using 2.00 ml of reference solution (a) in place
of the substance to be examined.
The assay is not valid unless :
the wavelength of the maximum absorption lies between
323 nm and 327 nm ;
the absorbance at 300 nm relative to that at 325 nm is
at most 0.73.
METHOD B
Liquid chromatography (2.2.29).
Test solution. Prepare duplicates. To 2.00 g in a
round-bottomed flask, add 5 ml of a freshly prepared 100 g/l
solution of ascorbic acid R, 10 ml of a freshly prepared
800 g/l solution of potassium hydroxide R and 100 ml of
anhydrous ethanol R. Boil under a reflux condenser on
a water-bath for 15 min. Add 100 ml of a 10 g/l solution
of sodium chloride R and cool. Transfer the solution to a
500 ml separating funnel, rinsing the round-bottomed flask
with about 75 ml of a 10 g/l solution of sodium chloride R
and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution
of potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R and then with 3 quantities, each of
50 ml, of a 10 g/l solution of sodium chloride R. Filter the
upper layer through 5 g of anhydrous sodium sulphate R
on a fast filter paper into a 250 ml flask suitable for a rotary
evaporator. Wash the funnel with 10 ml of fresh extraction
mixture, filter and combine the upper layers. Distil them at
a temperature not exceeding 30 C under reduced pressure
(water ejector) and fill with nitrogen R when evaporation
is completed. Alternatively, evaporate the solvent under a
gentle current of nitrogen R at a temperature not exceeding
30 C. Dissolve the residue in 2-propanol R, transfer to a
25 ml volumetric flask and dilute to 25 ml with 2-propanol R.
Gentle heating in an ultrasonic bath may be required. A large
fraction of the white residue is cholesterol, constituting
approximately 50 per cent m/m of the unsaponifiable
matter of cod-liver oil.
Reference solution (a). Prepare a solution of retinol
acetate CRS in 2-propanol R1 so that 1 ml contains about
1000 IU of all-trans-retinol.
The exact concentration of reference solution (b) is assessed

by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (b) with 2-propanol R1 to a presumed
all-trans-retinol concentration of 10-15 IU/ml and measure
the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International
Units per millilitre of reference solution (b), using the
following expression :
A325 =
V3 =
V4
volume of reference solution (b) used ;
1830 =
1821

Column :
size : l = 0.25 m, = 4.6 mm ;
chromatography R (film thickness 5-10 m).
Mobile phase : water R, methanol R (3:97 V/V).
Injection : 10 l ; inject in triplicate the test solution and
reference solution (b).
Retention time : all-trans-retinol = 5 1 min.
the chromatogram obtained with the test solution shows a
peak that corresponds to the peak due to all-trans-retinol
in the chromatogram obtained with reference solution (b) ;
when using the method of standard additions to the test
solution there is greater than 95 per cent recovery of the
added retinol acetate CRS,
The exact concentration of reference solution (a) is assessed

by ultraviolet absorption spectrophotometry (2.2.25). Dilute the results obtained with the duplicate test solutions do
not differ by more than 5 per cent ;
reference solution (a) with 2-propanol R1 to a presumed
concentration of 10-15 IU/ml and measure the absorbance
the recovery of all-trans-retinol in reference solution (b)
at 326 nm in matched 1 cm cells using 2-propanol R1 as the
as assessed by direct absorption spectrophotometry is
greater than 95 per cent.
603
Cod-liver oil
Calculate the content of vitamin A using the following

expression :
PURIFICATION
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : nitrile silica gel for chromatography R
(film thickness 10 m).
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
A1 = area of the peak due to all-trans-retinol in the
Inject 350 l of reference solution (b). Collect the eluate
A2 = area of the peak due to all-trans-retinol in
from 2 min before until 2 min after the retention time of
cholecalciferol, in a ground-glass-stoppered tube containing
solution (b) ;
1 ml of a 1 g/l solution of butylhydroxytoluene R in
C
= concentration of retinol acetate CRS in
hexane R. Repeat the procedure with test solutions (a)
reference solution (a) as assessed prior to the
saponification, in International Units per millilitre and (b). Evaporate the eluates obtained from reference
solution (b) and from test solutions (a) and (b), separately,
(= 1000 IU/ml) ;
V
= volume of reference solution (a) treated (2.00 ml) ; to dryness at a temperature not exceeding 30 C under a
gentle current of nitrogen R. Dissolve each residue in 1.5 ml
m
= mass of the substance to be examined in the test
of acetonitrile R.
solution (2.00 g).
DETERMINATION
Vitamin D3. Liquid chromatography (2.2.29). Carry out the Column :
size : l = 0.15 m, = 4.6 mm ;
assay as rapidly as possible, avoiding exposure to actinic
light and air.
chromatography R (film thickness 5 m).
Internal standard solution. Dissolve 0.50 mg of
Mobile phase : phosphoric acid R, 96 per cent V/V solution
ergocalciferol CRS in 100 ml of anhydrous ethanol R.
of acetonitrile R (0.2:99.8 V/V).
Test solution (a). Prepare duplicates. To 4.00 g in a
round-bottomed flask, add 5 ml of a freshly prepared 100 g/l Flow rate : 1.0 ml/min.
Injection : 2 quantities not exceeding 200 l of each of the
3 solutions obtained under Purification.
of sodium chloride R and cool the solution to room
temperature. Transfer the solution to a 500 ml separating
ergocalciferol and cholecalciferol in the chromatogram
funnel, rinsing the round-bottomed flask with about 75 ml of
obtained with reference solution (b) ;
a 10 g/l solution of sodium chloride R and then with 150 ml
of a mixture of equal volumes of light petroleum R1 and
when using the method of standard additions to test
ether R. Shake for 1 min. When the layers have separated
solution (a) there is greater than 95 per cent recovery of
completely, discard the lower layer and wash the upper
the added cholecalciferol CRS when due consideration
layer, first with 50 ml of a 30 g/l solution of potassium
has been given to correction by the internal standard.
hydroxide R in a 10 per cent V/V solution of anhydrous
the results obtained with the test solution (a) duplicates
ethanol R, and then with 3 quantities, each of 50 ml, of
do not differ by more than 5 per cent.
a 10 g/l solution of sodium chloride R. Filter the upper
Calculate the content of vitamin D3 in International Units
layer through 5 g of anhydrous sodium sulphate R on a
per gram using the following expression, taking into account
fast filter paper into a 250 ml flask suitable for a rotary
the assigned content of cholecalciferol CRS :
30 C. Dissolve the residue in 1.5 ml of the mobile phase
described under Purification. Gentle heating in an ultrasonic m1 = mass of the sample in test solution (b), in grams ;
bath may be required. A large fraction of the white residue m2 = total mass of cholecalciferol CRS used for
is cholesterol, constituting approximately 50 per cent m/m
the preparation of reference solution (a), in
of the unsaponifiable matter of cod-liver oil.
micrograms (500 g) ;
A1 = area (or height) of the peak due to cholecalciferol
Test solution (b). To 4.00 g add 2.0 ml of the internal
in the chromatogram obtained with test
standard solution and proceed as described for test
solution (a) ;
solution (a).
A2 = area (or height) of the peak due to cholecalciferol
Reference solution (a). Dissolve 0.50 mg of
cholecalciferol CRS in 100.0 ml of anhydrous
solution (b) ;
ethanol R.
A3 = area (or height) of the peak due to ergocalciferol
Reference solution (b). Into a round-bottomed flask,
solution (b) ;
add 2.0 ml of reference solution (a) and 2.0 ml of the
internal standard solution and proceed as described for test A4 = area (or height) of the peak due to ergocalciferol in
the chromatogram obtained with test solution (b) ;
solution (a).
604
A5
A6
V1
V2
area (or height) of a possible peak in the

chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting
with ergocalciferol in test solution (b) ;
area (or height) of the peak due to cholecalciferol
solution (b) ;
total volume of reference solution (a) (100 ml) ;
volume of reference solution (a) used for preparing
reference solution (b) (2.0 ml).
STORAGE
In an airtight and well-filled container, protected from light.
If no antioxidant is added, store under an inert gas.
Once the container has been opened, its contents are used
as soon as possible and any part of the contents not used at
once is protected by an atmosphere of inert gas.
IDENTIFICATION
A. Examine the 13C NMR spectra obtained in the test for
positional distribution ((2)-acyl) of fatty acids (see Tests).
The spectra contain peaks between 172 ppm and 173 ppm
with shifts similar to those in the typical spectrum (see
Figure 2398-1).
The positional distribution ((2)-acyl) for cervonic
(docosahexaenoic) acid (C22:6 n-3 ; DHA), timnodonic
(eicosapentaenoic) acid (C20:5 n-3 ; EPA) and moroctic
acid (C18:4 n-3) complies with the limits.
B. Linoleic acid (see Tests).
TESTS
Acid value (2.5.1) : maximum 2.0.
Anisidine value (2.5.36) : maximum 10.0.
Peroxide value (2.5.5, Method B) : maximum 5.0.
Unsaponifiable matter (2.5.7) : maximum 1.5 per cent,
LABELLING
determined on 2.0 g, and extracting with 3 quantities, each
The label states :
of 50 ml, of peroxide-free ether R.
the number of International Units of vitamin A ;
Stearin. Heat 10 ml to 60-130 C then allow to cool in a
the number of International Units of vitamin D3 ;
bath of iced water or a thermostatically controlled bath at
type A or B ;
0 0.5 C for 3 h. If necessary, to eliminate insoluble matter,
filter the sample after heating. The sample remains clear.
the name and concentration of any added antioxidant.
Positional distribution ((2)-acyl) of fatty acids (2.2.33).
Test solution. Dissolve 190-210 mg of fresh farmed cod-liver
oil in 500 l of deuterated chloroform R. Prepare at least
3 samples and examine within 3 days.
Apparatus : high resolution FT-NMR spectrometer operating
at not less than 300 MHz.
Monographs on cod-liver oil (wild cod) are included in the
Acquisition of 13C NMR spectra. The following parameters
Ph. Eur.
may be used :
The following monograph describes cod-liver oil obtained
sweep width : 200 ppm ( 5 ppm to 195 ppm) ;
from farmed fish.
The methods proposed for the determination of vitamins A irradiation frequency offset : 95 ppm ;
time domain : 64 K ;
and D3 are taken directly from the monographs on wild
cod-liver oil (see revision in this issue).
pulse delay : 2 s ;
XXXX:2398
pulse program : zgig 30 (inverse gated, 30 excitation
pulse) ;
COD-LIVER OIL, FARMED
dummy scans : 4 ;
number of scans: 4096.
Iecoris aselli oleum domestici
Processing and plotting. The following parameters may be
DEFINITION
used :
Purified fatty oil obtained from the fresh livers of farmed
size : 64 K (zero-filling) ;
Gadus morhua L., solid substances being removed by
window multiplication : exponential ;
cooling and filtering.
Lorentzian broadening factor : 0.2 Hz.
Content :
Use the CDCl3 signal for shift referencing. The shift of the
sum of the contents of EPA and DHA (expressed as
central
peak of the 1:1:1 triplet is set to 77.16 ppm.
triglycerides) : 10.0 per cent to 28.0 per cent ;
Plot
the
spectral region 171.5-173.5 ppm. Compare the
vitamin A : 50 IU (15 g) to 500 IU (150 g) per gram ;
spectrum with the reference spectrum in Figure 2398.-1. The
vitamin D3 : maximum 50 IU (1.3 g) per gram.
shift values lie within the ranges given in Table 2398.-1.
Authorised antioxidants in concentrations not exceeding the
Table 2398.-1 - Shift values
levels specified by the competent authority may be added.
PRODUCTION
The fish shall only be given feed with a composition that
is in accordance with the relevant EU or other applicable
regulations.
CHARACTERS
Appearance : clear, pale yellowish liquid.
Solubility : practically insoluble in water, slightly soluble in
alcohol (96 per cent), miscible with light petroleum.
Signal
Shift range (ppm)
DHA
172.05 - 172.09
DHA
172.43 - 172.47
EPA
172.52 - 172.56
EPA
172.90 - 172.94
C18:4
172.56 - 172.60
C18:4
172.95 - 172.99
605
1. C18:4
2. EPA
3. C18:4
4. EPA
5. DHA
6. DHA
Figure 2398.-1. 13C NMR spectrum carbonyl region of farmed cod-liver oil
System suitability : calculate the signal-to-noise ratio for the
smallest relevant peak corresponding to C18:4 signal (in
the range 172.95-172.99 ppm) ; measure the peak width at
half-height for the central CDCl3 signal (at 77.16 ppm) :
signal-to-noise ratio of the smallest peak: minimum 5 ;
peak width : maximum 0.02 ppm.
Calculation of positional distribution ((2)-acyl) : use the
Linoleic acid (2.4.29) : 3.0 per cent to 11.0 per cent.
ASSAY
EPA and DHA (2.4.29). See Figure 2398.-2.
Vitamin A. Carry out the test as rapidly as possible,
avoiding exposure to actinic light and air, oxidising agents,
oxidation catalysts (for example, copper and iron) and
acids.
Use method A. If method A is found not to be valid, use
method B.
METHOD A
Ultraviolet absorption spectrophotometry (2.2.25).
Test solution. To 1.00 g in a round-bottomed flask, add
= peak area of the corresponding -carbonyl peak ;

3 ml of a freshly prepared 50 per cent m/m solution of
= peak area of -carbonyl peak from C22:6 n-3,
potassium hydroxide R and 30 ml of anhydrous ethanol R.

C20:5 n-3 or C18:4 n-3, respectively.
Boil under reflux in a current of nitrogen R for 30 min.
Cool rapidly and add 30 ml of water R. Extract with 50 ml
Limits :
of ether R. Repeat the extraction 3 times and discard the
The positional distribution ((2)-acyl) is 71 per cent to 81 per lower layer after complete separation. Wash the combined
cent for cervonic (docosahexaenoic) acid (C22:6 n-3 ; DHA), upper layers with 4 quantities, each of 50 ml, of water R, and
32 per cent to 40 per cent for timnodonic (eicosapentaenoic) evaporate to dryness under a gentle current of nitrogen R
acid (C20:5 n-3 ; EPA) and 28 per cent to 38 per cent for
at a temperature not exceeding 30 C or in a rotary
moroctic acid (C18:4 n-3).
evaporator at a temperature not exceeding 30 C under
Composition of fatty acids (2.4.29). See the chromatogram reduced pressure (water ejector). Dissolve the residue in
sufficient 2-propanol R1 to give an expected concentration
in Figure 2398.-2.
of vitamin A equivalent to 10-15 IU/ml.
The 24 largest peaks of the methyl esters account for more
Measure the absorbances of the solution at 300 nm,
than 90 per cent of the total area (these correspond to, in
310 nm, 325 nm and 334 nm and at the wavelength of
common elution order : 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, 18:4 n-3, 20:1 n-11, maximum absorption with a suitable spectrophotometer in
1 cm specially matched cells, using 2-propanol R1 as the
20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 n-6, 20:3 n-3, 20:4 n-3,
20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3, 22:6 n-3).
606
1. C14:0
5. C16:4 n-1
9. C18:2 n-6
13. C20:1 n-9
17. C20:3 n-3
21. C22:1 n-9
2. C15:0
6. C18:0
10 C18:3 n-3
14. C20:1 n-7
18. C20:4 n-3
22. C21:5 n-3
3. C16:0
7. C18:1 n-9
11. C18:4 n-3
15. C20:2 n-6
19. C20:5 n-3
23. C22:5 n-3
4. C16:1 n-7
8. C18:1 n-7
12. C20:1 n-11
16. C20:4 n-6
20. C22:1 n-11
24. C22:6 n-3
Figure 2398.-2. Chromatogram for the test for composition of fatty acids of farmed cod-liver oil
If A325 has a value greater than A325,corr /0.970, calculate the
Calculate the content of vitamin A, as all- trans -retinol, in
International Units per gram, using the following expression : content of vitamin A using the following expression :
The assay is not valid unless :

the wavelength of maximum absorption lies between
323 nm and 327 nm ;
the
absorbance at 300 nm relative to that at 325 nm is
A325 = absorbance at 325 nm ;
at most 0.73.
m
= mass of the substance to be examined, in grams ;
METHOD B
= total volume of solution containing 10-15 IU of
V
vitamin A per millilitre ;
Test solution. Prepare duplicates. To 2.00 g in a
1821 = conversion factor for the specific absorbance of
all- trans -retinol, in International Units.
The above expression can be used only if A325 has a value
of not greater than A325,corr /0.970, where A325,corr is the
corrected absorbance at 325 nm and is given by the following of sodium chloride R and cool. Transfer the solution to a
equation :
500 ml separating funnel, rinsing the round-bottomed flask
with about 75 ml of a 10 g/l solution of sodium chloride R
and then with 150 ml of a mixture of equal volumes of light
petroleum R1 and ether R. Shake for 1 min. When the
layers have separated completely, discard the lower layer and
wash the upper layer, first with 50 ml of a 30 g/l solution of
potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R and then with 3 quantities, each of
50 ml, of a 10 g/l solution of sodium chloride R. Filter the
A designates the absorbance at the wavelength indicated
upper layer through 5 g of anhydrous sodium sulphate R
by the subscript.
607
on a fast filter paper into a 250 ml flask suitable for a rotary

30 C. Dissolve the residue in 2-propanol R, transfer to a
25 ml volumetric flask and dilute to 25 ml with 2-propanol R.
Gentle heating in an ultrasonic bath may be required. A large
fraction of the white residue is cholesterol, constituting
approximately 50 per cent m/m of the unsaponifiable
matter of cod-liver oil.
Reference solution (a). Prepare a solution of retinol
acetate CRS in 2-propanol R1 so that 1 ml contains about
1000 IU of all-trans-retinol.
The exact concentration of reference solution (a) is assessed
reference solution (a) with 2-propanol R1 to a presumed
concentration of 10-15 IU/ml and measure the absorbance
at 326 nm in matched 1 cm cells using 2-propanol R1 as the
Calculate the content of vitamin A in International Units
per millilitre of reference solution (a) using the following
expression, taking into account the assigned content of
retinol acetate CRS :
A326 =
V1 =
V2
1900 =
volume of reference solution (a) used ;

retinol acetate CRS, in International Units.
Reference solution (b). Proceed as described for the test

solution but using 2.00 ml of reference solution (a) in place
of the substance to be examined.
The exact concentration of reference solution (b) is assessed
reference solution (b) with 2-propanol R1 to a presumed
all-trans-retinol concentration of 10-15 IU/ml and measure
the absorbance at 325 nm in matched 1 cm cells using
2-propanol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in International
Units per millilitre of reference solution (b), using the
A325 =
V3 =
V4
volume of reference solution (b) used ;
1821 =

Column :
size : l = 0.25 m, = 4.6 mm ;
chromatography R (film thickness 5-10 m).
608

Injection : 10 l ; inject in triplicate the test solution and
reference solution (b).
Retention time : all-trans-retinol = 5 1 min.
the chromatogram obtained with the test solution shows a
peak that corresponds to the peak due to all-trans-retinol
in the chromatogram obtained with reference solution (b) ;
the results obtained with the duplicate test solutions do
not differ by more than 5 per cent ;
the recovery of all-trans-retinol in reference solution (b)
as assessed by direct absorption spectrophotometry is
greater than 95 per cent.
Calculate the content of vitamin A using the following
expression :
A1
A2
V
m
=
=
area of the peak due to all-trans-retinol in the

area of the peak due to all-trans-retinol in
solution (b) ;
concentration of retinol acetate CRS in
reference solution (a) as assessed prior to the
saponification, in International Units per millilitre
(= 1000 IU/ml) ;
volume of reference solution (a) treated (2.00 ml) ;
mass of the substance to be examined in the test
solution (2.00 g).
Vitamin D3. Liquid chromatography (2.2.29). Carry out the

assay as rapidly as possible, avoiding exposure to actinic
light and air.
Internal standard solution. Dissolve 0.50 mg of
ergocalciferol CRS in 100 ml of anhydrous ethanol R.
Test solution (a). Prepare duplicates. To 4.00 g in a
of sodium chloride R and cool the solution to room
temperature. Transfer the solution to a 500 ml separating
funnel, rinsing the round-bottomed flask with about 75 ml of
a 10 g/l solution of sodium chloride R and then with 150 ml
of a mixture of equal volumes of light petroleum R1 and
ether R. Shake for 1 min. When the layers have separated
completely, discard the lower layer and wash the upper
layer, first with 50 ml of a 30 g/l solution of potassium
hydroxide R in a 10 per cent V/V solution of anhydrous
ethanol R , and then with 3 quantities, each of 50 ml, of
a 10 g/l solution of sodium chloride R. Filter the upper
layer through 5 g of anhydrous sodium sulphate R on a
fast filter paper into a 250 ml flask suitable for a rotary
30 C. Dissolve the residue in 1.5 ml of the mobile phase
described under Purification. Gentle heating in an ultrasonic
bath may be required. A large fraction of the white residue
is cholesterol, constituting approximately 50 per cent m/m
of the unsaponifiable matter of cod-liver oil.
Crospovidone
Test solution (b). To 4.00 g add 2.0 ml of the internal

standard solution and proceed as described for test
solution (a).
cholecalciferol CRS in 100.0 ml of anhydrous
ethanol R.
Reference solution (b). In a round-bottomed flask, add 2.0 ml
of reference solution (a) and 2.0 ml of the internal standard
solution and proceed as described for test solution (a).
PURIFICATION
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : nitrile silica gel for chromatography R
(film thickness 10 m).
Mobile phase : isoamyl alcohol R, hexane R (1.6:98.4 V/V).
Inject 350 l of reference solution (b). Collect the eluate
from 2 min before until 2 min after the retention time of
cholecalciferol, in a ground-glass-stoppered tube containing
1 ml of a 1 g/l solution of butylhydroxytoluene R in
hexane R. Repeat the procedure with test solutions (a)
and (b). Evaporate the eluates obtained from reference
solution (b) and from test solutions (a) and (b), separately,
to dryness at a temperature not exceeding 30 C under a
gentle current of nitrogen R. Dissolve each residue in 1.5 ml
of acetonitrile R.
DETERMINATION
Column :
size : l = 0.15 m, = 4.6 mm ;
chromatography R (film thickness 5 m).
Mobile phase : phosphoric acid R, 96 per cent V/V solution
of acetonitrile R (0.2:99.8 V/V).
Injection: 2 quantities not exceeding 200 l of each of the
3 solutions obtained under Purification.
ergocalciferol and cholecalciferol in the chromatogram
the results obtained with the test solution (a) duplicates
do not differ by more than 5 per cent.
Calculate the content of vitamin D3 in International Units
per gram using the following expression, taking into account
the assigned content of cholecalciferol CRS:
A2
A3
A4
A5
A6
V1
V2

solution (b) ;
area (or height) of the peak due to ergocalciferol
solution (b) ;
area (or height) of the peak due to ergocalciferol in
the chromatogram obtained with test solution (b) ;
area (or height) of a possible peak in the
chromatogram obtained with test solution (a) with
the same retention time as the peak co-eluting
with ergocalciferol in test solution (b) ;
solution (b) ;
total volume of reference solution (a) (100 ml) ;
volume of reference solution (a) used for preparing
reference solution (b) (2.0 ml).
STORAGE
In an airtight and well-filled container, protected from light.
If no antioxidant is added, store under an inert gas.
Once the container has been opened, its contents are used
as soon as possible and any part of the contents not used at
once is protected by an atmosphere of inert gas.
LABELLING
The label states :
the concentration of EPA and DHA ;
the number of International Units of vitamin A ;
the number of International Units of vitamin D3 ;
the name and concentration of any added antioxidant.

The revision proposal for this monograph corresponds
to step 4 of the international harmonisation process
with the Japanese Pharmacopoeia and the United States
Pharmacopeia (see International Harmonisation section
of this issue).
The European Pharmacopoeia is the coordinating
pharmacopoeia for this monograph.
The draft presented below is a revised version of the
proposal that was published in Pharmeuropa 10.4. The
following changes have been made :
m1
mass of the sample in test solution (b), in grams ;
m2
A1
total mass of cholecalciferol CRS used for

the preparation of reference solution (a), in
micrograms (500 g) ;
solution (a) ;
identification : the former identification test D has been

replaced by a determination of the screening residue
after wet sieving ;
test for water soluble substances : the limit has been
increased to 1.5 per cent as the content of water can
increase during storage to more than 1.0 per cent ;
assay : the method has been revised and harmonised
with the USP proposal for the harmonisation of the
monograph on povidone.
609
Crospovidone
XXXX:0892
CROSPOVIDONE
Calculate the screening residue fraction of sample

particles having a diameter of more than 63 m, in grams
per 100 g, using the following expression :
Crospovidonum
m1
m2
m3
(C6H9NO)n
Mr (111.1)n
= mass of the screen and sample residue, after

drying for 5 h, in grams ;
= mass of the sample, in grams ;
= mass of the screen, in grams.
If the screening residue is 50 per cent or more, the

substance is classified as type A ; if the screening residue is
less than 40 per cent, the substance is classified as type B.
TESTS
Peroxides. Type A : maximum 400 ppm expressed as H2O2 ;
Cross-linked homopolymer of 1-ethenylpyrrolidin-2-one. It
type B : maximum 1000 ppm expressed as H2O2.
is available in different degrees of powder fineness (type A
Suspend 2.0 g in 50 ml of water R. To 25 ml of this
and type B).
suspension add 2 ml of titanium trichloride-sulphuric
acid reagent R. Allow to stand for 30 min and filter. The
Content : 11.0 per cent to 12.8 per cent of nitrogen (N ;
absorbance (2.2.25) of the filtrate, measured at 405 nm
Ar 14.01) (dried substance).
using a mixture of 25 ml of a filtered 40 g/l suspension of
the substance to be examined and 2 ml of a 13 per cent V/V
CHARACTERS
solution of sulphuric acid R as the compensation liquid, has
a maximum of 0.35.
Appearance : hygroscopic, white or yellowish-white powder
For type B use 10 ml of the suspension diluted to 25 ml with
or flakes.
water R for the test.
2 grades of crospovidone are available, depending on the
Water-soluble substances : maximum 1.0 1.5 per cent.
powder fineness : type A and type B.
Place 25.0 g in a 400 ml beaker, add 200 ml of water R and
Solubility : practically insoluble in water, in ethanol 96 per
stir for 1 h using a magnetic stirrer. Transfer the suspension
cent and in methylene chloride.
to a 250.0 ml volumetric flask, rinsing with water R, and
dilute to volume with the same solvent. Allow the bulk of
the solids to settle. Filter about 100 ml of the almost clear
IDENTIFICATION
supernatant liquid through a 0.45 m membrane filter,
protected by superimposing a 3 m membrane filter. While
filtering, stir the liquid above the filter manually or by means
Comparison : Ph. Eur. reference spectrum of
of a mechanical stirrer, taking care not to damage the filter.
crospovidone CRS.
Transfer 50.0 ml of the clear filtrate to a tared 100 ml beaker,
evaporate to dryness and dry at 105-110 C for 3 h. The
B. Suspend 1 g in 10 ml of water R, add 0.1 ml of 0.05 M
residue weighs a maximum of 50 75 mg.
iodine and shake for 30 s. Add 1 ml of starch solution R
Impurity
A. Liquid chromatography (2.2.29).
and shake. No blue colour develops within 30 s.
C. To 10 ml of water R, add 0.1 g and shake. A suspension is Test solution. Suspend 1.250 g in 50.0 ml of methanol R
and shake for 60 min. Leave the bulk to settle and filter
formed and no clear solution is obtained within 15 min.
through a 0.2 m filter.
D. Weigh a suitable quantity of the substance to be examined Reference solution (a). Dissolve 50 mg of
(for example 10 mg to 100 mg) and suspend it in 10.0 ml 1-vinylpyrrolidin-2-one R in methanol R and dilute
of water R, adding a wetting agent. Observe under a
microscope at a suitable magnification using a calibrated solution to 100.0 ml with methanol R. Dilute 5.0 ml of this
ocular micrometer. If the majority of particles are in
solution to 100.0 ml with the mobile phase.
the range 50 m to 300 m, the product is classified as
Reference solution (b). Dissolve 10 mg of
type A. If almost all the particles are below 50 m, the
1-vinylpyrrolidin-2-one R and 0.50 g of vinyl acetate R in
product is classified as type B. The analytical screens
must be clean and dry. For this purpose the screens are methanol R and dilute to 100 ml with the same solvent.
Dilute 1.0 ml of this solution to 100.0 ml with the mobile
washed in hot water and allowed to dry overnight in a
phase.
drying cabinet at 105 C.
Precolumn :
Place 20.00 g in a 1000 ml conical flask, add 500 ml of
size : l = 0.025 m, = 4 mm ;
water R and stir the suspension for 30 min. Pour the
suspension through a 63 m analytical screen and rinse
stationary phase : octadecylsilyl octylsilyl silica gel for
the screen with water R until the filtrate is clear. Dry the
chromatography R (5 m).
screen and sample residue at 105 C for 5 h in a drying
Column :
cabinet without circulating air. Cool in a desiccator for
size : l = 0.25 m, = 4 mm ;
30 min and weigh.
DEFINITION
610
Crospovidone
stationary phase: octadecylsilyl octylsilyl silica gel for

temperature : 40 C.
Mobile phase : acetonitrile R, water R (10:90 V/V).
Flow rate : adjusted so that the retention time of the peak
due to impurity A is about 10 min.
Injection: 50 l. After each injection of the test solution,
wash the precolumn by passing the mobile phase backwards,
at the same flow rate as applied in the test, for 30 min.
40 g/l solution of boric acid R and 0.05 ml of bromocresol

green-methyl red solution R and enough water R to cover
the tip of the condenser. Towards the end of the distillation
lower the receiver so that the tip of the condenser is above
the surface of the acid solution and rinse the end part of
the condenser with a small quantity of water R. Titrate the
distillate with 0.025 M sulphuric acid until the colour of the
solution changes from green through pale greyish-blue to
pale greyish-red-purple (n1 ml of 0.025 M sulphuric acid).
Repeat the test using about 100 mg of glucose R in place of
the substance to be examined (n2 ml of 0.025 M sulphuric
acid).
impurity A and vinyl acetate in the chromatogram
Gradually heat the flask until the solution has a clear,

yellowish-green colour, and the inside wall of the flask is free
from carbonised material, and then heat for a further 45 min.
After cooling, cautiously add 20 ml of water R, and connect
repeatability : maximum relative standard deviation of
the flask to the distillation apparatus previously washed by
2.0 per cent after 5 injections of reference solution (a).
passing steam through it. To the absorption flask add 30 ml
of a 40 g/l solution of boric acid R, 3 drops of bromocresol
green-methyl red solution R and sufficient water R to
Limits :
immerse the lower end of the condenser tube. Add 30 ml of
impurity A : not more than the area of the principal peak a solution of strong sodium hydroxide solution R through
in the chromatogram obtained with reference solution (a) a funnel, cautiously rinse the funnel with 10 ml of water R,
immediately close the clamp attached to the rubber tube,
(10 ppm).
then start the distillation with steam to obtain 80-100 ml
of distillate. Remove the absorption flask from the lower
end of the condenser tube, rinsing the end part with a small
2.0 g complies with test D. Prepare the reference solution
quantity of water R, and titrate the distillate with 0.025 M
using 2 ml of lead standard solution (10 ppm Pb) R.
sulphuric acid until the color of the solution changes from
green through pale greyish-blue to pale greyish red-purple.
Carry out a blank determination and make any necessary
correction.
1 ml of 0.025 M sulphuric acid is equivalent to 0.7004 mg
on 1.0 g.
of N.
ASSAY
STORAGE
Place 100.0 mg of the substance to be examined (m mg)

in a combustion flask and add 5 g of a mixture of 1 g of
copper sulphate R, 1 g of titanium dioxide R and 33 g
of dipotassium sulphate R, and 3 glass beads. Wash any
adhering particles from the neck into the flask with a
small quantity of water R. Add 7 ml of sulphuric acid R,
allowing it to run down the sides of the flask, and mix the
contents by rotation. Close the mouth of the flask loosely,
for example by means of a glass bulb with a short stem, to
avoid excessive loss of sulphuric acid. Heat gradually at
first, then increase the temperature until there is vigorous
boiling with condensation of sulphuric acid in the neck of
the flask ; precautions are to be taken to prevent the upper
part of the flask from becoming overheated. Continue the
heating for 45 min. Cool, dissolve the solid material by
cautiously adding to the mixture 20 ml of water R, cool
again and place in a steam-distillation apparatus. Add
30 ml of strong sodium hydroxide solution R through the
funnel, rinse the funnel cautiously with 10 ml of water R and
distil immediately by passing steam through the mixture.
Collect 80-100 ml of distillate in a mixture of 30 ml of a
In an airtight container.
LABELLING
The label states the type (type A or type B).
IMPURITIES
A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).
(8) LiChrospher 60 RP8 Select B is suitable.
611
Equine influenza vaccine (inactivated)
Reference: PA/PH/Exp. 15V/T (06) 17 ANP

The monograph was revised in 1996. At that time, it had
been decided to keep the haemagglutination-inhibition (HI)
test, even if the collaborative study for the establishment
of 3 European Pharmacopoeia reference preparations
for equine influenza horse antiserum showed that the
reproducibility of the HI test is weak (see Pharmeuropa
Bio 2000-1). The EDQM symposium on equine influenza
vaccines held in Budapest in December 2001 confirmed
the weak reproducibility of the HI test. More recently,
in April 2006, at the meeting of control authorities and
official control laboratories involved in the control of
immunological veterinary medicinal products, it became
apparent that the HI test is indeed poorly reproducible.
It is therefore proposed to delete the HI test from the
monograph, keeping only the single radial haemolysis test.
Furthermore, the new BRP recently established and
adopted by the European Pharmacopoeia Commission
has been included, as well as the new nomenclature of the
biological reference preparations (BRP).
XXXX:0249
EQUINE INFLUENZA VACCINE

(INACTIVATED)
Vaccinum influenzae equi inactivatum
DEFINITION
Equine influenza vaccine (inactivated) is a preparation of
one or more suitable strains of equine influenza virus,
inactivated in such a manner that immunogenic activity is
maintained. Suitable strains contain both haemagglutinin
and neuraminidase.
PRODUCTION
Each strain of virus is propagated separately in fertilised
hen eggs from healthy flocks or in suitable cell cultures
(5.2.4). The viral suspensions may be purified and
concentrated. The antigen content of the vaccine is based
on the haemagglutinin content of the viral suspensions
determined as described under In-process Tests ; the amount
of haemagglutinin for each strain is not less than that in the
vaccine shown to be satisfactory in the test for potency.
The test for residual infectious influenza virus is carried
out using method A or method B whichever is the more
sensitive. The quantity of inactivated virus used is equivalent
to not less than 10 doses of vaccine.
A. The vaccine is inoculated into suitable cells ; after
incubation for 8 days, a subculture is made. It is
incubated for a further 6 to 8 days. Harvest about
0.1 ml of the supernatant and examine for live virus by a
haemagglutination test. If haemagglutination is found,
carry out a further passage in cell culture and test for
haemagglutination ; no haemagglutination occurs.
B. Inoculate 0.2 ml into the allantoic cavity of each of
10 fertilised eggs and incubate at 33 C to 37 C for
3 to 4 days. The test is not valid unless not fewer than
8 of the 10 embryos survive. Harvest 0.5 ml of the
allantoic fluid from each surviving embryo and pool the
fluids. Inoculate 0.2 ml of the pooled fluid into a further
10 fertilised eggs and incubate at 33 C to 37 C for 3
to 4 days. The test is not valid unless at least 8 of the
10 embryos survive. Harvest about 0.1 ml of the allantoic
fluid from each surviving embryo and examine each
612
individual harvest for live virus by a haemagglutination

test. If haemagglutination is found for any of the fluids,
carry out a further passage of that fluid in eggs and test
for haemagglutination ; no haemagglutination occurs.
The vaccine may contain suitable adjuvants.
CHOICE OF VACCINE COMPOSITION
The choice of strains used in the vaccine is based on
epidemiological data. The Office international des pizooties
reviews the epidemiological data periodically and if
necessary recommends new strains corresponding to
prevailing epidemiological evidence. Such strains are used
in accordance with the regulations in force in the signatory
States of the Convention on the Elaboration of a European
Pharmacopoeia.
The vaccine is shown to be satisfactory with respect to safety
and immunogenicity in horses. Where a particular breed
of horse is known to be especially sensitive to the vaccine,
horses from that breed are included in the tests for safety.
The following tests may be used during demonstration of
safety (5.2.6) and efficacy (5.2.7).
Safety. A test is carried out in each category of animals for
which the vaccine is intended and by each recommended
route. Use animals that preferably have no equine influenza
antibodies or, where justified, use animals with a low level
of such antibodies as long as they have not been vaccinated
against equine influenza and administration of the vaccine
does not cause an anamnestic response. 2 doses of vaccine
are injected by the intended route into each of not fewer
than 10 animals. After 14 days, 1 dose of vaccine is injected
into each of the animals. The animals are observed for a
further 14 days. During the 28 days of the test, no abnormal
local or systemic reaction occurs. If the vaccine is intended
for use in pregnant horses, for the test in this category of
animal, horses are vaccinated during the relevant trimester
or trimesters of pregnancy and the observation period is
prolonged up to foaling ; any effects on gestation or the
offspring are noted.
Immunogenicity. The test described under Potency is
suitable to demonstrate the immunogenicity of the strains
present in the vaccine.
A test with virulent challenge is carried out for at least
one vaccine strain. For other strains in the vaccine,
demonstration of immunogenicity may, where justified, be
based on the serological response induced in horses by the
vaccine ; justification for protection against these strains may
be based on published data on the correlation of the antibody
titre with protection against antigenically related strains.
Where serology is used, the test is carried out as described
under Potency but instead of virulent challenge, a blood
sample is drawn 2 weeks after the last vaccination and the
antibody titre of each serum is determined by a suitable
immunochemical method (2.7.1), such as the single radial
haemolysis test or the haemagglutination-inhibition test
shown below ; a reference serum is used to validate the
test. The acceptance criteria depend on the strain and are
based on available data ; for A/equine-2 virus, vaccines have
usually been found to be satisfactory if the antibody titre of
each serum is not less than 85 mm2 where the single radial
haemolysis test is used, or not less than 1:64 (before mixture
with the suspension of antigen and erythrocytes) where the
haemagglutination-inhibition test is used.
Equine influenza subtype I horse antiserum BRP, equine
influenza subtype 2 American-like horse antiserum BRP
and equine influenza subtype 2 European-like horse
antiserum BRP are suitable for use as reference sera for the
single radial haemolysis test.
Equine influenza vaccine (inactivated)
The claims for the product reflect the type of immunogenicity

demonstrated (protection against challenge or antibody
production).
Single radial haemolysis. Heat each serum at 56 C for
30 min. Perform tests on each serum using respectively
the antigen or antigens prepared from the strain(s) used
in the production of the vaccine. Mix 1 ml of sheep
erythrocyte suspension in barbital buffer solution (1 volume
of erythrocytes in 10 volumes of final suspension) with 1 ml
of a suitable dilution of the influenza virus strain in barbital
buffer solution and incubate the mixture at 4 C for 30 min.
To 2 ml of the virus/erythrocyte mixture, add 1 ml of a
3 g/l solution of chromium(III) trichloride hexahydrate R,
mix and allow to stand for 10 min. Heat the sensitised
erythrocytes to 47 C in a water-bath. Mix 15 ml of a 10 g/l
solution of agarose for electrophoresis R in barbital buffer
solution, 0.7 ml of sensitised erythrocyte suspension and
the appropriate amount of diluted guinea-pig complement in
barbital buffer solution at 47 C. Pour the mixture into Petri
dishes and allow the agar to set. Punch holes in the agar
layer and place in each hole, 5 l of the undiluted serum
to be tested or control serum. Incubate the Petri dishes at
37 C for 18 h. Measure the diameter of the haemolysis zone
and calculate its area, which expresses the antibody titre,
in square millimetres.
Equine influenza subtype 1 strain A/eq/Newmarket/77
horse antiserum BRP, equine influenza subtype 2
European-like strain A/eq/Newmarket/2/93 horse
antiserum BRP, equine influenza subtype 2 American-like
strain A/eq/Newmarket/1/93 horse antiserum BRP
and equine influenza subtype 2 American-like strain
A/eq/South Africa/4/03 horse antiserum BRP are suitable
for use as reference sera for the single radial haemolysis test.
Haemagglutination-inhibition test. Inactivate each serum
by heating at 56 C for 30 min. To one volume of each
serum add three volumes of phosphate-buffered saline
pH 7.4 R and four volumes of a 250 g/l suspension of
light kaolin R in the same buffer solution. Shake each
mixture for 10 min. Centrifuge, collect the supernatant
liquid and mix with a concentrated suspension of chicken
erythrocytes. Allow to stand at 37 C for 60 min and
centrifuge. The dilution of the serum obtained is 1:8.
Perform tests on each serum using each antigen prepared
from the strains used in the production of the vaccine. Using
each diluted serum, prepare a series of twofold dilutions. To
0.025 ml of each of the latter dilutions add 0.025 ml of a
suspension of antigen treated with ether R and containing
four haemagglutinating units. Allow the mixture to stand
for 30 min and add 0.05 ml of a suspension of chicken
erythrocytes containing 2 107 erythrocytes/ml. Allow to
stand for 1 h and note the last dilution of serum that still
completely inhibits haemagglutination.
IN-PROCESS TESTS
The content of haemagglutinin in the inactivated virus
suspension, after purification and concentration where
applicable, is determined by a suitable immunochemical
method (2.7.1), such as single radial immunodiffusion, using
a suitable haemagglutinin reference preparation ; the content
is shown to be within the limits shown to allow preparation
of a satisfactory vaccine. For vaccines produced in eggs, the
content of bacterial endotoxins is determined on the virus
harvest to monitor production.
BATCH TESTING
The test described under Potency is not carried out for
routine testing of batches of vaccine. It is carried out, for
a given vaccine, on one or more occasions, as decided by
or with the agreement of the competent authority ; where
the test is not carried out, an alternative validated method is
used, the criteria for acceptance being set with reference to a

batch of vaccine that has given satisfactory results in the test
described under Potency. The following test may be used.
Batch potency test. Inject one dose of vaccine
subcutaneously into each of 5 guinea-pigs free from
specified antibodies. 21 days later, collect blood samples
and separate the serum. Carry out tests on the serum for
specific antibodies by a suitable immunochemical method
(2.7.1) such as single radial haemolysis or haemagglutinin
inhibition, using reference sera to validate the test. The
antibody titres are not significantly lower than those
obtained in guinea-pigs with a reference batch of vaccine
shown to have satisfactory potency in horses.
IDENTIFICATION
In susceptible animals, the vaccine stimulates the production
of specific antibodies.
TESTS
Safety. Use horses that preferably have no equine influenza
virus antibodies or, where justified, use horses with a low
level of such antibodies as long as they have not been
vaccinated against equine influenza and administration
of the vaccine does not cause an anamnestic response.
Administer by a recommended route a double dose of vaccine
to each of not fewer than 2 horses. After 2 weeks, administer
a single dose to each horse. Observe the animals for a further
10 days. No abnormal local or systemic reaction occurs.
Inactivation. Inoculate 0.2 ml of the vaccine into the
allantoic cavity of each of 10 fertilised eggs and incubate
at 33 C to 37 C for 3 to 4 days. The test is not valid
unless at least 8 of the 10 embryos survive. Harvest 0.5 ml
of the allantoic fluid from each surviving embryo and
pool the fluids. Inoculate 0.2 ml of the pooled fluid into a
further 10 fertilised eggs and incubate at 33 C to 37 C
for 3 to 4 days. The test is not valid unless not fewer than
8 of the 10 embryos survive. Harvest about 0.1 ml of the
allantoic fluid from each surviving embryo and examine
each individual harvest for live virus by a haemagglutination
test. If haemagglutination is found for any of the fluids,
carry out for that fluid a further passage in eggs and test for
haemagglutination ; no haemagglutination occurs.
Sterility. The vaccine complies with the test for sterility
prescribed in the monograph on Vaccines for veterinary
use (0062).
POTENCY
Carry out the potency test using a challenge strain against
which the vaccine is stated to provide protection. Use
where possible a recent isolate.
Use 10 horses, at least 6 months old, that do not have specific
antibodies against equine influenza virus. Draw a blood
sample from each animal and test individually for antibodies
against equine influenza virus to determine seronegativity.
Vaccinate 6 animals using the recommended schedule.
Draw a second blood sample from each animal 7 days after
the first vaccination and test individually for antibodies
against equine influenza virus, to detect an anamnestic
sero-response. Animals showing sero-conversion at this
stage are excluded from the test. At least 2 weeks after the
last vaccination, administer by aerosol to the 10 animals
a quantity of equine influenza virus sufficient to produce
characteristic signs of disease such as fever, nasal discharge
and coughing in a susceptible animal. Observe the animals
for 14 days. Collect nasal swabs daily from each individual
animal to isolate the virus. The vaccinated animals show
no more than slight signs ; the controls show characteristic
613
signs. The average number of days on which virus is

excreted, and the respective virus titres are significantly
lower in vaccinated animals than in control animals.
LABELLING
The label states :
the age at which animals are to be vaccinated,
the period of time that is to elapse between the first and
second injections,
any booster injections required,
the strains of virus included in the vaccine.
IDENTIFICATION
A. Specific optical rotation (2.2.7) : 126 to 134.
Dissolve 0.250 g in methanol R and dilute to 25.0 ml
Comparison : esomeprazole magnesium trihydrate CRS.
C. Atomic absorption spectrometry (2.2.23) as described in
the test for magnesium.
The test solution shows the absorption maximum at
285.2 nm.
TESTS
Absorbance (2.2.25) : maximum 0.2 at 440 nm.

Dissolve 0.5 g in methanol R and dilute to 25.0 ml with the
Reference: PA/PH/Exp. 10 C/T (06) 11 ANP
same solvent. Filter the solution through a membrane filter
(0.45 m)(9).
Identification : atomic absorption spectrometry is used for Related substances. Liquid chromatography (2.2.29).
the identification of magnesium, making the incineration Use the normalisation procedure. Use freshly prepared
of the substance unnecessary ; the Ph. Eur. standard
solutions.
test (2.3.1) for magnesium does not work properly for
reasons of solubility in water ; the blank is positive in
examined in the mobile phase and dilute to 25.0 ml with the
methanol.
mobile phase.
Related substances : the method is harmonised with the
Reference solution (a). Dissolve 1 mg of omeprazole CRS
method described for Omeprazole sodium (1032) and the
and 1 mg of omeprazole impurity D CRS in the mobile
same octylsilyl silica gel is described ; the method also works phase and dilute to 10.0 ml with the mobile phase.
with octadecylsilyl silica gel and this piece of information
Reference solution (b). Dilute 1.0 ml of the test solution
is given in a foot-note for the convenience of the users;
to 100.0 ml with the mobile phase. Dilute 1.0 ml of this
impurity C is also covered by the LC method making
the use of a separate TLC, as described in Omeprazole
Column:
(0942) and Omeprazole sodium (1032) monographs,
size : l = 0.125 m, = 4.6 mm ;
unnecessary ; a revision of these monographs is foreseen
for harmonisation.
stationary phase : octylsilyl silica gel for
chromatography R (5 m)(10).
Assay : no suitable titrimetric method has been found and
the assay is done by LC using omeprazole as a reference
Mobile phase : mix 27 volumes of acetonitrile R and
standard.
73 volumes of a 1.4 g/l solution of disodium hydrogen
XXXX:2372 phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
ESOMEPRAZOLE MAGNESIUM
TRIHYDRATE
Injection : 40 l.
Esomeprazolum magnesicum trihydricum Run time : 5 times the retention time of esomeprazole.
Relative retention with reference to esomeprazole
(retention time = about 9 min) : impurity E = about 0.6;
impurity D = about 0.8.
System suitability : reference solution (a) :
impurity D and esomeprazole. If necessary, adjust the pH
of the mobile phase or its proportion of acetonitrile ; an
increase in the pH will improve the resolution.
C34H36N6O6S2Mg,3H2O
Mr 761 Limits :
impurity D : maximum 0.2 per cent ;
DEFINITION
impurity E : maximum 0.1 per cent ;
Magnesium bis[5-methoxy-2-[(S)-[(4-methoxy-3,5 unspecified impurities : for each impurity, maximum
dimethylpyridin-2-yl)methyl]sulphinyl]benzimidazol-1-ide]
0.10 per cent ;
trihydrate.
total : maximum 0.5 per cent ;
substance).
in the chromatogram obtained with reference solution (b)
(0.05 per cent).
CHARACTERS
Appearance : white or slightly coloured powder.
Impurity F. Liquid chromatography (2.2.29).
Phosphate buffer solution pH 6. Mix 70 ml of a 156.0 g/l
Solubility : slightly soluble in water, soluble in methanol,
solution of sodium dihydrogen phosphate R with 20 ml of
practically insoluble in heptane.
(9) Millipore Millex-HV 0.45 m is suitable.
(10) Symmetry C8, LiChrosorb C8, Zorbax SB-C8 and Novapac C18 are suitable.
614
1. impurity A
3. impurity D
5. esomeprazole
2. impurity E
4. impurity B
6. impurity C
Figure 2372.-1. Chromatogram for the test for related substances of esomeprazole magnesium trihydrate : esomeprazole
magnesium trihydrate spiked with about 0.1 per cent of impurities A, B, C, D and E
a 179.1 g/l solution of disodium hydrogen phosphate R.
Dilute to 1000 ml with water R, and dilute 250 ml of this
solution to 1000 ml with water R.
solution of trisodium phosphate dodecahydrate R with
22 ml of a 179.1 g/l solution of disodium hydrogen
phosphate R, and dilute to 1000 ml with water R.
Calculate the percentage content of impurity F using the

ri

examined in 5 ml of methanol R and dilute to 25 ml with
rs
phosphate buffer solution pH 11. Dilute 1 ml of this solution
to 50 ml with phosphate buffer solution pH 11.
area of the peak due to impurity F in the

chromatogram obtained with the test solution;
sum of the areas of the peaks due to esomeprazole
and impurity F in the chromatogram obtained
with the test solution.
Reference solution (a). Dissolve 2 mg of omeprazole CRS in Limits :

phosphate buffer solution pH 11 and dilute to 10 ml with the impurity F : maximum 0.2 per cent.
same buffer solution. Dilute 1 ml of this solution to 50 ml
Magnesium : 3.30 per cent to 3.55 per cent (anhydrous
with phosphate buffer solution pH 11.
substance).
Reference solution (b). Dilute 1.0 ml of reference solution (a) Atomic absorption spectrometry (2.2.23, Method I).
to 50.0 ml with phosphate buffer solution pH 11.
Test solution. Dissolve 0.250 g in 20 ml of a 103 g/l solution
Column :
of hydrochloric acid R, adding the acid slowly, and dilute
size : l = 0.1 m, = 4.0 mm ;
200.0 ml with water R. To 10.0 ml of the solution obtained
stationary phase : silica gel AGP for chiral
add 4 ml of lanthanum chloride solution R and dilute to
Reference solutions. Prepare the reference solutions using
Mobile phase : acetonitrile R, phosphate buffer solution
magnesium standard solution (1000 ppm Mg) R, diluted
pH 6 (65:435 V/V).
as necessary with water R.
Wavelength : 285.2 nm.
0.200 g.
Injection: 20 l.
Elution order : impurity F, esomeprazole.
Retention time : esomeprazole = about 4 min.
impurity F and esomeprazole in the chromatogram
obtained with reference solution (a) ;
signal-to-noise ratio : minimum 10 for the peak due to
impurity F in the chromatogram obtained with reference
solution (b).
ASSAY
examined in about 10 ml of methanol R, add 10 ml of
phosphate buffer solution pH 11 and dilute to 200.0 ml with
water R.
(11) Chiral AGP is suitable.
615
Reference solution. Dissolve 10.00 mg of omeprazole CRS

in about 10 ml of methanol R, add 10 ml of phosphate buffer
solution pH 11 and dilute to 200.0 ml with water R.
Column :
size : l = 0.125 m, = 4 mm ;
stationary phase: octylsilyl silica gel for
Mobile phase : mix 35 volumes of acetonitrile R with
phosphate R previously adjusted to pH 7.6 with phosphoric
acid R.
Injection: 20 l.
Run time : until complete elution of the peak due to
esomeprazole.
System suitability : reference solution :
retention time : esomeprazole = minimum 4 min.
Calculate the percentage content of C34H36N6O6S2Mg from
the declared content of omeprazole CRS.
1 g of omeprazole is equivalent to 1.032 g of esomeprazole
magnesium.
A. 5-methoxy-1H-benzimidazole-2-thiol,
B. R = H, X = SO : 2-[(RS)-[(3,5-dimethylpyridin-2yl)methyl]sulphinyl]-5-methoxy-1H-benzimidazole,
C. R = OCH3, X = S : 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulphanyl]-1H-benzimidazole
(ufiprazole),
D. R = OCH3, X = SO2 : 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole
(omeprazole sulphone),
STORAGE
IMPURITIES
E. 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2yl)sulphinyl]methyl]-3,5-dimethylpyridine 1-oxide.
Specified impurities : D, E, F.
F. 5-methoxy-2-[(R)-[(4-methoxy-3,5-dimethylpyridin-2pharmaceutical use) : A, B, C.
yl)methyl]sulphinyl]-1H-benzimidazole.
1. esomeprazole
Figure 2372.-2. Chromatogram for the assay of esomeprazole magnesium

616
Etamsylate
Reagents
Magnesium standard solution (1000 ppm Mg). XXXXXXX.
Dissolve 5.275 g of magnesium nitrate R(12) in 16 ml of
dilute nitric acid R and dilute to 500.0 ml with water R.
Standardisation : carry out the determination of magnesium
by complexometry (2.5.11).

Related substances : TLC for hydroquinone has been
replaced by an LC for related substances in accordance
XXXX:1204
ETAMSYLATE
Etamsylatum
C10H17NO5S
Mr 263.3
DEFINITION
N-Ethylethanamine 2,5-dihydroxybenzenesulphonate.
CHARACTERS
Solubility : very soluble in water, freely soluble in methanol,
soluble in anhydrous ethanol, practically insoluble in
methylene chloride.
It shows polymorphism.
IDENTIFICATION
A. Melting point (2.2.14) : 127 C to 134 C.
Preparation : discs.
Comparison : etamsylate CRS.
C. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
Test solution. Dissolve 0.100 g in water R and dilute to
200.0 ml with the same solvent. Dilute 5.0 ml of this
solution to 100.0 ml with water R. Examine immediately.
Absorption maxima: at 221 nm and 301 nm.
Specific absorbance at the absorption maximum at
301 nm : 145 to 151.
D. Into a test-tube, introduce 2 ml of freshly prepared
solution S (see Tests) and 0.5 g of sodium hydroxide R.
Warm the mixture and place a wet strip of red litmus
paper R near the open end of the tube. The colour of the
paper becomes blue.
TESTS
Appearance of solution. Solution S, when freshly prepared,
is clear (2.2.1) and colourless (2.2.2, Method II).
Hydroquinone. Examine by thin-layer chromatography
(2.2.27), using as the coating substance a suitable silica gel
with a fluorescent indicator having an optimal intensity at
254 nm.
Reference solution. Dissolve 10 mg of hydroquinone R in
Apply to the plate 10 l of each solution and dry the starting
points in a current of cool air. Develop over a path of 15 cm
using a mixture of 20 volumes of methylene chloride R,
30 volumes of methyl acetate R and 50 volumes of ethyl
acetate R. Dry the plate in a current of hot air and examine
in ultraviolet light at 254 nm. Any spot corresponding to
hydroquinone in the chromatogram obtained with the test
solution is not more intense than the principal spot in the
chromatogram obtained with the reference solution (0.1 per
cent).
freshly prepared solutions, maintaining them at 6 C.
Buffer solution. Dissolve 1.2 g of anhydrous sodium
dihydrogen phosphate R in 900 ml of water for
chromatography R. Adjust to pH 6.5 with disodium
hydrogen phosphate solution R and dilute to 1000 ml with
water for chromatography R.
solvent.
hydroquinone CRS in water R and dilute to 25.0 ml
Reference solution (c). Mix 1 ml of reference solution (b)
and 4 ml of the test solution.
to 20.0 ml with water R.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: spherical end-capped octadecylsilyl
silica gel for chromatography R (5 m)(13).
Mobile phase : acetonitrile R1, buffer solution (10:90 V/V).
Injection : 10 l of the test solution and reference
Run time : 2.5 times the retention time of etamsylate.
Relative retention with reference to etamsylate (retention
time = about 6 min) : impurity A = about 1.7.
System suitability : reference solution (c) :
etamsylate and impurity A.
(12) Merck 105855 is suitable.

(13) Atlantis dC18 and Hypersil C18 are suitable.
617
0.40
hydroquinone - 9.353
etamsylate - 5.523
0.50
0.30
0.20
0.10
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Min
Figure 1204.-1. Chromatogram for the test for related substances of etamsylate : solution of etamsylate spiked with
impurity A
Limits:
XXXX:2346
in the chromatogram obtained with reference solution (d)
(0.1 per cent) ;
FLUCLOXACILLIN MAGNESIUM
OCTAHYDRATE
Flucloxacillinum magnesicum octahydricus
total: not more than 0.2 per cent ;
(0.05 per cent).
Iron (2.4.9) : maximum 10 ppm, determined on solution S.
using 1.5 ml of lead standard solution (10 ppm Pb) R.
Loss on drying (2.2.32) : maximum 0.5 per cent, determined Mg(C19H16ClFN3O5S)2,8H2O
Mr 1074
on 1.000 g by drying in vacuo in an oven at 60 C.
DEFINITION
Magnesium bis[(2S,5R,6R)-6-[[[3-(2-chloro-6-fluorophenyl)on 1.0 g.
5-methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4thia-1-azabicyclo[3.2.0]heptane-2-carboxylate] octahydrate.
ASSAY
Semi-synthetic product derived from a fermentation product.
Dissolve 0.200 g in a mixture of 10 ml of water R and 40 ml of
dilute sulphuric acid R. Titrate with 0.1 M cerium sulphate, Content : 95.0 per cent to 102.0 per cent (anhydrous
substance).
determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M cerium sulphate is equivalent to 13.16 mg of CHARACTERS
C10H17NO5S.
Solubility : slightly soluble in water, freely soluble in
STORAGE
methanol.
IDENTIFICATION
First identification : A, C.
IMPURITIES
Second identification : B, C.
Comparison : flucloxacillin magnesium CRS.
B. Thin-layer chromatography (2.2.27).
examined in 5 ml of water R.
Reference solution (a). Dissolve 25 mg of flucloxacillin
A. benzene-1,4-diol (hydroquinone).
sodium CRS in 5 ml of water R.
618

Reference solution (c). To 10 mg of the substance to be
examined add 1 ml of sodium carbonate solution R and
dilute to 25 ml with water R. Place the solution in an oven at
70 C for 20 min (in situ preparation of impurity A).
Reference solution (d). Dilute 1 ml of reference solution (c)
to 10 ml with a 27 g/l solution of dipotassium hydrogen
phosphate R previously adjusted to pH 3.5 with dilute
phosphoric acid R.
Reference solution (e). To 10 ml of reference solution (c)
add 5 ml of dilute hydrochloric acid R and dilute to 25 ml
with water R. Place the solution in an oven at 70 C for 1 h.
Dilute 1 ml of this solution to 5 ml with a 27 g/l solution
of dipotassium hydrogen phosphate R previously adjusted
to pH 7.0 with phosphoric acid R (in situ preparation of
impurity B).
Reference solution (f). Dilute 2 ml of reference solution (a)
to 10 ml with reference solution (e).
Reference solution (g). Dissolve 1.5 mg of flucloxacillin
impurity C CRS in 1 ml of the mobile phase and dilute to
50 ml with the mobile phase.
TESTS
Reference solution (h). Dissolve 1 mg of flucloxacillin
pH (2.2.3) : 4.5 to 6.5.
impurity D CRS in 100 ml of the mobile phase. To 1 volume
Dissolve 0.25 g in carbon dioxide-free water R and dilute to of this solution add 2 volumes of the mobile phase.
Reference solution (i). Dissolve 1 mg of flucloxacillin
Specific optical rotation (2.2.7) : + 163 to + 173 (anhydrous impurity E CRS in 100 ml of the mobile phase. To 1 volume
of this solution add 2 volumes of the mobile phase.
substance).
Column :
Dissolve 0.250 g in water R and dilute to 50.0 ml with the
size : l = 0.25 m, = 4 mm ;
same solvent.
Prepare the solutions immediately before use.
temperature
: 40 C.
Test solution (a). Dissolve 50.0 mg of the substance to be
examined in the mobile phase and dilute to 50.0 ml with the Mobile phase : mix 25 volumes of acetonitrile R and
75 volumes of a 2.7 g/l solution of potassium dihydrogen
mobile phase.
Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml phosphate R previously adjusted to pH 5.0 with dilute
sodium hydroxide solution R.
with the mobile phase.
Reference solution (a). Dissolve 50.0 mg of flucloxacillin
sodium CRS in the mobile phase and dilute to 50.0 ml with Detection : spectrophotometer at 225 nm.
the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml
Injection : 20 l of test solution (a) and reference
solutions (b), (d), (e), (f), (g), (h) and (i).
Reference solution (b). Dissolve 25 mg of cloxacillin
sodium CRS, 25 mg of dicloxacillin sodium CRS and
25 mg of flucloxacillin sodium CRS in 5 ml of water R.
Plate : TLC silanised silica gel plate R.
Mobile phase : 30 volumes of acetone R and 70 volumes
of a 154 g/l solution of ammonium acetate R previously
adjusted to pH 5.0 with glacial acetic acid R.
Application : 1 l.
Development : over 2/3 of the plate.
Drying : in air.
Detection : expose the plate to iodine vapour until the
spots appear.
the chromatogram shows 3 clearly separated spots.
with the test solution is similar in position, colour and
size to the principal spot in the chromatogram obtained
with reference solution (a).
C. It gives the reaction of magnesium (2.3.1).
1. impurity C
4. impurity A2
7. impurity B1
2. impurity A1
5. impurity D
8. impurity B2
3. unknown impurity
6. unknown impurity
9. flucloxacillin
Figure 2346.-1. Chromatogram for the test for related substances of flucloxacillin magnesium octahydrate : test
solution (a)
(14) Hypersil ODS is suitable.
619
Run time : 7 times the retention time of flucloxacillin.
IMPURITIES
Identification of impurities : use the chromatograms

obtained with reference solutions (d), (e), (g), (h) and (i)
to identify the peaks due to impurities A, B, C, D and E
respectively.
Specified impurities : A, B, C, D, E.
Relative retention with reference to flucloxacillin (retention

time = about 8 min) : impurity C = about 0.2 ; impurity A
(isomer 1) = about 0.3 ; impurity A (isomer 2) = about 0.5 ;
impurity D = about 0.6 ; impurity B (isomer 1) = about 0.8 ;
impurity B (isomer 2) = about 0.9 ; impurity E = about 6.
System suitability : reference solution (f) :
resolution : minimum 2.0 between the 2nd peak due to
impurity B (isomer 2) and the peak due to flucloxacillin.
Limits :
correction factor : for the calculation of the content,
multiply the peak area of impurity C by 3.3 ;
impurity A (sum of the 2 isomers) : the sum of the
areas of the 2 peaks is not more than twice the area of
the principal peak in the chromatogram obtained with
reference solution (b) (2.0 per cent) ;
A. R = CO2H : (4S)-2-[carboxy[[[3-(2-chloro-6-fluorophenyl)5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids

of flucloxacillin),
B. R = H : (2RS,4S)-2-[[[[3-(2-chloro-6-fluorophenyl)5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of

flucloxacillin),
impurity B (sum of the 2 isomers) : the sum of the areas

of the 2 peaks is not more than the area of the principal
peak in the chromatogram obtained with reference
solution (b) (1.0 per cent) ;
impurity C : not more than the area of the principal peak
in the chromatogram obtained with reference solution (b) C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(1.0 per cent) ;
(6-aminopenicillanic acid),
impurities D, E : for each impurity, not more than 0.3 times
the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.3 per cent) ;
any other impurity : for each impurity, not more
chromatogram obtained with reference solution (b)
(0.3 per cent) ;
(3.0 per cent) ;
(0.05 per cent).
D. 3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carboxylic
acid,
2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent m/m.

on 0.100 g.
ASSAY
related substances with the following modifications.
Injection: test solution (b) and reference solution (a).
Calculate the percentage content of Mg(C19H16ClFN3O5S)2
from the declared content of flucloxacillin sodium CRS,
multiplying by 0.9773.
620
E. (2S,5R,6R)-6-[[[(2S,5R,6R)-6-[[[3-(2-chloro-6fluorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept2-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid.
Fludeoxyglucose (18F) (prepared by nucleophilic substitution) injection
carbonate, which is then evaporated to dryness. Addition

of a phase-transfer catalyst, such as an aminopolyether or
a tetra-alkyl ammonium salt in dry acetonitrile, may be
18
The monograph has been revised to replace the TLC test for used to enhance the nucleophilicity of the [ F]fluoride
so
that
it
reacts
easily
with
the
tetra-acetylated
mannose
aminopolyether by a more efficient and practical spot test.
triflate at elevated temperature. A variation of the method
18
In view of the fact that 2-[ F]fluoro-2-deoxy-D-glucose
uses solid-phase-catalysed nucleophilic substitution on
nowadays is prepared almost exclusively by a
derivatised anion-exchange resin, e.g. derivatised with
nucleophilic substitution reaction with [18F]fluoride
4-(4-methylpiperidino)pyridine.
on tetraacetylmannose triflate, the monograph now
Electrophilic pathways for production of 2-[18F]fluoro-2-deoxy18
applies only to 2-[ F]fluoro-2-deoxy-D-glucose prepared
D-glucose proceed by the reaction of molecular [18F]fluorine
by nucleophilic substitution. For this reason, the title, the
or [18F]acetylhypofluorite with 3,4,6-tri-O-acetyl-D-glucal.
definition and the paragraph on radiochemical synthesis
[18F]Acetylhypofluorite is obtained by conversion of
have been modified accordingly.
molecular [18F]fluorine on a solid complex of acetic acid and
The paragraph on starting materials has been deleted
potassium acetate. The production of molecular [18F]fluorine
because separate monographs on starting materials for
requires the addition of small amounts of fluorine to the neon
radiochemical synthesis are being elaborated.
target gas, usually from 0.1 per cent to 1 per cent, resulting in
XXXX:1325 the reduction of the specific radioactivity of the end-product.
Hydrolysis of the O-acetyl protected [18F]fluorinated sugar
18
18
D-glucose and usually small
FLUDEOXYGLUCOSE ( F) (PREPARED yields 2-[ F]fluoro-2-deoxyamounts of 2-[18F]fluoro-2-deoxy-D-mannose.
BY NUCLEOPHILIC SUBSTITUTION) Hydrolysis under either alkaline or acidic conditions yields
INJECTION
2-[18F]fluoro-2-deoxy-D-glucose. Depending on the conditions
of hydrolysis, variable amounts of 2-chloro-2-deoxy-D-glucose
and/or 2-[18F]fluoro-2-deoxy-D-mannose may be formed as
Fludeoxyglucosi (18F) nucleophilo
a by-product.
substitutione preparati solutio iniectabilis The preparation can be purified by serial chromatography
on combinations of ion-retardation resin, ion-exchange resin,
alumina and octadecyl derivatised silica gel.
Production systems and their performance comply with the
requirements set by the competent authority.
STARTING MATERIALS
1. Target materials
Each batch of target material must be tested in special
production runs before its use in routine fluorine-18
production and manufacture of the preparation, to ensure
DEFINITION
that under specified conditions, the target yields fluorine-18
Sterile solution containing 2-[18F]fluoro-2-deoxy-Din the desired quantity and quality.
18
glucopyranose (2-[ F]fluoro-2-deoxy-D-glucose), which may
2. Precursors for organic synthesis
also contain 2-[18F]fluoro-2-deoxy-D-mannose.
It is recommended to test the precursors in production runs
Content :
before their use for the manufacture of the preparation,
fluorine-18 : 90 per cent to 110 per cent of the declared
to ensure that under specified production conditions, the
fluorine-18 radioactivity at the date and hour stated on
precursors yield the preparation in the desired quantity and
the label ;
quality.
fluorine-18 in the form of 2-[18F]fluoro-2-deoxy-D-glucose 1,3,4,6-Tetra-O-acetyl-2-O-trifluoromethanesulphonyland 2-[18F]fluoro-2-deoxy-D-mannose : minimum 95 per
-D-mannopyranose. Examine by infrared absorption
cent of the total radioactivity ;
spectrophotometry (2.2.24), comparing with the Ph. Eur.
2-[18F]fluoro-2-deoxy-D-mannose : maximum 10 per cent reference spectrum of 1,3,4,6-tetra-O-acetyl-2-Otrifluoromethanesulphonyl--D-mannopyranose.
of the total radioactivity ;
Melting point (2.2.14) : 119 C to 122 C.
2-fluoro-2-deoxy-D-glucose : maximum 10 0.5 mg per
maximum recommended dose, in millilitres.
3,4,6-Tri-O-acetyl-D-glucal. Examine by infrared absorption
spectrophotometry (2.2.24), comparing with the Ph. Eur.
PRODUCTION
reference spectrum of 3,4,6-tri-O-acetyl-D-glucal.
RADIONUCLIDE PRODUCTION
Melting point (2.2.14) : 53 C to 55 C.
Fluorine-18 is a radioactive isotope of fluorine which may be
CHARACTERS
produced by various nuclear reactions induced by proton
irradiation of oxygen-18, deuteron irradiation of neon-20,
Appearance : clear, colourless or slightly yellow solution.
helium-3 or helium-4 irradiation of oxygen-16.
Half-life and nature of radiation of fluorine-18 : see Table of
The radionuclide fluorine-18 is most commonly produced by physical characteristics of radionuclides (5.7).
proton irradiation of water enriched in oxygen-18.
IDENTIFICATION
RADIOCHEMICAL SYNTHESIS
18
2-[ F]Fluoro-2-deoxy-D-glucose is mostly prepared by phase Record the gamma-ray spectrum using a suitable instrument.
The only gamma photons have an energy of 0.511 MeV ; and
transfer catalysed nucleophilic substitution of 1,3,4,6-tetraO-acetyl-2-O-trifluoromethanesulphonyl--D-mannopyranose depending on the measurement geometry, a sum peak of
with [18F]fluoride. Generally, [18F]fluoride is adsorbed on an 1.022 MeV may be observed.
anion-exchange resin and eluted with a solution of potassium A. It complies with test A for radionuclidic purity (see Tests).
621
System suitability : chromatogram obtained with reference

solution (c) using the carbohydrate detector :
resolution : minimum 2.0 between the peaks due
to 2-fluoro-2-deoxy-D-glucose and 2-fluoro-2-deoxy-Dmannose.
Limits : chromatogram obtained with the carbohydrate
detector :
2-fluoro-2-deoxy-D-glucose : not more than the area of the
corresponding peak in the chromatogram obtained with
reference solution (a) (10 0.5 mg/V) ;
TESTS
solution (b) (0.5 mg/V).
Particular tests for chemical impurities may be omitted if
Impurity B. Spot test.
the substances mentioned are not used or cannot be formed
Test
solution. The preparation to be examined.
in the production process.
Reference solution (a) : water R
pH (2.2.3) : 4.5 to 8.5.
Reference solution (b). Dissolve 11 mg of aminopolyether R
Sterility. It complies with the test for sterility prescribed in (impurity B) in water R and dilute to 5 ml with the same
the monograph Radiopharmaceutical preparations (0125). solvent. Dilute 1 ml of this solution to V with water R, V
The injection may be released for use before completion of
being the maximum recommended dose, in millilitres.
the test.
Plate : TLC silica gel plate for aminopolyether test R.
Bacterial endotoxins (2.6.14) : less than 175/V IU/ml, V
Application : 2.5 l ; as an additional spot, apply 2.5 l of
being the maximum recommended dose, in millilitres. The
the test solution and then 2.5 l of reference solution (b)
injection may be released for use before completion of the
at the same place.
test.
Detection : visually compare the spots 1 min after application.
2-Fluoro-2-deoxy-D-glucose and impurity A. Liquid
chromatography (2.2.29).
the spot made by the application of both the test solution
Test solution. The preparation to be examined.
and reference solution (b) has a similar appearance to
the spot of reference solution (b), which is characterised
2-fluoro-2-deoxy-D-glucose R(15) in water R and
by a number of concentric circles ; the darker innermost
dilute to 2.0 ml with the same solvent. Dilute 1.0 ml of
circle (of intensity proportional to the concentration of
this solution to V with water R, V being the maximum
impurity B) may be surrounded by a blue-black ring,
recommended dose, in millilitres.
outside of which is a lighter circle surrounded by a
peripheral dark edge ;
the spot made by the application of reference solution (a)
2-chloro-2-deoxy-D-glucose R(15) (impurity A) in
has a more diffuse inner circle, which is brownish-pink and
water R and dilute to 2.0 ml with the same solvent. Dilute
without a distinct margin between it and the surrounding
1.0 ml of this solution to V with water R, V being the
lighter zone ;
maximum recommended dose, in millilitres.
the
spot made with reference solution (b) is clearly
Reference solution (c). Dissolve 1.0 mg of
different from the spot made with reference solution (a).
2-fluoro-2-deoxy-D-mannose R(16) in water R and
dilute to 2.0 ml with the same solvent. Mix 0.5 ml of this
Limit :
solution and 0.5 ml of reference solution (a).
the central portion of the spot made with the test solution
Column :
is less intense than that of the spot made with reference
solution (b) (2.2 mg/V).
size : l = 0.25 m, = 4.0 mm ;
stationary phase: strongly basic anion-exchange resin
The following figure is shown for information but will not
be published in the European Pharmacopoeia.
temperature : at a constant temperature between 20 C
and 25 C.
Mobile phase : 4 g/l solution of sodium hydroxide R,
protected from atmospheric carbon dioxide.
Detection : a detector suitable for carbohydrates in the
required concentration range and a radioactivity detector
connected in series.
1. reference solution (a)
2. reference solution (b)
Injection: 20 l.
Run time : twice the retention time of 2-fluoro-2-deoxy-DFigure 1325.-1. Spot test for impurity B. Visualisation of
glucose.
spots obtained with reference solution (a) and reference
Relative retention with reference to 2-fluoro-2solution (b)
deoxy-D-glucose (retention time = about 12 min) :
2-fluoro-2-deoxy-D-mannose = about 0.9 ; impurity A = about (b) Aminopolyether. This test is performed only on the
1.1.
bulk solution before addition of sodium chloride by the
B. Determine the approximate half-life by at least
3 measurements of the activity of a sample in the same
geometrical conditions within a suitable period of time
(for example, 30 min).
Results : 105 to 115 min.
C. Examine the chromatograms obtained in test A for
radiochemical purity (see Tests).
Results : the principal peak in the radiochromatogram
obtained with the test solution has approximately
the same retention time as the principal peak in the
chromatogram obtained with reference solution (a).
(15) Available from ABX Germany.

(16) Available from Glycoteam GmbH and Fluorochem.
(17) Carbopack PA10 from Dionex is suitable.
622
producer and it is not intended for the final preparation to

be injected. Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel plate R.
Reference solution. Dissolve 0.110 g of aminopolyether R in
water R and dilute to 10.0 ml with the same solvent. Dilute
0.2 ml of this solution to V with the same solvent, V being
the maximum recommended dose in millilitres.
Apply separately to the plate 2 l of the test solution and 2 l
of the reference solution. Develop over a path of about 8 cm
using a mixture of 1 volume of ammonia R and 9 volumes
of methanol R. Allow the plate to dry in air for 15 min.
Expose the plate to iodine vapour for at least 10 min. In
the chromatogram obtained with the test solution the spot
corresponding to aminopolyether is not more intense than
the spot in the chromatogram obtained with the reference
solution (2.2 mg per V).
(e) Residual solvents (2.4.24). The concentration of
acetonitrile does not exceed 4.1 mg per V, V being the
maximum recommended dose in millilitres. The injection
may be released for use before completion of the test.
Impurity C. Liquid chromatography (2.2.29).
Reference solution (a). Dilute 2.1 ml of a 25.95 g/l solution
of tetrabutylammonium hydroxide R (impurity C) to 20 ml
with water R. Dilute 1 ml of this solution to V with water R,
V being the maximum recommended dose, in millilitres.
Reference solution (b). Dilute 1.0 ml of a 25.95 g/l solution
of tetrabutylammonium hydroxide R to 10.0 ml with
water R. Dilute 1.0 ml of this solution to 25.0 ml with
water R.
Column :
size : l = 0.10 m, = 4.6 mm ;
temperature : constant temperature between 20 C and
25 C.
Mobile phase : 25 volumes of a 0.95 g/l solution of
toluenesulphonic acid R and 75 volumes of acetonitrile R.
Injection: 20 l.
Run time : twice the retention time of tetrabutylammonium
ions.
Retention time : tetrabutylammonium hydroxide =
about 3.3 min.
symmetry factor : maximum 1.8 for the principal peak ;
signal-to-noise ratio : minimum 10 for the principal peak.
Limit :
impurity C : not more than the area of the corresponding
solution (a) (2.75 mg/V).
Impurity D. Ultraviolet spectrophotometry (2.2.25).
Reference solution. Dissolve 20 mg of 4-(4methylpiperidino)pyridine R (impurity D) in water R and
dilute to 100.0 ml with the same solvent. Dilute 0.1 ml
of this solution to V with water R, V being the maximum
recommended dose, in millilitres.
Measure the absorbance of the test solution and the

reference solution at the absorption maximum of 263 nm.
Limit : the absorbance of the test solution is not greater than
that of the reference solution (0.02 mg/V).
RADIONUCLIDIC PURITY
Limit :
fluorine-18 : minimum 99.9 per cent of the total
radioactivity.
The preparation may be released for use before completion
of test B
A. Gamma-ray spectrometry.
Results : the only gamma photons have an energy of
0.511 MeV and, depending on the measurement geometry,
a sum peak of 1.022 MeV may be observed.
B. Gamma-ray spectrometry.
Determine the amount of fluorine-18 and radionuclidic
impurities with a half-life longer than 2 h. For the
detection and quantification of impurities, retain the
preparation to be examined for a sufficient time to
allow the fluorine-18 to decay to a level that permits the
detection of impurities.
Results : the spectrum obtained with the preparation to
be examined does not differ significantly from that of a
background spectrum.
Record the gamma-ray spectrum using a suitable instrument.
The half-life is between 105 min and 115 min. The injection
may be released for use before completion of the test.
RADIOCHEMICAL PURITY
Limits :
sum of fluorine-18 in the form of 2-[18F]fluoro-2-deoxy-Dglucose and 2-[18F]fluoro-2-deoxy-D-mannose : minimum
95 per cent of the total radioactivity ;
2-[18F]fluoro-2-deoxy-D-mannose : maximum 10 per cent
of the total radioactivity.
A. Liquid chromatography (2.2.29) as described in the
test for 2-fluoro-2-deoxy-D-glucose and impurity A. If
necessary, dilute the test solution with water R to obtain
a radioactivity concentration suitable for the radioactivity
detector.
Injection : test solution, reference solutions (a) and (c).
Relative retention with reference to 2-[18F]fluoro2-deoxy-D-glucose (retention time = about 13 min) :
2-[18F]fluoro-2-deoxy-D-mannose = about 0.9. Partially
and fully acetylated derivatives of both compounds
hydrolyse under the chromatographic conditions and
therefore elute as 2-[18F]fluoro-2-deoxy-D-glucose and
2-[18F]fluoro-2-deoxy-D-mannose.
Locate the peaks due to 2-[18F]fluoro-2-deoxy-D-glucose
and 2-[18F]fluoro-2-deoxy-D-mannose by comparison with
the chromatograms obtained with the carbohydrate
detector and reference solutions (a) and (c).
Calculate the relative percentage of 2-[18F]fluoro-2deoxy-D-mannose plus its acetyl derivatives and of
2-[18F]fluoro-2-deoxy-D-glucose plus its acetyl derivatives
using the chromatogram obtained with the radioactivity
detector.
B. Thin-layer chromatography (2.2.27).
Reference solution : dissolve, with gentle heating, 30 mg
of 1,2,3,4-tetra-O-acetyl--D-glucopyranose R and 20 mg
of glucose R in 1 ml of water R.
(18) Nucleosil C18 is suitable.
623
Plate : TLC silica gel plate R(19).

Mobile phase : water R, acetonitrile R (5:95 V/V).
Application : about 2 l.
Drying : in air for 15 min.
Detection : determine the distribution of radioactivity
using a suitable detector, then immerse the plate in a
75 g/l solution of sulphuric acid R in methanol R and
dry with a heat gun or at 150 C until the appearance
of dark spots in the chromatogram obtained with the
reference solution.
Retention factors : 2-[18F]fluoro-2-deoxy-D-glucose and
2-[18F]fluoro-2-deoxy-D-mannose = about 0.45 ; partially
or fully acetylated 2-[18F]fluoro-2-deoxy-D-glucose
derivatives = about 0.8 to 0.95.
the chromatogram shows 2 clearly separated spots.
Calculate the relative percentage of partially or fully
acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives.
Calculate the radiochemical purity using the following
expression :
using a suitable detector. Not less than 95 per cent of the

total radioactivity is found in the spot corresponding to
2-fluoro-2-deoxy-D-glucose (Rf about 0.45).
Possible contaminants are [18F]fluoride (Rf 0.0) ; partially
acetylated 2-[18F]fluoro-2-deoxy-D-glucose derivatives (Rf
about 0.8-0.95).
RADIOACTIVITY
Measure the radioactivity using suitable counting equipment
by comparison with a standardised fluorine-18 solution
or using an instrument calibrated with the aid of such a
solution. Standardised fluorine-18 solutions are available
from laboratories recognised by the competent authority.
Determine the radioactivity using a calibrated instrument.
LABELLING
The accompanying information specifies the particular
synthetic pathway of production. The label on the actual
container states the maximum recommended dose in
millilitres.
The label states the maximum recommended dose, in
millilitres.
IMPURITIES
Specified impurities
A. 2-chloro-2-deoxy-D-glucose,
= relative percentage of the sum

of 2-[18F]fluoro-2-deoxy-D-glucose,
2-[18F]fluoro-2-deoxy-D-mannose and
their partially or fully acetylated derivatives
found in test A for radiochemical purity ;
= relative percentage of partially or fully
acetylated 2-[18F]fluoro-2-deoxy-D-glucose
derivatives found in test B for radiochemical
purity.
If the radiochromatogram obtained in test A for

radiochemical purity shows a peak at the retention time
of 2-[18F]fluoro-2-deoxy-D-mannose, calculate the relative
percentage of 2-[18F]fluoro-2-deoxy-D-mannose using the
= relative percentage of radioactivity

eluting at the retention time of
2-[18F]fluoro-2-deoxy-D-mannose found in
test A for radiochemical purity ;
= relative percentage of partially or fully
acetylated 2-[18F]fluoro-2-deoxy-D-glucose
derivatives found in test B for radiochemical
purity.
B. Examine by thin-layer chromatography (2.2.27) using a

TLC silica gel plate R.
B. aminopolyether,
C. tetrabutylammonium,
D. 4-(4-methylpiperidino)pyridine,
E. [18F]fluoride.
Reagents
TLC silica gel plate for aminopolyether test. XXXXXXX.
Immerse a TLC silica gel plate R in iodoplatinate reagent R1
for 5-10 s. Dry the plate at room temperature for 12 h,
protected from light.
Storage : protected from light, in an open container ; use
within 30 days after preparation.
Iodoplatinate reagent R1. XXXXXXX.
Mix 2.5 ml of a 50 g/l solution of chloroplatinic acid R,
22.5 ml of a 100 g/l solution of potassium iodide R and
50 ml of water R.
Storage : protected from light, at a temperature of 2 C to
8 C.
1,2,3,4-Tetra-O-acetyl--D-glucopyranose. C14H20O10.
(Mr 348.3). XXXXXXX. [13100-46-4].
A white or almost white powder, soluble in water with gentle
heating.
: + 11, c = 6 g/l in CHCl3.
mp : 126 C to 128 C.
Apply 2 l to 10 l to the plate. Develop over a path

of 8 cm using a mixture of 5 volumes of water R and
2-Fluoro-2-deoxy-D-mannose. C6H11FO5. (Mr 182.1).
95 volumes of acetonitrile R. Allow the plate to dry in air XXXXXXX. [31077-88-0].
Colourless semi-solid.
for 15 min. Determine the distribution of radioactivity
(19) Merck silica F60 is suitable.
624
Human -1-antitrypsin
CHARACTERS
Appearance : freeze-dried products are hygroscopic, white
XXXX:2387 or pale yellow to pale brown powders or friable solids ; liquid
products are clear or slightly opalescent, and colourless, pale
yellow, pale green or pale brown.
HUMAN -1-ANTITRYPSIN
If the preparation to be examined is freeze-dried,
reconstitute it as stated on the label immediately before
-1-Antitrypsinum humanum
carrying out the identification, tests (except those for
solubility and water) and assay.
DEFINITION
Human -1-antitrypsin is a plasma protein fraction containing
mainly -1-antitrypsin (also known as -1-prote(in)ase
inhibitor or -1-antiproteinase). Human -1-antitrypsin is a
glycoprotein existing in isoforms with different isoelectric
points, and is the most abundant multifunctional serine
proteinase inhibitor in human plasma. It is purified
from human plasma that complies with the monograph
Human plasma for fractionation (0853), using a suitable
fractionation process and further purification steps. Other
plasma proteins may be present.
PRODUCTION
GENERAL PROVISIONS
The method of preparation includes steps that have been
shown to remove or to inactivate known agents of infection.
The subsequent purification procedure must be validated
to demonstrate that the concentration of any substances
used for inactivation of viruses during production is reduced
to a suitable level and that any residues are such as not to
compromise the safety of the preparation for patients.
The specific activity is not less than 0.35 mg of active human
-1-antitrypsin per milligram of total protein.
The human -1-antitrypsin is dissolved in a suitable liquid.
Buffering and other auxiliary substances such as a stabiliser
may be included. No antimicrobial preservative is added.
The solution is passed through a bacteria-retentive filter and
distributed aseptically into the final containers. The product
may subsequently be freeze-dried.
CONSISTENCY OF THE METHOD OF PRODUCTION
The consistency of the method of production, including
demonstration that the manufacturing process yields a
product with a consistent composition and maintains the
functional integrity of human -1-antitrypsin, is evaluated by
suitable analytical procedures that are determined during
process development, and which include :
assay of human -1-antitrypsin activity ;
determination of specific human -1-antitrypsin activity,
expressed as the ratio of active human -1-antitrypsin to
total protein (2.5.33) ;
characterisation of isoform composition and protein
structure by suitable methods such as isoelectric
focusing (2.2.54) or spectroscopic methods (e.g. mass
spectrometry) ;
determination of the ratio of human -1-antitrypsin
activity to human -1-antitrypsin antigen ;
characterisation of accompanying plasma proteins that
might be present, by a set of suitable methods such as
SDS-PAGE, cellulose acetate electrophoresis or capillary
zone electrophoresis, and quantitative determination of
relevant accompanying plasma proteins ;
determination of molecular-size distribution, used to
quantify the polymeric forms of human -1-antitrypsin ;
consideration is given to the potential presence of
accompanying proteins that might affect the results.
IDENTIFICATION
The assay of human -1-antitrypsin activity and the human
-1-antitrypsin antigen determination also serve to identify
the preparation.
TESTS
pH (2.2.3) : 6.5 to 7.8.
Solubility. To a container of the preparation to be examined
add the volume of the liquid stated on the label at room
temperature. The preparation dissolves completely with
gentle swirling within 15 min, giving a clear, colourless to
slightly greenish or slightly yellowish to brownish solution.
Osmolality (2.2.35) : minimum 210 mosmol/kg.
Ratio of human -1-antitrypsin activity to human
-1-antitrypsin antigen : minimum 0.7.
Suitable quantitative methods for the specific determination
of human -1-antitrypsin antigen such as enzyme-linked
immunosorbent assay (ELISA), immunonephelometry or
radial immunodiffusion, may be used.
A reference preparation with an assigned concentration of
human -1-antitrypsin antigen as defined by an international
reference preparation is used in the test and serves as
the basis for calculating the antigen concentration in test
samples.
Total protein. Carry out the test using a sufficient number
of pooled units. Dilute the pool with a 9 g/l solution of
sodium chloride R to obtain a solution containing about
15 mg of protein in 2 ml. To 2.0 ml of this solution in a
round-bottomed centrifuge tube add 2 ml of a 75 g/l solution
of sodium molybdate R and 2 ml of a mixture of 1 volume
of nitrogen-free sulphuric acid R and 30 volumes of
water R. Shake, centrifuge for 5 min, decant the supernatant
liquid and allow the inverted tube to drain on filter paper.
Determine the nitrogen in the residue by the method of
sulphuric acid digestion (2.5.9) and calculate the protein
content by multiplying the quantity of nitrogen by 6.25.
Water. Determined by a suitable method, such as the
semi-micro determination of water (2.5.12), loss on drying
(2.2.32) or near-infrared spectrophotometry (2.2.40), the
water content is within the limits approved by the competent
authority.
Sterility (2.6.1). It complies with the test.
Pyrogens (2.6.8). It complies with the test. Inject per
kilogram of the rabbits mass a volume equivalent to not less
than 60 mg of human -1-antitrypsin.
ASSAY
Carry out the assay of human -1-antitrypsin (2.7.32)(20). The
estimated potency is not less than 80 per cent and not more
than 120 per cent of the stated potency. The confidence
limits (P = 0.95) are not less than 80 per cent and not more
than 120 per cent of the estimated potency.
(20) See draft in this issue.
625
The product shall have been shown, by suitable tests in

animals and evaluation during clinical trials, to be well
tolerated when administered intramuscularly.
Human normal immunoglobulin is prepared from pooled
LABELLING
material from at least 1000 donors by a method that has
been shown to yield a product that :
The label states :
does not transmit infection ;
the potency of active (functional) human -1-antitrypsin
at a protein concentration of 160 g/l, contains antibodies
per container ;
for at least 2 of which (1 viral and 1 bacterial) an
the name and quantity of any added substances ;
International Standard or Reference Preparation is
where applicable, the name and volume of the liquid to
available, the concentration of such antibodies being at
be used for reconstitution ;
least 10 times that in the initial pooled material.
that the transmission of infectious agents cannot be
If the human normal immunoglobulin is intended for
totally excluded when medicinal products prepared from subcutaneous infusion, the production method shall have
human blood or plasma are administered.
been shown to yield consistently products that comply with
the test for Fc function of immunoglobulin (2.7.9).
Human normal immunoglobulin is prepared as a stabilised
solution, for example in a 9 g/l solution of sodium chloride,
a 22.5 g/l solution of glycine or, if the preparation is to
Reference: PA/PH/Exp. 6B/T (05) 25 ANP 1R
be freeze-dried, a 60 g/l solution of glycine. Multidose
preparations contain an antimicrobial preservative.
Single-dose preparations do not contain an antimicrobial
This monograph was revised and published in
preservative. Any antimicrobial preservative or stabilising
Pharmeuropa 17.4 to add the subcutaneous route, and to
agent used shall have been shown to have no deleterious
require to test for Fc function if it is intended for that use. effect on the final product in the amount present. The
The reason is that subcutaneous infusion of human normal solution is passed through a bacteria-retentive filter. The
immunoglobulin is being employed to an increasing extent preparation may subsequently be freeze-dried and the
as an alternative to intravenous immunoglobulin in the
containers closed under vacuum or under an inert gas.
treatment of primary immunoglobulin deficiencies. Both
The stability of the preparation is demonstrated by suitable
the capacity to bind appropriate antigens and to mediate
tests carried out during development studies.
Fc-dependent functions have to be demonstrated to prove
functional integrity of the antibodies in the preparations,
CHARACTERS
since both capacities are necessary conditions of a
The liquid preparation is clear and pale-yellow or light-brown ;
satisfactory therapeutic efficacy. Furthermore, the test for
during storage it may show formation of slight turbidity
Fc function is already carried out by the manufacturers
or a small amount of particulate matter. The freeze-dried
and is therefore not an additional requirement in practice.
preparation is a hygroscopic, white or slightly yellow powder
In light of the comments received, the text is published
or solid, friable mass.
again with 2 additional tests to be performed when human For the freeze-dried preparation, reconstitute as stated on
normal immunoglobulin is intended for subcutaneous
the label immediately before carrying out the identification
infusion : a test for anti-A and anti-B haemagglutination
and the tests, except those for solubility and water.
(2.6.20) and a test for anti-D antibodies (2.6.26). This is in
line with EMEA guidelines.
IDENTIFICATION
XXXX:0338 Examine by a suitable immunoelectrophoresis technique.
Using antiserum to normal human serum, compare normal
HUMAN NORMAL IMMUNOGLOBULIN human serum and the preparation to be examined, both
diluted to a protein concentration of 10 g/l. The main
of the preparation to be examined corresponds
Immunoglobulinum humanum normale component
to the IgG component of normal human serum. The solution
may show the presence of small quantities of other plasma
DEFINITION
proteins.
Human normal immunoglobulin is a liquid or freeze-dried
preparation containing immunoglobulins, mainly
TESTS
immunoglobulin G (IgG). Other proteins may be present.
Solubility. For the freeze-dried preparation, add the volume
Human normal immunoglobulin contains the IgG antibodies of the liquid stated on the label. The preparation dissolves
of normal subjects. It is intended for intramuscular injection completely within 20 min at 20-25 C.
or for subcutaneous infusion.
pH (2.2.3) : 5.0 to 7.2.
Human normal immunoglobulin is obtained from plasma
Dilute the preparation to be examined with a 9 g/l solution
that complies with the requirements of the monograph on
of sodium chloride R to a protein concentration of 10 g/l.
Human plasma for fractionation (0853). No antibiotic is
added to the plasma used.
Total protein. Dilute the preparation to be examined with
a 9 g/l solution of sodium chloride R to obtain a solution
PRODUCTION
containing about 15 mg of protein in 2 ml. To 2.0 ml of
The method of preparation includes a step or steps that have this solution in a round-bottomed centrifuge tube add 2 ml
of a 75 g/l solution of sodium molybdate R and 2 ml of a
been shown to remove or to inactivate known agents of
infection ; if substances are used for inactivation of viruses, it mixture of 1 volume of nitrogen-free sulphuric acid R and
shall have been shown that any residues present in the final 30 volumes of water R. Shake, centrifuge for 5 min, decant
product have no adverse effects on the patients treated with the supernatant liquid and allow the inverted tube to drain
on filter paper. Determine the nitrogen in the residue by
the immunoglobulin.
STORAGE
Unless otherwise justified and authorised, in an airtight
container, at a temperature not exceeding 25 C.
626
the method of sulphuric acid digestion (2.5.9) and calculate

the content of protein by multiplying the result by 6.25. The
preparation has a protein concentration of not less than
100 g/l and not more than 180 g/l and contains not less
than 90 per cent and not more than 110 per cent of the
quantity of protein stated on the label.
Protein composition. Examine by zone electrophoresis
(2.2.31).
Use strips of suitable cellulose acetate gel or suitable agarose
gel as the supporting medium and barbital buffer solution
pH 8.6 R1 as the electrolyte solution.
If cellulose acetate is the supporting material, the method
described below can be used. If agarose gels are used, and
because they are normally part of an automated system, the
manufacturers instructions are followed instead.
Test solution. Dilute the preparation to be examined
with a 9 g/l solution of sodium chloride R to a protein
concentration of 50 g/l.
Reference solution. Reconstitute human immunoglobulin
for electrophoresis BRP and dilute with a 9 g/l solution of
sodium chloride R to a protein concentration of 50 g/l.
To a strip apply 2.5 l of the test solution as a 10 mm band
or apply 0.25 l per millimetre if a narrower strip is used. To
another strip apply in the same manner the same volume of
the reference solution. Apply a suitable electric field such
that the albumin band of normal human serum applied
on a control strip migrates at least 30 mm. Stain the strip
with amido black 10B solution R for 5 min. Decolourise
with a mixture of 10 volumes of glacial acetic acid R and
90 volumes of methanol R so that the background is just
free of colour. Develop the transparency of the strips
with a mixture of 19 volumes of glacial acetic acid R and
81 volumes of methanol R. Measure the absorbance of the
bands at 600 nm in an instrument having a linear response
over the range of measurement. Calculate the result as the
mean of 3 measurements of each strip.
System suitability : in the electropherogram obtained with
the reference solution on cellulose acetate or on agarose
gels, the proportion of protein in the principal band is within
the limits stated in the leaflet accompanying the reference
preparation.
Results : in the electropherogram obtained with the test
solution on cellulose acetate or on agarose gels, not more
than 10 per cent of protein has a mobility different from that
of the principal band.
Distribution of molecular size. Liquid chromatography
(2.2.29).
Test solution. Dilute the preparation to be examined with
a 9 g/l solution of sodium chloride R to a concentration
suitable for the chromatographic system used. A
concentration in the range of 4-12 g/l and injection of
50-600 g of protein are usually suitable.
Reference solution. Dilute human immunoglobulin
(molecular size) BRP with a 9 g/l solution of sodium
chloride R to the same protein concentration as the test
solution.
Column :
size : l = 0.6 m, = 7.5 mm ; or l = 0.3 m, = 7.8 mm ;
stationary phase: hydrophilic silica gel for
chromatography R(21), of a grade suitable for fractionation
of globular proteins with relative molecular masses in the
range 10 000 to 500 000.
Mobile phase : dissolve 4.873 g of disodium hydrogen

phosphate dihydrate R, 1.741 g of sodium dihydrogen
phosphate monohydrate R, 11.688 g of sodium chloride R
and 50 mg of sodium azide R in 1 litre of water R.
In the chromatogram obtained with the reference solution,
the principal peak corresponds to the IgG monomer and
there is a peak corresponding to the dimer with a relative
retention to the principal peak of about 0.85. Identify
the peaks in the chromatogram obtained with the test
solution by comparison with the chromatogram obtained
with the reference solution ; any peak with a retention time
shorter than that of the dimer corresponds to polymers
and aggregates. The preparation to be examined complies
with the test if, in the chromatogram obtained with the test
solution :
relative retention : for the monomer and for the dimer,
the relative retention to the corresponding peak in the
chromatogram obtained with the reference solution is
1 0.02 ;
peak area : the sum of the peak areas of the monomer
and the dimer represent not less than 85 per cent of the
total area of the chromatogram and the sum of the peak
areas of polymers and aggregates represents not more
than 10 per cent of the total area of the chromatogram.
Anti-A and anti-B haemagglutinins (2.6.20). If human
normal immunoglobulin is intended for subcutaneous
infusion, carry out the tests for anti-A and anti-B
haemagglutinins. If the preparation to be examined has an
immunoglobulin concentration greater than 30 g/l, dilute
it to this concentration before preparing the dilutions to
be used in the test. The 64-fold dilutions do not show
agglutination.
Anti-D antibodies (2.6.26). If human normal
immunoglobulin is intended for subcutaneous infusion,
it complies with the test for anti-D antibodies in human
immunoglobulin for intravenous administration.
Antibody to hepatitis B surface antigen. Not less than
0.5 IU/g of immunoglobulin, determined by a suitable
immunochemical method (2.7.1).
Antibody to hepatitis A virus. If intended for use in the
prophylaxis of hepatitis A, it complies with the following
additional requirement. Determine the antibody content
by comparison with a reference preparation calibrated in
International Units, using an immunoassay of suitable
sensitivity and specificity (2.7.1).
The International Unit is the activity contained in a stated
amount of the International Standard for anti-hepatitis A
immunoglobulin. The equivalence in International Units of
the International Standard is stated by the World Health
Organisation.
Human hepatitis A immunoglobulin BRP is calibrated in
International Units by comparison with the International
Standard.
The stated potency is not less than 100 IU/ml. The estimated
potency is not less than the stated potency. The confidence
limits (P = 0.95) of the estimated potency are not less than
80 per cent and not more than 125 per cent.
Water. Determined by a suitable method, such as the
semi-micro determination of water (2.5.12), loss on drying
(2.2.32) or near infrared spectrophotometry (2.2.40), the
water content is within the limits approved by the competent
authority.
(21) TSK G3000 SW (l = 0.6 m, = 7.5 mm) and TSK G3000 SWXL (l = 0.3 m, = 7.8 mm) are suitable.
627
Sterility (2.6.1). It complies with the test for sterility.

Pyrogens (2.6.8). It complies with the test for pyrogens.
Inject 1 ml per kilogram of the rabbits mass.
The pool of plasma is tested for hepatitis B surface

antigen (HBsAg), for hepatitis C virus antibodies and for
HIV antibodies using test methods of suitable sensitivity and
specificity ; the pool must give negative results in these tests.
STORAGE
The plasma pool is also tested for hepatitis C virus RNA using
For the liquid preparation, in a colourless glass container,
a validated nucleic acid amplification technique (2.6.21).
protected from light. For the freeze-dried preparation, in an A positive control with 100 IU of hepatitis C virus RNA
airtight colourless glass container, protected from light.
per millilitre and, to test for inhibitors, an internal control
prepared by addition of a suitable marker to a sample of the
LABELLING
plasma pool are included in the test. The test is invalid if
The label states :
the positive control is non-reactive or if the result obtained
for liquid preparations, the volume of the preparation in with the internal control indicates the presence of inhibitors.
the container and the protein content expressed in grams The pool complies with the test if it is found non-reactive for
hepatitis C virus RNA.
per litre ;
Hepatitis C virus RNA for NAT testing BRP is suitable for
for freeze-dried preparations, the quantity of protein in
use as a positive control.
the container ;
the route of administration ;
To limit the potential burden of B19 virus in plasma pools,
the plasma pool is also tested for B19 virus using a validated
for freeze-dried preparations, the name or composition
and the volume of the reconstituting liquid to be added ; nucleic acid amplification technique (2.6.21).
where applicable, that the preparation is suitable for use B19 virus DNA. The plasma pool contains not more than
10.0 IU/l.
in the prophylaxis of hepatitis A infection ;
where applicable, the anti-hepatitis A virus activity in
A positive control with 10.0 IU of B19 virus DNA per
International Units per millilitre ;
microlitre and, to test for inhibitors, an internal control
where applicable, the name and amount of antimicrobial prepared by addition of a suitable marker to a sample of the
plasma pool are included in the test. The test is invalid if the
preservative in the preparation.
positive control is non-reactive or if the result obtained with
the internal control indicates the presence of inhibitors.
B19 virus DNA for NAT testing BRP is suitable for use as a
positive control.
The method of preparation is designed to minimise
activation of any coagulation factor (to minimise potential
Production : the monograph presented below has been
thrombogenicity) and includes a step or steps that have been
revised in order to require that the production process
shown to inactivate known agents of infection ; if substances
guarantees the integrity of coagulation proteins ; indeed,
are used for the inactivation of viruses during production,
certain inactivation methods reduce the levels of some of
the subsequent purification procedure must be validated to
these proteins, sometimes significantly, which may result
demonstrate that the concentration of these substances is
in severe complications in some patients.
reduced to a suitable level and that any residues are such as
not to compromise the safety of the preparation for patients.
Assay : limits for protein C, protein S and
alpha-2-antiplasmin are proposed below and assays are
A typical method to inactivate enveloped viruses is the
described in separate chapters.
solvent-detergent process, which uses treatment with a
XXXX:1646 combination of tributyl phosphate and octoxinol 10(22) ;
these reagents are subsequently removed by oil extraction
or by solid phase extraction so that the amount in the final
HUMAN PLASMA (POOLED AND
is less than 2 g/ml for tributyl phosphate and less
TREATED FOR VIRUS INACTIVATION) product
than 5 g/ml for octoxinol 10.
No antimicrobial preservative is added.
Plasma humanum coagmentatum
The solution is passed through a bacteria-retentive filter,
conditumque ad exstinguendum virum
distributed aseptically into the final containers and
immediately frozen ; it may subsequently be freeze-dried.
DEFINITION
Human plasma (pooled and treated for virus inactivation) is Plastic containers comply with the requirements for sterile
plastic containers for human blood and blood components
a frozen or freeze-dried, sterile, non-pyrogenic preparation
obtained from human plasma derived from donors belonging (3.2.3).
to the same ABO blood group. The preparation is thawed or Glass containers comply with the requirements for glass
containers for pharmaceutical use (3.2.1).
reconstituted before use to give a solution for infusion.
The human plasma used complies with the monograph on
CHARACTERS
Human plasma for fractionation (0853).
The frozen preparation, after thawing, is a clear or slightly
PRODUCTION
opalescent liquid free from solid and gelatinous particles.
The freeze-dried preparation is an almost white or slightly
The units of plasma to be used are cooled to 30 C or
lower within 6 h of separation of cells and in any case within yellow powder or friable solid.
24 h of collection.
Thaw or reconstitute the preparation to be examined as
The pool is prepared by mixing units of plasma belonging to stated on the label immediately before carrying out the
identification, tests and assay.
the same ABO blood group.
(22) Triton X-100.
628
Calcium : maximum 5.0 mmol/l.

Atomic absorption spectrometry (2.2.23, Method I).
A. Examine by electrophoresis (2.2.31) comparing with
normal human plasma. The electropherograms show the Source : calcium hollow-cathode lamp using a transmission
same bands.
band preferably of 0.5 nm.
B. It complies with the test for anti-A and anti-B
Wavelength : 622 nm.
haemagglutinins (see Tests).
Atomisation device : air-acetylene or acetylene-propane
flame.
TESTS
Potassium : maximum 5.0 mmol/l.
pH (2.2.3) : 6.5 to 7.6.
Atomic emission spectrometry (2.2.22, Method I).
Osmolality (2.2.35) : minimum 240 mosmol/kg.
Total protein : minimum 45 g/l.
Sodium : maximum 2.00 102 mmol/l.
Dilute with a 9 g/l solution of sodium chloride R to obtain Atomic emission spectrometry (2.2.22, Method I).
a solution containing about 15 mg of protein in 2 ml. Place
Wavelength : 589 nm.
2.0 ml of this solution in a round-bottomed centrifuge tube
and add 2 ml of a 75 g/l solution of sodium molybdate R
Water : for the freeze-dried product : determined by a
and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric suitable method, such as the semi-micro determination
acid R and 30 volumes of water R. Shake, centrifuge for
of water (2.5.12), loss on drying (2.2.32) or near-infrared
5 min, decant the supernatant liquid and allow the inverted spectrometry (2.2.40), the water content is within the limits
tube to drain on filter paper. Determine the nitrogen in the approved by the competent authority.
residue by the method of sulphuric acid digestion (2.5.9)
Sterility (2.6.1). It complies with the test for sterility.
and calculate the quantity of protein by multiplying the
Pyrogens (2.6.8). It complies with the test for pyrogens.
result by 6.25.
Inject 3 ml per kilogram of the rabbits mass.
Activated coagulation factors (2.6.22). It complies with the
test for activated coagulation factors. Carry out the test with ASSAY
0.1 ml of the preparation to be examined instead of 1 to 10
Factor VIII. Carry out the assay of coagulation factor VIII
and 1 to 100 dilutions. The coagulation time for the tube
(2.7.4) using a reference plasma calibrated against the
containing the preparation to be examined is not less than
International Standard for blood coagulation factor VIII in
150 s.
plasma.
Anti-A and anti-B haemagglutinins (2.6.20). The presence
of haemagglutinin (anti-A or anti-B) corresponds to the blood The estimated potency is not less than 0.5 IU/ml. The
confidence limits (P = 0.95) are not less than 80 per cent and
group stated on the label.
not more than 120 per cent of the estimated potency.
Hepatitis A virus antibodies : minimum 2 IU/ml, determined
Factor V. Using imidazole buffer solution pH 7.3 R, prepare
by a suitable immunochemical method (2.7.1).
3 twofold dilutions of the preparation to be examined,
Human hepatitis A immunoglobulin BRP is suitable for use preferably in duplicate, from 1 in 10 to 1 in 40. Test each
as a reference preparation.
dilution as follows : mix 0.1 ml of plasma substrate deficient
in factor V R, 0.1 ml of the dilution to be examined, 0.1 ml of
Irregular erythrocyte antibodies. The preparation to
thromboplastin R and 0.1 ml of a 3.5 g/l solution of calcium
be examined does not show the presence of irregular
chloride R ; measure the coagulation times, i.e. the interval
erythrocyte antibodies when examined without dilution by
between the moment at which the calcium chloride solution
an indirect antiglobulin test.
is added and the first indication of the formation of fibrin,
Citrate. Liquid chromatography (2.2.29).
which may be observed visually or by means of a suitable
Test solution. Dilute the preparation to be examined with an apparatus.
equal volume of a 9 g/l solution of sodium chloride R. Filter In the same manner, determine the coagulation time of
the solution using a filter with 0.45 m pores.
4 twofold dilutions (1 in 10 to 1 in 80) of human normal
Reference solution. Dissolve 0.300 g of sodium citrate R in plasma in imidazole buffer solution pH 7.3 R. 1 unit of
factor V is equal to the activity of 1 ml of human normal
water R and dilute to 100.0 ml with the same solvent.
plasma. Human normal plasma is prepared by pooling
Column :
plasma units from not fewer than 30 donors and stored at
30 C or lower.
size : l = 0.3 m, = 7.8 mm ;
Check the validity of the assay and calculate the potency
stationary phase: cation exchange resin R (9 m)(23).
of the test preparation by the usual statistical methods for
Mobile phase : 0.51 g/l solution of sulphuric acid R.
a parallel-line assay (for example, 5.3).
The estimated potency is not less than 0.5 IU/ml. The
Equilibration: 15 min.
Factor XI. Carry out the assay of human coagulation
Injection: 10 l.
factor XI (2.7.22) using as reference human normal plasma
(see above under Factor V).
Retention time : citrate = about 10 min.
Limit :
citrate : maximum 25 mmol/l.
IDENTIFICATION
(23) Aminex HPX-87H is suitable.
629
Protein C. Carry out the assay of protein C (2.7.30)(24) using

a reference plasma calibrated against the International
Standard for protein C in plasma.
Protein S. Carry out the assay of protein S (2.7.31)(24) using
a reference plasma calibrated against the International
Standard for protein S in plasma.
2-Antiplasmin. Carry out the assay of 2-antiplasmin
(2.7.25)(24) using a reference plasma calibrated against a
suitable reference preparation for 2-antiplasmin in plasma.
B. Dissolve 20 mg in 0.01 M hydrochloric acid and dilute to

100.0 ml with the same acid. Dilute 1.0 ml of this solution
to 10.0 ml with 0.01 M hydrochloric acid. Examined
between 230 nm and 350 nm, the solution shows a single
absorption maximum (2.2.25), at 251 nm, and a shoulder
at 270 nm. The specific absorbance at the maximum is
about 260.
C. Infrared absorption spectrophotometry (2.2.24).
Comparison : imipramine hydrochloride CRS.
D. Dissolve about 5 mg in 2 ml of nitric acid R. An intense
blue colour develops.
E. Dissolve about 50 mg in 3 ml of water R and add 0.05 ml
of a 25 g/l solution of quinhydrone R in methanol R. No
red colour develops within 15 min.
F. About 20 mg gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. To 3.0 g add 20 ml of carbon dioxide-free
LABELLING
water R, dissolve rapidly by shaking and triturating with a
The label states :
glass rod and dilute to 30 ml with the same solvent.
the ABO blood group ;
Appearance of solution. Solution S is clear (2.2.1).
the method used for virus inactivation.
Immediately after preparation, dilute solution S with an
equal volume of water R. This solution is not more intensely
coloured than reference solution BY6 (2.2.2, Method II).
pH (2.2.3) : 3.6 to 5.0 for solution S, measured immediately
after preparation.
(2.2.27), using a TLC silica gel G plate R.
Related substances : TLC replaced by an LC in accordance Test solution. Dissolve 0.25 g of the substance to be
with current policy. A scientific note published in this issue examined in methanol R and dilute to 10 ml with the same
describes the development and validation of the method.
solvent. Prepare immediately before use.
XXXX:0029
Reference solution (a). Dilute 1 ml of the test solution to
10 ml with methanol R. Dilute 1 ml of this solution to 50 ml
IMIPRAMINE HYDROCHLORIDE
with methanol R.
Reference solution (b). Dissolve 5 mg of iminodibenzyl R
Imipramini hydrochloridum
in methanol R and dilute to 100 ml with the same solvent.
Prepare immediately before use.
Apply to the plate 10 l of each solution. Develop over a
path of 12 cm using a mixture of 5 volumes of hydrochloric
acid R, 5 volumes of water R, 35 volumes of glacial acetic
acid R and 55 volumes of ethyl acetate R. Allow the plate
to dry in air for 5 min and spray with a 5 g/l solution
of potassium dichromate R in a mixture of 1 volume of
sulphuric acid R and 4 volumes of water R. Examine the
plate immediately. The chromatogram obtained with the test
C19H25ClN2
Mr 316.9 solution shows a blue principal spot. In the chromatogram
obtained with the test solution : any spot corresponding
DEFINITION
to iminodibenzyl is not more intense than the spot in the
3-(10,11-Dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,Nchromatogram obtained with reference solution (b) (0.2 per
dimethylpropan-1-amine hydrochloride.
cent) ; any spot apart from the principal spot and any spot
corresponding to iminodibenzyl, is not more intense than
the spot in the chromatogram obtained with reference
CHARACTERS
solution (a) (0.2 per cent).
Appearance : white or slightly yellow, crystalline powder.
Solubility : freely soluble in water and in ethanol (96 per
cent).
mobile phase.
IDENTIFICATION
Reference solution. Dissolve 5.0 mg of imipramine
First identification : A, C, F.
impurity B CRS in 5.0 ml of the test solution and dilute to
Second identification : A, B, D, E, F.
50.0 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100.0 ml with the mobile phase.
(24) See draft in this issue.
630
Figure 0029.-1. Chromatogram for the test for related substances of imipramine hydrochloride : imipramine
hydrochloride spiked with impurities A, B, C and D
Column :
size : length = 0.15 m, = 4.6 mm ;
stationary phase : end-capped polar-embedded
octadecylsilyl amorphous organosilica polymer R
(5 m)(25) ;
temperature : 40 C.
Mobile phase : mix 40 volumes of acetonitrile R1 with
60 volumes of a 5.2 g/l solution of dipotassium hydrogen
acid R.
Injection : 10 l.
Run time : 5 times the retention time of imipramine.
Relative retention with reference to imipramine (retention
time = about 7 min) : impurity B = about 0.7.
impurity B and imipramine.
Limits :
impurity B : not more than the area of the corresponding
peak in the chromatogram obtained with the reference
solution (0.1 per cent) ;
than the area of the peak due to imipramine in the
chromatogram obtained with the reference solution
(0.10 per cent) ;
total : not more than 3 times the area of the peak due
to imipramine in the chromatogram obtained with the
reference solution (0.3 per cent) ;
disregard limit : 0.5 times the area of the peak due to
imipramine in the chromatogram obtained with the
reference solution (0.05 per cent).
on 1.0 g.
ASSAY
Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R
and add 5.0 ml of 0.01 M hydrochloric acid. Carry out
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
1 ml of 0.1 M sodium hydroxide is equivalent to 31.69 mg
of C19H25ClN2.
STORAGE
Protected from light.
(25) X-Terra RP18 (hybrid particles) is suitable.
631
Lauromacrogol 400
IMPURITIES
Specified impurities : B.
pharmaceutical use) : A, C, D.
macrogols have been added as well as an assay by NMR

(average number of EO moles and average chain length of
the fatty alcohol).
The International Non-proprietary Name (INN) of this
substance is Lauromacrogol 400 ; since the substance is
also known as Polidocanol 9 and macrogol 9 lauryl ether,
the latter names are proposed as synonyms.
XXXX:2046
LAUROMACROGOL 400
Lauromacrogolum 400
A. 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-Nmethylpropan-1-amine,
B. 3-(5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1amine,
C. 10-[3-(dimethylamino)propyl]acridin-9(10H)-one,
D. 10,11-dihydro-5H-dibenzo[b,f]azepine.

Lauromacrogol 400 corresponds to macrogol lauryl ether
with 9 ethylene oxide (EO) moles per mole of substance.
A monograph on Macrogol lauryl ether (1124) is already
included in the Ph. Eur., covering grades having 3 to
23 EO moles per mole of substance. These grades are used
as excipients.
The present draft covers macrogol lauryl ether with 9
EO units used as the active substance, mainly for vein
sclerosis. The quality control tests proposed are therefore
more sophisticated : tests for free lauryl alcohol and free
632
DEFINITION
Mixture of lauryl alcohol (dodecanol) monoethers of mixed
macrogols. It may contain some free macrogols and it
contains various amounts of free lauryl alcohol. The number
of moles of ethylene oxide reacted per mole of lauryl alcohol
is 9.
Synonyms : polidocanol 9, macrogol 9 lauryl ether.
Content :
average chain length of the fatty alcohol : 10.0 to 14.0,
average number of moles of ethylene oxide: 7.0 to 11.0.
CHARACTERS
Appearance : white or almost white, unctuous and
hygroscopic mass, melting at 24 C into a clear white or
yellowish, viscous liquid.
Solubility : freely soluble in water, very soluble in ethanol
(96 per cent) and in acetone.
IDENTIFICATION
A. Hydroxyl value (see Tests).
B. Saponification value (see Tests).
C. Warm the substance to be examined in an incubator at
40 C for 1 h until fully molten and clear. Transfer 50 ml
to a warmed cloud point tube (flat-bottomed glass tube
30-33.5 mm in internal diameter and 115-125 mm high).
Insert the tube into a cooling bath which allows the
outer surface of the tube to be in contact with chilled air,
contained within a cylindrical metal container (internal
diameter 9.5-12.5 mm greater than the external diameter
of the sample tube, 115 mm high) that is surrounded by
iced water. The base of the glass tube rests on a 6 mm
thick cork disc which prevents direct thermal contact
with the cooled metal cylinder. Stir the substance to
be examined continuously with a thermometer so that
the temperature is constant throughout the substance.
Periodically lift the tube out of the cooling bath to
check for signs of cloudiness at the bottom of the tube.
Examine the tube against a bright light source. When
cloudiness is first observed, check more frequently
until the substance becomes completely cloudy and the
thermometer, suspended in the centre of the substance,
is only just visible when viewed horizontally. Record the
temperature : this is the cloud point. It is 20 C to 25 C.
TESTS
Appearance : the molten substance is clear (2.2.1) and not
more intensely coloured than reference solution GY6 (2.2.2,
Method I).
Alkalinity. Dissolve 2.0 g in a hot mixture of 10 ml of
water R and 10 ml of ethanol (96 per cent) R. Add 0.1 ml of
bromothymol blue solution R1. Not more than 0.5 ml of
0.1 M hydrochloric acid is required to change the colour of
the indicator to yellow.
Lauromacrogol 400
Acid value (2.5.1) : maximum 1.0, determined on 5.0 g.

Hydroxyl value (2.5.3, Method B) : 90 to 105, determined
on 0.35 g.
Iodine value (2.5.4, Method A) : maximum 2.0.
Peroxide value (2.5.5, Method A) : maximum 5.0.
Introduce 10.0 g into a 100 ml beaker, dissolve with glacial
acetic acid R and dilute to 20 ml with the same solvent. Add
1 ml of saturated potassium iodide solution R and allow to
stand for 1 min. Add 50 ml of carbon dioxide-free water R
and a magnetic stirring bar. Titrate with 0.01 M sodium
thiosulphate, determining the end-point potentiometrically
(2.2.20). Carry out a blank titration.
Determine the peroxide value using the following expression:
n1
n2
volume of 0.01 M sodium thiosulphate required

for the substance to be examined, in millilitres ;
volume of 0.01 M sodium thiosulphate requred
for the blank, in millilitres ;
molarity of the sodium thiosulphate solution, in
moles per litre ;
mass of substance to be examined, in grams.
Saponification value (2.5.6) : maximum 3.0.

Free lauryl alcohol (dodecanol). Gas chromatography
(2.2.28).
examined in acetone R and dilute to 10.0 ml with the same
solvent.
Reference solution. Dissolve 2.0 g of dodecanol R in
acetone R and dilute to 100.0 ml with the same solvent.
Dilute 1.0 ml of this solution to 50.0 ml with acetone R.
Column :
size : l = 30 m, = 0.25 mm ;
stationary phase : poly(dimethyl)(diphenyl)siloxane R
(film thickness 0.1 m)(26).
Carrier gas : helium for chromatography R.
Split ratio : 50:1.
Temperature :
Column
Time
(min)
0-1
Temperature
(C)
120
1 - 23
120 350
23 - 33
350
Injection port
300
Detector
350

Injection: 1.0 l.
Retention time : dodecanol = about 5 min.
Limit :
free dodecanol : maximum 2.0 per cent.
Free macrogols. Size-exclusion chromatography (2.2.30).

in the mobile phase and dilute to 250.0 ml with the mobile
phase.
Reference solution (a). Dissolve about 0.4 g of macrogol
1000 R in the mobile phase and dilute to 250.0 ml with the
mobile phase.
Reference solution (b). Dilute 50.0 ml of reference
solution (a) to 100.0 ml with the mobile phase.
Precolumns (2) :
size : l = 0.125 m, = 4 mm ;
stationary phase : spherical octadecylsilyl silica gel for
chromatography R (5 m)(27) with a pore size of 10 nm.
Column :
size : l = 0.30 m, = 7.8 mm ;
stationary phase : hydroxylated polymethacrylate gel R
(6 m)(28) with a pore size of 12 nm.
Connect both precolumns to the column using a 3-way valve
and switch the mobile phase flow according to the following
programme :
0 to 114 s : precolumn 1 and column ;
115 s to the end : precolumn 2 and column ;
115 s to 7 min : flow back of precolumn 1.
Detection : refractometer.
Injection : 20 l.
Calculate the percentage content of free macrogols using the
m1
m2
A1
A2
A3
mass of the substance to be examined in the test

solution, in grams ;
mass of macrogol 1000 R in reference solution (a),
in grams ;
area of the peak due to free macrogols in the
substance to be examined in the chromatogram
obtained with the test solution ;
area of the peak due to macrogol 1000 in
solution (a) ;
area of the peak due to macrogol 1000 in
solution (b).
Limit :
free macrogols : maximum 3.0 per cent.
Ethylene oxide and dioxan (2.4.25, Method A) : maximum
1 ppm of ethylene oxide and maximum 10 ppm of dioxan.
Water (2.5.12) : maximum 2.0 per cent, determined on
1.000 g.
Total ash (2.4.16) : maximum 0.2 per cent, determined on
2.0 g.
(26) Rtx-5 column is suitable.

(27) Lichospher 100-RP-18, 5 m (Merck) is suitable.
(28) Ultrahydrogel 120, 6 m (Waters) is suitable.
633
Lauromacrogol 400
Table 2046.-1. - Shift values
ASSAY
Use a high resolution Fourier transform nuclear magnetic
resonance (FT-NMR) spectrometer (2.2.33) operating at
minimum 300 MHz.
Test solution. If the substance is in the solid state at room
temperature, heat gently before sampling. Dissolve 0.4 ml
of the substance to be examined in 0.3 ml of a mixture
of 2 volumes of deuterated chloroform R and 1 volume
of methanol R, containing 0.1 mol/l of chromium(III)
acetylacetonate R as a relaxation aid.
Acquisition of 13C NMR spectra. The following parameters
may be used:
sweep width : 250 ppm ( 15 ppm to 235 ppm) ;
irradiation frequency offset : 110 ppm ;
time domain : 64 K ;
pulse delay : 3 s ;
pulse program : zgig 30 (inverse gated, 30 excitation
pulse) ;
dummy scans : 4 ;
number of scans: 2048.
Processing and plotting. The following parameters may be
used :
size : 64 K (zero-filling) ;
window multiplication : exponential ;
Signal
Shift (ppm)
Normalized integrals
CH3
14.4
0.989
CH2 (alkyl chain)
23.2
1.000
CH2 (alkyl chain)
25.5
1.001
30
7.410
CH2s (alkyl chain)

CH2 (alkyl chain)
32.5
0.963
CH2 (-CH2-OH)
(final CH2-group of
macrogol)
61.6
1.001
CH2s (macrogol)
70.7
16.25
CH2 (R-CH2-Omacrogol) (first

CH2-group of fatty
alcohol)
72.6
0.998
CH2 (macrogol)
73.1
0.929
signal-to-noise ratio : minimum 150, for the smallest
relevant peak (CH2 at 73.1 ppm) ;
peak width at half-height : maximum 0.05 ppm, for the
central CDCl3 signal (at 78.6 ppm).
Calculation of the average chain length of the fatty alcohol
and the average number of moles of ethylene oxide: define
the signal at 23.2 ppm as 1.000 and normalise the integrals
of the other signals listed in Table 2046.- 1.
Average chain length of the fatty alcohol : it is calculated
Lorentzian broadening factor : 1 Hz.

Use the CD3OD signal for shift referencing. The shift of the
central peak of the multiplet is set to 49.0 ppm.
Plot the spectral region 0.0-80.0 ppm. Compare the
spectrum with the spectrum in Figure 2046.-1. The shift
values lie near the ranges given in Table 2046.-1.
14-33 In,i
In,72.6
= sum of the normalised integrals of the

signals from 14 ppm to 33 ppm ;
= normalised integral of the signal at
72.6 ppm.
Figure 2046.-1 13C NMR spectrum of lauromacrogol 400

634
Levodropropizine
Average number of moles of ethylene oxide : it is calculated

Dissolve 1.50 g in a 21 g/l solution of hydrochloric acid R

and dilute to 50.0 ml with the same acid.
Comparison : levodropropizine CRS.
TESTS
pH (2.2.3) : 9.2 to 10.2.
Suspend 2.5 g in carbon dioxide-free water R, heat to
dissolve, cool to room temperature and dilute to 100 ml with
the same solvent.
Impurity B and related substances. Liquid chromatography
(2.2.29).
Reagents
Test
solution. Dissolve 24.0 mg 25.0 mg of the substance
Dodecanol. C12H26O. (Mr 186.3). XXXXXXX. [112-53-8].
to
be
examined in the mobile phase and dilute to 100.0 ml
Dodecan-1-ol. Lauryl alcohol.
bp : about 261 C.
Reference solution (a). Dissolve 12.0 mg 25.0 mg of
mp : about 24 C.
Content : minimum 98.0 per cent of C12H26O, determined by 1-phenylpiperazine R (impurity B) in methanol R and dilute
to 100.0 ml with the same solvent. Dilute 1.0 ml of this
gas chromatography.
Chromium(III) acetylacetonate. Cr (C5H7O2)3. (Mr 349.3).
Reference solution (b). Mix 2 ml 1 ml of the test solution
XXXXXXX. [21679-31-2].
with 2 ml 1 ml of reference solution (a) and dilute to 10 ml
Column :
size : l = 0.15 m, = 4.6 mm ;
Reference: PA/PH/Exp. 10C/T (06) 54 ANP
stationary phase : base-deactivated octylsilyl end-capped
octadecylsilyl silica gel for chromatography R (5 m)(29).
Impurity B and related substances :
Mobile phase : mix 12 volumes of methanol R and
test solution : increase of the concentration to improve
phosphate R adjusted to pH 3.0 with phosphoric acid R.
the sensitivity of the method ;
reference solution (b) : increase of the concentration to Flow rate : 1.5 ml/min.
give a better idea of the separation at the concentration Detection : spectrophotometer at 254 nm.
of the test solution ;
Injection : 20 l.
stationary phase : a better separation is obtained with
the stationary phase proposed ;
limits : introduction of a limit for the total and of the
levodropropizine and impurity B.
usual value of 0.05 per cent for the disregard limit.
Limits
:
XXXX:1535
impurity B : not more than the area of the corresponding
LEVODROPROPIZINE
solution (a) (0.5 per cent) ;
unspecified
impurities : for each impurity, not more
Levodropropizinum
than 0.2 times the area of the peak due to impurity B in
the chromatogram obtained with reference solution (a)
(0.10 per cent) ;
total : not more than 1.2 times the area of the peak due to
impurity B in the chromatogram obtained with reference
solution (a) (0.6 per cent) ;
disregard limit : 0.02 times 0.1 times the area of the peak
C13H20N2O2
Mr 236.3
due to impurity B in the chromatogram obtained with
reference solution (a) (0.01 per cent) (0.05 per cent).
DEFINITION
Impurity C. Gas chromatography (2.2.28). Prepare the
(2S)-3-(4-Phenylpiperazin-1-yl)propane-1,2-diol.
solutions immediately before use.
examined in methylene chloride R and dilute to 2.5 ml with
CHARACTERS
the same solvent.
Appearance : white or almost white powder.
Reference solution (a). Dissolve 0.20 g of glycidol R
Solubility : slightly soluble in water, freely soluble in dilute
(impurity
C) in methylene chloride R and dilute to 100.0 ml
acetic acid and in methanol, slightly soluble in ethanol
with the same solvent. Dilute 0.5 ml of this solution to
(96 per cent).
100.0 ml with methylene chloride R.
IDENTIFICATION
Reference solution (b). Dissolve 0.50 g of the substance to be
A. Specific optical rotation (2.2.7) : 30.0 to 33.5 (dried
examined in methylene chloride R, add 0.5 ml of reference
substance).
solution (a) and dilute to 2.5 ml with methylene chloride R.
In,62, In,71, In,73
= normalised integral of the signals at

62 ppm, 71 ppm and 73 ppm respectively.
The sum of the normalised integrals of the signals at 62 ppm,
71 ppm and 73 ppm corresponds to the average number of
methylene groups in the macrogol part of lauromacrogol 400.
(29) Zorbax Eclipse XDB-C18 is suitable.
635
Levodropropizine
Figure 1535.-1. Chromatogram for the test for related substances and impurity B of levodropropizine : impurity B at
0.05 per cent
Column :
size : l = 30 m, = 0.53 mm ;
stationary phase : poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R (film thickness 3 m).
Split ratio : 1:8.
Temperature :
column : 140 C ;
injection port : 170 C ;
detector : 250 C.
Injection: 1 l of the test solution and reference solution (b).
Use a split-liner consisting of a column about 1 cm long
packed with glass wool.
At the end of a series of tests, heat the column at 250 C
for 4-6 h.
Limit :
impurity C : not more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
reference solution (b) (10 ppm).
Enantiomeric purity. Liquid chromatography (2.2.29).
Solvent mixture : anhydrous ethanol R, hexane R
(40:60 V/V).
examined in 10.0 ml of the solvent mixture. Dilute 1.0 ml of
this solution to 50.0 ml with the solvent mixture.
levodropropizine CRS in 10.0 ml of the solvent
mixture. Dilute 1.0 ml of this solution to 50.0 ml with the
solvent mixture.
Reference solution (b). Dissolve 10.0 mg of levodropropizine
impurity A CRS in 10.0 ml of the solvent mixture. Dilute
1.0 ml of this solution to 50.0 ml with the solvent mixture.
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 50.0 ml with the solvent mixture.
to 25 ml with reference solution (a).
636
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : silica gel OD for chiral separations R.
Mobile phase : diethylamine R, anhydrous ethanol R,
hexane R (0.2:5:95 V/V/V).
Injection : 20 l of the test solution and reference
Elution order : impurity A, levodropropizine.
retention times : the retention times of the principal
peaks in the chromatograms obtained with the test
solution and reference solution (a) are similar ;
impurity A and levodropropizine in the chromatogram
obtained with reference solution (d).
Limit :
solution (c) (2 per cent).
on 0.500 g by drying in vacuo at 60 C over diphosphorus
pentoxide R at a pressure of 0.15-0.25 kPa for 4 h.
on 1.0 g.
ASSAY
Dissolve 0.100 g in 50 ml of anhydrous acetic acid R.
Carry out a potentiometric titration (2.2.20), using 0.1 M
perchloric acid. Read the volume added at the 2nd point of
inflexion.
1 ml of 0.1 M perchloric acid is equivalent to 11.82 mg of
C13H20N2O2.
STORAGE
IMPURITIES
Specified impurities : A, B, C.
2.9.9. Measurement of consistency by penetrometry
A. (2R)-3-(4-phenylpiperazin-1-yl)propane-1,2-diol
(dextrodropropizine),
B. 1-phenylpiperazine,
C. [(2RS)-oxiran-2-yl]methanol (glycidol).

NOTE ON THE GENERAL METHOD
The method is revised to take account of the specifications
of the cone (Figure 2.9.9.-2) mentioned in ASTM
standard D217-02.
XXXX:20909
Figure 2.9.9.-1. Penetrometer

A. Scale showing the depth of penetration, graduated in
tenths of millimetres.
2.9.9. MEASUREMENT OF
B.
Vertical shaft to maintain and guide the penetrating
CONSISTENCY BY PENETROMETRY
object.
This test is intended to measure, under determined and
C. Device to retain and to release the penetrating object
validated conditions, the penetration of an object into the
automatically and for a constant time.
product to be examined in a container with a specified shape D. Device to ensure that the penetrating object is vertical
and size.
and that the base is horizontal.
E.
Penetrating
object (see Figures 2.9.9.-2 and 3).
APPARATUS
F.
Container.
The apparatus consists of a penetrometer made up of a stand
G. Horizontal base.
and a penetrating object. A suitable apparatus is shown in
Figure 2.9.9.-1.
H. Control for the horizontal base.
Figure 2.9.9.-2. Cone (m = 102.5 0.05 g), suitable container (d = 102 mm or 75 mm, h 62 mm)
and shaft (l = 162 mm ; m = 47.5 0.05 g).
Dimensions in millimetres
637
2.9.9. Measurement of consistency by penetrometry
The stand is made up of :

a vertical shaft to maintain and guide the penetrating
object ;
a horizontal base ;
a device to ensure that the penetrating object is vertical ;
a device to check that the base is horizontal ;
a device to retain and release the penetrating object ;
a scale showing the depth of penetration, graduated in
tenths of a millimetre.
The penetrating object, made of a suitable material, has a
smooth surface, and is characterised by its shape, size and
mass (m).
Suitable penetrating objects are shown in Figures 2.9.9.-2
and 2.9.9.-3.
PROCEDURE
B. Store 3 samples at 25 0.5 C for 24 h. Apply a suitable

shear to the samples for 5 min. Carefully and completely
fill 3 containers, without forming air bubbles, and level
if necessary to obtain a flat surface.
C. Melt 3 samples and carefully and completely fill
3 containers, without forming air bubbles. Store the
samples at 25 0.5 C for 24 h, unless otherwise
prescribed.
Determination of penetration. Place the test sample
on the base of the penetrometer. Verify that its surface
is perpendicular to the vertical axis of the penetrating
object. Bring the temperature of the penetrating object to
25 0.5 C and then adjust its position such that its tip just
touches the surface of the sample. Release the penetrating
object and hold it free for 5 s. Clamp the penetrating object
and measure the depth of penetration. Repeat the test with
the 2 remaining containers.
EXPRESSION OF THE RESULTS
Prepare the test samples according to one of the following

procedures.
The penetration is expressed in tenths of a millimetre as

the arithmetic mean of the 3 measurements. If any of
the individual results differ from the mean by more than
A. Carefully and completely fill 3 containers, without
3 per cent, repeat the test and express the results of the
forming air bubbles. Level if necessary to obtain a flat
surface. Store the samples at 25 0.5 C for 24 h, unless 6 measurements as the mean and the relative standard
deviation.
otherwise prescribed.
Figure 2.9.9.-3 Micro-cone (m = 7.0 g), suitable container and shaft (l = 116 mm ; m = 16.8 g)
Dimensions in millimetres
638
Methylphenidate hydrochloride
C. It gives reaction (a) of chlorides (2.3.1).
TESTS
mobile phase.
Reference solution (a). Dissolve 5.0 mg of methylphenidate
for peak identification CRS (containing impurities A and B)
phase.
METHYLPHENIDATE
HYDROCHLORIDE
Column :
Methylphenidati hydrochloridum
size : l = 0.25 m, = 4.6 mm ;
Mobile phase : mix 1 volume of methanol R2 with 2 volumes
of a 1.82 g/l solution of potassium dihydrogen phosphate R
and adjust to pH 4.6 with glacial acetic acid R.
C14H20ClNO2
Mr 269.8 Detection : spectrophotometer at 209 nm.
Injection : 10 l.
DEFINITION
Relative retention with reference to methylphenidate
Methyl-(2RS)-phenyl[(2RS)-piperidin-2-yl]acetate
(retention time = about 11 min) : impurity B = about 0.6 ;
hydrochloride.
impurity A = about 0.8.
peak-to-valley ratio : minimum 2.5, where Hp = height
CHARACTERS
above the baseline of the peak due to impurity A and
Appearance : white or almost white, fine crystalline powder.
Hv = height above the baseline of the lowest point of
Solubility : freely soluble in water and in methanol, soluble
the curve separating this peak from the peak due to
in anhydrous ethanol, slightly soluble in acetone.
methylphenidate.
Limits
:
IDENTIFICATION
impurity A : not more than 5 times the area of the
principal peak in the chromatogram obtained with
Second identification : B, C.
impurity B : not more than 1.5 times the area of the
Comparison : methylphenidate hydrochloride CRS.
B. Thin layer chromatography (2.2.27).
examined in ethanolic hydrochloric acid R and dilute to
Reference solution. Dissolve 5 mg of methylphenidate
total : not more than 10 times the area of the principal
hydrochloride CRS in ethanolic hydrochloric acid R and
dilute to 1 ml with the same solvent.
Plate : TLC silica gel plate R.
Mobile phase : concentrated ammonia R, methanol R,
(0.05 per cent).
methylene chloride R (1:4:95 V/V/V).
Application : 5 l.
1.0 g complies with test A. Prepare the reference solution
Development : over 2/3 of the plate.
Drying : at 100-105 C for 15 min.
Detection : expose the plate to chlorine vapour for
1 min, by placing a beaker containing 0.5 g of potassium on 1.000 g by drying in an oven in vacuo at 60 C for 4 h.
permanganate R and 10 ml of dilute hydrochloric acid R Sulphated ash (2.4.14) : maximum 0.1 per cent, determined
into the chromatographic chamber. Allow the excess of
on 1.0 g.
chlorine to evaporate from the plate for 2 min. Spray the
ASSAY
plate with potassium iodide and starch solution R.
Results : the principal spot in the chromatogram obtained Dissolve 0.250 g in 50 ml of ethanol (96 per cent) R
with the test solution is similar in position, colour and size and add 5.0 ml of 0.01 M hydrochloric acid. Carry out
to the principal spot obtained with the reference solution. a potentiometric titration (2.2.20), using 0.1 M sodium
Methylphenidate hydrochloride is a central nervous system
stimulant and used to treat attention-deficit hyperactivity
disorder. It is a controlled substance and has been known
for more than 25 years. A typical daily dose is 20-30 mg.
The substance is already described in the USP and the Ph.
Helv.. The draft below introduces a related substances test
by LC and an assay by potentiometric titration.
XXXX:2235
(30) Waters Symmetry is suitable.
639
Methyltestosterone
1. impurity B
2. impurity A
3. methylphenidate
Figure 2235.-1. Chromatogram for the test for related substances of methylphenidate hydrochloride
hydroxide and an electrode for non-aqueous acid-base
titrations(31). Read the volume added between the 2 points
of inflexion.

Related substances : TLC replaced by an LC in accordance
1 ml of 0.1 M sodium hydroxide is equivalent to 26.98 mg of
with current policy.
C14H20ClNO2.
Only comments on the test for related substances are
requested.
STORAGE
XXXX:0410
METHYLTESTOSTERONE
IMPURITIES
A. (2RS)-phenyl[(2RS)-piperidin-2-yl]acetic acid,
Methyltestosteronum
C20H30O2
Mr 302.5
DEFINITION
(17)-17-Hydroxy-17-methylandrost-4-en-3-one.
B. methyl-(2RS)-phenyl[(2RS)-piperidin-2-yl]acetate.
CHARACTERS
Appearance : white or slightly yellowish-white, crystalline
powder.
Solubility : practically insoluble in water, freely soluble in
ethanol (96 per cent).
(31) Metrohm solvotrode (combined Metrosensor glass electrode) is suitable.
640
Methyltestosterone
IDENTIFICATION
Second identification : A, C.
Comparison : methyltestosterone CRS.
examined in a mixture of 1 volume of methanol R and
9 volumes of chloroform R and dilute to 10 ml with the
same mixture of solvents.
Reference solution. Dissolve 20 mg of methyltestosterone CRS in 1 ml of a mixture of 1 volume of
methanol R and 9 volumes of chloroform R.
Plate : TLC silica gel F254 plate R.
Mobile phase : anhydrous acetic acid R, light
petroleum R, butyl acetate R (1:30:70 V/V/V).
Application : 5 l.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm
and spray with a saturated solution of potassium
dichromate R in a mixture of 30 volumes of water R and
70 volumes of sulphuric acid R. Examine immediately in
daylight.
with the reference solution.
TESTS
Specific optical rotation (2.2.7) : + 79 to + 85 (dried
substance).
Dissolve 0.250 g in ethanol (96 per cent) R and dilute to
25.0 ml with the same solvent.

(2.2.27), using as the coating substance a suitable silica
gel containing a fluorescent indicator having an optimal
intensity at 254 nm.
in a mixture of 1 volume of methanol R and 9 volumes of
chloroform R and dilute to 10 ml with the same mixture of
solvents.
methyltestosterone CRS in 1 ml of a mixture of
1 volume of methanol R and 9 volumes of chloroform R.
Reference solution (b). Dilute 1 ml of the test solution
to 100 ml with a mixture of 1 volume of methanol R and
9 volumes of chloroform R.
Reference solution (c). Dilute 5 ml of reference solution (b)
to 10 ml with a mixture of 1 volume of methanol R and
9 volumes of chloroform R.
Reference solution (d). Dissolve 10 mg of testosterone CRS
in 0.5 ml of reference solution (a) and dilute to 10 ml with
a mixture of 1 volume of methanol R and 9 volumes of
chloroform R.
Apply separately to the plate 5 l of each solution. Develop
over a path of 15 cm using a mixture of 1 volume of
anhydrous acetic acid R, 30 volumes of light petroleum R
and 70 volumes of butyl acetate R. Allow the plate to dry
in air and examine in ultraviolet light at 254 nm. Any spot
in the chromatogram obtained with the test solution, apart
from the principal spot, is not more intense than the spot
(1.0 per cent) and at most one such spot is more intense
than the spot in the chromatogram obtained with reference
solution (c) (0.5 per cent). The test is not valid unless the
chromatogram obtained with reference solution (d) shows
two clearly separated spots.
examined in methanol R and dilute to 100.0 ml with the
same solvent.
Reference solution (a). Dilute 0.5 ml of the test solution to
100.0 ml with methanol R.
1. methyltestosterone
2. impurity A
Figure 0410.-1. Chromatogram for the test for related substances of methyltestosterone : solution of methyltestosterone
spiked with impurity A
641
Reference solution (b). Dissolve 5.0 mg of methyltestosterone

for system suitability CRS (containing impurity A) in
methanol R and dilute to 10.0 ml with the same solvent.
Column :
size : l = 0.15 m, = 3.9 mm ;
stationary phase : end-capped octadecylsilyl silica gel
for chromatography R (4 m)(32).
A. (17)-17-hydroxy-17-methylandrost-4-en-3-one.
Mobile phase :
mobile phase A : methanol R ;
mobile phase B : water R ;
Time
(min)
0 - 15
Mobile phase A
(per cent V/V)
70
Mobile phase B
(per cent V/V)
30
15 - 45
70 100
30 0
45 - 50
100

Injection : 20 l.
Identification of impurities : use the chromatogram
supplied with methyltestosterone for system suitability CRS
and the chromatogram obtained with reference solution (b)
to identify the peak due to impurity A.
Relative retention with reference to methyltestosterone
(retention time = about 8 min) : impurity A = about 1.5.
System suitability : reference solution (b):
methyltestosterone and impurity A.
Limits :
(0.5 per cent) ;
chromatogram obtained with reference solution (a)
(0.10 per cent) ;
total: not more than twice the area of the principal peak
(1.0 per cent) ;
(0.05 per cent).
on 0.50 g by drying in an oven at 100-105 C for 2 h.

Related substances : TLC has been replaced by LC in
accordance with current policy.
requested.
XXXX:0846
MIANSERIN HYDROCHLORIDE
Mianserini hydrochloridum
C18H21ClN2
Mr 300.8
DEFINITION
(RS)-2-Methyl-1,2,3,4,10,14b- hexahydrodibenzo[c,
f]pyrazino[1,2-a]azepine hydrochloride.
CHARACTERS
Appearance : white or almost white, crystalline powder or
crystals.
Solubility : sparingly soluble in water, soluble in methylene
chloride, slightly soluble in ethanol (96 per cent).
IDENTIFICATION
First identification : B, D.
ASSAY
Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
(2.2.25).
50.0 ml with the same solvent. Dilute 10.0 ml of the solution
Test solution. Dissolve 50.0 mg in water R and dilute
to 100.0 ml with ethanol (96 per cent) R. Dilute 10.0 ml
of this solution to 100.0 ml with ethanol (96 per cent) R.
solution to 50.0 ml with water R.
Measure the absorbance (2.2.25) at the maximum at 241 nm.
Calculate the content of C20H30O2 , taking the specific
absorbance to be 540.
Absorption maximum : at 279 nm.
Specific absorbance at the absorption maximum : 64
STORAGE
to 72.
IMPURITIES
Preparation : discs of potassium chloride R.
Comparison : mianserin hydrochloride CRS.
(32) Novapak C18 or the 10 8 mm cartridge, Symmetry C18, Hypersil 5 C18 and Spherisorb ODS 2 are suitable.
642
If the spectra obtained show differences, dissolve the

substance to be examined and the reference substance
separately in methanol R, evaporate to dryness and
record new spectra using the residues.
The following chromatogram is shown for information but

will not be published in the European Pharmacopoeia.

examined in methylene chloride R and dilute to 5 ml
Reference solution (a). Dissolve 10 mg of mianserin
hydrochloride CRS in methylene chloride R and dilute
Reference solution (b). Dissolve 10 mg of mianserin
hydrochloride CRS and 10 mg of cyproheptadine
hydrochloride CRS in methylene chloride R and dilute
Plate : silica gel GF254 R.
Mobile phase : diethylamine R, ether R, cyclohexane R
(5:20:75 V/V/V).
Application : 2 l.
the chromatogram shows 2 clearly separated principal
spots.
1. impurity B
3. impurity A
5. mianserin
2. impurity C
4. impurity D
6. impurity E
7. impurity F
Figure 0846.-1. Chromatogram for the test for related

substances of mianserin hydrochloride : solution of
mianserin hydrochloride spiked with impurities A, B, C,
D, E and F
Buffer solution pH 3.0. Dissolve 5.0 g of sodium
D. It gives reaction (a) of chlorides (2.3.1).
octanesulphonate R in water R and dilute to 350 ml with
the same solvent. Stir until complete dissolution. Adjust to
TESTS
pH 3.0 with a mixture of 1 volume of phosphoric acid R and
pH (2.2.3) : 4.0 to 5.5.
3 volumes of water R.
Dissolve 0.10 g in carbon dioxide-free water R and dilute to Test solution. Dissolve 20.0 mg of the substance to be
mobile phase.
(2.2.27), using silica gel G R as the coating substance.
examined in a mixture of 1 volume of ammonia R and
4 volumes of methanol R and dilute to 10 ml with the same Reference solution (b). Dissolve 5.0 mg of mianserin
hydrochloride for system suitability CRS (containing
mixture of solvents.
impurities A, B, D and E) in the mobile phase and dilute to
Reference solution (a). Dilute 1 ml of the test solution
to 200 ml with a mixture of 1 volume of ammonia R and
Column :
4 volumes of methanol R.
size : l = 0.15 m, = 3.9 mm ;
Reference solution (b). Dilute 5 ml of reference solution (a)
stationary phase : end-capped octylsilyl silica gel for
to 25 ml with a mixture of 1 volume of ammonia R and
4 volumes of methanol R.
Mobile phase : buffer solution pH 3.0, methanol R
Apply to the plate 5 l of each solution. Develop over a
path of 15 cm using a mixture of 10 volumes of methanol R (370:630 V/V).
and 90 volumes of methylene chloride R. Dry the plate in
a current of cold air. Expose the plate to iodine vapour for
20 min. Any spot in the chromatogram obtained with the test
Injection : 10 l.
solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference Run time : twice the retention time of mianserin.
solution (a) (0.5 per cent) and at most one such spot is more
intense than the spot in the chromatogram obtained with
with mianserin hydrochloride for system suitability CRS
reference solution (b) (0.1 per cent).
and the chromatogram obtained with reference solution (b)
to identify the peaks due to impurities A, B, D and E.
reference solution (a).
(33) Symmetry C8 and Zorbax eclipse XDB C8 are suitable.
643
Relative retention with reference to mianserin (retention

time = about 18 min) : impurity B = about 0.2 ;
impurity A = about 0.5 ; impurity D = about 0.9 ;
impurity E = about 1.1.

pharmaceutical use) : C, F.
peak-to-valley ratio : minimum 5, where Hp = height

above the baseline of the peak due to impurity E and
Hv = height above the baseline of the lowest point of the
curve separating the peak due to mianserin from the peak
due to impurity E.
Limits :
correction factor : for the calculation of content,
the corresponding correction factor : impurity D = 2.1 ;
impurity A = 2.4 ;
impurity B : not more than 3 times the area of the
reference solution (a) (0.3 per cent) ;
A. [2-[(2RS)-4-methyl-2-phenylpiperazin-1-yl]phenyl]methanol,
B. unknown structure,
impurities A, D : for each impurity, not more than twice

the area of the principal peak in the chromatogram
impurity E : not more than the area of the principal peak
(0.1 per cent) ;
C. (2-aminophenyl)methanol,
(0.5 per cent) ;
(0.05 per cent).
on 1.000 g by drying over diphosphorus pentoxide R at
65 C at a pressure not exceeding 700 Pa for 3 h.
on 1.0 g.
D. [2-[(2RS)-2,4-diphenylpiperazin-1-yl]phenyl]methanol,
ASSAY
Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric
acid and 50 ml of ethanol (96 per cent) R. Carry out
a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points
of inflexion.
E. (14bRS)-1,2,3,4,10,14b-hexahydrodibenzo[c,f]pyrazino[1,
2-a]azepine,
l ml of 0.1 M sodium hydroxide is equivalent to 30.08 mg
of C18H21ClN2.
STORAGE
IMPURITIES
Specified impurities : A, B, D, E.
644
F. (14bRS)-2-phenyl-1,2,3,4,10,14b-hexahydrodibenzo[c,
f]pyrazino[1,2-a]azepine.
Norfloxacin
diethylamine R, 20 volumes of toluene R, 40 volumes of

chloroform R and 40 volumes of methanol R. Dry the plate
in a current of air and examine in ultraviolet light at 254 nm
Related substances : TLC replaced by LC in accordance
and then 365 nm. Any spot in the chromatogram obtained
with current policy.
with test solution (a), apart from the principal spot, is not
more intense than the principal spot in the chromatogram
obtained with reference solution (a) (0.2 per cent) and
requested.
XXXX:1248 there are no more than three such spots. The test is not
valid unless, in the chromatogram obtained with reference
solution (b), the ratio of the Rf value of impurity A to the Rf
NORFLOXACIN
value of norfloxacin is at least 1.2.
Norfloxacinum
Solution A : water R previous adjusted to pH 2.0 with
phosphoric acid R.
examined in 1.0 ml of acetonitrile R and dilute to 50.0 ml
with solution A.
to 100.0 ml with solution A. Dilute 1.0 ml of this solution
to 10.0 ml with solution A.
C16H18FN3O3
Mr 319.3 Reference solution (b). Dissolve 20.0 mg of norfloxacin
for peak identification CRS (containing impurities A, E
DEFINITION
and H) in 1.0 ml of acetonitrile R and dilute to 50.0 ml with
1-Ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline- solution A.
3-carboxylic acid.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase : hexadecylamidylsilyl silica gel for
CHARACTERS
Appearance : white or pale-yellow, hygroscopic,
temperature : 60 C.
photosensitive, crystalline powder.
Solubility : very slightly soluble in water, slightly soluble in Mobile phase :
acetone and in ethanol (96 per cent).
mobile phase A : solution A ;
mobile phase B : acetonitrile R ;
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : norfloxacin CRS.
TESTS
Appearance of solution. Dissolve 0.5 g in a previously
filtered 4 g/l solution of sodium hydroxide R in methanol R
and dilute to 50 ml with the same solution. The solution
is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than reference solution B7
(2.2.2, Method II).
(2.2.27), using a TLC silica gel GF254 plate R, previously
washed with methanol R and dried in air.
Test solution (a). Dissolve 40 mg of the substance to be
examined in a mixture of equal volumes of methanol R and
methylene chloride R and dilute to 5 ml with the same
mixture of solvents.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with
a mixture of equal volumes of methanol R and methylene
chloride R.
Reference solution (a). Dilute 1 ml of test solution (b) to
50 ml with a mixture of equal volumes of methanol R and
methylene chloride R.
Reference solution (b). Dissolve 4.0 mg of norfloxacin
impurity A CRS in a mixture of equal volumes of methanol R
and methylene chloride R and dilute to 5 ml with the same
mixture of solvents. Dilute 1 ml of this solution to 2 ml with
test solution (b).
Apply to the plate 5 l of test solution (a) and 5 l of
each reference solution. Develop over a path of 18 cm
using a mixture of 8 volumes of water R, 14 volumes of
Time
(min)
05
Mobile phase A
(per cent V/V)
95
Mobile phase B
(per cent V/V)
5
57
95 93
57
7 10
93 87
7 13
10 15
87 47
13 53
15.0 20.0
47.0 10.0
53.0 90.0

Injection : 20 l.
Run time : 2.5 times the retention time of norfloxacin.
supplied with norfloxacin for peak identification CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A, E and H.
Relative retention with reference to norfloxacin
(retention time = about 11 min) : impurity E = about 1.0 ;
impurity A = about 1.5 ; impurity H = about 1.6.
resolution :
minimum 1.5 between the peaks due to impurity E
and norfloxacin ;
minimum 3.0 between the peaks due to impurities A
and H.
Limits :
(0.1 per cent) ;
(34) Supelcosil LC-ABZ is suitable.
645
Norfloxacin
1. impurity C
4. impurity E
7. impurity G
10. impurity I
2. impurity D
5. norfloxacin
8. impurity A
11. impurity J
3. impurity B
6. impurity F
9. impurity H
Figure 1248.-1. Chromatogram for the test for related substances of norfloxacin : solution of norfloxacin spiked
with impurities A to J
(0.5 per cent) ;
(0.05 per cent).
on 1.000 g by drying in an oven at 100-105 C under high
vacuum for 2 h.
on 1.0 g in a platinum crucible.
ASSAY
C16H18FN3O3.
STORAGE

pharmaceutical use) : A, B, C, D, F, G, H, I, J.
A. R = Cl : 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid,
B. R = NH-CH2-CH2-NH2 : 7-[(2-aminoethyl)amino]-1-ethyl-6fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid,
C. 1-ethyl-4-oxo-6,7-dipiperazin-1-yl-1,4-dihydroquinoline-3carboxylic acid,
IMPURITIES
Specified impurities : E.
impurities and/or by the general monograph Substances for D. 1-ethyl-6-fluoro-7-piperazin-1-ylquinolin-4(1H)-one,
646
E. 7-chloro-1-ethyl-4-oxo-6-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid,
F. 6-chloro-1-ethyl-4-oxo-7-piperazin-1-yl-1,4dihydroquinoline-3-carboxylic acid,

Identification : atomic absorption spectrometry is used for
the identification of magnesium, making the incineration
of the substance unnecessary ; the Ph. Eur. standard test
(2.3.1) for magnesium does not work properly for reasons
of solubility in water ; the blank is positive in methanol.
Related substances : the method is harmonised with the
method described for Omeprazole sodium (1032) and
the same octylsilyl silica gel is described ; the method
also works with octadecylsilyl silica gel and this piece of
information is given in a foot-note for the convenience
of the users. Impurity C is also covered by the LC
method making the use of a separate TLC, as described
in Omeprazole (0942) and Omeprazole sodium (1032)
monographs, unnecessary ; a revision of these monographs
is foreseen for harmonisation.
Assay : no suitable titrimetric method has been found and
the assay is done by LC.
XXXX:2374
OMEPRAZOLE MAGNESIUM
Omeprazole magnesicum
G. 1-ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4dihydroquinoline-3-carboxylic acid,
C34H36N6O6S2Mg
H. 7-[4-(ethoxycarbonyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo1,4-dihydroquinoline-3-carboxylic acid,
Mr 713
DEFINITION
Magnesium bis[5-methoxy-2-[(RS)-[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulphinyl]-1H-benzimidazole]. It
contains a variable quantity of water.
substance).
CHARACTERS
Solubility : very slightly soluble in water, sparingly soluble
in methanol, practically insoluble in heptane.
I. 7-chloro-6-[4-(ethoxycarbonyl)piperazin-1-yl]-1-ethyl-4-oxo1,4-dihydroquinoline-3-carboxylic acid,
J. 6,7-bis[4-(ethoxycarbonyl)piperazin-1-yl]-1-ethyl-4-oxo-1,4dihydroquinoline-3-carboxylic acid.
IDENTIFICATION
A. Optical rotation (2.2.7) : 0.10 to + 0.10.
Dissolve 0.250 g in methanol R and dilute to 25.0 ml
Comparison : omeprazole magnesium CRS.
C. Atomic absorption spectrometry (2.2.23) as described in
the test for magnesium.
The test solution shows the absorption maximum at
285.2 nm.
TESTS
Absorbance (2.2.25) : maximum 0.1 at 440 nm.
Dissolve 0.5 g in methanol R and dilute to 25.0 ml with the
same solvent. Filter the solution through a membrane filter
(0.45 m)(35).
(35) Millipore Millex-HV 0.45 m is suitable.
647

Use the normalisation procedure. Use freshly prepared
solutions.
mobile phase.
Reference solution (a). Dissolve 1 mg of omeprazole CRS
and 1 mg of omeprazole impurity D CRS in the mobile
phase and dilute to 10.0 ml with the mobile phase.
Column :
size : l = 0.125 m, = 4.6 mm ;
stationary phase: octylsilyl silica gel for
acid R.
Injection: 40 l.
Run time : 5 times the retention time of omeprazole.
Relative retention with reference to omeprazole
(retention time = about 9 min) : impurity E = about 0.6 ;
impurity D = about 0.8.
impurity D and omeprazole. If necessary, adjust the pH
of the mobile phase or its proportion of acetonitrile ; an
increase in the pH will improve the resolution.
Limits :
impurity D : maximum 0.1 per cent ;
impurity E : maximum 0.1 per cent ;
unspecified impurities : for each impurity, maximum
0.10 per cent ;
total: maximum 0.5 per cent ;

(0.05 per cent).
Magnesium : 3.30 per cent to 3.55 per cent (anhydrous
substance).
Atomic absorption spectrometry (2.2.23, Method I).
Test solution. Dissolve 0.250 g in 20 ml of a 103 g/l solution
of hydrochloric acid R, adding the acid slowly, and dilute
200.0 ml with water R. To 10.0 ml of the solution obtained
add 4 ml of lanthanum chloride solution R and dilute to
Reference solutions. Prepare the reference solutions using
magnesium standard solution (1000 ppm Mg) R, diluted
as necessary with water R.
on 0.200 g.
ASSAY
Liquid chromagraphy (2.2.29).
examined in about 10 ml of methanol R, add 10 ml of
phosphate buffer solution pH 11 and dilute to 200.0 ml with
water R.
Reference solution. Dissolve 10.00 mg of omeprazole CRS
in about 10 ml of methanol R, add 10 ml of phosphate buffer
solution pH 11 and dilute to 200.0 ml with water R.
Column :
size : l = 0.125 m, = 4 mm ;
Mobile phase : mix 35 volumes of acetonitrile R with
acid R.
1. impurity A
3. impurity D
5. omeprazole
2. impurity E
4. impurity B
6. impurity C
Figure 2374.-1. Chromatogram for the test for related substances of omeprazole magnesium : omeprazole magnesium
spiked with about 0.1 per cent of impurities A, B, C, D and E
(36) Symmetry C8, LiChrosorb C8, Zorbax SB-C8 and Novapac C18 are suitable.
648

Injection: 20 l.
A. 5-methoxy-1H-benzimidazole-2-thiol,
Run time : until complete elution of the peak due to

omeprazole.
retention time : omeprazole = minimum 4 min.
Calculate the percentage content of C34H36N6O6S2Mg from
the declared content of omeprazole CRS.
B. R = H, X = SO : 2-[(RS)-[(3,5-dimethylpyridin-2yl)methyl]sulphinyl]-5-methoxy-1H-benzimidazole,
1 g of omeprazole is equivalent to 1.032 g of omeprazole

magnesium.
C. R = OCH3, X = S : 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulphanyl]-1H-benzimidazole
(ufiprazole),
STORAGE
D. R = OCH3, X = SO2 : 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulphonyl]-1H-benzimidazole

(omeprazole sulphone),
IMPURITIES
Specified impurities : D, E.
pharmaceutical use) : A, B, C.
E. 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol-2yl)sulphinyl]methyl]-3,5-dimethylpyridine 1-oxide.
Reagents
Magnesium standard solution (1000 ppm Mg). XXXXXXX.
Dissolve 5.275 g of magnesium nitrate R(37) in 16 ml of
dilute nitric acid R and dilute to 500.0 ml with water R.
Standardisation : carry out the determination of magnesium
by complexometry (2.5.11).
1. omeprazole
Figure 2374.-2. Chromatogram for the assay of omeprazole magnesium
(37) Merck 105855 is suitable.
649
Reference solution (a). Dissolve 10 mg of orciprenaline

sulphate CRS in ethanol (96 per cent) R and dilute to
Definition, Identification A : the monograph Substances
Reference solution (b). Dissolve 10 mg of orciprenaline
for pharmaceutical use (2034) states that the content of
sulphate CRS and 10 mg of salbutamol CRS in ethanol
residual solvent is taken into account for calculation of
(96 per cent) R and dilute to 10 ml with the same solvent.
the assay content and of the specific absorbance. As a
Plate : TLC silica gel G plate R.
consequence, this has been deleted from the monograph.
Mobile
phase : ammonia R, water R, aldehyde-free
Related substances : TLC replaced by LC in accordance
methanol
R (1.5:10:90 V/V/V).
Application : 2 l.
XXXX:1033
Drying : in air.
ORCIPRENALINE SULPHATE
Detection : spray with a 10 g/l solution of potassium
permanganate R.
Orciprenalini sulfas
the chromatogram shows 2 clearly separated principal
spots.
with reference solution (a).
D. Dissolve about 20 mg in 2 ml of ethanol (96 per
cent) R. Add 2 ml of a 1 g/l solution of
C22H36N2O10S
Mr 520.6
dichloroquinonechlorimide R in ethanol (96 per
cent) R and 1 ml of sodium carbonate solution R. A violet
DEFINITION
colour is produced, turning to brown.
Di[(RS)-1-(3,5-dihydroxyphenyl)-2-[(1-methylethyl)amino]ethanol] sulphate.
E. It gives reaction (a) of sulphates (2.3.1).
TESTS
solvent-free substance).
CHARACTERS
Appearance : white or almost white, crystalline powder,
Appearance of solution. Solution S is clear (2.2.1) and
slightly hygroscopic.
Solubility : freely soluble in water and in ethanol (96 per
cent), practically insoluble in methylene chloride.
IDENTIFICATION
(2.2.27), using silica gel G R as the coating substance.
First identification : B, E.
Second identification : A, C, D, E.
examined in a mixture of 1 volume of water R and 5 volumes
of methanol R and dilute to 10 ml with the same mixture
of solvents.
(2.2.25).
Reference solution (a). Dilute 1 ml of the test solution to
Test solution. Dissolve 50.0 mg in a 0.04 per cent V/V
solution of hydrochloric acid R and dilute to 50.0 ml with 100 ml with a mixture of 1 volume of water R and 5 volumes
of methanol R.
the same acid solution. Dilute 5.0 ml of this solution to
50.0 ml with a 0.04 per cent V/V solution of hydrochloric Reference solution (b). Dilute 5 ml of reference solution (a)
acid R.
to 10 ml with a mixture of 1 volume of water R and 5 volumes
of methanol R.
Reference solution (c). Dilute 5 ml of reference solution (a)
Specific absorbance at the absorption maxima : 68.5 to to 20 ml with a mixture of 1 volume of water R and 5 volumes
of methanol R.
76.0 (anhydrous and solvent-free substance).
Apply separately to the plate 10 l of each solution.
Develop over a path of 15 cm using a mixture of 4 volumes
of concentrated ammonia R, 16 volumes of water R,
Comparison : orciprenaline sulphate CRS.
30 volumes of 2-propanol R and 50 volumes of ethyl
If the spectra obtained show differences, dissolve
acetate R. Allow the plate to dry in air. Expose the plate
separately, with heating, 50 mg of the substance to be
to iodine vapour. Any spot in the chromatogram obtained
examined and 50 mg of the reference substance, in the
with the test solution, apart from the principal spot, is not
minimum volume of water R. Add 10 ml of acetone R
more intense than the principal spot in the chromatogram
and centrifuge. Dry the precipitates at 40 C under
obtained with reference solution (a) (1.0 per cent) and at
reduced pressure for 3 h and record new spectra using
most 1 such spot is more intense than the principal spot
the residues.
(0.5 per cent). The test is not valid unless the spot in the
chromatogram obtained with reference solution (c) is clearly
examined in ethanol (96 per cent) R and dilute to 10 ml visible.
650

mobile phase.
Reference solution (b). Dissolve 20.0 mg of orciprenaline
for system suitability CRS (containing impurities A and B) in
the mobile phase and dilute to 20.0 ml with the mobile phase.
Column :
size : l = 0.125 m, = 4.0 mm ;
stationary phase : spherical end-capped octadecylsilyl
silica gel for chromatography R (5 m)(38) ;
temperature : 45 C.
Mobile phase. Dissolve 18.16 g of potassium dihydrogen
phosphate R and 9.2 g of sodium octanesulphonate R in
2000 ml of water R. Adjust to pH 4.0 with dilute phosphoric
acid R and add 280 ml of acetonitrile R.
Injection : 10 l.
Run time : twice the retention time of orciprenaline.
supplied with orciprenaline for system suitability CRS and
the chromatogram obtained with reference solution (b) to
identify the peaks due to impurities A and B.
Relative retention with reference to orciprenaline
(retention time = about 7 min) : impurity A = about 0.9 ;
impurity B = about 1.3.
impurity A and orciprenaline.
Limits :
correction factor : for the calculation of content, multiply
the peak area of impurity B by 0.3 ;
impurities A, B : for each impurity, not more than the

total : not more than twice the area of the principal peak
(0.2 per cent) ;
(0.05 per cent).
Methanol and 2-propanol. Gas chromatography (2.2.28).
Internal standard solution. Dilute 2 ml of ethanol R1 to
100 ml with water R. Dilute 1 ml of this solution to 10 ml
with water R.
solvent.
Test solution (b). Dissolve 1.0 g of the substance to be
examined in water R, add 1.0 ml of the internal standard
solution and dilute to 10.0 ml with water R.
Reference solution. Dilute a mixture of 1.0 ml of methanol R
and 3.0 ml of 2-propanol R to 100.0 ml with water R. Dilute
1.0 ml of this solution to 10.0 ml with water R. To 1.0 ml of
the solution obtained add 1.0 ml of the internal standard
solution and dilute to 10.0 ml with water R.
Column :
material : glass ;
size : l = 2 m, = 2 mm ;
stationary phase : ethylvinylbenzene-divinylbenzene
copolymer R (150-180 m).
Carrier gas : nitrogen for chromatography R.
Temperature :
column : 140 C ;
injection port and detector: 180 C.
Injection : 1 l.
1. impurity C
2. impurity A
3. orciprenaline
4. impurity B
Figure 1033.-1. Chromatogram for the test for related substances of orciprenaline sulphate : solution of orciprenaline
sulphate spiked with impurities A, B and C
(38) Inertsil ODS 2 is suitable.
651
System suitability : in the chromatogram obtained with

test solution (a), verify that there is no peak with the same
retention time as the internal standard.
Calculate the content of methanol and 2-propanol taking
their density at 20 C to be 0.792 g/ml and 0.785 g/ml,
respectively.
Limits :
methanol : maximum 0.1 per cent m/m ;
B. 1-(3,5-dihydroxyphenyl)-2-[(1-methylethyl)amino]ethanone,
2-propanol : maximum 0.3 per cent m/m.

Phenone : maximum 0.1 per cent.
Dissolve 0.50 g in a 0.04 per cent V/V solution of
hydrochloric acid R and dilute to 25.0 ml with the same acid
solution. The absorbance (2.2.25) of the solution measured
at 328 nm is not greater than 0.16.
Iron (2.4.9) : maximum 20 ppm.
The residue obtained in the test for sulphated ash complies
with the test. Prepare the reference solution using iron
standard solution (2 ppm Fe) R.
C. 3-hydroxy-5-[1-hydroxy-2-(isopropylamino)ethyl]cyclohex2-en-1-one.
Heavy metals : maximum 20 ppm.

1.000 g.
on 1.0 g.
ASSAY
Dissolve 0.400 g in 5 ml of anhydrous formic acid R and
add 30 ml of anhydrous acetic acid R. Titrate with 0.1 M
perchloric acid using 0.1 ml of crystal violet solution R as
indicator.
1 ml of 0.1 M perchloric acid is equivalent to 52.06 mg
of C22H36N2O10S.

Residual solvents : addition of a test for ethyl acetate, which
is used instead of butyl acetate in approved products ;
a method allowing quantification of the 2 solvents is
proposed.
XXXX:2260
OXACILLIN SODIUM MONOHYDRATE

Oxacillinum natricum monohydricum
STORAGE
IMPURITIES
pharmaceutical use) : C.
C19H18N3NaO5S,H2O
Mr 441.4
DEFINITION
Sodium (2S,5R,6R)-3,3-dimethyl-6-[[(5-methyl-3phenylisoxazol-4-yl)carbonyl]amino]-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate monohydrate.
Semi-synthetic product derived from a fermentation product.
substance).
CHARACTERS
Solubility : freely soluble in water, soluble in methanol,
insoluble or practically insoluble in methylene chloride.
A. 2-(1-methylethyl)-1,2,3,4-tetrahydroisoquinoline-4,6,8triol,
652
IDENTIFICATION
Comparison : oxacillin sodium monohydrate CRS.
B. It gives reaction (a) of sodium (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and its
absorbance (2.2.25) at 430 nm is maximum 0.10.
same solvent.
pH (2.2.3) : 4.5 to 7.5.
Dissolve 0.30 g in carbon dioxide-free water R and dilute to
Specific optical rotation (2.2.7) : + 196 to + 212 (anhydrous
substance).
same solvent.
mobile phase.
Test solution (b). Dilute 5.0 ml of test solution (a) to 50.0 ml
Reference solution (a). Dissolve 50.0 mg of oxacillin sodium
monohydrate CRS in the mobile phase and dilute to 50.0 ml
with the mobile phase. Dilute 5.0 ml of this solution to
Reference solution (c). Dissolve 5 mg of cloxacillin
sodium CRS and 5 mg of oxacillin sodium monohydrate CRS
phase.
Reference solution (d). Dissolve 25 mg of the substance to

be examined in 1 ml of 0.05 M sodium hydroxide. Wait for
3 min then dilute to 100 ml with the mobile phase. Inject
immediately (in situ degradation to obtain impurities B
and D).
Reference solution (e). Dissolve 5 mg of oxacillin for peak
identification CRS (containing impurities E, F, G, I and J)
in 5 ml of the mobile phase.
Column :
size : l = 0.25 m, = 4.0 mm ;
phosphate R previously adjusted to pH 5.0 with dilute
sodium hydroxide solution R.
Injection : 20 l of test solution (a) and reference
solutions (b), (c), (d) and (e).
Run time : 7 times the retention time of oxacillin.
Identification of impurities: in the chromatogram obtained
with reference solution (d), the 2 principal peaks eluting
before the main peak correspond to impurities B and D
respectively. Use the chromatogram obtained with reference
solution (e) and the chromatogram supplied with oxacillin
for peak identification CRS to identify the peaks due to
impurities E, F, G, I and J.
1. impurity B (isomer 1)
4. oxacillin
7. impurity G
2. impurity B (isomer 2)
5. impurity E
8. impurity I
3. impurity D
6. impurity F
9. impurity J
Figure 2260.-1. Chromatogram for the test for related substances of oxacillin sodium monohydrate : test solution
(39) Hypersil ODS is suitable.
653
1. impurity B
2. impurity D
3. oxacillin
Figure 2260.-2. Chromatogram for the test for related substances of oxacillin sodium monohydrate : reference
solution (d)
Relative retention with reference to oxacillin (retention
time = about 5 min) : impurity A = about 0.3 ; impurity B
(isomer 1) = about 0.4 ; impurity B (isomer 2) = about 0.5 ;
impurity C = about 0.65 ; impurity D (2 isomers) = about 0.9 ;
impurity E = about 1.5 ; impurity F = about 1.9 ;
impurity G = about 2.1 ; impurity H = about 3.5 ;
impurity I = about 3.8 ; impurity J = about 5.8.
oxacillin and impurity E in the chromatogram obtained
with reference solution (c),
the chromatogram obtained with reference solution (e) is
similar to the chromatogram supplied with oxacillin for
peak identification CRS.
Limits :
impurity B : for the sum of the areas of the 2 isomer
peaks, not more than 1.5 times the area of the principal
(1.0 per cent) ;
impurities D (sum of the 2 isomers), F, G, I, J : for each
impurity, not more than 0.5 times the area of the principal
any other impurity : for each impurity, not more
(0.5 per cent) ;
total : not more than 3 times the area of the principal peak
(3.0 per cent) ;
(0.05 per cent).
Butyl acetate (2.4.24) : maximum 1.0 per cent.
Ethyl acetate and butyl acetate. Head-space gas
solvent.
Reference solution. Dissolve 83 mg of ethyl acetate R and
83 mg of butyl acetate R in water R and dilute to 250.0 ml
with the same solvent. Use 6.0 ml of this solution.
Close the vials immediately with a rubber membrane
stopper coated with polytetrafluoroethylene and secure
with an aluminium crimped cap. Mix to obtain a
homogeneous solution.
Column :
material: fused silica ;
size : l = 50 m, = 0.32 mm ;
stationary phase : poly(dimethyl)siloxane R (film
thickness 5 m)(40).
Static head-space conditions that may be used :
equilibration temperature : 80 C ;
equilibration time : 60 min ;
(40) Varian is suitable.
654
transfer line temperature : 140 C ;

pressurisation time : 30 s.
Temperature :
Column
D. R = H : (2RS,4S)-5,5-dimethyl-2-[[[(5-methyl-3phenylisoxazol-4-yl)carbonyl]amino]methyl]thiazolidine-4carboxylic acid (penilloic acids of oxacillin),
Time
(min)
0 - 16
Temperature
(C)
70
6 - 16
70 220
16 - 18
220
Injection port
140
Detector
250

C. 5-methyl-3-phenylisoxazole-4-carboxylic acid,
Retention time : ethyl acetate = about 10 min ; butyl
acetate = about 15.5 min.
E. cloxacillin,
Limits :
ethyl acetate : maximum 1.0 per cent,
butyl acetate : maximum 1.0 per cent.
N,N-Dimethylaniline (2.4.26, Method B) : maximum 20 ppm.
2-Ethylhexanoic acid (2.4.28) : maximum 0.8 per cent.
0.300 g.
Bacterial endotoxins (2.6.14) : less than 0.20 IU/mg, if
intended for use in the manufacture of parenteral dosage
forms without a further appropriate procedure for the
F. R1 = SH, R2 = H : (2S,5R,6R)-3,3-dimethyl-6-[[(5removal of bacterial endotoxins.
methyl-3-phenylisoxazol-4-yl)carbonyl]amino]-7-oxo-4thia-1-azabicyclo[3.2.0]heptane-2-thiocarboxylic acid
ASSAY
(thiooxacillin),
related substances with the following modification.
Injection: test solution (b) and reference solution (a).
G. R1 = OH, R2 = Cl : (2S,5R,6R)-6-[[[3-(chlorophenyl)Calculate the percentage content of C19H18N3NaO5S from the
5-methylisoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7declared content of oxacillin sodium monohydrate CRS.
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(cloxacillin isomer),
IMPURITIES
Specified impurities : B, D, E, F, G, I, J.
pharmaceutical use) : A, C, H.
H. (3S,7R,7aR)-2,2-dimethyl-5-(5-methyl-3-phenylisoxazol4-yl)-2,3,7,7a-tetrahydroimidazo[5,1-b]thiazole-3,7dicarboxylic acid (penillic acid of oxacillin),
A. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenicillanic acid),
B. R = CO2H : (4S)-2-[carboxy[[(5-methyl-3-phenylisoxazol4-yl)carbonyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic acid (penicilloic acids of oxacillin),

I. (2S,5R,6R)-6-[[(2S,5R,6R)-3,3-dimethyl-6-[[(5-methyl3-phenylisoxazol-4-yl)carbonyl]amino]-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(6APA oxacillin amide),
655
2.8.13. Pesticide residues
successive updates. Limits for pesticides that are not listed

in Table 2.8.13.-1 nor in EU texts are calculated using the
ADI
= acceptable daily intake, as published by

FAO-WHO, in milligrams per kilogram of body
mass,
= body mass in kilograms (60 kg),
J. (2S,5R,6R)-6-[[(2R)-[(2R,4S)-4-carboxy-5,5M
dimethylthiazolidin-2-yl][[(5-methyl-3-phenylisoxazol-4yl)carbonyl]amino]acetyl]amino]-3,3-dimethyl-7-oxo-4-thia- MDD
daily dose of the herbal drug, in kilograms.
HD =
1-azabicyclo[3.2.0]heptane-2-carboxylic acid (ozolamide
of 6-APA dimer).
If the herbal drug is intended for the preparation of extracts,
tinctures, essential oils or other pharmaceutical forms whose
preparation method modifies the content of pesticides
in the finished product, the limits for pesticides in these
preparations are calculated using the following expression :
Reference: PA/PH/Exp. PST/T (06) 2 ANP
This general chapter has been revised for the following
reasons :
to take account of the regulation EU 396/2005 replacing
directives EC 76/895 and EC 90/642 ;
to extend the list of pesticides frequently observed in
herbal drugs ;
to limit the pesticides in view of toxicology data and
according to a 90 per cent percentile approach ;
to modify the formula for the calculation of the
limit for the pesticide content in extracts and other
pharmaceutical forms prepared from herbal drugs : the
extraction factor had been taken out of the formula ; it
had not been clear if the extraction factor was related to
the active principle or to the pesticide in question ; the
new formula results in more transparent limits ;
to give more details on the validation procedure for a
chosen analytical method ;
to delete the method for the determination of pesticides
in herbal drugs, given for information. This method was
misinterpreted as reference method.
XXXX:20813
2.8.13. PESTICIDE RESIDUES

Definition. For the purposes of the Pharmacopoeia,
a pesticide is any substance or mixture of substances
intended for preventing, destroying or controlling any
pest, unwanted species of plants or animals causing harm
during or otherwise interfering with the production,
processing, storage, transport or marketing of vegetable
herbal drugs. The item includes substances intended for
use as growth-regulators, defoliants or desiccants and any
substance applied to crops either before or after harvest to
protect the commodity from deterioration during storage
and transport.
Limits. Unless otherwise indicated in the monograph, the
herbal drug to be examined at least complies with the
limits indicated in Table 2.8.13.-1. The limits applying to
pesticides that are not listed in the table and whose presence
is suspected for any reason comply with the limits set by
European Community Union directives 76/895 and 90/642,
Regulation (EC) NO. 396/2005, including annexes and
656
(Deleted)
(Insert)
extraction factor of the method of preparation,

determined experimentally.
MDDPF = daily dose of the pharmaceutical form, in

kilograms.
Higher limits can also be authorised, in exceptional cases,
especially when a plant requires a particular cultivation
method or has a metabolism or a structure that gives rise to
a higher than normal content of pesticides.
The competent authority may grant total or partial
exemption from the test when the complete history (nature
and quantity of the pesticides used, date of each treatment
during cultivation and after the harvest) of the treatment of
the batch is known and can be checked precisely according
to good agricultural practice (GAP).
Sampling
Method. For containers up to 1 kg, take one sample from
the total content, thoroughly mixed, sufficient for the tests.
For containers between 1 kg and 5 kg, take 3 samples,
equal in volume, from the upper, middle and lower parts of
the container, each being sufficient to carry out the tests.
Thoroughly mix the samples and take from the mixture an
amount sufficient to carry out the tests. For containers
of more than 5 kg, take 3 samples, each of at least 250 g
from the upper, middle and lower parts of the container.
Thoroughly mix the samples and take from the mixture an
amount sufficient to carry out the tests.
Size of sampling. If the number (n) of containers is 3 or
fewer, take samples from each container as indicated above
under Method. If the number of containers is more than 3,
take at least
samples from containers as indicated
under Method, rounding up to the nearest unit if necessary.
Prepare the bulk sample by combining and thoroughly
mixing the samples taken from each of the randomly selected
containers.
The samples are to be analysed immediately to Fresh material

is stored under conditions that avoid possible degradation of
the residues. If this is not possible, the samples are stored in
airtight containers suitable for food contact, at a temperature
below 0 C, protected from light.
Reagents. All reagents and solvents are free from any
contaminants, especially pesticides, that might interfere
with the analysis. It is often necessary to use special quality
solvents or, if this is not possible, solvents that have recently
been re-distilled in an apparatus made entirely of glass. In
any case, suitable blank tests must be carried out.
Apparatus. Clean the apparatus and especially glassware to
ensure that they are free from pesticides, for example, soak
for at least 16 h in a solution of phosphate-free detergent,
rinse with large quantities of distilled water R and wash with
acetone and hexane or heptane.
Qualitative and quantitative analysis of pesticide
residues. The analytical procedures used are validated
according to the regulations in force (e.g. according to
Document N SANCO/10232/2006(41)). In particular, they
satisfy the following criteria :
the chosen method, especially the purification steps, is
suitable for the combination pesticide residue/substance
to be analysed, and not susceptible to interference from
co-extractives ; the limits of detection and quantification
are measured for each pesticide-matrix combination to
be analysed, ;
natural occurrence of analytes is considered in
the interpretation of results (e.g. disulfide from
Cruciferaceae) ;
the concentration of test and reference solutions and the
setting of the apparatus are such that the responses used
for quantification of the pesticide residues are within
the dynamic range of the detector. Test solutions of
samples containing pesticide residues at a level outside
the dynamic range, may be diluted within the calibration
range, provided that the concentration of the matrix in
the solution is adjusted in the case where the calibration
solutions must be matrix-matched ;
between 70 per cent to 110 per cent of each pesticide is
recovered ;
the repeatability of the method is not less than the values
indicated in Table 2.8.13.-2 ;
the reproducibility of the method is not less than the
values indicated in Table 2.8.13.-2.
the concentration of test and reference solutions and the
setting of the apparatus are such that a linear response is
obtained from the analytical detector.
Table 2.8.13.-1
Substance
Alachlor
Limit
(mg/kg)
0.02
Substance
DDT (sum of p,p-DDT, o,p-DDT, p,p-DDE and p,p-TDE)
Limit
(mg/kg)
1.0
Deltamethrin
0.5
Diazinon
0.5
Dichlorvos
1.0
Dithiocarbamates (as CS2)
2.0
Endosulfan (sum of isomers and Endosulfan sulphate)
3.0
Endrin
0.05
Ethion
2.0
Fenitrothion
0.5
Fenvalerate
1.5
Fonofos
0.05
Heptachlor (sum of Heptachlor and Heptachlorepoxide)
0.05
Hexachlorobenzene
0.1
Hexachlorocyclohexane isomers (other than )
0.3
Lindane (-Hexachlorocyclohexane)
0.6
Malathion
1.0
Methidathion
0.2
Parathion
0.5
Parathion-methyl
0.2
Permethrin
1.0
Phosalone
0.1
Piperonyl butoxide
3.0
Pirimiphos-methyl
4.0
Pyrethrins (sum of)
3.0
Quintozene (sum of quintozene, pentachloroaniline and

methyl pentachlorophenyl sulphide)
1.0
Table 2.8.13.-1
Substance
Limit
(mg/kg)
Acephate
0.1
Alachlor
0.05
Aldrin and dieldrin (sum of)
0.05
Azinphos-ethyl
0.1
Azinphos-methyl
Bromine
50
Bromophos-ethyl
0.05
Bromophos-methyl
0.05
Brompropylate
Aldrin and Dieldrin (sum of)
0.05
Chlordane (sum of cis-, trans - and oxychlordane)
0.05
Azinphos-methyl
1.0
Chlorfenvinphos
0.5
Bromopropylate
3.0
Chlorpyriphos (ethyl)
0.2
Chlordane (sum of cis-, trans - and Oxythlordane)
0.05
Chlorpyriphos-methyl
0.1
Chlorfenvinphos
0.5
Chlorthal-dimethyl
0.01
Chlorpyrifos
0.2
Cyfluthrin, sum
0.1
Chlorpyrifos-methyl
0.1
-Cyhalothrin
Cypermethrin (and isomers)
1.0
Cypermethrin (and isomers)
(41) http : //ec.europa.eu/food/plant/resources/qualcontrol_en.pdf.
657
Substance
DDT (sum of o,p-DDE, p,p-DDE, o,p-DDT, p,p-DDT,
o,p-TDE and p,p-TDE)
Limit
(mg/kg)
1
Deltamethrin
0.5
Diazinon
0.5
Dichlofluanid
0.1
Dichlorvos
Dicofol
0.5
Dimethoate and omethoate (sum of)
0.1
Dithiocarbamates (as CS2)
Endosulfan (sum of isomers and endosulfan sulphate)
Endrin
0.05
Ethion
Etrimphos
0.05
Fenchlorophos (sum of fenchlorophos and

fenchlorophos-oxon)
0.1
Fenitrothion
0.5
Fenpropathrin
0.03
Fensulfothion (sum of fensulfothion, fensulfothion-oxon,

fensulfothion-oxonsulfon and fensulfothion-sulfon)
0.05
Fenthion (sum of fenthion, fenthion-oxon,

fenthion-oxonsulfon, fenthion-oxon-sulfoxid,
fenthion-sulfon and fenthion-sulfoxid)
0.05
Fenvalerate
1.5
Flucytrinate
-Fluvalinate
Fonophos
0.05
Heptachlor (sum of heptachlor, cis-heptachlorepoxide

and trans-heptachlorepoxide)
0.05
Hexachlorbenzene
0.1
Hexachlorocyclohexane (sum of isomers -, -, - and

-hexachlorocyclohexane)
0.3
Lindan (-hexachlorocyclohexane)
0.6
Malathion and malaoxon (sum of)
Prothiophos
0.05
3
Pyrethrum (sum of cinerin I, cinerin II, jasmolin I,

jasmolin II, pyrethrin I and pyrethrin II)
0.05
Quinalphos
Quintozene (sum of quintozene, pentachloraniline and

methyl penthachlorphenyl sulfide)
S-421
0.02
Tecnazene
0.05
Tetradifon
0.3
Vinclozolin
0.4
Table 2.8.13.-2
0.005
0.01
0.100
0.025
0.05
1.000
0.125
0.25
0.001 - 0.01
30
60
> 0.01 - 0.1
20
40
0.05
> 0.1 - 1
15
30
0.05
>1
10
20
Methamidophos
0.05
Methidathion
0.2
Methoxychlor
0.05
Mirex
0.01
Monocrotophos
0.1
Parathion-ethyl and Paraoxon-ethyl (sum of)
0.5
Parathion-methyl and Paraoxon-methyl (sum of)
0.2
Pendimethalin
0.1
Pentachloranisol
0.01
1
Phosalone
0.1
Phosmet
0.05
658
0.1
0.010
0.05
Pirimiphos-methyl (sum of pirimiphos-methyl and

N-desethyl-pirimiphos-methyl)
Profenophos
Reproducibility
(difference,
mg/kg)
(per cent)
Methacriphos
Pirimiphos-ethyl
0.1
Repeatability
(difference,
mg/kg)
(per cent)
0.05
Piperonyl butoxide
Procymidone
Concentration
range of
the pesticide
(mg/kg)
Mecarbam
Permethrin (and isomers) (sum of)
Limit
(mg/kg)
Substance
3
0.05
4
The following section is published for information.
Test for pesticides

ORGANOCHLORINE, ORGANOPHOSPHORUS AND
PYRETHROID INSECTICIDES
The following methods may be used, in connection with
the general method above. Depending on the substance
being examined, it may be necessary to modify, sometimes
extensively, the procedure described hereafter. In any case,
it may be necessary to use, in addition, another column
with a different polarity or another detection method (mass
spectrometry...) or a different method (immunochemical
methods...) to confirm the results obtained.
This procedure is valid only for the analysis of samples of
vegetable drugs containing less than 15 per cent of water.
Samples with a higher content of water may be dried,
provided it has been shown that the drying procedure does
not affect significantly the pesticide content.
1. EXTRACTION
To 10 g of the substance being examined, coarsely
powdered, add 100 ml of acetone R and allow to stand
for 20 min. Add 1 ml of a solution containing 1.8 g/ml
of carbophenothion R in toluene R. Homogenise using a
high-speed blender for 3 min. Filter and wash the filter cake
with two quantities, each of 25 ml, of acetone R. Combine
the filtrate and the washings and heat using a rotary
evaporator at a temperature not exceeding 40 C until the
solvent has almost completely evaporated. To the residue
add a few millilitres of toluene R and heat again until the
acetone is completely removed. Dissolve the residue in 8 ml
of toluene R. Filter through a membrane filter (45 m), rinse
the flask and the filter with toluene R and dilute to 10.0 ml
with the same solvent (solution A).
2. PURIFICATION
2.1. Organochlorine, organophosphorus and
pyrethroid insecticides. Examine by size-exclusion
The chromatographic procedure may be carried out using :
a stainless steel column 0.30 m long and 7.8 mm in
internal diameter packed with styrene-divinylbenzene
copolymer R (5 m),
as mobile phase toluene R at a flow rate of 1 ml/min.
Performance of the column. Inject 100 l of a solution
containing 0.5 g/l of methyl red R and 0.5 g/l of oracet blue
2R R in toluene R and proceed with the chromatography.
The column is not suitable unless the colour of the eluate
changes from orange to blue at an elution volume of about
10.3 ml. If necessary calibrate the column, using a solution
containing, in toluene R, at a suitable concentration, the
insecticide to be analysed with the lowest molecular mass
(for example, dichlorvos) and that with the highest molecular
mass (for example, deltamethrin). Determine which fraction
of the eluate contains both insecticides.
Purification of the test solution. Inject a suitable volume
of solution A (100 l to 500 l) and proceed with the
chromatography. Collect the fraction as determined above
(solution B). Organophosphorus insecticides are usually
eluted between 8.8 ml and 10.9 ml. Organochlorine and
pyrethroid insecticides are usually eluted between 8.5 ml
and 10.3 ml.
2.2. Organochlorine and pyrethroid insecticides. In
a chromatography column, 0.10 m long and 5 mm in
internal diameter, introduce a piece of defatted cotton and
0.5 g of silica gel treated as follows : heat silica gel for
chromatography R in an oven at 150 C for at least 4 h.
Allow to cool and add dropwise a quantity of water R
corresponding to 1.5 per cent of the mass of silica gel used ;
shake vigorously until agglomerates have disappeared
and continue shaking for 2 h using a mechanical shaker.
Condition the column using 1.5 ml of hexane R. Prepacked
columns containing about 0.50 g of a suitable silica gel may
also be used provided they are previously validated.
Concentrate solution B in a current of helium for
chromatography R or oxygen-free nitrogen R almost to
dryness and dilute to a suitable volume with toluene R
(200 l to 1 ml according to the volume injected in the
preparation of solution B). Transfer quantitatively onto
the column and proceed with the chromatography using
1.8 ml of toluene R as the mobile phase. Collect the eluate
(solution C).
3. QUANTITATIVE ANALYSIS
3.1. Organophosphorus insecticides. Examine by gas
chromatography (2.2.28), using carbophenothion R as
internal standard. It may be necessary to use a second
internal stan-dard to identify possible interference with the
peak corresponding to carbophenothion.
Test solution. Concentrate solution B in a current of helium
for chromatography R almost to dryness and dilute to 100 l
with toluene R.
Reference solution. Prepare at least three solutions in
toluene R containing the insecticides to be determined and
carbophenothion at concentrations suitable for plotting a
calibration curve.
a fused-silica column 30 m long and 0.32 mm in internal
diameter the internal wall of which is covered with a layer
0.25 m thick of poly(dimethyl)siloxane R,
hydrogen for chromatography R as the carrier gas. Other

gases such as helium for chromatography R or nitrogen
for chromatography R may also be used provided the
chromatography is suitably validated.
a phosphorus-nitrogen flame-ionisation detector or a
atomic emission spectrometry detector,
maintaining the temperature of the column at 80 C for
1 min, then raising it at a rate of 30 C/min to 150 C,
maintaining at 150 C for 3 min, then raising the temperature
at a rate of 4 C/min to 280 C and maintaining at this
temperature for 1 min, and maintaining the temperature
of the injector port at 250 C and that of the detector at
275 C. Inject the chosen volume of each solution. When the
chromatograms are recorded in the prescribed conditions,
the relative retention times are approximately those listed
in Table 2.8.13.-3. Calculate the content of each insecticide
from the peak areas and the concentrations of the solutions.
3.2. Organochlorine and pyrethroid insecticides. Examine
by gas chromatography (2.2.28), using carbophenothion as
the internal standard. It may be necessary to use a second
internal standard to identify possible interference with the
peak corresponding to carbophenothion
Test solution. Concentrate solution C in a current of helium
for chromatography R or oxygen-free nitrogen R almost to
dryness and dilute to 500 l with toluene R.
Reference solution. Prepare at least three solutions in
toluene R containing the insecticides to be determined and
carbophenothion at concentrations suitable for plotting a
calibration curve.
Table 2.8.13.-3
Substance
Relative retention times
Dichlorvos
0.20
Fonofos
0.50
Diazinon
0.52
Parathion-methyl
0.59
Chlorpyrifos-methyl
0.60
Pirimiphos-methyl
0.66
Malathion
0.67
Parathion
0.69
Chlorpyrifos
0.70
Methidathion
0.78
Ethion
0.96
Carbophenothion
1.00
Azinphos-methyl
1.17
Phosalon
1.18

a fused silica column 30 m long and 0.32 mm in internal
diameter the internal wall of which is covered with a layer
0.25 m thick of poly(dimethyl)(diphenyl)siloxane R,
hydrogen for chromatography R as the carrier gas. Other
gases such as helium for chromatography R or nitrogen
for chromatography R may also be used, provided the
chromatography is suitably validated,
an electron-capture detector,
a device allowing direct cold on-column injection,
maintaining the temperature of the column at 80 C for
1 min, then raising it at a rate of 30 C/min to 150 C,
maintaining at 150 C for 3 min, then raising the temperature
at a rate of 4 C/min to 280 C and maintaining at this
temperature for 1 min, and maintaining the temperature
659
Racecadotril
of the injector port at 250 C and that of the detector at

275 C. Inject the chosen volume of each solution. When the
chromatograms are recorded in the prescribed conditions,
the relative retention times are approximately those listed
in Table 2.8.13.-4. Calculate the content of each insecticide
from the peak areas and the concentrations of the solutions.
Table 2.8.13.-4
Substance
Relative rentention times
-Hexachlorocyclohexane
0.44
Hexachlorobenzene
0.45
0.49
DEFINITION
Benzyl N-[(2S)-3-(acetylthio)-2-benzylpropanoyl]glycinate.
CHARACTERS
Solubility : insoluble in water, freely soluble in methanol
and in methylene chloride.
IDENTIFICATION
Comparison : racecadotril CRS.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y6 (2.2.2,
0.56
Method I).
0.61
Heptachlor
Dissolve 1.0 g in 2 ml of acetone R.
0.68
Aldrin
0.76
cis-Heptachlor-epoxide
Solvent mixture : mobile phase A, mobile phase B
(50:50 V/V).
o,p-DDE
0.81
0.82
-Endosulfan
examined in the solvent mixture and dilute to 25.0 ml with
0.87
the solvent mixture.
Dieldrin
Test solution (b). Dilute 5.0 ml of test solution (a) to 25.0 ml
p,p-DDE
0.87
with the solvent mixture.
o,p-DDD
0.89
Reference solution (a). Dilute 1.0 ml of test solution (a)
0.91
Endrin
to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this
solution to 10.0 ml with the solvent mixture.
0.92
-Endosulfan
Reference solution (b). Dilute 0.5 ml of racecadotril
o,p-DDT
0.95
impurity A CRS in the solvent mixture and dilute to 250.0 ml
1.00
Carbophenothion
with the solvent mixture. Dilute 2.0 ml of this solution
to 20.0 ml with the solvent mixture. Dilute 1.0 ml of this
p,p-DDT
1.02
solution to 100.0 ml with the solvent mixture.
1.29
cis-Permethrin
Reference solution (c). Dissolve 5.0 mg of racecadotril
1.31
trans-Permethrin
impurity G CRS in the solvent mixture and dilute to 50.0 ml
with the solvent mixture. To 5.0 ml of this solution, add
1.40
Cypermethrin*
1.0 ml of test solution (b) and dilute to 100.0 ml with the
1.47 and 1.49
Fenvalerate*
solvent mixture.
1.54
Reference solution (d). Dissolve 20.0 mg of racecadotril CRS
Deltamethrin
in the solvent mixture and dilute to 50.0 ml with the solvent
* The substance shows several peaks.
mixture.
Column:
size : l = 0.25 m, = 4.0 mm ;
temperature : 30 C.
XXXX:2171 Mobile phase:
mobile phase A : acetonitrile for chromatography R ;
RACECADOTRIL
mobile phase B : dissolve 1.0 g of potassium dihydrogen
phosphate R in water R, adjust to pH 2.5 with phosphoric
acid R and dilute to 1000 ml with water R ;
Racecadotrilum
Lindane
0.49
0.54
C21H23NO4S
Time
(min)
0-5
Mobile phase A
(per cent V/V)
40
Mobile phase B
(per cent V/V)
60
5 - 25
40 80
60 20
25 - 35
80
20
35 - 50
80 40
20 60

Mr 385.48 Detection : spectrophotometer at 210 nm.
(42) liChrospher RP18 is suitable.
660
Racecadotril
Figure 2171.-1. Chromatogram for the test for related substances of racecadotril
Injection: 10 l of test solution (a) and reference
solutions (a), (b) and (c).
Relative retention with reference to racecadotril
(retention time = about 16 min) : impurity A = about
0.2 ; impurity C = about 0.26 ; impurity E = about 0.45 ;
impurity F = about 0.92.
System suitability : reference solution (c):
resolution : minimum of 1.5 between the peaks due to
racecadotril and impurity G.
Limits:
correction factors : for the calculation of contents,
the corresponding correction factor : impurity C = 1.4 ;
impurity E = 0.6 ; impurity F = 0.7 ;
impurities C, E, F : for each impurity, not more twice the
(0.5 per cent) ;
(0.05 per cent).
on 1.000 g in an oven in vacuo at 60 C for 4h.
on 1.0 g.
ASSAY
related substances with the following modification.
Injection : test solution (b) and reference solution (d).

Calculate the percentage content of C21H23NO4S from the
declared content of racecadotril CRS.
IMPURITIES
Specified impurities : A, C, E, F.
pharmaceutical use) : B, G, H.
A. ethanethioic acid,
B. N-[(2S)-2-benzyl-3-mercaptopropanoyl]glycine,
C. N-[(2S)-3-(acetylthio)-2-benzylpropanoyl]glycine,
661
XXXX:0132
SEMI-SOLID PREPARATIONS FOR

CUTANEOUS APPLICATION
Praeparationes molles ad usum dermicum
The requirements of this monograph apply to all
semi-solid preparations for cutaneous application. Where
D. [[(2R)-2-benzyl-3-[[(2S)-2-benzyl-3-[(carboxymethyl)amino]- appropriate, additional requirements specific to semi-solid
3-oxopropyl]dithio]propanoyl]amino]acetic acid,
preparations intended to be applied to particular surfaces
or mucous membranes may be found in other general
monographs, for example Ear preparations (0652), Nasal
preparations (0676), Rectal preparations (1145), Eye
preparations (1163) and Vaginal preparations (1164).
DEFINITION
Semi-solid preparations for cutaneous application are
intended for local or transdermal delivery of active
substances, or for their emollient or protective action. They
are of homogeneous appearance.
Semi-solid preparations for cutaneous application consist
of a simple or compound basis in which, usually, 1 or more
active substances are dissolved or dispersed. According to
F. benzyl N-(2-benzylacryloyl)glycinate,
its composition, the basis may influence the activity of the
preparation.
The basis may consist of natural or synthetic substances
and may be single phase or multiphase. According to the
nature of the basis, the preparation may have hydrophilic or
hydrophobic properties ; it may contain suitable excipients
such as antimicrobial preservatives, antioxidants, stabilisers,
emulsifiers, thickeners and penetration enhancers.
G. benzyl N-[(2S)-2-benzyl-3-mercaptopropanoyl]glycinate,
Semi-solid preparations for cutaneous application intended
for use on severely injured skin are sterile.
Where applicable, containers for semi-solid preparations for
cutaneous application comply with the requirements for
Materials used for the manufacture of containers (3.1 and
subsections) and Containers (3.2 and subsections).
Several categories of semi-solid preparations for cutaneous
application may be distinguished :
ointments ;
creams ;
gels ;
pastes ;
H. benzyl (7R,12S)-7,12-dibenzyl-3,6,13-trioxo-1-phenyl-2-oxa- poultices ;
medicated plasters.
9,10-dithia-5,14-diazahexadecan-16-oate.
According to their structure, ointments, creams and gels
generally show viscoelastic behaviour and are non-Newtonian
in character e.g. plastic, pseudoplastic or thixotropic type
flow at high shear rates. Pastes frequently exhibit dilatancy.
PRODUCTION
During development of semi-solid preparations for cutaneous
According to the definition in the monograph, semi-solid
application whose formulation contains an antimicrobial
preparations are intended for local or transdermal delivery. preservative, the need for and the efficacy of the chosen
preservative shall be demonstrated to the satisfaction of
Some pharmaceutical products (in particular
the competent authority. A suitable test method together
hormone-based products) can be found in the form of
semi-solid preparations for transdermal release. Supplying with criteria for judging the preservative properties of
the formulation are provided in Efficacy of antimicrobial
them in single-dose containers is intended to improve
preservation (5.1.3). In the manufacture, packaging,
compliance, and the content of a single-dose container
corresponds to a precise dose of the medicinal product. It storage and distribution of semi-solid preparations for
cutaneous application, suitable steps are taken to ensure
is therefore necessary to introduce into the monograph a
their microbiological quality ; recommendations on this are
test for uniformity of dosage units (2.9.40) for single-dose
provided in Microbiological Quality of Pharmaceutical
preparations intended for transdermal delivery and for
Preparations (5.1.4). Sterile semi-solid preparations
which the entire contents correspond to one dose of the
for cutaneous application are prepared using materials
medicinal product.
E. 2-benzylacrylic acid,
662
and methods designed to ensure sterility and to avoid

the introduction of contaminants and the growth of
micro-organisms ; recommendations on this are provided in
Methods of Preparation of Sterile Products (5.1.1).
During development, it must be demonstrated that the
nominal content can be withdrawn from the container of
semi-solid preparations for cutaneous application presented
in single-dose containers.
In the manufacture of semi-solid preparations for cutaneous
application, suitable measures are taken to ensure that
the defined rheological properties are fulfilled. Where
appropriate, the following non-mandatory tests may be
carried out : measurement of consistency by penetrometry
(2.9.9), viscosity (apparent viscosity) (2.2.10) and a suitable
test to demonstrate the appropriate release of the active
substance(s).
application containing 1 or more active substances that are
not dissolved in the basis (e.g. emulsions or suspensions),
measures are taken to ensure appropriate homogeneity of
the preparation to be delivered.
application containing dispersed particles, measures are
taken to ensure a suitable and controlled particle size with
regard to the intended use.
TESTS
Uniformity of dosage units. Semi-solid preparations
supplied in single-dose containers which represent one
dose of medicinal product and are intended for transdermal
delivery of the active substance(s) in view of a systemic
effect, comply with the test for uniformity of dosage units
(2.9.40). Herbal drugs and herbal drug preparations present
in the dosage form are not subject to the provisions of this
paragraph.
Sterility (2.6.1). Where the label indicates that the
preparation is sterile, it complies with the test for sterility.
STORAGE
If the preparation contains water or other volatile
ingredients, store in an airtight container. If the preparation
is sterile, store in a sterile, airtight, tamper-proof container.
LABELLING
The label states :
the name of any added antimicrobial preservative ;
where applicable, that the preparation is sterile.
appropriate, additional requirements specific to semi-solid
Ointments
DEFINITION
An ointment consists of a single-phase basis in which solids
or liquids may be dispersed.
Hydrophobic Ointments
Hydrophobic ointments can absorb only small amounts of
water. Typical bases used for their formulation are hard,
liquid and light liquid paraffins, vegetable oils, animal fats,
synthetic glycerides, waxes and liquid polyalkylsiloxanes.
Water-emulsifying Ointments
Water-emulsifying ointments can absorb larger amounts

of water and thereby produce water-in-oil or oil-in-water
emulsions depending on the nature of the emulsifiers :
water-in-oil emulsifying agents such as wool alcohols,
sorbitan esters, monoglycerides and fatty alcohols, or
oil-in-water emulsifying agents such as sulphated fatty
alcohols, polysorbates, macrogol cetostearyl ether or esters
of fatty acids with macrogols may be used for this purpose.
Their bases are those of the hydrophobic ointments.
Hydrophilic Ointments
Hydrophilic ointments are preparations having bases that are
miscible with water. The bases usually consist of mixtures of
liquid and solid macrogols (polyethylene glycols). They may
contain appropriate amounts of water.
Creams
DEFINITION
Creams are multiphase preparations consisting of a lipophilic
phase and an aqueous phase.
Lipophilic Creams
Lipophilic creams have as the continuous phase the lipophilic
phase. They contain water-in-oil emulsifying agents such as
wool alcohols, sorbitan esters and monoglycerides.
Hydrophilic Creams
Hydrophilic creams have as the continuous phase the
aqueous phase. They contain oil-in-water emulsifying agents
such as sodium or trolamine soaps, sulphated fatty alcohols,
polysorbates and polyoxyl fatty acid and fatty alcohol esters
combined, if necessary, with water-in-oil emulsifying agents.
Gels
DEFINITION
Gels consist of liquids gelled by means of suitable gelling
agents.
Lipophilic Gels
Lipophilic gels (oleogels) are preparations whose bases
usually consist of liquid paraffin with polyethylene or fatty
oils gelled with colloidal silica or aluminium or zinc soaps.
Hydrophilic Gels
Hydrophilic gels (hydrogels) are preparations whose bases
usually consist of water, glycerol or propylene glycol
gelled with suitable gelling agents such as starch, cellulose
derivatives, carbomers and magnesium-aluminium silicates.
663
Sevoflurane

Pastes
DEFINITION
Pastes are semi-solid preparations for cutaneous application
containing large proportions of solids finely dispersed in
the basis.
Poultices
DEFINITION
Poultices consist of a hydrophilic heat-retentive basis in
which solid or liquid active substances are dispersed. They
are usually spread thickly on a suitable dressing and heated
before application to the skin.
Medicated plasters
DEFINITION
Medicated plasters are flexible preparations containing 1 or
more active substances. They are intended to be applied
to the skin. They are designed to maintain the active
substance(s) in close contact with the skin such that these
may be absorbed slowly, or act as protective or keratolytic
agents.
Medicated plasters consist of an adhesive basis, which may
be coloured, containing 1 or more active substances, spread
as a uniform layer on an appropriate support made of natural
or synthetic material. It is not irritant or sensitising to the
skin. The adhesive layer is covered by a suitable protective
liner, which is removed before applying the plaster to the
skin. When removed, the protective liner does not detach
the preparation from the outer, supporting layer.
Medicated plasters are presented in a range of sizes directly
adapted to their intended use or as larger sheets to be cut
before use. Medicated plasters adhere firmly to the skin
when gentle pressure is applied and can be peeled off without
causing appreciable injury to the skin or detachment of the
preparation from the outer, supporting layer.
TESTS
Dissolution. A suitable test may be required to demonstrate
the appropriate release of the active substance(s), for
example one of the tests described in Dissolution test for
transdermal patches (2.9.4).
Reference: PA/PH/Exp. P4/T (05) 14 ANP

Identification : the determination of the refractive index
of sevoflurane requires an apparatus that is not widely
present and the determination of the density is described
as an alternative.
XXXX:2269
SEVOFLURANE
Sevofluranum
C 4H 3F 7O
Mr 200.1
DEFINITION
1,1,1,3,3,3-Hexafluoro-2-(fluoromethoxy)propane.
CHARACTERS
Appearance : clear, colourless, volatile liquid.
Solubility : slightly soluble in water. It is miscible with
ethanol (96 per cent).
Boiling point: about 58.5 C.
It is non-flammable.
IDENTIFICATION
Second identification : A, B.
Preparation : examine the substance in the gaseous state.
Comparison : sevoflurane CRS.
B. Relative density (2.2.5) : 1.5204 to 1.5207.
C. Refractive index (2.2.6) : 1.2745 to 1.2760(43).
TESTS
Acidity or alkalinity. Introduce 20.0 ml of the substance to
be examined and 20.0 ml of carbon dioxide-free water R
into a separating funnel, shake for 3 min and allow to
stand. Collect the aqueous upper layer and add 0.2 ml of
bromocresol purple solution R. Not more than 0.10 ml
of 0.01 M sodium hydroxide or not more than 0.60 ml of
0.01 M hydrochloric acid is required to change the colour
of the indicator.
Related substances. Gas chromatography (2.2.28).
Internal standard : methylal R.
Test solution. Introduce 20.0 ml of the substance to be
examined into a vial, and seal with a cap and septum. Using
a microsyringe, add 5 l of internal standard through the
septum and mix thoroughly.
Reference solution (a). Introduce 2.0 ml of ethylene
chloride R into a screw-capped vial, immediately seal
with a cap and septum, and place on a balance. Using
a microsyringe, add about 20 l of the substance to be
examined through the septum. Record the quantity added,
in milligrams, of the substance to be examined (M2). Then,
using a microsyringe, add about 20 l of the internal
standard through the septum. Record the quantity added, in
milligrams, of the internal standard (M1).
Reference solution (b) : sevoflurane CRS (containing
impurities A and B).
(43) A Bellingham Stanley Abbe 60 refractometer model 60/LR is suitable.
664
Sevoflurane
Reference solution (c). Place a 40 ml vial with a septum lid

onto an analytical balance and tare the balance. Add 20.0 ml
of ethylene chloride R into the vial and seal tightly. Record
the weight of the ethylene chloride R, and tare. Using a
microsyringe, add 20 l of the substance to be examined
through the septum, record the weight, and mix thoroughly.
Dilute 0.5 ml of the solution obtained to 100.0 ml with
ethylene chloride R.
Reference solution (d). Introduce 2.0 ml of the substance
to be examined into a vial, and seal with a cap and septum.
Using a microsyringe, add about 20 l of ethylene chloride R
through the septum and mix thoroughly. Use this solution
to identify the peak due to ethylene chloride.

Injection : 2 l.
Wash the syringe with a solution containing ethylene
chloride R before the injection of the reference solutions.
Wash the syringe with the test solution before the injection
of the test solution.
with sevoflurane CRS and the chromatogram obtained
with reference solution (b) to identify the peaks due to
impurities A and B.
Column :
Relative retention with reference to sevoflurane (retention

time = about 6.6 min) : impurity A = about 0.78 ;
impurity B = about 0.83 ; internal standard = about 1.35.
size : l = 30 m, = 0.32 mm ;

impurities A and B.
stationary phase : poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R(44) (film thickness 1.8-3 m).

Calculate the relative response factor( F1) for reference

solution (a), using the following expression :

Split ratio : 1:20.
Temperature :
Column
Time
(min)
0 - 10
Temperature
(C)
40
10 - 26
40 200
26 - 40
200
Injection port
200
Detector
225
M1
M2
mass of the internal standard in reference

mass of the substance to be examined in reference
ratio of the area of the peak due to sevoflurane to
the area of the peak due to the internal standard
from the chromatogram obtained with reference
solution (a).
Figure 2269.-1. Chromatogram for the test for related substances of sevoflurane : test solution.
(44) Quadrex Corporation, fused silica column, P/N 007-624-30W-3.0F is suitable.
665
Spanish sage oil
Calculate the quantity of each impurity in the substance to

be examined, in ppm, using the following expression :
0.859 =
specific density of the internal standard ;
1.525 =
R1 =
specific density of sevoflurane ;
F1
ratio of the area of the peak due to the impurity to

the area of the peak due to the internal standard
from the chromatogram obtained with the test
solution ;
relative response factor for reference solution (a).
Limits :
impurity A : maximum 25 ppm ;
impurity B : maximum 50 ppm ;
unspecified impurities : for each impurity, maximum
25 ppm ;
sum of impurities other than A and B : maximum 50 ppm ;
disregard limit : disregard any peak with an area less
obtained with reference solution (c) (5 ppm), and any
peak due to ethylene chloride.
Fluorides : maximum 2 g/ml.
Potentiometry (2.2.36, Method I). Use plastic utensils
throughout this test.
Buffer solution. Transfer 110 g of sodium chloride R and
1 g of sodium citrate R to a 2000.0 ml volumetric flask,
and dissolve with 700 ml of water R. Carefully add 150 g of
sodium hydroxide R and shake to dissolve. Cool to room
temperature, and carefully add 450 ml of glacial acetic
acid R while stirring. Cool and add 600 ml of isopropyl
alcohol R. Dilute with water R to 2000.0 ml. The pH of this
solution is between 5.0 and 5.5. This solution may be used
for 6 weeks when stored at room temperature.
Test solution. Introduce 50.0 ml of the substance to be
examined and 50.0 ml of water R into a separating funnel,
shake vigorously for 3 min, and allow the layers to separate
completely. Dilute 25.0 ml of the aqueous upper layer to
50.0 ml with the buffer solution.
Fluoride standard solution (1000 ppm F). Dissolve 221.0 mg
of sodium fluoride R, previously dried at 150 C for 4 h, in
water R. Add 1.0 ml of 0.01 M sodium hydroxide and dilute
to 100.0 ml with water R. Store in a tightly closed plastic
container. This solution may be used for 2 weeks when
stored at 2-8 C.
Reference stock solutions. Prepare the reference stock
solutions using the fluoride standard solution (1000 ppm F)
diluted with water R to obtain solutions having known
concentrations of about 5, 2, 0.5, and 0.2 g of fluoride per
millilitre.
Reference solutions. Dilute 25.0 ml of each of the reference
stock solutions to 50.0 ml with the buffer solution.
indicator electrode : fluoride-selective.
reference electrode : glass-sleeved calomel.
Apparatus: voltmeter capable of a minimum reproducibility
of 0.2 mV.
Carry out the measurements on the reference stock
solutions, reference solutions and test solution. When taking
measurements, transfer the solution under test to a 100 ml
beaker containing a polytetrafluoroethylene-coated magnetic
stirring bar, and immerse the electrodes. Allow to stir on a
magnetic stirrer with an insulated top until equilibrium is
666
attained in about 2-3 min, and record the potential. Rinse

the electrodes with the buffer solution and dry, taking care
to avoid damaging the crystal of the specific-ion electrode.
System suitability : the difference between the potentials
obtained with the reference stock solutions having fluoride
concentrations of 5 g/ml and of 0.5 g/ml is between
50 mV and 60 mV.
Calculate the concentration of fluorides using the calibration
curve.
Non-volatile residue : maximum 100 mg/l.
Transfer 10.0 ml to a tared evaporating dish, evaporate to
dryness on a steam bath, and dry the residue at 105 C for
2 h. The residue weighs a maximum of 1.0 mg.
Water (2.5.12) : 0.030 per cent m/m to 0.050 per cent m/m,
determined on 10.0 ml.
STORAGE
In an airtight, stainless steel container, protected from light.
IMPURITIES
A. 1,1,3,3,3-pentafluoro-2-(fluoromethoxy)prop-1-ene,
B. 1,1,1,3,3,3-hexafluoro-2-methoxypropane.
Reagents
Methylal. C3H8O2. (Mr 76.1). XXXXXXX. [109-87-5].
Formaldehyde dimethyl acetal. 1,1-Dimethoxymethane.
Clear, colourless, volatile, flammable liquid, soluble in water
and miscible with ethanol (96 per cent).
: about 0.860.
: about 1.354.
bp : about 41 C.
Methylal used in gas chromatography, complies with the
following additional test:
Content : minimum 99.5 per cent determined by gas
chromatography.

XXXX:1849
SPANISH SAGE OIL

Salviae lavandulifoliae aetheroleum
DEFINITION
Spanish sage oil is obtained by steam distillation from the
aerial parts of Salvia lavandulifolia Vahl collected at the
flowering stage.
CHARACTERS
A clear, colourless to pale yellow, mobile liquid.
It has a camphorlike odour.
IDENTIFICATION
Second identification : A.
Spanish sage oil
A. Thin-layer chromatography (2.2.27).

Test solution. Dissolve 0.1 ml of the substance to be
examined in 10 ml of toluene R.
Reference solution. Dissolve 20 l of thujone R and 30 l
of cineole R in 10 ml of toluene R.
Plate : TLC silica gel plate R (5-40 m) [or TLC silica gel
plate R (2-10 m)].
Mobile phase : ethyl acetate R, toluene R (5:95 V/V).
Application : 10 l [or 3 l], as bands.
Development : over a path of 15 cm [or 6 cm].
Drying : in air.
Detection : spray with a freshly prepared 200 g/l solution
of phosphomolybdic acid R in ethanol (96 per cent) R
and heat at 105 C for 10 min ; examine in daylight.
Results : see below the sequence of the zones present in
the chromatograms obtained with the reference solution
and the test solution. Furthermore, other zones may
be present in the chromatogram obtained with the test
solution.
Top of the plate
A blue zone
_______
_______
Thujone : 2 pink-violet zones

Cineole : a blue zone
A blue zone (cineole)
_______
_______
TESTS
Relative density (2.2.5) : 0.907 to 0.932.
Refractive index (2.2.6) : 1.4650 to 1.4730.
Optical rotation (2.2.7) : + 7 to + 17.
Acid value (2.5.1) : maximum 2.0, determined on 5.00 g.
Solubility in alcohol (2.8.10). 1 volume of the oil is soluble
in 2 volumes or more of ethanol (80 per cent V/V) R.
Chromatographic profile. Gas chromatography (2.2.28) :
use the normalisation procedure.
examined in ethanol (96 per cent) R and dilute to 10.0 ml
Reference solution (a). Dissolve 0.200 g of spanish sage oil
for peak identification CRS in ethanol (96 per cent) R and
dilute to 10.0 ml with the same solvent.
Reference solution (b). Dissolve 5 l of limonene R in
50.0 ml of heptane R. Dilute 0.5 ml of this solution to 5.0 ml
Column :
material : fused-silica ;
size : l = 60 m, = 0.25 mm ;
stationary phase : macrogol 20 000 R (film thickness
0.25 m).
Split ratio : 1:50.
Temperature :
3 blue zones
Reference solution
Test solution
B. Examine the chromatograms obtained in the test for

chromatographic profile.
Results : the characteristic peaks in the chromatogram
obtained with the test solution are similar in retention
time to those in the chromatogram obtained with the
reference solution.
Column
Time
(min)
0 - 43
Temperature
(C)
60 232
Injection port
250
Detector
250

Injection : 1 l.
1. -pinene
4. 1,8-cineole
7. linalool
10. sabinyl acetate
2. sabinene
5. thujone
8. linalyl acetate
11. -terpinyl acetate
3. limonene
6. camphor
9. terpinen-4-ol
12. borneol
Figure 1849.-1. Chromatogram for the test for chromatographic profile of Spanish sage oil
667
the chromatogram obtained with the reference solution is

similar to the chromatogram provided with spanish sage
oil for peak identification CRS ;
limonene and 1,8-cineole.
Use the chromatogram supplied with spanish sage oil for
peak identification CRS and the chromatogram obtained
with the reference solution to locate the peaks due to
-pinene, sabinene, limonene, 1,8-cineole, thujone, linalool,
camphor, linalyl acetate, terpinen-4-ol, sabinyl acetate,
-terpinyl acetate and borneol in the test solution.
Determine the percentage content of each of these
components. The limits are within the following ranges :
a-pinene : 4.0 per cent to 11.0 per cent ;
sabinene : 0.1 per cent to 3.5 per cent ;
limonene : 2.0 per cent to 6.0 per cent ;
1,8-cineole : 10.0 per cent to 30.0 per cent ;
thujone : less than 0.5 per cent ;
linalool : 0.3 per cent to 4.0 per cent ;
camphor : 11.0 per cent to 36.0 per cent ;
linalyl acetate : less than 5.0 per cent ;
terpinen-4-ol: less than 2.0 per cent ;
sabinyl acetate : 0.5 per cent to 9.0 per cent ;
-terpinyl acetate : 0.5 per cent to 9.0 per cent ;
borneol: 1.0 per cent to 7.0 per cent ;
disregard limit : the area of the principal peak in the
(0.05 per cent).
STORAGE
In a well-filled, airtight container, protected from light and
at a temperature not exceeding 25 C.

Related substances : TLC has been replaced by LC in
accordance with current policy as part of a special revision
programme. The limits proposed are based on batch results.
Impurities : section has been introduced showing
impurities controlled by the LC test.
XXXX:0107
SULFACETAMIDE SODIUM
Sulfacetamidum natricum
C8H9N2NaO3S,H2O
Mr 254.2
DEFINITION
Sodium derivative of N-[(4-aminophenyl)sulphonyl]acetamide.
substance).
668
CHARACTERS
Appearance : white or yellowish-white, crystalline powder.
Solubility : freely soluble in water, slightly soluble in
anhydrous ethanol.
IDENTIFICATION
First identification : B, F.
Second identification : A, C, D, E, F.
(2.2.25).
Test solution. Dissolve 0.1 g in phosphate buffer solution
pH 7.0 R and dilute to 100.0 ml with the same buffer
solution. Dilute 1.0 ml of this solution to 100.0 ml with
phosphate buffer solution pH 7.0 R.
Specific absorbance at the absorption maximum : 660 to
720 (anhydrous substance).
B. Infrared absorption spectrophotometry (2.2.24),
Comparison : sulfacetamide sodium CRS.
C. Melting point (2.2.14) : 191 C to 185 C.
Dissolve 1 g in 10 ml of water R, add 6 ml of dilute acetic
acid R and filter. Wash the precipitate with a small
quantity of water R and dry at 100-105 C for 4 h.
D. Dissolve 0.1 g of the precipitate obtained in identification
test C in 5 ml of ethanol (96 per cent) R. Add 0.2 ml of
sulphuric acid R and heat. The odour of ethyl acetate is
perceptible.
E. Dissolve about 1 mg of the precipitate obtained in
identification test C, with heating, in 1 ml of water R. The
solution gives the reaction of primary aromatic amines
(2.3.1) with formation of an orange-red precipitate.
F. Solution S (see Tests) gives the reactions of sodium
(2.3.1).
TESTS
Appearance of solution. Solution S is clear (2.2.1) and not
more intensely coloured than reference solution GY4 (2.2.2,
Method II).
(2.2.27), using silica gel HF254 R as the coating substance.
Reference solution (a). Dissolve 5 mg of sulfanilamide R in
Reference solution (b). Dilute 5 ml of reference solution (a)
to 10 ml with water R.
Reference solution (c). Dissolve 5 mg of sulfanilamide R
in 10 ml of the test solution.
Apply to the plate 5 l of each solution. Develop over a path
of 15 cm using a mixture of 10 volumes of concentrated
ammonia R, 25 volumes of ethanol R, 25 volumes of water R
and 50 volumes of butanol R. Allow the plate to dry in air
and spray with dimethylaminobenzaldehyde solution R2.
Any spot in the chromatogram obtained with the test
solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with reference
solution (a) (0.5 per cent), and not more than one such spot
is more intense than the spot in the chromatogram obtained
with reference solution (b) (0.25 per cent). The test is not
valid unless the chromatogram obtained with reference
solution (c) shows two clearly separated spots.
Limits :
impurity A : not more than twice the area of the principal
Liquid chromatography (2.2.29). Prepare the solutions
immediately before use and carry out the test protected
from light.
total
: not more than 5 times the area of the principal peak
in
the
mobile phase.
(0.5 per cent) ;
Reference solution (a). Dissolve 20 mg of sulfacetamide
sodium CRS and 20 mg of sulfanilamide CRS in the mobile
phase and dilute to 1.0 ml with the mobile phase.
(0.05 per cent).
Sulphates (2.4.13) : maximum 200 ppm.
Dissolve 2.5 g in distilled water R and dilute to 25 ml with
the same solvent. Add 25 ml of dilute acetic acid R, shake
for 30 min and filter. 15 ml of the filtrate complies with the
Column :
limit test for sulphates.
size : l = 0.125 m, = 4 mm ;
12 ml of the filtrate obtained in the test for sulphates
for chromatography R (5 m)(45).
complies with test A. Prepare the reference solution using
Mobile phase : glacial acetic acid R, methanol R, water for lead standard solution (1 ppm Pb) R.
chromatography R (1:10:89 V/V/V).
Water (2.5.12). 6.0 per cent to 8.0 per cent, determined on
0.200 g.
ASSAY
Dissolve 0.500 g in a mixture of 50 ml of water R and 20 ml
of dilute hydrochloric acid R. Cool the solution in a bath
of iced water and carry out the determination of primary
aromatic amino-nitrogen (2.5.8), determining the end-point
electrometrically.
1 ml of 0.1 M sodium nitrite is equivalent to 23.62 mg of
C8H9N2NaO3S.

impurity A and sulfacetamide sodium.
STORAGE
Injection : 10 l.
Run time : 7 times the retention time of sulfacetamide
sodium.
Relative retention with reference to sulfacetamide sodium
(retention time = about 5 min) : impurity A = about 0.5.
1. impurity A
3. impurity B
2. sulfacetamide sodium
4. impurity C
5. impurity D
Figure 0107.-1. Chromatogram for the test for related substances of sulfacetamide sodium : solution of sulfacetamide
sodium spiked with impurities A, B, C and D
(45) LiChrospher ODS 1, Kromasil C18 and Spherisorb ODS 2C18 C18 are suitable.
669
Swine-fever vaccine (live, prepared in cell cultures), classical
IMPURITIES
pharmaceutical use) : B, C, D.
A. sulfanilamide,
B. N-[4-(aminosulphonyl)phenyl]acetamide,
C. N-[(4-acetamidophenyl)sulphonyl]acetamide,
D. dapsone.
Reference: PA/PH/Exp. 15V/T (04) 31 ANP 1R

The monograph on freeze-dried classical swine-fever
vaccine (live) has not been revised for many years. In the
meantime, the epidemiological situation and the disease
control strategies in Europe have changed. Routine
vaccination has not been used for more than 10 years. It
has nevertheless been decided to revise the monograph to
bring it into line with current concepts since member states
may choose to use the vaccine for disease control if there is
a widespread outbreak of disease.
The first proposed revised text was published in
Pharmeuropa 17.4. We received a lot of comments, and the
new revised text below takes them into account (mainly
the exclusion of vaccines produced in rabbits from the
scope of the monograph, revision of the test for increase in
virulence, and immune status of pigs mentioned for each
test).
To summarise, the monograph has been revised as follows
to exclude vaccines produced in rabbits from its scope, and
to align it with the current policy.
670
The piglets used in the relevant tests are 6-10 weeks old
(instead of 6-7 weeks old) and free from antibodies against
pestiviruses (instead of swine-fever virus and bovine viral
diarrhoea virus).
Production/Safety :
the test has been changed : it is now a test with 10 piglets
not older than the minimum age recommended for
vaccination, with 10 doses of vaccine, and the body
temperature is recorded ;
the group of piglets with administration of prednisolone
has been deleted ;
primiparous sows have been replaced by sows or gilts
and the test is now done with only 1 dose of vaccine
instead of 2 ; administration of sodium chloride in the
controls has been deleted ; the body temperature is
recorded and piglets born are controlled.
Non transmissibility : the challenge has been deleted and
alternative methods are used to detect classical swine-fever
virus in the controls.
Increase in virulence : the passage requirement has been
lowered to 5 (like in other monographs) ; the quantity of
blood collected and administered has been lowered to 2 ml;
blood samples are collected daily between day 2 and day 7
post-vaccination.
Immunogenicity : the PD50 test has been changed.
Identification : test A has been deleted since it was used to
identify the vaccine produced in rabbits.
Extraneous agents : the test in mice has been deleted since
it does not provide extra information.
Safety : 2 healthy piglets are used instead of 3, and the
observation period is now 14 days instead of 21 days ; the
phrase in apparent good health has been clarified.
Virus titre : a titration of the vaccine virus for vaccines
prepared in cell cultures has been added.
Furthermore, the monograph is presented in the new
format to be used in future for all veterinary vaccines. The
editorial re-arrangement does not entail technical change.
XXXX:0065
SWINE-FEVER VACCINE (LIVE,

PREPARED IN CELL CULTURES),
CLASSICAL, FREEZE-DRIED
Vaccinum pestis classicae suillae vivum
cryodesiccatum ex cellulis
1. DEFINITION
Freeze-dried Classical swine-fever vaccine (live, prepared
in cell cultures) is a preparation obtained from a strain of
classical swine-fever virus that has lost its pathogenicity for
the pig by adaptation either to cell cultures or to the rabbit.
2. PRODUCTION
2-1. PREPARATION OF THE VACCINE
The vaccine virus is grown in cell cultures or in rabbits. The
vaccine is freeze-dried.
2-2. SUBSTRATE FOR VIRUS PROPAGATION
2-2-1. Cell cultures. If the vaccine virus is grown in c Cell
cultures, they comply with the requirements for cell cultures
for production of veterinary vaccines (5.2.4).
2-2-2. Rabbits. If the vaccine virus is grown in rabbits, the

seed-lot (or the vaccine) is made from the homogenised
organs and/or blood of rabbits from healthy colonies,
euthanised at the peak of the temperature rise following
intravenous inoculation of the virus.
2-3. CHOICE OF VACCINE VIRUS
The vaccine virus is shown to be satisfactory with respect
to safety (5.2.6) and efficacy (5.2.7) for the swine for which
it is intended.
The following tests for safety described under Safety test
in piglets (section 2-3-1), Safety test in pregnant sows
and test for transplacental transmission (section 2-3-2),
Non-transmissibility (section 2-3-3), Increase in virulence
(2-3-4) and Immunogenicity (2-3-5) may be used during the
demonstration of safety and immunogenicity.
The dose of vaccine used throughout the following tests
is determined by the manufacturer on the basis of prior
experiments.
The animals used must have a week in which to adapt
themselves to the new quarters where the tests are to be
carried out.
2-3-1. Safety test in piglets. Carry out the test for the
intramuscular each recommended route using in each case
piglets of 6-7 weeks old not older than the minimum age
recommended for vaccination. Use vaccine virus at the least
attenuated passage level that will be present between the
master seed lot and a batch of the vaccine.
vaccine virus at the least attenuated passage level that will

be present between the master seed lot and in a batch of the
vaccine.
Use for the test not fewer than 20 healthy sows or gilts
of the same age and origin, that have had no contact
with swine-fever virus and that do not have antibodies
against swine-fever virus and bovine viral diarrhoea virus.
Administer to not fewer than 10 sows or gilts a quantity of
the vaccine virus equivalent to not less than 2 times the
maximum virus titre likely to be contained in 1 dose of the
vaccine. Maintain not fewer than 10 sows as controls and
administer to them an equal volume of a 9 g/l solution of
sodium chloride R. Record the body temperature on at least
the 3 days preceding administration of the vaccine, at the
time of administration, 4 h after and then daily for at least
15 days. Observe until farrowing.
The vaccine virus complies with the test if. Carry out tests
for serum antibodies against classical swine-fever virus. No
antibodies against classical swine-fever virus are found in
sera taken before ingestion of colostrum. The test is invalid
if the vaccinated sows do not seroconvert. The vaccine virus
complies with the test if no abnormalities in the gestation or
in the piglets are noted, no sow or gilt shows a temperature
rise greater than 1.5 C for a period exceeding 5 days, and
no sow or gilt shows notable signs of swine fever or dies
from causes attributable to the vaccine virus.
2-3-3. Non-transmissibility. Keep together for the test not

fewer than 12 healthy piglets, 6-7 6-10 weeks old and of
Use not fewer than 10 healthy piglets that do not have
the same origin, and that do not have antibodies against
antibodies against pestiviruses. Administer to not fewer
pestiviruses that have had no contact with swine-fever virus
than 10 piglets a quantity of the vaccine virus equivalent
and that do not have antibodies against swine-fever virus
to not less than 10 times the maximum virus titre likely
and bovine viral diarrhoea virus. Vaccinate Administer by a
to be contained in 1 dose of the vaccine. Observe the
recommended route to not fewer than 6 piglets a quantity of
piglets at least daily for 21 days. The body temperature of
the vaccine virus equivalent to not less than the maximum
each vaccinated animal is measured on at least the 3 days
virus titre likely to be contained in 1 dose of the vaccine.
preceding administration of the vaccine, at the time of
Maintain not fewer than 6 piglets as contact controls.
administration, 4 h after and then daily for at least 14 days. Challenge each piglet after 40 days by the intramuscular
No animal shows a temperature rise greater than 1.5 C for route with a sufficient quantity of virulent swine-fever virus
a period exceeding 3 days. No animal shows notable signs of to kill an unvaccinated piglet in 7 days. The challenge virus
swine fever or dies from causes attributable to the vaccine
preparation consists of blood of pigs infected experimentally
virus.
by virus that has not been submitted to passage in cell
cultures. The mixing of vaccinated piglets and contact
For each test, Use not fewer than 15 healthy piglets that
piglets is done 24 h after vaccination.
have had no contact with swine-fever virus and that do not
have antibodies against swine-fever virus and bovine viral
The control piglets show typical signs of swine fever, and
diarrhoea virus . Divide them randomly into 3 groups of
the vaccinated piglets survive and show no notable signs
not fewer than 5 piglets.
of swine fever.
Administer to each piglet of the first group (a) a quantity
After 45 days, euthanise all piglets. Carry out appropriate
of the vaccine virus equivalent to not less than 10 times
tests on the piglets to detect antibodies to classical
the maximum virus titre likely to be contained in 1 dose of
swine-fever. Carry out appropriate tests on the control
the vaccine. Administer to each piglet of a second group
piglets to detect classical swine-fever antigen in the tonsils
(b) 2 mg/kg of body mass of prednisolone daily, for 5
consecutive days and on the 3rd day, 1 dose of the vaccine. and the mandibular lymph nodes. The vaccine complies with
the test if antibodies are found in all vaccinated piglets and if
Maintain not fewer than 5 piglets as controls. Observe the
no antibodies and no antigen are found in the control piglets.
piglets at least daily for 21 days.
2-3-4. Increase in virulence. The test for increase in
The test is invalid if the temperature curve and the weight
virulence consists of the administration of the vaccine virus
curve differ significantly from those of control animals.
at the least attenuated passage level that will be present
The vaccine virus complies with the test if no piglet shows
between the master seed lot and a batch of the vaccine to
notable signs of swine fever or dies from causes attributable piglets that do not have antibodies against pestiviruses that
to the vaccine virus.
have had no contact with swine-fever virus and that do not
2-3-2. Safety test in pregnant sows and test for
have antibodies against swine-fever virus and bovine viral
transplacental transmission. Carry out the test by the
diarrhoea virus, followed by sequential passaging, 7 times
intramuscular a recommended route using not fewer than
where possible, to further similar groups, and testing of
10 healthy primiparae sows or gilts of the same age and
the final recovered virus for increase in virulence. If the
origin, between the 25th 55th and 35th 80th days of gestation, properties of the vaccine virus allow sequential passage to
and that do not have antibodies against pestiviruses. Use
7 groups via natural spreading, this method may be used,
671
otherwise passage as described below is carried out and the

maximally passaged virus that has been recovered is tested
for increase in virulence.
Administer to each of 2 healthy piglets, 6-7 6-10 weeks old,
by the intramuscular a recommended route, a quantity of the
vaccine virus equivalent to not less than the maximum virus
titre likely to be contained in 1 dose of the vaccine. Collect 5
2 ml of blood from each piglet daily between day 2 and day
7 7 days after administration of the vaccine virus, and pool
these samples taken on the same day. Administer 5 2 ml of
the pooled blood of the day with the highest virus titre by
the intramuscular a recommended route to each of 2 other
piglets of the same age and origin. If no virus is found, repeat
the test once again with another 2 piglets. If virus is found,
carry out a 2nd series of passages by administering 2 ml of
positive blood by a recommended route to each of 2 other
piglets of the same age and origin. Carry out this passage
operation not fewer than 7 5 times, verifying the presence
of the virus at each passage. If the virus is not found at a
passage level, carry out a second series of passages. Care
must be taken to avoid contamination by virus from previous
passages.
The vaccine virus complies with the test if no indication
of increasing virulence of the maximally passaged virus
compared with the unpassaged virus is observed. If virus is
not recovered at any passage level in the 1st and 2nd series of
passages, the vaccine virus also complies with the test.
2-3-5. Immunogenicity
Potency is expressed as the number of 50 per cent protective
doses (PD50) for pigs contained in the dose indicated on the
label.
A test is carried out for the intramuscular route using in
each case 6-7 week-old piglets. The vaccine virus to be
administered to each piglet is at the most attenuated passage
level that will be present in a batch of the vaccine.
Use for the test not fewer than 12 healthy piglets of the same
origin, that have had no contact with swine-fever virus and
that do not have antibodies against swine-fever virus and
bovine viral diarrhoea virus. Divide them randomly into 3
groups among which there are 2 groups of not fewer than
5 piglets. Vaccinate each piglet of the 1st group with 1/40
of a dose and each piglet of the 2nd group with 1/160 of
a dose of the vaccine to be examined, diluted in buffered
salt solution pH 7.2 R. Maintain not fewer than 2 piglets
as controls. Challenge each piglet after 14 days by the
intramuscular route with a sufficient quantity of virulent
swine-fever virus to kill an unvaccinated piglet in 7 days.
The challenge virus preparation consists of blood of pigs
infected experimentally by virus that has not been submitted
to passage in cell cultures. Observe the piglets at least daily
for 14 days after challenge.
Calculate the number of PD50 contained in the vaccine using
the usual statistical methods from the number of piglets that
survive in each vaccinated group without showing signs of
swine fever.
The test is invalid if fewer than 100 per cent of the control
piglets die within 7 days of challenge. The vaccine complies
with the test if the minimum dose stated on the label
corresponds to not less than 100 PD50.
2-3-5-1. Protective dose. The efficacy of the vaccine is
expressed by the number of 50 per cent protective doses
(PD50) for pigs contained in the vaccinal dose as indicated on
the label. The vaccine contains at least 100 PD50 per dose.
Use 1 or more groups of piglets aged 6-10 weeks and that
do not have antibodies against pestiviruses, and use an
additional group of piglets of the same age as controls. Each
672
group of piglets is vaccinated with 1 dilution of the vaccine

dose. 14 days after the single injection of vaccine, challenge
the piglets by a suitable route with a suitable strain of
virulent virus and a dose that kills not fewer than 50 per cent
of the non-vaccinated piglets in less than 21 days. Observe
the piglets for 21 days and record the body temperature
3 days before challenge and daily after challenge for 21 days.
The PD50 is calculated by the usual statistical methods (for
example, 5.3), taking into account the surviving piglets that
have no clinical signs of swine fever, including cutaneous
lesions and an increase in body temperature.
Piglets displaying typical signs of serious infection
with swine-fever virus, including cutaneous lesions, are
euthanised to avoid unnecessary suffering.
The test is invalid if fewer than 50 per cent of the control
piglets display typical signs of serious infection with
swine-fever virus, including cutaneous lesions, or die, and if
fewer than 100 per cent of the control piglets show clinical
signs of disease within the 21 days following challenge. The
vaccine complies with the test if the minimum dose stated
on the label corresponds to not less than 100 PD50.
2-3-5-2. Protection against transplacental infection. Use
8 sows that do not have antibodies against pestiviruses,
randomly allocated to either the vaccine group (n = 6) or the
control group (n = 2).
Between the 40th and 50th day of gestation, all sows allocated
to the vaccine group are vaccinated once with 1 dose of
vaccine containing not more than the minimum titre stated
on the label. On day 60 of gestation, all sows are challenged
by a recommended route with a suitable strain of virulent
virus. Just before farrowing and about 5-6 weeks after
challenge, the sows are euthanised and their foetuses are
examined for classical swine-fever virus. Serum samples from
sows and foetuses are tested for the presence of antibodies
against classical swine-fever virus. Isolation of classical
swine-fever virus is carried out from blood of the sows
(collected 3, 6, and 9 days after challenge and at euthanasia),
and from homogenised organ material (spleen, kidneys,
lymph nodes) of the foetuses.
The test is invalid if one or more of the vaccinated sows
do not seronconvert, or if no virus is found in more than
50 per cent of the foetuses from the control sows (excluding
mummified foetuses).
The vaccine complies with the test if no virus is found in the
blood of vaccinated sows and in foetuses from the vaccinated
sows, and no antibodies against classical swine-fever virus are
found in the serum of the foetuses from the vaccinated sows.
3. BATCH TESTS
3-1. Identification
For non-lapinised vaccines prepared in cell cultures, the
serum of pigs immunised with the vaccine neutralises
the virus used in the preparation of the vaccine. Specific
classical swine-fever monoclonal antibodies can be used to
identify the vaccinal strain.
3-2. Bacteria and fungi. The vaccine complies with the
test for sterility prescribed in the monograph Vaccines for
veterinary use (0062).
3-3. Mycoplasmas (2.6.7). The vaccine complies with the
test for mycoplasmas.
3-4. Extraneous agents. Mix Neutralize the vaccine
using monoclonal antibodies to the vaccine virus.with a
monospecific antiserum and i Inoculate into susceptible cell
cultures known to be sensitive to viruses pathogenic for pigs
and to pestiviruses. Maintain these cultures for not less than
14 days and carry out at least 3 passages during this period.
Teicoplanin
The vaccine complies with the test if no cytopathic effect

is produced ; the cells show no evidence of the presence of
haemadsorbing agents.
Carry out a haemagglutination test using chicken red blood
cells and the supernatant liquid of the cell cultures. The test
is negative. Carry out a haemadsorption test on the cell
cultures. The test is negative. Use monoclonal antibodies
that can identify possible contamination with pestiviruses.
No virus is detected by an appropriate method.
Use 10 mice each weighing 11 g to 15 g. Inject intracerebrally
into each mouse 0.03 ml of the vaccine reconstituted so that
1 ml contains 1 dose. Observe the mice at least daily for
21 days. The test is invalid and must be repeated if more than
2 mice die within the first 48 h. The vaccine complies with
the test if, from the 3 rd to the 21st day after the injection, no
mouse shows abnormalities attributable to the vaccine.
3-5. Safety. Use 3 2 healthy piglets, 6-7 6-10 weeks old
and that do not have antibodies against pestiviruses, that
have had no contact with swine-fever virus and that do
not have antibodies against swine-fever virus and bovine
viral diarrhoea virus. Administer to each piglet by the
intramuscular a recommended route 10 doses of the vaccine.
Observe the piglets at least daily for 21 14 days. The test is
invalid if a piglet shows notable signs of swine fever or dies
from causes attributable to the vaccine. The vaccine complies
with the test if the body temperature curve remains normal
in each animal and the animals remain in apparent good
health and display normal growth if no animal shows notable
clinical signs of disease or dies from causes attributable to
the vaccine.
3-6. Virus titre. Titrate the vaccine virus in suitable cell
cultures (5.2.4). The vaccine complies with the test if 1 dose
contains not less than the minimum virus titre stated on
the label.
3-7. Pyrogenic character. For vaccines prepared in rabbits
and lapinised vaccines prepared in cell cultures, administer
by the intravenous route 0.5 ml of the vaccine reconstituted
as stated on the label into 1 or more non-immunised rabbits
and 1 or more rabbits immunised either with an identical
dose of a vaccine of the same type injected by the same
route at least 10 days and at most 2 months beforehand or
with a sufficient dose of antiserum administered a few hours
before the injection of the vaccine. Measure the temperature
of the rabbits in the morning and the evening starting 24 h
after the injection and continuing until the 5th day after the
injection. The vaccine is identified by its specific pyrogenic
character leading to a rise in temperature of at least 1.5 C
in the non-immunised rabbits only.
3-8 7. Potency. The vaccine complies with the requirements
of the test prescribed under Immunogenicity (section 2-3-5)
when administered by a recommended route and method. It
is not necessary to carry out the potency test for each batch
of the vaccine if it has been carried out on a representative
batch using a vaccinating dose containing not more than the
minimum virus titre stated on the label.
4. LABELLING
The label states that the vaccine has been prepared in cell
cultures or in rabbits as appropriate.
Reference: PA/PH/Exp. P4/T (04) 26 ANP

At the end of this text there is an annex relating to the
general method Microbiological assay of antibiotics (2.7.2).
This has been included because the use of teicoplanin CRS
as the reference substance in the assay requires that the
general method be revised.
XXXX:2358
TEICOPLANIN
Teicoplaninum
DEFINITION
Mixture of glycopeptide molecules produced by the growth
of certain strains of Actinoplanes teichomyceticus sp. ; the
6 principal components of the mixture are teicoplanin A2-1
to A2-5 and teicoplanin A3-1.
Potency : minimum 900 IU/mg (anhydrous and sodium
chloride-free substance).
CHARACTERS
Appearance : yellowish, amorphous powder.
Solubility : freely soluble in water, sparingly soluble in
N,N-dimethylformamide, practically insoluble in methanol
and in ethanol (96 per cent V/V).
IDENTIFICATION
Comparison : teicoplanin CRS.
B. Examine the chromatograms obtained in the test for
composition.
673
Teicoplanin
Results : the principal peaks in the chromatogram

the chromatogram obtained is similar to the
obtained with the test solution are similar in retention
chromatogram supplied with teicoplanin CRS.
time and size to the principal peaks in the chromatogram Calculate the percentage content of the different components
obtained with reference solution (a).
using the following equations :
TESTS
Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution BY3 or B4
(2.2.2, Method I).
Dissolve 0.8 g in 10 ml of water R.
pH (2.2.3) : 6.5 to 7.5.
Dissolve 0.5 g in carbon dioxide-free water R and dilute to
Composition. Liquid chromatography (2.2.29). Use the
normalisation procedure.
solvent.
Reference solution (a). Dissolve the contents of a vial of
teicoplanin CRS in water R to obtain a concentration of
2 g/l.
Reference solution (c). Dissolve 50.0 mg of mesityl oxide R
in water R and dilute to 25.0 ml with the same solvent.
Dilute 1.0 ml of the solution to 10.0 ml with water R. Dilute
1.0 ml of this solution to 100.0 ml with water R.
Column :
size : l = 0.25 m, = 4.6 mm ;
stationary phase: spherical octadecylsilyl silica gel for
Mobile phase :
mobile phase A : mix 100 ml of acetonitrile R and
900 ml of a 3.0 g/l solution of anhydrous sodium
dihydrogen phosphate R adjusted to pH 6 with 1 M
sodium hydroxide ;
mobile phase B : mix 300 ml of a 3.0 g/l solution of
anhydrous sodium dihydrogen phosphate R adjusted
to pH 6 with 1 M sodium hydroxide, and 700 ml of
acetonitrile R ;
Time
(min)
0 - 30
Mobile phase A
(per cent V/V)
100 50
Mobile phase B
(per cent V/V)
0 50
30 - 31
50 10
50 90
31 - 35
10
90
35 - 40
10 100
90 0

Injection: 20 l.
Relative retention with reference to teicoplanin A2-2 (retention
time = about 18 min) : teicoplanin A3-1 = about 0.43 ;
teicoplanin A3 group 0.70 ; 0.74 < tecoplainin A2
group 1.25 ; teicoplanin A2-1 = about 0.93 ;
teicoplanin A2-3 = about 1.04 ; teicoplanin A2-4 = about 1.12 ;
teicoplanin A2-5 = about 1.14 ; other impurities > 1.25.
teicoplanin A2-4 and teicoplanin A2-5 ;
teicoplanin A2 group
teicoplanin A2-1 group
teicoplanin A2-2
teicoplanin A2-4
teicoplanin A3 group
other impurities
Sa
Sb
Sc
S1
S2
S3
S4
S5
sum of the areas of the peaks due to teicoplanin

A2 in the chromatogram obtained with the test
solution ;
sum of the areas of the peaks due to teicoplanin A3
in the chromatogram obtained with the test
solution ; disregard any peak due to mesityl oxide ;
sum of the areas of any peaks other than those
due to teicoplanin A2 and teicoplanin A3, in the
sum of the areas of the peaks due to teicoplanin A2-1
solution ;
area of the peak due to teicoplanin A2-2 in the
solution ;
area of the peak due to teicoplanin A2-4 in the
solution.
Limits :
teicoplanin A2 group : minimum 80.0 per cent ;
teicoplanin A2-1 group : maximum 20.0 per cent ;
teicoplanin A2-2 : 35.0 to 50.0 per cent ;
teicoplanin A2-4 : maximum 20.0 per cent ;
teicoplanin A3 group : maximum 15.0 per cent ;
any other impurity : maximum 5.0 per cent ;
disregard limit : the area of the peak due to teicoplanin A2-2
(0.5 per cent) ; disregard any peak due to mesityl oxide.
(46) Hypersil ODS-RP 18 is suitable.
674
Teicoplanin
1. teicoplanin A3-1
3. teicoplanin A2-1
5. teicoplanin A2-3
2. mesityl oxide
4. teicoplanin A2-2
6. teicoplanin A2-4
7. teicoplanin A2-5
Figure 2358.-1. Chromatogram for the test for composition of teicoplanin : teicoplanin CRS
Sodium chloride : maximum 5 per cent (anhydrous
substance).
STORAGE
Protected from light, at a temperature of 2 C to 8 C.
Dissolve 1 g in 300 ml of water R, stir and acidify with 2 ml

of nitric acid R. Carry out a potentiometric titration (2.2.20),
using 0.1 M silver nitrate.
Annex
The adoption of the monograph Teicoplanin (2358)

1 ml of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl. will necessitate the revision of the general method 2.7.2.
Microbiological assay of antibiotics with an additional
Heavy metals : maximum 20 ppm.
line in Table 2.7.2.-1 and the introduction of a new culture
0.50 g complies with test G. Prepare the reference solution
medium (medium H), as shown below.
using 100 l of lead standard solution (100 ppm Pb) R.
Medium H
Filter the solutions through a membrane filter (0.45 m).
0.300 g.
Bacterial endotoxins (2.6.14) : less than 0.31 IU/mg.
Peptone
5.0 g
Agar
15.0 g
3.0 g
Beef extract powder

Water to produce
ASSAY
Carry out the microbiological assay of antibiotics (2.7.2),
using the diffusion method. Use teicoplanin CRS as the
reference substance.
1000 ml
pH 7.8 - 8.0 adjusted with 0.1 M sodium hydroxide.
Addition to Table 2.7.2.-1.

Antibiotic
Teicoplanin
Reference substance
Teicoplanin CRS
Solvent to be used
in preparing the
stock solution
pH 6.0 (0.05 M)
Buffer solution
(pH)
Micro-organism
Medium and final

pH ( 0.1 pH unit)
Incubation
temperature
pH 6.0 (0.05 M)
Bacillus subtilis
NCTC 10400
CIP 5262
ATCC 6633
H - pH 7.8-8.0
35-37 C
675
2.6.26. Test for anti-D antibodies in intravenous immunoglobulin

It is proposed in the revised draft below to modify
requirements regarding supply of:
METHOD
The test described in this chapter is performed at room
temperature on the reference solutions, the negative control
solutions and the test solutions at the same time and under
identical conditions.
D-positive red blood cells by allowing, in addition to

OR2R2 donors, the use of OR1R1 and OR1R2 donors,
although OR2R2 donors are preferred ;
Reference solutions and negative control solutions.

Reconstitute the reference preparation and the
negative control according to instructions. Dilute The
D-negative red blood cells by allowing a single Orr
immunoglobulin G (IgG) concentration is 50 g/l in each
donor, although the use of 3 Orr donors is preferred ;
of the reconstituted preparations. Make a twofold dilution
because some blood groups are rare in some countries and of each reconstituted preparation with an equal volume of
PBS containing bovine albumin R at 2 g/l, to give solutions
this leads to supply difficulties.
containing IgG at 25 g/l. Prepare 7 further serial twofold
Furthermore, clarifications are given regarding :
dilutions of each preparation using PBS containing bovine
the use of sodium azide in the PBS buffer used to
albumin R at 2 g/l to give a total extend the dilution range
prepare the reference and test solutions ;
from 1/2 to 1/256 (0.195 g/l IgG). Make 2 independent sets
of
dilutions for each preparation. Add 20 l of each dilution
the calculation of titres of the preparation to be
to
the microtitre plate.
examined and the reference preparation at an IgG
concentration of 25 g/l ; an assigned nominal dilution
of 1 in 2 is given to this 25 g/l solutions in order to allow Test solutions. Initially Dilute the test samples preparation
to be examined with PBS containing bovine albumin R at
comparison of both preparations ;
2 g/l to give a starting IgG concentration of 25 g/l. For
the introduction of requirements regarding the negative 50 g/l products, this is a twofold dilution ; adjust the dilution
control preparation.
factor accordingly for samples that are not 50 g/l to give
XXXX:20626 a starting concentration of 25 g/l for testing. This 25 g/l
solution is assigned a nominal dilution factor of twofold for
comparison with the reference preparations, even if this
2.6.26. TEST FOR ANTI-D ANTIBODIES does not reflect the true dilution factor used to achieve
25 g/l. Prepare 7 further serial twofold dilutions of each
IN HUMAN IMMUNOGLOBULIN FOR
preparation using PBS containing bovine albumin R at 2 g/l
INTRAVENOUS ADMINISTRATION
to give a total extend the nominal dilution range from 1/2
to 1/256 (0.195 g/l IgG) for comparison with the reference
MATERIALS
preparations over the same IgG concentration range. Make 2
Phosphate-buffered saline (PBS). Dissolve 8.0 g of sodium independent sets of dilutions for each test sample. Add 20 l
of each dilution to the microtitre plate.
chloride R, 0.76 g of anhydrous disodium hydrogen
phosphate R, 0.2 g of potassium chloride R and 0.2 g of
Prepare 3 per cent V/V suspensions of papain-treated
potassium dihydrogen phosphate R and 0.2 g of sodium
D-positive (preferably OR2R2, but OR1R1 or OR1R2 may also be
azide R in water R and dilute to 1000 ml with the same
solvent. If the solution has to be kept for several days, 0.2 g used) and D-negative (Orr) red blood cells in PBS containing
of sodium azide R may be added in order to avoid microbial bovine albumin R at 2 g/l. Add 20 l of D-positive cells to
one dilution series of each of the test sample preparation
contamination.
to
be examined, the reference preparation and the negative
Papain solution(47). Use serological grade papain from a
control, and 20 l of D-negative cells to the other dilution
commercial source, the activity of which has been validated. series of each of the test sample preparation to be examined,
the reference preparation and the negative control. Mix by
Red blood cells. Use pooled D-positive red blood cells
shaking the plate on a shaker for 10 s.
from not fewer than 3 donors, preferably of group OR2R2.
D-positive red blood cells may also be obtained from OR1R1
Centrifuge the plate at 80 g for 1 min to pack the cells.
or OR1R2 donors. Mixing phenotypes has not been tested
Place the plate at an angle of approximately 70. Read
and is therefore not recommended.
after 4-5 min (or once the cells have streamed in the wells
Use pooled D-negative red blood cells, preferably from at
containing the negative control and the wells where the
least 3 donors of group Orr. When only 1 donor of group
D-negative cells have been added). A cell button at the
Orr is available, D-negative red blood cells of only 1 donor
bottom of the well indicates a positive result. A stream of
may be used.
cells represents a negative result.
Wash the cells 4 times with PBS or until the supernatant
Record the endpoint titre as the reciprocal of the highest
is clear. Centrifuge the cells at 1800 g for 5 min to pack.
Treat the packed cells with papain solution according to the dilution that gives rise to a positive result.
manufacturers instructions. Store in Alsevers solution for
The negative control must have a titre of less than 2,
not more than 1 week.
otherwise
an investigation of the test reagents and conditions
Microtitre plates(48). Use V-bottomed rigid micro-titre plates. has to be performed.
Reference standards. Immunoglobulin (anti-D antibodies
The titre of the preparation to be examined is not greater
test) BRP and Immunoglobulin (anti-D antibodies test
negative control) BRP are suitable for use as the reference than the titre of the reference preparation when all
preparations are titrated from 25 g/l.
preparation and negative control, respectively.
(47) Papain from LSEZ Diagnostics, catalogue no. PN090, is suitable ; follow manufacturers instructions.
(48) V-bottomed microtitre plates from Greiner, catalogue no. 651101, are suitable.
676
Tinidazole
Result : the principal spot in the chromatogram obtained

the reference solution.
Identification : the TLC test formerly used for both
identification and related substances is now described
E. To about 10 mg add about 10 mg of zinc powder R,
under Identification.
0.3 ml of hydrochloric acid R and 1 ml of water R. Heat
in a water-bath for 5 min and cool. The solution gives the
Related substances : the TLC has been replaced by an LC in
reaction of primary aromatic amines (2.3.1).
accordance with current policy as part of a special revision
programme. The limits proposed are based on batches
TESTS
results.
XXXX:1051 Appearance of solution. The solution is clear (2.2.1) and not
more intensely coloured than reference solution Y5 (2.2.2,
Method
II).
TINIDAZOLE
Dissolve 1.0 g in acetone R and dilute to 20 ml with the
same solvent.
Tinidazolum
(2.2.27), using silica gel GF254 R as the coating substance.
examined in methanol R with the aid of ultrasound and
Test solution (b). Dilute 1.0 ml of test solution (a) to 10 ml
with methanol R.
C8H13N3O4S
Mr 247.3
Reference solution (a). Dissolve 20 mg of tinidazole CRS in
DEFINITION
methanol R and dilute to 10 ml with the same solvent.
1-[2-(Ethylsulphonyl)ethyl]-2-methyl-5-nitro-1H-imidazole.
20 ml with methanol R.
Reference solution (c). Dilute 4 ml of reference solution (b)
CHARACTERS
to 10 ml with methanol R.
Appearance : almost white or pale yellow, crystalline powder. Reference solution (d). Dissolve 10 mg of
Solubility : practically insoluble in water, soluble in acetone 2-methyl-5-nitroimidazole R (impurity A) in methanol R and
and in methylene chloride, sparingly soluble in methanol.
Reference solution (e). Dissolve 10 mg of tinidazole
IDENTIFICATION
impurity B CRS in methanol R and dilute to 100 ml with
the same solvent.
Second identification : A, B, D, E.
Heat the plate at 110 C for 1 h and allow to cool.
Apply separately to the plate 10 l of each solution. Develop
B. Ultraviolet and visible absorption spectrophotometry
over a path of 15 cm using a mixture of 25 volumes of
(2.2.25).
butanol R and 75 volumes of ethyl acetate R. Allow the plate
Test solution. Dissolve 10.0 mg in methanol R and dilute to dry in air and examine in ultraviolet light at 254 nm.
Any spots corresponding to tinidazole impurity A and
tinidazole impurity B in the chromatogram obtained with
solution to 10.0 ml with methanol R.
test solution (a) are not more intense than the corresponding
spots in the chromatogram obtained with reference
Absorption maximum : 310 nm.
solutions (d) and (e) (0.5 per cent), respectively. Any spot,
Specific absorbance at the absorption maximum : 340
apart from the principal spot and any spots corresponding
to 360.
to tinidazole impurity A and tinidazole impurity B in the
chromatogram obtained with test solution (a), is not more
C. Infrared absorption spectrophotometry (2.2.24).
intense than the spot in the chromatogram obtained with
reference solution (b) (0.5 per cent) and at most one such
Comparison : tinidazole CRS.
spot is more intense than the spot in the chromatogram
obtained with reference solution (c) (0.2 per cent).
D. Thin-layer chromatography (2.2.27).
Liquid chromatography (2.2.29). Prepare the solutions
examined in methanol R and dilute to 10 ml with the
immediately before use.
same solvent.
Reference solution. Dissolve 20 mg of tinidazole CRS in examined in 10.0 ml of methanol R and dilute to 100.0 ml
Plate : silica gel GF254 R ; heat the plate at 110 C for 1 h Reference solution (a). Dilute 1.0 ml of the test solution
and allow to cool.
Mobile phase : butanol R, ethyl acetate R (25:75 V/V).
Reference solution (b). Dissolve 5.0 mg of tinidazole
Application : 10 l.
impurity A CRS and 5.0 mg of tinidazole impurity B CRS
in 10.0 ml of methanol R and dilute to 100.0 ml with the
Drying : in air.
mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with
the mobile phase.
677
Tinidazole
1. impurity A
2. impurity B
3. tinidazole
Figure 1051.-1. Chromatogram for the test for related substances of tinidazole : solution of tinidazole spiked with
impurities A and B
Reference solution (c). Dilute 1.0 ml of reference solution (b)
to 50.0 ml with the mobile phase.
Column :
size : l = 0.25 m, = 3.0 mm ;
Mobile phase : acetonitrile R, methanol R, water R
(10:20:70 V/V/V).
Injection : 20 l.
Run time : 1.5 times the retention time of tinidazole.
Relative retention with reference to tinidazole
(retention time = about 6 min) : impurity A = about 0.6 ;
impurity B = about 0.7.
impurities A and B.
Limits :
impurities A, B : for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with reference solution (c) (0.2 per cent) ;
(0.4 per cent) ;
(0.05 per cent).

on 1.0 g.
ASSAY
C8H13N3O4S.
STORAGE
IMPURITIES
A. 2-methyl-5-nitro-1H-imidazole,
B. 1-[2-(ethylsulphonyl)ethyl]-2-methyl-4-nitro-1H-imidazole.
(49) Zorbax SB C8, Symmetry C8 and LiChrospher C8 are suitable.
678
Capsicum
Illustrations of Powdered Drugs

in Herbal Monographs
It has been decided to introduce progressively illustrations of powdered drugs into herbal monographs in order to
complement the corresponding identification section (usually Identification B). These drawings will be published
in Pharmeuropa as they become available.

This monograph is currently published in the 5th Edition.
XXXX:1174
XXXX:1859
BIRCH LEAF
CAPSICUM
Betulae folium
Capsici fructus
A. Epicarp: cells with striated

cuticle (Aa); cells arranged in
rows (Ab) and cells close to the
peduncle (Ac)
F. Fragment of endocarp
in transverse section with
sclerenchymatous cells (Fa)
and thin-walled parenchymatous
cells (Fb)
G. Mesophyll of the calyx with
prisms (Ga), microcrystals (Gb) and
cluster crystals of calcium oxalate
A. Upper epidermis of the

lamina accompanied by palisade
parenchyma
E. Crystal sheaths containing

prisms of calcium oxalate, spongy
parenchyma (Ea) and cells
containing cluster crystals of
calcium oxalate (Eb)
B. Lower epidermis of the lamina

with anomocytic stomata
F. Transverse section of the lamina,

showing a peltate gland in side
view
B. Epicarp in transverse section

showing cuticle (Ba), accompanied
by parenchyma containing
microcrystals of calcium
oxalate (Bb)
C. Peltate gland, in surface view
G. Covering trichomes on
the margin of the lamina (B.
pubescens)
C. Outer epidermis of the calyx
H. Inner epidermis of the calyx

with glandular trichomes
D. Episperm
J. Vessels
D. Xylem vessels accompanied by

sclerenchyma fibres
H. Covering trichomes on the

upper epidermis of the lamina (B.
pubescens)
E. Endosperm
Figure 1174.-1. Illustration of powdered herbal drug of

birch leaf (see identification B)

capsicum (see identification B)
679
Cinchona bark



XXXX:0174
XXXX:1095
CINCHONA BARK
DEVILS CLAW ROOT
Cinchonae cortex
Harpagophyti radix
A. Cork cells in surface view (Aa)

and in transverse section (Ab)
E. Parenchymatous cells
containing starch granules
B. Phloem fibres
F. Starch granules
C. Parenchymatous cells
G. Phloem parenchyma with

medullary ray in tangential section
D. Parenchymatous idioblasts
filled with microprisms of calcium
oxalate

cinchona bark (see identification B)
680
A. Fragments of cork layer in

surface view (Aa) and in transverse
section (Ab)
D. Lignified parenchyma from the

central cylinder
B. Fragments of cortical
parenchyma; fragment containing
yellow droplets (Ba)
E. Parenchyma containing prisms

of calcium oxalate
C. Vessels with reticulate or pitted

thickenings
F. Rectangular or polygonal
sclereids

devils claw root (see identification B)
Ginger

XXXX:1522
GINGER
Zingiberis rhizoma
A. Groups of septate fibres,

sometimes with dentate walls
D. Fragments of cork, in surface

view (Da) and in transverse
section (Db)
B. Vessels with reticulate

thickening (Ba), sometimes
accompanied by cells with brown
contents (Bb)
E. Starch granules, free (Ea) and

in parenchymatous cells (Eb)
C. Parenchyma with cells

containing oleoresin (Ca)

ginger (see identification B)
681
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682
Crospovidone
This section contains proposals for monographs and general texts, new or revised, elaborated under the international
harmonisation procedure (see chapter 5.8 of the European Pharmacopoeia). After these texts have undergone the
harmonisation procedure and have been adopted, they will be included in the European Pharmacopoeia and the
pharmacopoeias of the United States and Japan.
The draft harmonised texts are published for comment (stage 4: official public enquiry in the forum of each of the three
pharmacopoeias). You may send your comments through the appropriate national pharmacopoeia authority at the
address listed on the back cover page of this issue. Readers whose country is not a signatory state of the European
Pharmacopoeia Convention can send their comments directly to the European Directorate for the Quality of Medicines
(see address of the EDQM on the cover of this issue). To facilitate the processing of comments received by the Secretariats
of the national authorities and the EDQM please mention in any correspondence the PA/PH reference number at
the beginning of each text. If you are requesting a change in the limits or are proposing other methods of analysis,
please support your proposal by providing appropriate analytical data obtained on a significant number of samples
and the results of a comparative study between the official method and the proposed method. Comments sent before
31 December 2006 will be considered for the preparation of the final version of the harmonised texts.
We wish to emphasise that these draft texts have not yet been adopted by the European Pharmacopoeia Commission
and therefore cannot be considered to be official texts.
It should be noted that for monographs, a version drafted in the European Pharmacopoeia style is usually published at
the same time in the section on Draft monographs and general texts for comments.

Stage 4
The European Pharmacopoeia is the coordinating
pharmacopoeia for this monograph.
The stage 4 proposal presented below is a revised version
of the proposal that was published in Pharmeuropa 10.4.
The following changes have been made :
identification : the former identification test D has been
replaced by a determination of the screening residue
after wet sieving ;
test for water soluble substances : the limit has been
increased to 1.5 per cent as the content of water can
increase during storage to more than 1.0 per cent ;
assay : the method has been revised and harmonised
with the USP proposal for the harmonisation of the
monograph on povidone.
XXXX:0892
CROSPOVIDONE
DEFINITION
Cross-linked homopolymer of 1-ethenylpyrrolidin-2-one. It
is available in different degrees of powder fineness (type A
and type B).
Content : 11.0 per cent to 12.8 per cent of nitrogen (N ;
Ar 14.01) (dried substance).
CHARACTERS
Appearance : hygroscopic, white or yellowish-white powder
or flakes.
2 grades of crospovidone are available, depending on the
powder fineness : type A and type B.
Solubility : practically insoluble in water, in ethanol 96 per
cent and in methylene chloride.
IDENTIFICATION
Crospovidonum
Comparison : Ph. Eur. reference spectrum of

crospovidone CRS.
B. Suspend 1 g in 10 ml of water R, add 0.1 ml of 0.05 M
iodine and shake for 30 s. Add 1 ml of starch solution R
and shake. No blue colour develops within 30 s.
(C6H9NO)n
Mr (111.1)n
C. To 10 ml of water R, add 0.1 g and shake. A suspension is

formed and no clear solution is obtained within 15 min.
683
Crospovidone

1-vinylpyrrolidin-2-one R in methanol R and dilute
solution to 100.0 ml with methanol R. Dilute 5.0 ml of this
Reference solution (b). Dissolve 10 mg of
1-vinylpyrrolidin-2-one R and 0.50 g of vinyl acetate R in
Dilute 1.0 ml of this solution to 100.0 ml with the mobile
phase.
Precolumn :
size : l = 0.025 m, = 4 mm ;
chromatography R (5 m).
Column :
size : l = 0.25 m, = 4 mm ;
temperature : 40 C.
Mobile phase : acetonitrile R, water R (10:90 V/V).
Flow rate : adjusted so that the retention time of the peak
due to impurity A is about 10 min.
Injection : 50 l. After each injection of the test solution,
m1 = mass of the screen and sample residue, after
wash the precolumn by passing the mobile phase backwards,
drying for 5 h, in grams ;
at the same flow rate as applied in the test, for 30 min.
m2 = mass of the sample, in grams ;
m3 = mass of the screen, in grams.
impurity A and vinyl acetate in the chromatogram
If the screening residue is 50 per cent or more, the
substance is classified as type A ; if the screening residue is repeatability : maximum relative standard deviation of
less than 40 per cent, the substance is classified as type B.
2.0 per cent after 5 injections of reference solution (a).
Limits
:
TESTS
Peroxides. Type A : maximum 400 ppm expressed as H2O2 ;
type B : maximum 1000 ppm expressed as H2O2.
(10 ppm).
Suspend 2.0 g in 50 ml of water R. To 25 ml of this
Heavy
metals (2.4.8) : maximum 10 ppm.
suspension add 2 ml of titanium trichloride-sulphuric
acid reagent R. Allow to stand for 30 min and filter. The
absorbance (2.2.25) of the filtrate, measured at 405 nm
using a mixture of 25 ml of a filtered 40 g/l suspension of
the substance to be examined and 2 ml of a 13 per cent V/V on 0.500 g by drying in an oven at 100-105 C.
solution of sulphuric acid R as the compensation liquid, has
a maximum of 0.35.
on 1.0 g.
For type B use 10 ml of the suspension diluted to 25 ml with
water R for the test.
ASSAY
Place 100.0 mg of the substance to be examined (m mg)
Water-soluble substances : maximum 1.0 1.5 per cent.
Place 25.0 g in a 400 ml beaker, add 200 ml of water R and in a combustion flask and add 5 g of a mixture of 1 g of
stir for 1 h using a magnetic stirrer. Transfer the suspension copper sulphate R, 1 g of titanium dioxide R and 33 g
of dipotassium sulphate R, and 3 glass beads. Wash any
to a 250.0 ml volumetric flask, rinsing with water R, and
adhering
particles from the neck into the flask with a
dilute to volume with the same solvent. Allow the bulk of
small quantity of water R. Add 7 ml of sulphuric acid R,
the solids to settle. Filter about 100 ml of the almost clear
allowing it to run down the sides of the flask, and mix the
supernatant liquid through a 0.45 m membrane filter,
protected by superimposing a 3 m membrane filter. While contents by rotation. Close the mouth of the flask loosely,
filtering, stir the liquid above the filter manually or by means for example by means of a glass bulb with a short stem, to
of a mechanical stirrer, taking care not to damage the filter. avoid excessive loss of sulphuric acid. Heat gradually at
Transfer 50.0 ml of the clear filtrate to a tared 100 ml beaker, first, then increase the temperature until there is vigorous
boiling with condensation of sulphuric acid in the neck of
evaporate to dryness and dry at 105-110 C for 3 h. The
the flask ; precautions are to be taken to prevent the upper
residue weighs a maximum of 50 75 mg.
part of the flask from becoming overheated. Continue the
Impurity A. Liquid chromatography (2.2.29).
heating for 45 min. Cool, dissolve the solid material by
Test solution. Suspend 1.250 g in 50.0 ml of methanol R
cautiously adding to the mixture 20 ml of water R, cool
and shake for 60 min. Leave the bulk to settle and filter
again and place in a steam-distillation apparatus. Add
through a 0.2 m filter.
30 ml of strong sodium hydroxide solution R through the
D. Weigh a suitable quantity of the substance to be examined
(for example 10 mg to 100 mg) and suspend it in 10.0 ml
of water R, adding a wetting agent. Observe under a
microscope at a suitable magnification using a calibrated
ocular micrometer. If the majority of particles are in
the range 50 m to 300 m, the product is classified as
type A. If almost all the particles are below 50 m, the
product is classified as type B. The analytical screens
must be clean and dry. For this purpose the screens are
washed in hot water and allowed to dry overnight in a
drying cabinet at 105 C.
Place 20.00 g in a 1000 ml conical flask, add 500 ml of
water R and stir the suspension for 30 min. Pour the
suspension through a 63 m analytical screen and rinse
the screen with water R until the filtrate is clear. Dry the
screen and sample residue at 105 C for 5 h in a drying
cabinet without circulating air. Cool in a desiccator for
30 min and weigh.
Calculate the screening residue fraction of sample
particles having a diameter of more than 63 m, in grams
per 100 g, using the following expression :
(1) LiChrospher 60 RP8 Select B is suitable.
684
Crospovidone
funnel, rinse the funnel cautiously with 10 ml of water R and

distil immediately by passing steam through the mixture.
Collect 80-100 ml of distillate in a mixture of 30 ml of a
40 g/l solution of boric acid R and 0.05 ml of bromocresol
green-methyl red solution R and enough water R to cover
the tip of the condenser. Towards the end of the distillation
lower the receiver so that the tip of the condenser is above
the surface of the acid solution and rinse the end part of
the condenser with a small quantity of water R. Titrate the
distillate with 0.025 M sulphuric acid until the colour of the
solution changes from green through pale greyish-blue to
pale greyish-red-purple (n1 ml of 0.025 M sulphuric acid).
Repeat the test using about 100 mg of glucose R in place of
the substance to be examined (n2 ml of 0.025 M sulphuric
acid).
a solution of strong sodium hydroxide solution R through

a funnel, cautiously rinse the funnel with 10 ml of water R,
immediately close the clamp attached to the rubber tube,
then start the distillation with steam to obtain 80-100 ml
of distillate. Remove the absorption flask from the lower
end of the condenser tube, rinsing the end part with a small
quantity of water R, and titrate the distillate with 0.025 M
sulphuric acid until the color of the solution changes from
green through pale greyish-blue to pale greyish red-purple.
Carry out a blank determination and make any necessary
correction.
1 ml of 0.025 M sulphuric acid is equivalent to 0.7004 mg
of N.
STORAGE
In an airtight container.
LABELLING
The label states the type (type A or type B).
Gradually heat the flask until the solution has a clear,

yellowish-green colour, and the inside wall of the flask is free IMPURITIES
from carbonised material, and then heat for a further 45 min.
After cooling, cautiously add 20 ml of water R, and connect
the flask to the distillation apparatus previously washed by
passing steam through it. To the absorption flask add 30 ml
of a 40 g/l solution of boric acid R, 3 drops of bromocresol
green-methyl red solution R and sufficient water R to
immerse the lower end of the condenser tube. Add 30 ml of A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).
685
STATE OF WORK
OF INTERNATIONAL HARMONISATION
(Updated June 2006)
Item
General Methods Relevant to Q6A
Dissolution (rev. 1)
Disintegration
Uniformity of Content/Mass
Microbial Contamination
Tests for Specified Microorganism
Microbial Enumeration
Microbial contamination limits for Non-Sterile Products
Bacterial Endotoxins (rev. 1)
Color (instrumental method)
Extractable Volume of Parenterals (rev. 1)
Test for particulate contamination: subvisible particles (rev. 1)
Residue on Ignition (rev. 2)
Sterility test
General Chapters
Analytical Sieving
Bulk Density and Tapped Density
Conductivity
Density of Solids
Flowability (Powder Flow)
Tablet Friability
Heavy Metals
Inhalation
Optical Microscopy
Powder Fineness
Specific Surface Area
Porosimetry by Mercury Intrusion
Laser Diffraction Measurement of Particle Size
X-Ray Powder Diffraction
Water-Solid Interaction
Thermal behaviour of powders
Methods for Biotechnology Products
Amino Acid Determination
Capillary Electrophoresis
Isoelectric Focusing
Protein Determination
Peptide Mapping
Polyacrylamide Gel Electrophoresis
Excipients
Alcohol (rev. 2)
Dehydrated Alcohol (rev. 2)
Benzyl Alcohol (rev. 1)
Calcium Disodium Edetate
Calcium Phosphate Dibasic (and Anhydrous)
Carmellose Calcium (rev. 1)
Carmellose Sodium
Croscarmellose Sodium
Microcrystalline Cellulose (rev. 1)
Cellulose, Powdered (rev. 1)
Cellulose Acetate (rev. 1)
686
CP
Stage
USP
USP
USP
EP
EP
EP
EP
JP
EP
EP
EP
JP
EP
6
6
6
6
6
6
6
2
2
6
6
6
6
USP
EP
EP
EP
USP
USP
USP
EP
USP
USP
EP
EP
EP
EP
EP
EP
6
4
2
4
6
6
3
4
6
4 rev.
6
4
4
4
3
2
USP
EP
EP
USP
USP
EP
6
6
6
6
6
6
EP
EP
EP
JP
JP
USP
USP
USP
USP
USP
USP
5A
5A
6
6
6
6
4
6
6
6
6
Cellulose Acetate Phthalate

Citric Acid, Anhydrous (rev. 1)
Citric Acid, Monohydrate (rev. 1)
Crospovidone
Ethylcellulose
Hydroxyethylcellulose
Hydroxypropylcellulose
Hydroxypropylcellulose, Low Substituted
Hydroxypropylmethylcellulose
Hypromellose Phthalate
Lactose, Anhydrous (rev. 2)
Lactose, Monohydrate
Magnesium Stearate
Methylcellulose (rev. 1)
Methyl Paraben
Petrolatum
Petrolatum, White
Polyethylene Glycol
Polysorbate 80
Povidone
Saccharin
Saccharin, Sodium (rev. 1)
Saccharin, Calcium
Silicon Dioxide
Silicon Dioxide, Colloidal
Sodium Chloride (rev. 2)
Sodium Starch Glycolate (rev. 1)
Starch, Corn (rev. 1)
Starch, Potato
Starch, Rice
Starch, Wheat
Stearic Acid
Sucrose
Talc
Titanium Dioxide
Ethyl Paraben
Propyl Paraben
Butyl Paraben
Glycerin
Carmellose
Calcium Carbonate
Copovidone
Gelatin
Glucose Monohydrate/Anhydrous
Glyceryl Monostearate
Mannitol
Propylene Glycol
Sodium Laurylsulfate
Starch, Pregelatinised
Water for Injection in Containers
Stage 1:
Stage 2:
Stage 3:
Stage 4:
Stage 5A:
Stage 5B:
Stage 6:
Stage 7:
USP
EP
EP
EP
EP
EP
USP
USP
JP
USP
USP
USP
USP
JP
EP
USP
USP
USP
EP
JP
USP
USP
USP
JP
JP
EP
USP
USP
EP
EP
EP
EP
EP
EP
JP
EP
EP
EP
USP
JP
USP
JP
EP
EP
USP
EP
EP
USP
JP
USP
6
6
6
4
6
4-2
4
4
6
5B
6
6
4 rev.
6
6
4
4
4 rev.
4 rev.
5A2
6
6
6
4 rev.
4 rev.
6
6
6
6
5A
6
5A
4
6
5A2
6
6
6
3
4
3
4
3
3
2
3
4
3
2
3
Identification
Investigation
Proposal for Expert Committee Review
Official Inquiry
Provisional Consensus
Draft sign-off Consensus
Regional adoption and implementation
Inter-regional implementation
687
PROJECTED TIMETABLE FOR PUBLICATION AND

IMPLEMENTATION OF TEXTS SIGNED OFF BY
THE PHARMACOPOEIAL DISCUSSION GROUP (PDG)
Effective harmonisation of monographs and general chapters requires that texts signed off by the PDG be published
and implemented by all three pharmacopoeias. This process of adoption and implementation takes place according
to the particular procedure of each pharmacopoeia. The table below shows the projected timetable for publication
and implementation of all signed-off texts, based on information provided by each pharmacopoeia.
Q6A GENERAL CHAPTERS (July 2006)
Item
Sign-off
Dissolution
Disintegration
Content uniformity
Extractable volume of
parenterals
2004/6
2004/6
2004/2
2000/7
EP
2005/6
2005/6
2004/12
2001/6
JP
2006/3
2006/3
2006/3
-
USP
2005/3
2005/3
2005/1
2000/7
Stage 6B
Regional Implementation
EP
JP
USP
2006/1
2006/4
2006/4
2006/1
2006/4
2006/4
2005/6
2006/4
2007/1
2002/1
2002/1
Rev. 1
Particulate matter in
injectables
2004/6
2001/5
2005/6
2002/6
2005/6
-
2005/2
2001/3
2003/1
Rev. 1
Sterility
Tests for specic
microorganisms
Microbiological
enumeration
Microbial contamination
limits for non-sterile
products
Bacterial endotoxins
2004/6
2002/09
2005/11
2003/6
2006/6
2006/3
2004/12
2005/2
2003/5
2006/11
2004/1
2007/1
2005/11
2006/6
2006/11
2007/1
2005/11
2006/6
2006/11
2007/1
2000/1
2000/6
2001/3
2000/7
Sulphated ash/Residue
on ignition
2000/2
2001/6
2002/12
Rev. 1
Rev. 2
Colour and clarity of
solution
2002/9
2004/10
2005/6
(2004/12)
2006/3
Publication
2005/7
-
2006/8
2002/1
2006/4
2005/1
2004/1
2001/1
2001/4
2001/1
2002/11
2002/1
2003/1
(2002/11)
2005/5
2006/1
2005/1
2006/4
(2003/8)
2006/4
Rev. 1
OTHER GENERAL CHAPTERS (July 2006)

Item
688
Publication
Sign-off
EP
JP
USP
Stage 6B
EP
JP
USP
Amino acid determination
2002/9
2003/6
2004/12
2001/12
2005/1
2005/1
2002/1
Capillary electrophoresis
2002/9
2003/6
2004/12
2003/7
2005/1
2005/1
2003/8
Isoelectric focusing
2002/9
2003/6
2004/12
2003/7
2004/1
2005/1
2003/8
Protein determination
2002/9
2003/6
2004/12
2002/12
2004/1
2005/1
2003/1
Peptide mapping
2002/9
2003/6
2004/12
2001/12
2004/1
2005/1
2002/1
Polyacrylamide gel
electrophoresis
1999/10
2001/9
2002/12
2001/1
2002/1
2003/1
2001/3
Tablet Friability
2004/2
2004/12
2006/3
2006/8
2005/7
2006/4
2006/8
Specic Surface Area
2003/11
2004/9
2006/3
2004/7
2005/4
2006/4
2005/4
Analytical Sieving
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
Powder Flow
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
Optical Microscopy
2004/6
2005/6
2006/3
2004/11
2006/1
2006/4
2005/8
MONOGRAPHS (July 2006)

Item
Sign-off
Alcohol (Rev. 1)
Alcohol,
dehydrated
(Rev. 1)
Benzyl alcohol
Rev. 1
Calcium
Disodium edetate
Calcium
phosphate Dibasic
dihydrate
Anhydrous
Calcium
phosphate dibasic
Carmellose
calcium (Rev. 1)
Croscarmellose
sodium
Cellulose acetate
(Rev. 1)
Cellulose acetate
phthalate
Cellulose, microcrystalline
Rev. 1
Cellulose powder
Rev. 1
Citric acid
anhydrous
Rev. 1
Citric acid
monohydrate
Rev. 1
Ethylcellulose
Hypromellose
Hypromellose
phthalate
Lactose
anhydrous
Rev. 2
Lactose
monohydrate
Methyl cellulose
Rev. 1
Methyl
parahydroxybenzoate
Saccharin
Saccharin calcium
(Rev. 1)
Saccharin sodium
(Rev.1)
Sodium chloride
Rev. 2
Sodium starch
glycolate
Rev .1
Starch, corn
Rev.1
Starch, potato
Starch, wheat
Talc
Ethyl
Propyl
parahydroxybenzoate
Butyl
parahydroxybenzoate
2002/9
2002/9
Publication
Stage 6B
EP
JP
USP
2004/1
2006/4
2005/8
2004/1
2006/4
2005/8
EP
2003/6
2003/6
JP
2006/3
2006/3
USP
2004/9
2004/9
2001/10
2004/12
2007/4
2005/1
2007/8
2007/8
2000/7
2005/11
2005/11
2006/10
2004/3
2006/9
2006/7
2005/11
2006/10
2006/7
2007/4
2007/8
2005/11
2006/10
2006/7
2007/4
2007/8
2003/7
2003/9
2004/12
2004/3
2004/4
2005/1
2005/1
2001/10
2001/9
2006/3
2004/3
2002/4
2006/4
2005/1
2003/2
2003/9
2004/3
2004/4
2001/10
2002/6
2004/3
2003/1
2004/12
2004/2
2002/4
2005/1
2005/1
2005/1
2004/7
2005/5
2004/2
2005/5
2001/5
2006/6
2006/3
2006/6
2003/6
2003/11
2001/5
2003/6
2003/11
2002/2
2003/11
2006/6
2002/11
2006/6
2007/1
2003/2
2005/1
2005/4
2007/1
2006/4
2006/3
2002/12
2006/3
2004/7
2006/3
2004/3
2007/1
2004/1
2006/4
2003/1
2007/1
2005/4
2007/1
2005/1
2006/3
2002/12
2004/9
2004/3
2004/1
2006/4
2003/1
2005/8
2005/1
2006/3
2006/4
2006/3
2004/9
2004/3
2006/9
2003/6
2006/3
2005/11
2002/9
2003/6
2003/11
2005/11
2004/2
2003/6
2007/1
2007/7
2006/4
2005/8
2005/1
2007/8
2006/4
2004/1
2006/4
2007/1
2006/3
2006/9
2006/9
2004/1
2006/4
2007/8
2007/8
2006/3
2002/1
2006/3
2006/3
2006/4
2006/9
2004/7
2002/7
2006/4
2006/4
2007/4
2007/8
2005/4
2003/2
2003/2
2005/6
2006/3
2004/7
2004/7
2006/1
2006/4
2006/4
2006/4
2004/2
2005/6
2006/3
2004/7
2006/1
2006/4
2006/4
2001/5
2003.11
2003/11
2003/6
2002/12
2006/3
2004/3
2004/1
2005/1
2004/7
2005/7
2003/1
2006/4
2006/4
2005/9
2004/3
2007/1
2004/12
2005/5
2001/10
2004/2
2001/10
2001/10
2003/11
2004/2
2004/2
2006/6
2002/6
2002/6
2002/6
2004/9
2002/1
2002/1
2004/2
2002/1
2004/12
2006/3
2004/12
2004/12
2007/1
2005/1
2006/4
2005/1
2005/1
2005/4
2006/4
2005/1
2006/3
2006/3
2004/3
2004/3
2004/9
2004/7
2004/7
2005/4
2002/7
2002/7
2006/4
2006/4
2005/1
2005/1
2005/8
2005/4
2005/4
2006/3
2004/7
2002/7
2006/4
2006/1
689
Contents of the JP Forum
PUBLICATION OF THE LISTS OF CONTENTS

APPEARING IN THE JP FORUM AND USP FORUM
Partners in International Harmonisation
JAPANESE PHARMACOPOEIAL FORUM (JP)
Vol. 15, No. 2
June 2006
CONTENTS
Cyanocobalamin Injection
Hydrocortisone Sodium Phosphate
Nicorandil
Roxithromycin
Drafts for revision
Sulbactam Sodium
Drafts for the First Supplement to JP 15
Sultamicillin Tosilate
1. General Tests, Processes and Apparatus
Teicoplanin
(1) Revision
9.
Reference Standards; Standard Solutions;

Reagents, Test Solutions; Measuring
Instruments, Appliances
9.41 Reagents, Test Solutions

2. Official Monographs
(1) Addition
(1) Revision
Alpinia Officinarum Rhizome
Anemarrhena Rhizome
Angelica Dahurica Root
Asiasarum Root
Acemetacin
Asparagus Tuber
Alminoprofen
Atractylodes Rhizome
Amlexanox
Powdered Atractylodes Rhizome
Amlexanox Tablets
Calumba
Azelastine Hydrochloride
Powdered Calumba
Buformine Hydrochloride
Cimicifuga Rhizome
Buformine Hydrochloride Tablets
Clematis Root
Cibenzoline Succinate
Cnidium Rhizome
Doxorubicin Hydrochloride for Injection
Powdered Cnidium Rhizome
Ibudilast
Coptis Rhizome
Labetalol Hydrochloride
Powdered Coptis Rhizome
Labetalol Hydrochloride Tablets
Corydalis Tuber
Manidipine Hydrochloride
Cyperus Rhizome
Manidipine Hydrochloride Tablets
Powdered Cyperus Rhizome
Mitomycin C for Injection
Dioscorea Rhizome
Mizoribine
Powdered Dioscorea Rhizome
Nizatidine
Fritillaria Bulb
Nizatidine Capsules
Gastrodia Tuber
Tobramycin Injection
Gentian
Ubenimex
Powdered Gentian
Zidovudine
Glehnia Root
(2) Revision
690
3. Ofcial Monographs - Crude Drugs
Imperata Rhizome
Calcium Pantothenate
Ipecac
Cefalotin Sodium
Powdered Ipecac
Chlorphenesin Carbamate
Japanese Gentian
Cyanocobalamin
Powdered Japanese Gentian
Contents of the USP Forum
Lindera Root
POLICIES AND ANNOUNCEMENTS
Lithospermum Root
Changes Adopted for the Rules and Procedures of the

20052010 Council of Experts
Lycium Bark
Implementation Period for Upcoming Oficial Revisions to

the USPNF Extended
Mulberry Bark
Notopterygium Rhizome
Coordination of PF Submissions and New USP Reference

Standards
Nuphar Rhizome
Residual Solvents: General Notices and General Chapters

<467> - Implementation Date Delayed
Panax Rhizome
Powdered Panax Rhizome
Immediate IRA for Tablets Containing at Least

Three of the Following Acetaminophen and Salts
of Chlorpheniramine, Dextromethorphan, and
Pseudoephedrine
Polygala Root
Powdered Polygala Root
Polygonum Root
Immediate IRA for Nitrofurantoin Capsules
Polyporus Sclerotium
Immediate IRA for Zinc Sulfate Tablets
Powdered Polyporus Sclerotium
USP Information Expert Committee Members Elected
Processed Aconite Root
USP Annual Scientific Meeting 2006
Powdered Processed Aconite Root
Pharmacopeial Education Courses
Processed Ginger
Visit the USP Web Site at (http://www.usp.org)
Saposhnikovia Root
International Correspondence
Saussurea Root
How to Submit Comments
Scopolia Rhizome
Scutellaria Root
Pharmacopeial Forum Public Review and Comment

Period Deadlines
Powdered Scutellaria Root
Priority New Monograph Items
Senega
FOURTH INTERIM REVISION
Powdered Senega
General Notices and Requirements
Smilax Rhizome
USP MONOGRAPHS
Powdered Smilax Rhizome
Tablets Containing at Least Three of the

Following Acetaminophen and Salts of
Chlorpheniramine, Dextromethorphan, and
Pseudoephedrine
Sophora Root
Powdered Sophora Root
Termeric
Amoxicillin Tablets
Japanese Valerian
Fluoxetine Delayed-Release Capsules
Powdered Japanese Valerian
Nifedipine Extended-Release Tablets
Zedoary
Sterile Water for Inhalation
PHARMACOPOEIAL FORUM (USP)

Vol. 32, No. 4
JulAug 2006
CONTENTS*
Sterile Water for Injection

Sterile Water for Irrigation
Sterile Purified Water
Water for Hemodialysis
STANDARDS DEVELOPMENT
Zinc Sulfate Tablets
HOW TO USE PF
DIETARY SUPPLEMENTS - MONOGRAPHS
Section Descriptions
Valerian
Committee Designations
Powdered Valerian
Staff Directory
ERRATA LIST FOR USP 29NF 24
* The USPNF (USP 30NF 25), the Supplement (Supp), or the Interim Revision Announcement (IRA) for which the revision proposal is targeted
is shown in parentheses next to each proposed item.
691
IN-PROCESS REVISION
Travoprost [new] (2nd Supp to USP 30)
USP MONOGRAPHS
Travoprost Ophthalmic Solution [new] (2nd Supp to

USP 30)
Bemotrizinol [new] (2nd Supp to USP 30)

Bupropion Hydrochloride Extended-Release Tablets
(2nd Supp to USP 30)
Butorphanol Tartrate Nasal Solution [new] (2nd Supp to
USP 30)
Capecitabine [new] (2nd Supp to USP 30)
Capecitabine Tablets [new] (2nd Supp to USP 30)
Carvedilol [new] (2nd Supp to USP 30)
Cipro.oxacin Injection (2nd Supp to USP 30)
Citalopram Hydrobromide (2nd Supp to USP 30)
Dantrolene Sodium Capsules [new] (2nd Supp to USP 30)
Triamcinolone Diacetate (2nd Supp to USP 30)

DIETARY SUPPLEMENTS - MONOGRAPHS
Cats Claw [new] (2nd Supp to USP 30)
Powdered Cats Claw [new] (2nd Supp to USP 30)
Powdered Cats Claw Extract [new] (2nd Supp to USP 30)
Cats Claw Capsules [new] (2nd Supp to USP 30)
Cats Claw Tablets [new] (2nd Supp to USP 30)
Black Cohosh [new] (2nd Supp to USP 30)
Powdered Black Cohosh [new] (2nd Supp to USP 30)
Doxazosin Mesylate [new] (2nd Supp to USP 30)
Powdered Black Cohosh Extract [new] (2nd Supp to

USP 30)
Edetate Disodium (2nd Supp to USP 30)
Black Cohosh Fluidextract [new] (2nd Supp to USP 30)
Edetate Disodium Injection (2 Supp to USP 30)
Black Cohosh Tablets [new] (2nd Supp to USP 30)
Estradiol Vaginal Tablets [new] (2 Supp to USP 30)
Glucosamine Tablets (2nd Supp to USP 30)
nd
nd
Conjugated Estrogens Tablets (2 Supp to USP 30)

nd
Glipizide and Metformin Hydrochloride Tablets [new]

Glyburide Tablets (2nd Supp to USP 30)
Hydrocortisone Tablets (2nd Supp to USP 30)
Hypromellose Ophthalmic Solution (2nd Supp to USP 30)
Glucosamine and Methylsulfonylmethane Tablets [new]

Glucosamine, Chondroitin Sulfate Sodium, and
Methylsulfonylmethane Tablets [new] (2nd Supp to
USP 30)
Maritime Pine (2nd Supp to USP 30)
Irbesartan (2nd Supp to USP 30)
Maritime Pine Extract (2nd Supp to USP 30)
Levodopa (2nd Supp to USP 30)
EXCIPIENTS
Lisinopril Tablets (2nd Supp to USP 30)
Excipients, USP and NF Excipients, Listed by Category

(2nd Supp to NF 25)
Magnesium Hydroxide (2nd Supp to USP 30)

Magnesium Hydroxide Paste (2nd Supp to USP 30)
Metoprolol Tartrate (2nd Supp to USP 30)
Netilmicin Sulfate (2nd Supp to USP 30)
Nevirapine Oral Suspension [new] (2nd Supp to USP 30)
Norgestimate (2nd Supp to USP 30)
Ondansetron Injection (2nd Supp to USP 30)
Pancuronium Bromide Injection [new] (2nd Supp to
USP 30)
NF MONOGRAPHS
Almond Oil (2nd Supp to NF 25)
High Fructose Corn Syrup [new] (2nd Supp to NF 25)
Isomalt (2nd Supp to NF 25)
Polydextrose [new] (2nd Supp to NF 25)
GENERAL CHAPTERS
<11> USP Reference Standards (2nd Supp to USP 30)
<621> Chromatography (2nd Supp to USP 30)
Permethrin [new] (2nd Supp to USP 30)
<660> Containers Glass [new] (2nd Supp to USP 30)
Permethrin Cream [new] (2nd Supp to USP 30)
<661> Containers Plastics (2nd Supp to USP 30)
PEG 3350 and Electrolytes for Oral Solution (2nd Supp to

USP 30)
<671> Containers Performance Testing (2nd Supp to

USP 30)
<681> Repackaging into Single-Unit Containers and

Unit-Dose Containers for Nonsterile Solids and Liquid
Promethazine Hydrochloride Tablets (2nd Supp to USP 30)
Dosage Forms [new] (2nd Supp to USP 30)
Risperidone Tablets [new] (2nd Supp to USP 30)
<721> Distilling Range (2nd Supp to USP 30)
nd
Ritonavir (2 Supp to USP 30)
<905> Uniformity of Dosage Units (2nd Supp to USP 30)
nd
Saccharin Calcium (2 Supp to USP 30)
GENERAL INFORMATION CHAPTERS
Saccharin Sodium (2nd Supp to USP 30)
<1079> Good Storage and Shipping Practices (2nd Supp
Tiamulin Fumarate (2nd Supp to USP 30)
to USP 30)
Promethazine Hydrochloride (2nd Supp to USP 30)
692
<1120> Raman Spectrophotometry (2nd Supp to USP 30)
Phthalic Acid (2nd Supp to USP 30)
<1121> Nomenclature (2nd Supp to USP 30)
Phthalic Anhydride (2nd Supp to USP 30)
<1150> Pharmaceutical Stability (2nd Supp to USP 30)
Phthalimide (2nd Supp to USP 30)
<1226> Verification of Compendial Procedures (2nd Supp

to USP 30)
2-Picoline (2nd Supp to USP 30)
REAGENTS, INDICATORS, AND SOLUTIONS
Picrolonic Acid (2nd Supp to USP 30)
Reagent Specifications
Pipemidic Acid (2nd Supp to USP 30)
n-Butyl Chloride (2nd Supp to USP 30)
Piperidine (2nd Supp to USP 30)
Casein, Hammersten [new] (2nd Supp to USP 30)
Platinic Chloride (2nd Supp to USP 30)
Diaveridine [new] (2nd Supp to USP 30)
Polyethylene Glycol 600 (2nd Supp to USP 30)
Eriochrome Black TSodium Chloride Indicator [new]

Polyethylene Glycol 20,000 (2nd Supp to USP 30)
1-Nonyl Alcohol (2nd Supp to USP 30)
Potassium Acetate (2nd Supp to USP 30)
Octadecyl Silane (2nd Supp to USP 30)
Potassium Bicarbonate (2nd Supp to USP 30)
Octanophenone (2nd Supp to USP 30)
Potassium Biphthalate (2nd Supp to USP 30)
Orange G (2 Supp to USP 30)
Potassium Bisulfate (2nd Supp to USP 30)
Orcinol (2nd Supp to USP 30)
Potassium Bromate (2nd Supp to USP 30)
Osmium Tetroxide (2nd Supp to USP 30)
Potassium Bromide (2nd Supp to USP 30)
Oxalic Acid (2nd Supp to USP 30)
Potassium Carbonate, Anhydrous (2nd Supp to USP 30)
3,3-Oxydipropionitrile (2nd Supp to USP 30)
Potassium Chlorate (2nd Supp to USP 30)
Palladium Chloride (2nd Supp to USP 30)
Potassium Chloride (2nd Supp to USP 30)
Pancreatin (2 Supp to USP 30)
Potassium Chromate (2nd Supp to USP 30)
Para-aminobenzoic Acid (2nd Supp to USP 30)
Potassium Cyanide (2nd Supp to USP 30)
Paraformaldehyde (2nd Supp to USP 30)
Potassium Dichromate (2nd Supp to USP 30)
Pentadecane (2nd Supp to USP 30)
Potassium Ferricyanide (2nd Supp to USP 30)
Pentane (2nd Supp to USP 30)
Potassium Ferrocyanide (2nd Supp to USP 30)
Pepsin (2nd Supp to USP 30)
Potassium Hydroxide (2nd Supp to USP 30)
Perchloric Acid (2 Supp to USP 30)
Potassium Iodate (2nd Supp to USP 30)
Periodic Acid (2nd Supp to USP 30)
Potassium Iodide (2nd Supp to USP 30)
Phenacetin (2nd Supp to USP 30)
Potassium Nitrate (2nd Supp to USP 30)
1,10-Phenanthroline (2nd Supp to USP 30)
Potassium Nitrite (2nd Supp to USP 30)
Phenol (2nd Supp to USP 30)
Potassium Perchlorate (2nd Supp to USP 30)
Phenoxybenzamine Hydrochloride (2nd Supp to USP 30)
Potassium Periodate (2nd Supp to USP 30)
2-Phenoxyethanol (2 Supp to USP 30)
Potassium Permanganate (2nd Supp to USP 30)
Phenyl Isocyanate (2nd Supp to USP 30)
Potassium Persulfate (2nd Supp to USP 30)
DL-Phenylalanine
Potassium Phosphate, Dibasic (2nd Supp to USP 30)
nd
nd
nd
nd
Picric Acid (2nd Supp to USP 30)
Polyvinyl Alcohol (2nd Supp to USP 30)
Phenylhydrazine (2nd Supp to USP 30)
Potassium Phosphate, Monobasic (2nd Supp to USP 30)
Phenylhydrazine Hydrochloride (2nd Supp to USP 30)
Potassium Phosphate, Tribasic (2nd Supp to USP 30)
3-Phenylphenol (2nd Supp to USP 30)
Potassium Pyroantimonate (2nd Supp to USP 30)
Phloroglucinol (2nd Supp to USP 30)
Potassium Pyrophosphate (2nd Supp to USP 30)
Phosphomolybdic Acid (2nd Supp to USP 30)
Potassium Pyrosulfate (2nd Supp to USP 30)
Phosphoric Acid (2nd Supp to USP 30)
Potassium Sodium Tartrate (2nd Supp to USP 30)
Phosphorous Pentoxide (2nd Supp to USP 30)
Potassium Sulfate (2nd Supp to USP 30)
Phthalazine (2nd Supp to USP 30)
Potassium Tellurite (2nd Supp to USP 30)
693
Potassium Thiocyanate (2nd Supp to USP 30)
Sodium Chloride (2nd Supp to USP 30)
Propionaldehyde (2nd Supp to USP 30)
Sodium Chromate (2nd Supp to USP 30)
Propionic Anhydride (2nd Supp to USP 30)
Sodium Cobaltinitrite (2nd Supp to USP 30)
n-Propyl Alcohol (2nd Supp to USP 30)
Sodium Cyanide (2nd Supp to USP 30)
Purine (2nd Supp to USP 30)
Sodium 1-Decanesulfonate (2nd Supp to USP 30)
Pyrazole (2nd Supp to USP 30)
Sodium Dichromate (2nd Supp to USP 30)
Pyrene (2nd Supp to USP 30)
Sodium Diethyldithiocarbamate (2nd Supp to USP 30)
Pyridine (2nd Supp to USP 30)
Sodium Dodecyl Sulfate (2nd Supp to USP 30)
Pyridine, Dried (2nd Supp to USP 30)
Sodium Ferrocyanide (2nd Supp to USP 30)
Pyridoxal Hydrochloride (2nd Supp to USP 30)
Sodium Fluoride (2nd Supp to USP 30)
Pyridoxal 5-Phosphate (2nd Supp to USP 30)
Sodium Glycocholate (2nd Supp to USP 30)
Pyridoxamine Dihydrochloride (2nd Supp to USP 30)
Sodium 1-Heptanesulfonate (2nd Supp to USP 30)
1-(2-Pyridylazo)-2-naphthol (2nd Supp to USP 30)
Sodium 1-Hexanesulfonate (2nd Supp to USP 30)
Pyrogallol (2nd Supp to USP 30)
Sodium Hydrosulfite (2nd Supp to USP 30)
Pyrrole (2nd Supp to USP 30)
Sodium Hydroxide (2nd Supp to USP 30)
Pyruvic Acid (2nd Supp to USP 30)
Sodium Hypochlorite Solution (2nd Supp to USP 30)
Quinhydrone (2nd Supp to USP 30)
Sodium Metabisulfite (2nd Supp to USP 30)
Resazurin (Sodium) (2nd Supp to USP 30)
Sodium Metaperiodate (2nd Supp to USP 30)
Rhodamine B (2nd Supp to USP 30)
Sodium Methoxide (2nd Supp to USP 30)
Rose Bengal Sodium (2nd Supp to USP 30)
Sodium Molybdate (2nd Supp to USP 30)
Ruthenium Red (2nd Supp to USP 30)
Sodium Nitrate (2nd Supp to USP 30)
Safranin O (2nd Supp to USP 30)
Sodium Nitrite (2nd Supp to USP 30)
Salicylaldehyde (2nd Supp to USP 30)
Sodium Nitroferricyanide (2nd Supp to USP 30)
Selenious Acid (2nd Supp to USP 30)
Sodium 1-Octanesulfonate (2nd Supp to USP 30)
Selenium (2nd Supp to USP 30)
Sodium Oxalate (2nd Supp to USP 30)
Selenomethionine (2nd Supp to USP 30)
Sodium (tri) Pentacyanoamino Ferrate (2nd Supp to

USP 30)
Silicic Acid (2nd Supp to USP 30)

Silicon Carbide (2nd Supp to USP 30)
Silicotungstic Acid, n-Hydrate (2nd Supp to USP 30)
Silver Diethyldithiocarbamate (2nd Supp to USP 30)
Silver Nitrate (2nd Supp to USP 30)
Silver Oxide (2nd Supp to USP 30)
Sodium (2nd Supp to USP 30)
Sodium 1-Pentanesulfonate (2nd Supp to USP 30)

Sodium Perchlorate (2nd Supp to USP 30)
Sodium Peroxide (2nd Supp to USP 30)
Sodium Phosphate, Dibasic (2nd Supp to USP 30)
Sodium Phosphate, Dibasic, Anhydrous (2nd Supp to
USP 30)
Sodium Acetate (2nd Supp to USP 30)
Sodium Phosphate, Dibasic, Dodecahydrate [new]

Sodium Acetate, Anhydrous (2nd Supp to USP 30)
Sodium Phosphate, Monobasic (2nd Supp to USP 30)
Sodium Arsenite (2nd Supp to USP 30)
Sodium Phosphate, Tribasic (2nd Supp to USP 30)
Sodium Azide (2nd Supp to USP 30)
Sodium Pyrophosphate (2nd Supp to USP 30)
Sodium Bicarbonate (2nd Supp to USP 30)
Sodium Pyruvate (2nd Supp to USP 30)
Sodium Bisulfite (2nd Supp to USP 30)
Sodium Salicylate (2nd Supp to USP 30)
Sodium Bitartrate (2nd Supp to USP 30)
Sodium Selenite (2nd Supp to USP 30)
Sodium Borate (2nd Supp to USP 30)
Sodium Sulfate (2nd Supp to USP 30)
Sodium Borohydride (2nd Supp to USP 30)
Sodium Sulfate, Anhydrous (2nd Supp to USP 30)
Sodium Bromide (2nd Supp to USP 30)
Sodium Sulfide (2nd Supp to USP 30)
Sodium Carbonate, Anhydrous (2nd Supp to USP 30)
Sodium Sulfite, Anhydrous (2nd Supp to USP 30)
694
Sodium Tartrate (2nd Supp to USP 30)
Tetramethylammonium Nitrate (2nd Supp to USP 30)
Sodium Tetraphenylborate (2nd Supp to USP 30)
4-4-Tetramethyldiaminodiphenylmethane (2nd Supp to

USP 30)
Sodium Thioglycolate (2nd Supp to USP 30)

Sodium Thiosulfate (2nd Supp to USP 30)
Sodium Tungstate (2nd Supp to USP 30)
Stannous Chloride (2nd Supp to USP 30)
Starch, Soluble (2nd Supp to USP 30)
Stearic Acid (2nd Supp to USP 30)
Stearyl Alcohol (2nd Supp to USP 30)
Strontium Acetate (2nd Supp to USP 30)
Strontium Hydroxide (2nd Supp to USP 30)
Strychnine Sulfate (2nd Supp to USP 30)
Sudan III (2nd Supp to USP 30)
Sudan IV (2nd Supp to USP 30)
Sulfamic Acid (2nd Supp to USP 30)
Sulfanilamide (2nd Supp to USP 30)
Sulfanilic Acid (2nd Supp to USP 30)
Sulfosalicylic Acid (2nd Supp to USP 30)
Sulfuric Acid (2nd Supp to USP 30)
Sulfuric Acid, Fuming (2nd Supp to USP 30)
Sulfurous Acid (2nd Supp to USP 30)
Tannic Acid (2nd Supp to USP 30)
Tetrabutylammonium Bromide (2nd Supp to USP 30)
Tetrabutylammonium Hydrogen Sulfate (2nd Supp to
USP 30)
Tetrabutylammonium Hydroxide, 1.0 M in Methanol
Tetrabutylammonium Hydroxide, 40 Percent in Water
Tetrabutylammonium Iodide (2nd Supp to USP 30)
Tetrabutylammonium Phosphate (2nd Supp to USP 30)
Tetracosane (2nd Supp to USP 30)
Tetradecane (2nd Supp to USP 30)
Tetraethylene Glycol (2nd Supp to USP 30)
Tetraethylenepentamine (2nd Supp to USP 30)
Tetraheptylammonium Bromide (2nd Supp to USP 30)
Tetrahydrofuran (2nd Supp to USP 30)
Tetrahydro-2-furnancarboxylic Acid (2nd Supp to USP 30)
1,2,3,4-Tetrahydronaphthalene (2nd Supp to USP 30)
Tetramethylammonium Bromide (2nd Supp to USP 30)
Tetramethylammonium Chloride (2nd Supp to USP 30)
Tetramethylammonium Hydroxide (2nd Supp to USP 30)
Tetramethylammonium Hydroxide, Pentahydrate
Tetramethylammonium Hydroxide Solution in Methanol
Tetramethylsilane (2nd Supp to USP 30)

Theobromine (2nd Supp to USP 30)
Thiazole Yellow (2nd Supp to USP 30)
Thioacetamide (2nd Supp to USP 30)
2-Thiobarbituric Acid (2nd Supp to USP 30)
2,2c-Thiodiethanol (2nd Supp to USP 30)
Thiourea (2nd Supp to USP 30)
Thorium Nitrate (2nd Supp to USP 30)
Thromboplastin (2nd Supp to USP 30)
Thymol (2nd Supp to USP 30)
Tin (2nd Supp to USP 30)
Titanium Tetrachloride (2nd Supp to USP 30)
Titanium Trichloride (2nd Supp to USP 30)
o-Tolidine (2nd Supp to USP 30)
Tolualdehyde (2nd Supp to USP 30)
p-Tolualdehyde (2nd Supp to USP 30)
Toluene (2nd Supp to USP 30)
p-Toluenesulfonic Acid (2nd Supp to USP 30)
p-Toluic Acid (2nd Supp to USP 30)
o-Toluidine (2nd Supp to USP 30)
p-Toluidine (2nd Supp to USP 30)
n-Triacontane (2nd Supp to USP 30)
Tributyl Phosphate (2nd Supp to USP 30)
Tributyrin (2nd Supp to USP 30)
Trichloroacetic Acid (2nd Supp to USP 30)
Trichlorofluoromethane (2nd Supp to USP 30)
n-Tricosane (2nd Supp to USP 30)
Triethylamine (2nd Supp to USP 30)
Triethylamine Hydrochloride (2nd Supp to USP 30)
Triethylene Glycol (2nd Supp to USP 30)
Trifluoroacetic Acid (2nd Supp to USP 30)
Trifluoroacetic Anhydride (2nd Supp to USP 30)
2,2,2-Trifluoroethanol (2nd Supp to USP 30)
5-(Trifluoromethyl)uracil (2nd Supp to USP 30)
Trimethylacethydrazide Ammonium Chloride (2nd Supp
to USP 30)
2,2,4-Trimethylpentane (2nd Supp to USP 30)
2,4,6-Trimethylpyridine (2nd Supp to USP 30)
N-(Trimethylsilyl)-imidazole (2nd Supp to USP 30)
2,4,6-Trinitrobenzenesulfonic Acid (2nd Supp to USP 30)
Trioctylphosphine Oxide (2nd Supp to USP 30)
1,3,5-Triphenylbenzene (2nd Supp to USP 30)
695
Triphenylmethane (2nd Supp to USP 30)
Magnesium Chloride, 0.1 M [new] (2nd Supp to USP 30)
Triphenylmethanol (2nd Supp to USP 30)
Chromatographic Reagents [new] (2nd Supp to USP 30)
Triphenyltetrazolium Chloride (2nd Supp to USP 30)
REFERENCE TABLES
Tris(2-aminoethyl)amine (2nd Supp to USP 30)
Container Specifications for Capsules and Tablets

Tris(hydroxymethyl)aminomethane (2 Supp to USP 30)

nd
Tropaeolin OO (2nd Supp to USP 30)

L-Tryptophane
Description and Solubility (2nd Supp to USP 30)

PREVIOUS PF PROPOSALS STILL PENDING
Tubocurarine Chloride [new] (2nd Supp to USP 30)
CANCELED PROPOSALS
Uracil (2nd Supp to USP 30)
HARMONIZATION
Uranyl Acetate (2nd Supp to USP 30)
USP MONOGRAPHS
Urea (2nd Supp to USP 30)
Dibasic Calcium Phosphate Dihydrate (2nd Supp to

USP 30)
Urethane (2nd Supp to USP 30)

Uridine (2nd Supp to USP 30)
Anhydrous Dibasic Calcium Phosphate (2nd Supp to

USP 30)
Valeric Acid (2nd Supp to USP 30)
Edetate Calcium Disodium (2nd Supp to USP 30)
Valerophenone (2nd Supp to USP 30)

Vanadium Pentoxide (2nd Supp to USP 30)
Vanadyl Sulfate (2nd Supp to USP 30)
Vinyl Acetate (2nd Supp to USP 30)
1-Vinyl-2-pyrrolidone (2nd Supp to USP 30)
Wrights Stain (2nd Supp to USP 30)
Xanthine (2nd Supp to USP 30)
Xanthydrol (2nd Supp to USP 30)
Xylene (2nd Supp to USP 30)
o-Xylene (2 Supp to USP 30)
nd
p-Xylene (2nd Supp to USP 30)

Xylene Cyanole FF (2nd Supp to USP 30)
Xylose (2nd Supp to USP 30)
Zinc (2nd Supp to USP 30)
Zinc Acetate (2nd Supp to USP 30)
Zirconyl Nitrate (2nd Supp to USP 30)
Volumetric Solutions
Bismuth Nitrate [new] (2nd Supp to USP 30)
PHARMACOPEIAL PREVIEWS
STIMULI TO THE REVISION PROCESS
Instructions to Authors
Proposed Monograph for Piroxicam Topical Cream 3%,
A. Ashley, K. Gilbert, C. Pilatti, H. Rowe, B. Voigt, P.
White, and J. Graham Nairn
Preparations for Nebulization: Characterization, Keith
Truman, Steve Nichols, Jolyon Mitchell, Caroline
Vanneste, Markus Tservistas and John Dennis
Correction Formula for the Boiling Point Temperatures
in USP General Chapter Distilling Range <721>, Oscar A.
Quattrocchi, Antonio Hernndez Cardoso and James E.
DeMuth
Bioassay Glossary, Robert Singer, David M. Lansky, and
Walter W. Hauck
Proposed Revisions to USP Standards for
Containers-Glass, C. Jeanne Taborsky, Edward McKinley,
Brian Reamer, Michael Rssler, Desmond Hunt, and
Claudia Okeke
NOMENCLATURE
INDEX
________________________________________________________________________________
CORRESPONDENCE
Individuals who wish to correspond with the USP or the JP concerning any of the monographs/articles mentioned
can do so at the following addresses:
Japanese Pharmacopeial Forum
Secretariat of the Japanese Pharmacopoeia
Evaluation and Licensing Division
Pharmaceutical and Medical Safety Bureau
Ministry of Health and Welfare
1-2-2, Kasumigaseki, Chiyoda-ku
Tokyo, 100-8045, JAPAN
696
Pharmacopeial Forum
The United States Pharmacopeia
Division of Standards Development, USP-NF
12601 Twinbrook Parkway
Rockville, Maryland 20852
USA

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PHARMEUROPA 18.

Pharmeuropa, Pharmeuropa Bio and

Publication of Supplements 5.7 and 5.8

Electronic version of the 5 Edition of the European

Draft Monographs and General Texts

PHARMEUROPA Vol. 18, No. 4, October 2006

THE EUROPEAN PHARMACOPOEIA

PHARMEUROPA Vol. 18, No. 4, October 2006

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PHARMEUROPA Vol. 18, No. 4, October 2006

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PHARMEUROPA Vol. 18, No. 4, October 2006

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PHARMEUROPA Vol. 18, No. 4, October 2006

LIST OF TEXTS ADOPTED

2.4.32. Total cholesterol in oils rich in omega-3-acids

Methods of preparation of homoeopathic stocks and

Castor oil, refined (2367)

Essential oils (2098)

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LIST OF TEXTS PUBLISHED IN SUPPLEMENT 5.7

PHARMEUROPA Vol. 18, No. 4, October 2006

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