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ANALYTICAL PROCEDURES_SUMMARY
The anal%tical metho&s use& to test 'u(irocin )ree *ci& to
s(eci+ication are the metho&s o+ the ,uro(ean Pharmaco(oeia-
metho&s e.ui/alent to the ,uro(ean Pharmaco(oiea metho&s or in
house metho&s#
Table 1 Methods Used to Test Mupio!i" Fee A!id to Spe!i#i!atio"
A"al$ti!al Method Method Details
0escri(tion 0escri(tion
1&enti+ication
1n+rare& a2sor(tion s(ectro(hotometr%
'u(irocin content 2% HP34 'u(irocin )ree *ci& 4ontent 2%
HP34
(H Potentiometric &etermination o+ (H
5elate& Su2stances content 2% HP34 0rug 5elate& 1m(urities 2% HP34
S(eci+ic 6PT14*3 rotation 6(tical rotation
Water content Water semi7micro &etermination
5esi&ual Sol/ents 2% 84 5esi&ual Sol/ent 2% 84
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DESCRIPTION
O##i!ial e#ee"!e i" Peu% Ph& Eu&
*s (er current Ph# ,ur monogra(h +or 'u(irocin#
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EUROPEAN P'ARMACOPOEIAL MONO(RAP' FOR INFRARED
A)SORPTION SPECTROP'OTOMETRY
O##i!ial e#ee"!e i" Peu% 4urrent Ph# ,ur
*&*&*+& A)SORPTION SPECTROP'OTOMETRY, INFRARED
1n+rare& s(ectro(hotometers are use& +or recor&ing s(ectra in the
region o+ 400079:0 cm
;1
<!#:71:#4 =m> or in some cases &o"n to !00
cm
;1
<:0 =m>#
APPARATUS
S(ectro(hotometers +or recor&ing s(ectra consist o+ a suita2le light
source- monochromator or inter+erometer an& &etector#
)ourier trans+orm s(ectro(hotometers use (ol%chromatic ra&iation an&
calculate the s(ectrum in the +re.uenc% &omain +rom the original &ata
2% )ourier trans+ormation#
S(ectro(hotometers +itte& "ith an o(tical s%stem ca(a2le o+ (ro&ucing
monochromatic ra&iation in the measurement region ma% also 2e use&#
Normall% the s(ectrum is gi/en as a +unction o+ transmittance- the
.uotient o+ the intensit% o+ the transmitte& ra&iation an& the inci&ent
ra&iation# 1t ma% also 2e gi/en in a2sor2ance#
The a2sor2ance <*> is &e+ine& as the logarithm to 2ase 10 o+ the
reci(rocal o+ the transmittance <T>?
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T @ 1
1o
1
1o @ intensit% o+ inci&ent ra&iation-
1 @ intensit% o+ transmitte& ra&iation#
PREPARATION OF T'E SAMPLE
FOR RECORDIN( )Y TRANSMISSION OR A)SORPTION
Pre(are the su2stance 2% one o+ the +ollo"ing metho&s#
Li-uids# ,xamine a li.ui& either in the +orm o+ a +ilm 2et"een ! (lates
trans(arent to in+rare& ra&iation- or in a cell o+ suita2le (ath length- also
trans(arent to in+rare& ra&iation#
Li-uids o solids i" solutio"# Pre(are a solution in a suita2le sol/ent#
4hoose a concentration an& a (ath length o+ the cell "hich gi/e a
satis+actor% s(ectrum# 8enerall%- goo& results are o2taine& "ith
concentrations o+ 107100 gAl +or a (ath length o+ 0#:70#1 mm#
*2sor(tion &ue to the sol/ent is com(ensate& 2% (lacing in the
re+erence 2eam a similar cell containing the sol/ent use&# 1+ an )T715
instrument is use&- the a2sor(tion is com(ensate& 2% recor&ing the
s(ectra +or the sol/ent an& the sam(le successi/el%# The sol/ent
a2sor2ance- correcte& 2% a com(ensation +actor- is su2tracte& using
calculation so+t"are#
Solids# ,xamine soli&s &is(erse& in a suita2le li.ui& <mull> or in a soli&
<hali&e &isc>- as a((ro(riate# 1+ (rescri2e& in the monogra(h- ma$e a
+ilm o+ a molten mass 2et"een ! (lates trans(arent to in+rare&
ra&iation#
A& Mull
Triturate a small .uantit% o+ the su2stance to 2e examine& "ith the
minimum .uantit% o+ li.ui& (ara++in 5 or other suita2le li.ui&B :710 mg o+
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the su2stance to 2e examine& is usuall% su++icient to ma$e an
a&e.uate mull using one &ro( o+ li.ui& (ara++in 5# 4om(ress the mull
2et"een ! (lates trans(arent to in+rare& ra&iation#
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)& Dis!
Triturate 17! mg o+ the su2stance to 2e examine& "ith 007400 mg-
unless other"ise s(eci+ie&- o+ +inel% (o"&ere& an& &rie& (otassium
2romi&e 5 or (otassium chlori&e 5# These .uantities are usuall%
su++icient to gi/e a &isc o+ 1071: mm &iameter an& a s(ectrum o+
suita2le intensit%# 1+ the su2stance is a h%&rochlori&e- it is
recommen&e& to use (otassium chlori&e 5# 4are+ull% grin& the mixture-
s(rea& it uni+orml% in a suita2le &ie- an& su2mit it to a (ressure o+
a2out 800 'Pa <8 tCcm
;!
># )or su2stances that are unsta2le un&er
normal atmos(heric con&itions or are h%grosco(ic- the &isc is (resse&
in vacuo# Se/eral +actors ma% cause the +ormation o+ +ault% &iscs- such
as insu++icient or excessi/e grin&ing- humi&it% or other im(urities in the
&is(ersion me&ium or an insu++icient re&uction o+ (article siDe# * &isc is
reEecte& i+ /isual examination sho"s lac$ o+ uni+orm trans(arenc% or
"hen transmittance at a2out !000 cm
;1
<: =m> in the a2sence o+ a
s(eci+ic a2sor(tion 2an& is less than 90 (ercent "ithout com(ensation-
unless other"ise (rescri2e&#
(ases# ,xamine gases in a cell trans(arent to in+rare& ra&iation an&
ha/ing an o(tical (ath length o+ a2out 100 mm#
,/acuate the cell an& +ill to the &esire& (ressure through a sto(coc$ or
nee&le /al/e using a suita2le gas trans+er line 2et"een the cell an& the
container o+ the gas to 2e examine&#
1+ necessar% a&Eust the (ressure in the cell to atmos(heric (ressure
using a gas trans(arent to in+rare& ra&iation <+or exam(le nitrogen 5
an& argon 5># To a/oi& a2sor(tion inter+erences &ue to "ater- car2on
&ioxi&e or other atmos(heric gases- (lace in the re+erence 2eam- i+
(ossi2le- an i&entical cell that is either e/acuate& or +ille& "ith the gas
trans(arent to in+rare& ra&iation#
FOR RECORDIN( )Y DIFFUSE REFLECTANCE
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Solids# Triturate a mixture o+ the su2stance to 2e examine& "ith +inel%
(o"&ere& an& &rie& (otassium 2romi&e 5 or (otassium chlori&e 5#
Fse a mixture containing a((roximatel% : (er cent o+ the su2stance-
unless other"ise s(eci+ie&# 8rin& the mixture- (lace it in a sam(le cu(
an& examine the re+lectance s(ectrum#
The s(ectrum o+ the sam(le in a2sor2ance mo&e ma% 2e o2taine&
a+ter mathematical treatment o+ the s(ectra 2% the Ku2el$a7'un$
+unction#
FOR RECORDIN( )Y ATTENUATED TOTAL REFLECTION
*ttenuate& total re+lection <inclu&ing multi(le re+lection> in/ol/es light
2eing re+lecte& internall% 2% a transmitting me&ium- t%(icall% +or a
num2er o+ re+lections# Ho"e/er- se/eral accessories exist "here onl%
one re+lection occurs#
Pre(are the su2stance as +ollo"s# Place the su2stance to 2e examine&
in close contact "ith an internal re+lection element <15,> such as
&iamon&- germanium- Dinc seleni&e- thallium 2romi&e7thallium io&i&e
<K5S7:> or another suita2le material o+ high re+racti/e in&ex# ,nsure
close an& uni+orm contact 2et"een the su2stance an& the "hole
cr%stal sur+ace o+ the internal re+lection element- either 2% a((l%ing
(ressure or 2% &issol/ing the su2stance in an a((ro(riate sol/ent- then
co/ering the 15, "ith the o2taine& solution an& e/a(orating to
&r%ness# ,xamine the attenuate& total re+lectance <*T5> s(ectrum#
IDENTIFICACION USIN( REFERENCE SU)STANCES
Pre(are the su2stance to 2e examine& an& the re+erence su2stance 2%
the same (roce&ure an& recor& the s(ectra 2et"een 400079:0 cm
;1
<!#:71:#4 =m> un&er the same o(erational con&itions# The transmission
minima <a2sor(tion maxima> in the s(ectrum o2taine& "ith the
su2stance to 2e examine& corres(on& in (osition an& relati/e siDe to
those in the s(ectrum o2taine& "ith the re+erence su2stance <CRS).
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When the s(ectra recor&e& in the soli& state sho" &i++erences in the
(ositions o+ the transmission minima <a2sor(tion maxima>- treat the
su2stance to 2e examine& an& the re+erence su2stance in the same
manner so that the% cr%stallise or are (ro&uce& in the same +orm- or
(rocee& as (rescri2e& in the monogra(h- then recor& the s(ectra#
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IDENTIFICATION USIN( REFERENCE SPECTRA
Co"tol o# esolutio" pe#o.a"!e# )or instruments ha/ing a
monochromator- recor& the s(ectrum o+ a (ol%st%rene +ilm
a((roximatel% : =m in thic$ness# The &i++erence x <see )igure !#!#!4#7
1> 2et"een the (ercentage transmittance at the transmission maximum
* at !8G0 cm
;1
<#48 =m> an& that at the transmission minimum B at
!849#: cm
;1
<#:1 =m> must 2e greater than 18# The &i++erence %
2et"een the (ercentage transmittance at the transmission maximum 4
at 1:89 cm
;1
<9#!9 =m> an& that at the transmission minimum 0 at
1:8 cm
;1
<9#! =m> must 2e greater than 10#
)or )ourier7trans+orm instruments- use suita2le instrument resolution
"ith the a((ro(riate a(o&isation (rescri2e& 2% the manu+acturer# The
resolution is chec$e& 2% suita2le means- +or exam(le 2% recor&ing the
s(ectrum o+ a (ol%st%rene +ilm a((roximatel% : =m in thic$ness# The
&i++erence 2et"een the a2sor2ances at the a2sor(tion minimum at
!8G0 cm
;1
an& the a2sor(tion maximum at !849#: cm
;1
is greater than
0## The &i++erence 2et"een the a2sor2ances at the a2sor(tion
minimum at 1:89 cm
;1
an& the a2sor(tion maximum at 1:8 cm
;1
is
greater than 0#08#
#
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Fi/ue *&*&*+&01& H T%(ical s(ectrum o+ (ol%st%rene use& to /eri+% the
resolution (er+ormance
Ieri+ication o+ the "a/e7num2er scale# The "a/e7num2er scale ma% 2e
/eri+ie& using a (ol%st%rene +ilm- "hich has transmission minima
<a2sor(tion maxima> at the "a/e num2ers <in cm
J1
> sho"n in Ta2le
*&*&*+&01&
Ta2le !#!#!4#71# H Transmission minima an& acce(ta2le tolerances o+
a (ol%st%rene +ilm
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Transmission minima <cm
;1
> *cce(ta2le tolerance <cm
71
>
'onochromator
instruments
)ourier7trans+orm
instruments
090#0 K 1#: K 1#0
!849#: K !#0 K 1#0
1901#! K 1#0 K 1#0
1:8#0 K 1#0 K 1#0
11:4#: K 1#0 K 1#0
10!8# K 1#0 K 1#0
Method# Pre(are the su2stance to 2e examine& accor&ing to the
instructions accom(an%ing the re+erence s(ectrumAre+erence
su2stance# Fsing the o(erating con&itions that "ere use& to o2tain the
re+erence s(ectrum- "hich "ill usuall% 2e the same as those +or
/eri+%ing the resolution (er+ormance- recor& the s(ectrum o+ the
su2stance to 2e examine&#
The (ositions an& the relati/e siDes o+ the 2an&s in the s(ectrum o+ the
su2stance to 2e examine& an& the re+erence s(ectrum are concor&ant
in the ! s(ectra#
Co.pe"satio" #o 1ate 2apou a"d at.osphei! !abo" dio3ide#
)or )ourier7trans+orm instruments- s(ectral inter+erence +rom "ater
/a(our an& car2on &ioxi&e is com(ensate& using suita2le algorithms
accor&ing to the manu+acturerLs instructions# *lternati/el%- s(ectra can
2e ac.uire& using suita2le (urge& instruments or ensuring that sam(le
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an& 2ac$groun& single 2eam s(ectra are ac.uire& un&er exactl% the
same con&itions#
IMPURITIES IN (ASES
)or the anal%sis o+ im(urities- use a cell trans(arent to in+rare&
ra&iation an& o+ suita2le o(tical (ath length <+or exam(le- 17!0 m># )ill
the cell as (rescri2e& un&er 8ases# )or &etection an& .uanti+ication o+
the im(urities- (rocee& as (rescri2e& in the monogra(h#
MUPIROCIN FREE ACID CONTENT )Y 'PLC
O##i!ial e#ee"!e i" Peu? 1n7house metho&
Pepaatio" o# Solutio"s
Mobille Phase
Mobile Phase
Potassium &ih%&rogen (hos(hate 0#0!: ' + &iso&ium
h%&rogen (hos(hate 0#0!: '# 0issol/e #9 g o+ KH!P64
an& #: g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at
1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to
1000#0 ml "ith the same sol/ent#
Potassium &ih%&rogen (hos(hate 0#008G ' + &iso&ium
h%&rogen (hos(hate 0#00 '# 0issol/e 1#18 g o+ KH!P64
an& 4#0 g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at
1!0 K ! M4- in car2on &ioxi&e7+ree "ater 5 an& &ilute to
1000#0 ml "ith the same sol/ent#
0iso&ium tetra2orate 0#01 '# 0issol/e #80 g o+
Na!B46G-10H!6 in car2on &ioxi&e7+ree "ater 5 an& &ilute
to 1000#0 ml "ith the same sol/ent# Store (rotecte& +rom
atmos(heric car2on &ioxi&e#
So&ium car2onate 0#0!: ' + so&ium h%&rogen car2onate
0#0!: '# 0issol/e !#94 g o+ Na!46 an& !#09 g o+ NaH46
in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith
the same sol/ent# Store (rotecte& +rom atmos(heric car2on
&ioxi&e#
4alcium h%&roxi&e- saturate& at !: M4# Sha$e an excess o+
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calcium h%&roxi&e 5 "ith car2on &ioxi&e7+ree "ater 5 an&
&ecant at !: M4# Store (rotecte& +rom atmos(heric car2on
&ioxi&e#
ST65*8,
Store 2u++er solutions in suita2le chemicall% resistant- tight
containers- such as t%(e 1 glass 2ottles or (lastic containers
suita2le +or a.ueous solutions
Pepaatio" o# Sa.ple Solutio"
*ccuratel% "eigh 4:7:: mg 'u(irocin )ree *ci& into a !00 m3 /olumetric Nas$#
0issol/e the material in : m3 o+ methanol# ,nsure com(lete &issolution- using
sonication i+ necessar%# 0ilute to /olume using 0#1 ' ammonium acetate solution
(H :#G an& mix thoroughl%#
Pepaatio" o# Sta"dad Solutio"
*ccuratel% "eigh 4:7:: mg 'u(irocin )ree *ci& <or :0790 mg calcium salt> into a
!00 m3 /olumetric Nas$# 0issol/e the material in : m3 o+ methanol# ,nsure
com(lete &issolution- using sonication i+ necessar%# 0ilute to /olume using 0#1 '
ammonium acetate solution (H :#G an& mix thoroughl%#
Cho.ato/aphi! Paa.etes
4olumn? *((rox !: cm containing a
suita2le 2on&e& 48 silica (ac$ing
<e#g# Oor2ax 48- 0u(ont 3t&>
'o2ile (hase? 0#1 ' ammonium acetate <(H
:#G>? TH) <98?!>
)lo" rate? 1#0 m3Amin
0etection? FI at !0 nm
*ttenuation? *2out 0#: *F)S
1nEection Iolume? 10 =3
Po!edue
Set u( the HP34 s%stem using the instrument (arameters &escri2e&
an& ensure the s%stem suita2ilit% criteria are met# 1nEect 10 =3 ali.uots
o+ sam(le an& stan&ar& solutions an& measure the (ea$ areas# The
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retention time o+ 'u(irocin an& &rug7relate& im(urities are gi/en in
Ta2le !
Table *& T$pi!al Rete"tio" Ti.es
Co.po"e"t Mi"utes
0ea& /olume 1#0
B53 :9G98 <Pseu&omonic aci& )> #8
B53 !!89 <Pseu&omonic aci& 0> 9#G
'u(irocin 8#9
Cal!ulatio"
'u(irocin content <P> @ Sam(le area x Stan&ar& Wt x P St& Purit%
Stan&ar& *rea x Sam(le Wt
S$ste. Suitabilit$
)rom six re(licate inEections o+ the stan&ar&- calculate the relati/e
stan&ar& &e/iation o+ the (ea$ area#
RSD @ Stan&ar& &e/iation o+ the (ea$ area x 100
*ritmetic mean o+ the (ea$ area
The 5S0 shoul& not 2e greater than !P#
4alculate the resolution +actor 2et"een (seu&omonic aci& 0
<B53 !!89> an& 'u(irocin- the tailing +actor o+ 'u(irocin an&
the num2er o+ theoretical (lates using a solution o+ the stan&ar&
<Note? 1n the a2sence o+ a la2orator% stan&ar& containing a
measura2le amount o+ (seu&omonic aci& 0- a reasona2l% (ure
s(ecimen o+ 'u(irocin containing a2out P (seu&ominic aci& 0
ma% 2e su2stitute& in the s%stem suita2ilit% tests>#
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5esolution +actor <5> @ ! <T
!
J T
1
>
W
!
+ W
1
Where
T
1
- T
!
@ 5etention time o+ the ! (ea$s
W
1
- W
!
@ Basal (ea$ "i&ths o+ the ! (ea$s
The resolution +actor shoul& not 2e less than #:#
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Tailing +actor <T> @ W 0#0:
!)
Where?
W4&45 @ Pea$ "i&th o+ 'u(irocin at :P (ea$ height#
) @ Wi&th along lea&ing e&ge o+ (ea$ to (oint
2eneath (ea$ a(ex along :P (ea$ height time#
The tailing +actor shoul& not 2e greater than 1#:#
Num2er o+ theoretical (lates <N> @ :#:4: 1
!

W
0#:
Where?
T @ 5etention time o+ 'u(irocin
W
0#:
@ Pea$ "i&th o+ 'u(irocin at hal+ (ea$ height#
Num2er o+ theoretical (lates shoul& not 2e less than 4000#
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EUROPEAN P'ARMACOPOEIAL MONO(RAP' FOR
POTENTIOMETRIC DETERMINATION OF P'
O##i!ial e#ee"!e i" Peu? 4urrent Ph# ,ur
*&*&6 POTENTIOMETRIC DETERMINATION OF P'
The (H is a num2er "hich re(resents con/entionall% the h%&rogen
ion concentration o+ an a.ueous solution# )or (ractical (ur(oses- its
&e+inition is an ex(erimental one# The (H o+ a solution to 2e
examine& is relate& to that o+ a re+erence solution <(H> 2% the
+ollo"ing e.uation?
(h @ (H J , 7 ,:
$
in "hich , is the (otential- ex(resse& in /olts- o+ the cell containing
the solution to 2e examine& an& Es is the (otential- ex(resse& in
/olts- o+ the cell containing the solution o+ $no"n (H <(Hs>- $ is the
change in (otential (er unit change in (H ex(resse& in /olts- an&
calculate& +rom the Nernst e.uation#
Table *&*&6&01& 7 8alues o# 9 at di##ee"t te.peatues
Te.peatue :;C< 9 :8<
1: 0#0:G!
!0 0#0:8!
!: 0#0:9!
0 0#0901
: 0#0911
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The (otentiometric &etermination o+ (H is ma&e 2% measuring the
(otential &i++erence 2et"een ! a((ro(riate electro&es immerse& in
the solution to 2e examine&? 1 o+ these electro&es is sensiti/e to
h%&rogen ions <usuall% a glass electro&e> an& the other is the
re+erence electro&e <+or exam(le- a saturate& calomel electro&e>#
Appaatus# The measuring a((aratus is a /oltmeter "ith an in(ut
resistance at least 100 times that o+ the electro&es use&#
1t is normall% gra&uate& in (H units an& has a sensiti/it% such that
&iscrimination o+ at least 0#0: (H unit or at least 0#00 I ma% 2e
achie/e&#
Method# Fnless other"ise (rescri2e& in the monogra(h- all
measurements are ma&e at the same tem(erature <!07!: M4>#
Table *&*&6&0* sho"s the /ariation o+ (H "ith res(ect to tem(erature
o+ a num2er o+ re+erence 2u++er solutions use& +or cali2ration# )or
the tem(erature correction- "hen necessar%- +ollo" the
manu+acturerLs instructions# The a((aratus is cali2rate& "ith the
2u++er solution o+ (otassium h%&rogen (hthalate <(rimar% stan&ar&>
an& 1 other 2u++er solution o+ &i++erent (H <(re+era2l% one sho"n in
Ta2le !#!##7!># The (H o+ a thir& 2u++er solution o+ interme&iate (H
rea& o++ on the scale must not &i++er 2% more than 0#0: (H unit +rom
the /alue corres(on&ing to this solution# 1mmerse the electro&es in
the solution to 2e examine& an& ta$e the rea&ing in the same
con&itions as +or the 2u++er solutions#
When the a((aratus is in +re.uent use- chec$s must 2e carrie& out
regularl%# 1+ not- such chec$s shoul& 2e carrie& out 2e+ore each
measurement#
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*ll solutions to 2e examine& an& the re+erence 2u++er solutions must
2e (re(are& using car2on &ioxi&e7+ree "ater 5#
PREPARATION OF REFERENCE )UFFER SOLUTIONS
Potassiu. tetao3alate 4&45 M# 0issol/e 1!#91 g o+ 4
4
H

K6
=
-!H
!
6
in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the
same sol/ent#
Potassiu. h$do/e" tatate, satuated at *5 >C# Sha$e an
excess o+ 4
+
H
:
K6
9
/igorousl% "ith !abo" dio3ide0#ee 1ate R at
!: M4# )ilter or &ecant# Pre(are imme&iatel% 2e+ore use#
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Potassiu. dih$do/e" phosphate 4&4*5 M ? disodiu.
h$do/e" phosphate 4&4*5 M# 0issol/e #9 g o+ KH!P64 an&
#: g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in
car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same
sol/ent#
Potassiu. dih$do/e" phosphate 4&44=@ M ? disodiu.
h$do/e" phosphate 4&4646 M# 0issol/e 1#18 g o+ KH!P64 an&
4#0 g o+ Na!HP64- 2oth (re/iousl% &rie& +or ! h at 1!0 K ! M4- in
car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same
sol/ent#
Disodiu. tetaboate 4&41 M# 0issol/e #80 g o+ Na!B46G-
10H!6 in car2on &ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith
the same sol/ent# Store (rotecte& +rom atmos(heric car2on &ioxi&e#
Sodiu. !abo"ate 4&4*5 M ? sodiu. h$do/e" !abo"ate 4&4*5
M# 0issol/e !#94 g o+ Na!46 an& !#09 g o+ NaH46 in car2on
&ioxi&e7+ree "ater 5 an& &ilute to 1000#0 ml "ith the same sol/ent#
Store (rotecte& +rom atmos(heric car2on &ioxi&e#
Cal!iu. h$do3ide, satuated at *5 >C# Sha$e an excess o+
calcium h%&roxi&e 5 "ith car2on &ioxi&e7+ree "ater 5 an& &ecant at
!: M4# Store (rotecte& +rom atmos(heric car2on &ioxi&e#
STORA(E
Store 2u++er solutions in suita2le chemicall% resistant- tight
containers- such as t%(e 1 glass 2ottles or (lastic containers suita2le
+or a.ueous solutions
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DRU( RELATED IMPURITIES )Y 'PLC
Pepaatio" o# Solutio"s
Pepaatio" o# Mobile Phase
Pre(are a 0#1 ' ammonium acetate solution an& a&Eust to (H :#G
"ith small amounts o+ acetic aci&# 'ix the 2u++er "ith
tetrah%&ro+uran in the ratio G0P 2u++er? 0P tetrah%&ro+uran# )ilter
an& &egas 2e+ore use#
Pepaatio" o# Dilue"t
Pre(are a 0#1 ' so&ium acetate solution an& a&Eust to (H 4#0 "ith
small amounts o+ acetic aci&# 'ix the 2u++er "ith methanol in the
ratio <:0?:0>#
Pepaatio" o# Sa.ple Solutio"
*ccuratel% "eigh a2out 0#1 g o+ sam(le into a 10m3 +las$# 0issol/e
in an& &ilute to /olume "ith &iluent#
Pepaatio" o# Sta"dad Solutio"
*ccuratel% "eight a2out 0#0: g o+ the la2orator% re+erence stan&ar&
into a !00 m3 /olumetric +las$# 0issol/e in an& &ilute to /olume "ith
&iluent#
Cho.ato/aphi! Paa.etes
4olumn? *((rox !: cm containing a
suita2le 2on&e& 48 silica (ac$ing
<e#g# Oor2ax 48- 0u(ont 3t&>
'o2ile (hase? 0#1 ' ammonium acetate <(H
:#G>? TH) <G0?0>
)lo" rate? 1#0 m3Amin
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0etection? FI at !40 nm
*ttenuation? 0#0! *F)S
1nEection Iolume? 10 =3
Po!edue
Set u( the HP34 s%stem using the instrument (arameters &escri2e&
an& ensure the s%stem suita2ilit% criteria are met# 1nEect six ali.uots
o+ the stan&ar& solution +ollo"e& 2% an ali.uot <10 =3> o+ each
sam(le#
'easure the (ea$ areas o+ all com(onents- exclu&ing those &ue to
&iluent e++ects- in the sam(le solutions# 'easure the (ea$ areas o+
'u(irocin in the stan&ar& solutions# T%(ical retention times are
gi/en in Ta2le #
Table 6& T$pi!al Rete"tio" Ti.es
4om(onent 'inutes 5elati/e to 'u(irocin
B53 :9G98 <Pseu&omonic aci& )>
B53 4:!94 <5earrangement 1>
B53 4:!9: <5earrangement !>
B53 !!89 <Pseu&omonic aci& 0>
B53 !0!9G <Pseu&omonic aci& B>
'u(irocin <Pseu&omonic aci& *>
B53 4:!99
B53 4:!9G
B53 4:!98
B53 !0!8G <Pseu&omonic aci& 4>
:
G#:
8
9
10
1!
14
1:
!:
!G
0#4!
0#9
0#9G
0#G:
0#8
7
1#1G
1#!:
!#08
!#!:
Cal!ulatio"
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P 1n&i/i&ual im(urit% @ * x St& Wt x S(l Iol x P St& Purit%
*1 x St& Iol x S(l Wt
P Total im(urities @ Q * x St& Wt x S(l Iol x P St& Purit%
*1 x St& Iol x S(l Wt
Where?
* @ *rea o+ an in&i/i&ual im(urit% in the sam(le
*
1
@ is the area o+ mu(irocin in the stan&ar&
Q * is the sum o+ the areas o+ the in&i/i&ual im(urities in the
sam(le#
S$ste. Suitabilit$
)rom at least six inEections o+ the stan&ar& calculate the relati/e
stan&ar& &e/iations o+ the (ea$ area o+ 'u(irocin#
5S0 @ Stan&ar& &e/iation o+ the (ea$ area x 100
*rithmetic mean o+ the (ea$ area
The 5S0 shoul& not 2e greater than :P#
4alculate a resolution +actor 2et"een (seu&omonic aci& 0 <B53
!!89> an& 'u(irocin- the tailing o+ 'u(irocin an& the num2er o+
theoretical (lates using a solution o+ the stan&ar&# <Note? 1n the
a2sence o+ a la2orator% stan&ar& containing a measura2le
amount o+ (seu&ominic aci& 0- a reasona2l% (ure s(ecimen o+
'u(irocin containing a2out P (sue&omonic aci& 0 ma% 2e
su2stitute& in the s%stem suita2ilit% tests>#
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5esolution +actor <5> @ ! <T
!
J T
1
>
W
!
+ W
1
Where?
T
1
- T
!
@ 5etention time o+ the ! (ea$s
W
1
- W
!
@ Basal (ea$ "i&ths o+ the ! (ea$s
The resolution +actor shoul& not 2e less than #:#
Tailing +actor <T> @ W
0#0:
!)
Where?
W
0#0:
@ Pea$ "i&th o+ 'u(irocin at :P (ea$ height
) @ Wi&th along lea&ing e&ge o+ (ea$ to (oint 2eneath
(ea$ a(ex along :P (ea$ height line#
Num2er o+ theoretical (lates <N> @ :#:4: 1
!

W
0#:
Where?
T @ retention time o+ 'u(irocin
W
0#:
@ (ea$ "i&th o+ 'u(irocin at hal+ (ea$ height#
The tailing +actor shoul& not 2e greater than !#0#
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Num2er o+ theoretical (lates shoul& not 2e less than 000#
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EUROPEAN P'ARMACOPEIAL MONO(RAP' FOR OPTICAL
ROTATION
O##i!ial e#ee"!e i" Peu? 4urrent Ph# ,ur
6(tical rotation is the (ro(ert% &is(la%e& 2% chiral su2stances o+
rotating the (lane o+ (olarisation o+ (olarise& light#
6(tical rotation is consi&ere& to 2e (ositi/e <+> +or &extrorotator%
su2stances <i#e# those that rotate the (lane o+ (olarisation in a
cloc$"ise &irection> an& negati/e <;> +or lae/orotator% su2stances#
The s(eci+ic o(tical rotation is the rotation RSmT
t
is the rotation-
ex(resse& in ra&ians <ra&>- measure& at the tem(erature t an& at the
"a/elength U gi/en 2% a 1 m thic$ness o+ li.ui& or a solution containing
1 $gAm o+ o(ticall% acti/e su2stance# )or (ractical reasons the s(eci+ic
o(tical rotation RSmT
t
is normall% ex(resse& in millira&ians metre
s.uare& (er $ilogram <mra&Cm
!
C$g
;1
>#
The Pharmaco(oeia a&o(ts the +ollo"ing con/entional &e+initions#
The angle o+ o(tical rotation o+ a neat li.ui& is the angle o+ rotation S-
ex(resse& in &egrees <M>- o+ the (lane o+ (olarisation at the "a/elength
o+ the 07line o+ so&ium <U @ :89# nm> measure& at !0 M4 using a
la%er o+ 1 &mB +or a solution- the metho& o+ (re(aration is (rescri2e& in
the monogra(h#
The s(eci+ic o(tical rotation RSmT
!0
0
o+ a li.ui& is the angle o+ rotation S-
ex(resse& in &egrees <M>- o+ the (lane o+ (olarisation at the "a/elength
o+ the 07line o+ so&ium <U @ :89# nm> measure& at !0 M4 in the li.ui&
su2stance to 2e examine&- calculate& "ith re+erence to a la%er o+ 1 &m
an& &i/i&e& 2% the &ensit% ex(resse& in grams (er cu2ic centimetre#
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The s(eci+ic o(tical rotation RSmT
!0
0
o+ a su2stance in solution is the
angle o+ rotation S- ex(resse& in &egrees <M>- o+ the (lane o+
(olarisation at the "a/elength o+ the 07line o+ so&ium <U @ :89# nm>
measure& at !0 M4 in a solution o+ the su2stance to 2e examine& an&
calculate& "ith re+erence to a la%er o+ 1 &m containing 1 gAml o+ the
su2stance# The s(eci+ic o(tical rotation o+ a su2stance in solution is
al"a%s ex(resse& "ith re+erence to a gi/en sol/ent an& concentration#
1n the con/entional s%stem a&o(te& 2% the Pharmaco(oeia the s(eci+ic
o(tical rotation is ex(resse& 2% its /alue "ithout unitsB the actual units-
&egree millilitres (er &ecimetre gram R<M>CmlC&m
;1
Cg
;1
T are un&erstoo&#
The con/ersion +actor +rom the 1nternational S%stem to the
Pharmaco(oeia s%stem is the +ollo"ing?
RSmT
t
@ RST
t

x 0#1G4:
1n certain cases s(eci+ie& in the monogra(h the angle o+ rotation ma%
2e measure& at tem(eratures other than !0 M4 an& at other
"a/elengths#
The (olarimeter must 2e ca(a2le o+ gi/ing rea&ings to the nearest
0#01M# The scale is usuall% chec$e& 2% means o+ certi+ie& .uartD (lates#
The linearit% o+ the scale ma% 2e chec$e& 2% means o+ sucrose
solutions#
Method# 0etermine the Dero o+ the (olarimeter an& the angle o+
rotation o+ (olarise& light at the "a/elength o+ the 07line o+ so&ium <U @
:89# nm> at !0 K 0#: M4# 'easurements ma% 2e carrie& out at other
tem(eratures onl% "here the monogra(h in&icates the tem(erature
correction to 2e ma&e to the measure& o(tical rotation# 0etermine the
Dero o+ the a((aratus "ith the tu2e close&B +or li.ui&s the Dero is
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&etermine& "ith the tu2e em(t% an& +or soli&s +ille& "ith the (rescri2e&
sol/ent#
4alculate the s(eci+ic o(tical rotation using the +ollo"ing +ormulae#
)or neat li.ui&s?
RSmT
!0
0
@

SVV
l !0
)or su2stances in solution?
RSmT
!0
0
@ 1000S
l c
"here c is the concentration o+ the solution in grams (er litre
4alculate the content c in grams (er litre or the content cWin (er cent
mAm o+ a &issol/e& su2stance using the +ollo"ing +ormulae?
S @ angle o+ rotation in &egrees rea& at !0 K 0#:M4-
l @ length in &ecimetres o+ the (olarimeter tu2e-
X!0 @ &ensit% at !0 M4 in grams (er cu2ic centimetre#
)or the (ur(oses o+ the Pharmaco(oeia- &ensit% is re(lace& 2%
relati/e &ensit% <!#!#:>-
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*&5&1* AATER% SEMI0MICRO DETERMINATION
The semi7micro &etermination o+ Water is 2ase& u(on the .uantitati/e
reaction o+ "ater "ith sul+ur &ioxi&e an& io&ine in a suita2le anh%&rous
me&ium in the (resence o+ a 2ase "ith su+Ycient 2u++ering ca(acit%#
Appaatus
The a((aratus consists o+ a titration /essel "ith?
H ! i&entical (latinum electro&esB
H tight inlets +or intro&uction o+ sol/ent an& titrantB
H an inlet +or intro&uction o+ air /ia a &esiccantB
H a sam(le inlet Ytte& "ith a sto((er or- +or li.ui&s- a se(tum#
1nlet s%stems +or intro&uction o+ &r% nitrogen or +or as(iration o+
sol/ents ma% also 2e Ytte&#
The titration is carrie& out accor&ing to the instrument su((lierLs
instructions# 4are is ta$en throughout the &etermination to a/oi&
ex(osure o+ reagents an& sol/ents to atmos(heric moisture# The en&7
(oint is &etermine& using ! i&entical in&icator electro&es comrecte& to
an electrical source that maintains 2et"een the electro&es either a
constant current <!#!#9:# Ioltametric titration> or a constant /oltage
<!#!#19# *m(erometric titration># Where &irect titration is use& <metho&
*>- a&&ition o+ titrant causes either a &ecrease in /oltage "here
constant current is maintaine& or an increase in current "here constant
/oltage is maintaine&- until the en&7(oint is reache&# 1nstrmnents "ith
automatic en&7(oint &etection are commonl% use&#
Stan&ar&isation# To the titration /essel- a&& methanol 5- &rie& i+
necessar%- or the sol/ent recommen&e& 2% the su((lier o+ the titrant#
Where a((lica2le +or the a((aratus use&- eliminate resi&ual "ater
Yrom the measurement cell or carr% out a (re7titration# 1ntro&uce a
suita2le amount o+ "ater in an a((ro(riate +orm <"ater 5 or a certiYe&
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re+erence material> an& carr% out the titration- stirring +or the necessar%
time# The "ater e.ui/alent is not less than 80 (er cent o+ that in&icate&
2% the su((lier# Stan&ar&ise the titrant 2e+ore the Yrst use an& at
suita2le inter/als therea+ter#
Fnless other"ise (rescri2e&- use 'etho& *#
Method A# 1ntro&uce into the titration /essel methanol 5- or the sol/ent
in&icate& in the monogra(h or recommen&e& 2% the su((lier o+ the
titrant# Where a((lica2le +or the a((aratus use&- eliminate resi&ual
"ater +rom the measurement cell or carr% out a (re7titration# 1ntro&uce
the su2stance to 2e examine& ra(i&l% an& carr% out the titration- stirring
+or the necessar% extraction time#
Method )# 1ntro&uce into the titration /essel methanol 5- or the sol/ent
in&icate& in the monogra(h or recommen&e& 2% the su((lier o+ the
titrant# Where a((lica2le +or the a((aratus use&- eliminate resi&ual
"ater +rom the measurement cell or carr% out a (re7titration# 1ntro&uce
the su2stance to 2e examine& ra(i&l% an& in a suita2le state o+
&i/ision# *&& an accuratel% measure& /olume o+ the titrant- su+Ycient to
gi/e an excess o+ a2out 1 m3 or the (rescri2e& /olume# *llo" to stan&
(rotecte& +rom light +or 1 min or the (rescri2e& time- "ith stirring#
Titrate the excess o+ reagent using methanol R or the (rescri2e&
sol/ent- containing an accuratel% $no"n .uantit% o+ "ater#
Suitabilit$ The accurac% o+ the &etermination "ith the chosen titrant
must 2e /eri+ie& +or each su2stance to 2e examine&# The +ollo"ing
(roce&ure- gi/en as an exam(le- is suita2le +or sam(les containing !#:7
!: mg o+ "ater#
The "ater content o+ the su2stance to 2e examine& is &etermine&
using the reagentAsol/ent s%stem chosen# Therea+ter- se.uential $no"n
amounts o+ water R are a&&e& in an a((ro(riate +orm <at least :
a&&itions> an& the cumulati/e "ater content &etermine& a+ter each
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%iddlese&# 'W 9"3
CONFIDENTIAL
SmithKline Beecham
Pharmaceuticals
Worthing
West Sussex
BN14 8QH
T +44 190 8!! 000
"""#gs$#com
a&&ition# 4alculate the (ercentage reco/er% <r> at each (oint using the
+ollo"ing ex(ression?
W
1
@ amount o+ "ater a&&e&- in milligramsB
W
!
@ amount o+ "ater +oun&- in milligrams#
4alculate the regression line o+ the cumulati/e "ater &etermine&
against the "ater a&&e&# 4alculate the slo(e <b>- the interce(t "ith
the y7axis <a> an& the interce(t o+ the extra(olate& cali2ration line "ith
the x7axis <d>#
4alculate the (ercentage mean reco/er% <># 4alculate the (ercentage
errors <e
1
an& e
!
> using the +ollo"ing ex(ressions?
a
@
the y7axis interce(t- in milligrams o+ "aterB
d
@
the x7axis interce(t- in milligrams o+ "aterB
M
@ "ater content o+ the su2stance- in milligrams o+ "ater#
The reagentAsol/ent s%stem is consi&ere& to 2e acce(ta2le i+?
H Ze
1
Z an& Ze
!
Z are not greater than !#: (er centB
H b is 2et"een 0#9G: an& 1#0!:
SmithKline Beecham Limited

Registered in England and Wales

No. 2337959

Registered Ofce

9! "reat West Road# Brent$ord

%iddlese&# 'W 9"3
CONFIDENTIAL
SmithKline Beecham
Pharmaceuticals
Worthing
West Sussex
BN14 8QH
T +44 190 8!! 000
"""#gs$#com
RESIDUAL SOL8ENTS )Y (C
O##i!ial e#ee"!e i" Peu% I"0house .ethod
Sa.ple a"d Sta"dad Pepaatio"
Sta"dad Pepaatio"
To a :0 m3 /olumetric +las$ a&& a2out 40 m3 o+ !7methox%ethanol# *&&
!: =3 o+ each o+ the +ollo"ing sol/ents?
He(tane
'eth%l iso2ut%l $etone
,th%l acetate
*cetone
'a$e to /olume "ith ! methox%ethanol#
Sa.ple Pepaatio"
Weigh a2out 1g o+ sam(le into a 10m3 /olumetric +las$- &issol/e an&
&ilute to /olume "ith ! methox%ethanol#
Po!edue
SmithKline Beecham Limited

Registered in England and Wales

No. 2337959

Registered Ofce

9! "reat West Road# Brent$ord

%iddlese&# 'W 9"3