2.4.19. Alkaline impurities in fatty oils
01/2008:20419 If no sample preparation and/or measurement method is
described in the individual monograph, a suitable sample
preparation and/or measurement method must be developed
and validated (see Figures 2.4.20.-1 and 2.4.20.-2).
2.4.19. ALKALINE IMPURITIES IN
FATTY OILS
In a test-tube mix 10 mL of recently distilled acetone R and
0.3 mL of water R and add 0.05 mL of a 0.4 g/L solution of
bromophenol blue R in alcohol R. Neutralise the solution if
necessary with 0.01 M hydrochloric acid or 0.01 M sodium
hydroxide. Add 10 mL of the oil to be examined, shake and
allow to stand. Not more than 0.1 mL of 0.01 M hydrochloric
acid is required to change the colour of the upper layer to
yellow.
07/2018:20420 peroxide, at various concentrations, can be used to dissolve
corrected 9.6 the samples. The viscosity of sulfuric acid is greater than that
2.4.20. DETERMINATION OF
ELEMENTAL IMPURITIES
INTRODUCTION
This chapter describes the general approach for the
determination of elemental impurities in medicinal products
or substances for pharmaceutical use. As the chemical
composition of the considered samples and the specification
limits for the element(s) of interest vary considerably, it is
not possible to describe all suitable sample preparation and
measurement methods. Therefore, any method that fulfils the
requirements described in this chapter may be used.
The results of the analysis are acceptable only if the system
suitability has been demonstrated by a suitable test. Before
the initial use of a method, the analyst must ensure that the
method is appropriate for the samples and instruments used.
This is accomplished by applying a validation procedure to
methods not described in the individual monograph or by
a system suitability test for methods which are described in
the monograph. Decision trees for the choice of the sample
preparation and the measurement procedures are presented in
Figures 2.4.20.-1 and 2.4.20.-2.
PROCEDURES
As a reference procedure is not provided for each element,
matrix and concentration, the choice of procedure according
to Figures 2.4.20.-1 and 2.4.20.-2, including sample
preparation, detection technique and instrument parameters,
is the responsibility of the user.
Use the flow chart in Figure 2.4.20.-1 to define the sample
preparation method and the flow chart in Figure 2.4.20.-2 to
define the measurement method. The sample preparation
method should yield a sufficient quantity of sample to allow
quantification of each element at the specified limit stated in
the individual monograph or the general chapter.
All suitable sample preparation methods and measurement
techniques (e.g. 2.2.22. Atomic emission spectrometry (AES),
2.2.23. Atomic absorption spectrometry (AAS), 2.2.37. X-ray
fluorescence spectrometry (XRFS), 2.2.57. Inductively
coupled plasma-atomic emission spectrometry (ICP-AES),
2.2.58. Inductively coupled plasma-mass spectrometry
(ICP-MS), 2.4.2. Arsenic, 2.4.8. Heavy metals, 2.4.9. Iron,
2.4.10. Lead in sugars, 2.4.15. Nickel in polyols, 2.4.31. Nickel in
hydrogenated vegetable oils) can be used for the determination
of elemental impurities, if the method has been verified before
the initial use by a system suitability test or a validation
procedure according to this chapter.
144
EUROPEAN PHARMACOPOEIA 10.
SAMPLE PREPARATION
Sample preparation is critical to the success of elemental
analysis. Many techniques not using direct measurement are
heavily dependent on sample transport.
If an atomisation system is used, the most conventional means
by which samples are introduced into the atomisation system
is by solution nebulisation. In this case, solid samples must
be dissolved in order to be introduced into the atomisation
system. Samples may be dissolved in any appropriate solvent.
The use of aqueous or dilute nitric acid solutions is strongly
recommended, due to minimal interference with these
solvents compared to other solvents. Hydrochloric acid,
hydrofluoric acid, perchloric acid, sulfuric acid and hydrogen
of the other acids and is to be taken into account as it can
affect the overall fluidity of the solution.
The choice of solvents also includes, but is not limited to,
the use of dilute bases, straight or diluted organic solvents,
combinations of acids or bases, and combinations of organic
solvents.
Acids, bases, and hydrogen peroxide of high purity must be
used, especially when ICP-MS is employed. For aqueous
solutions, use deionised distilled water R. Diluents must
be checked for interference if they are used in an analysis.
Because it is not always possible to obtain organic solvents
that are free from elemental impurities, organic solvents of
the highest purity possible with regard to these contaminants
must be used. Specifically for ICP techniques, where samples
are introduced into the plasma via solution nebulisation,
it is important to consider the potential matrix effects and
interferences that might arise from the solvent. The use of an
appropriate internal standard and/or matching the standard
matrix with samples should be applied for ICP-AES and
ICP-MS analyses in cases where accuracy and precision are
not sufficient. In any case, the selection of an appropriate
internal standard should take into account the element(s) of
interest, ionisation energy, wavelengths or masses, and the
nature of the sample matrix.
Where a sample is found not to be soluble in any acceptable
solvent, a variety of digestion or incineration techniques can
be employed. These include hot-plate digestion, incineration
and microwave-assisted digestions, using an open- or
closed-vessel.
The decision regarding the type of digestion technique to be
used depends on the nature of the sample being digested, as
well as on the element(s) of interest and the concentration
range of the elements to be quantified. Open-vessel digestion
is not recommended for the analysis of volatile elements.
The suitability of a digestion technique, whether open-
or closed-vessel, should be supported by spike recovery
experiments in order to verify that, within an acceptable
tolerance, volatile elements have not been lost during sample
preparation. The digestion cycle is suitable if a clear solution
is obtained.
It is important to consider the selection of the type, the
material of construction, the pretreatment, and the cleaning
of analytical labware used in elemental analyses. The material
must be inert and, depending on the specific application,
resistant to caustics, acids, and/or organic solvents. For some
analyses, care must be exercised to prevent the adsorption of
elemental impurities onto the surface of a vessel, particularly
in ultra-trace analyses. Contamination of sample solutions by
elemental impurities and ions present in the container can
also lead to inaccurate results.
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 10.0
Figure 2.4.20.-1. – Elemental impurities decision tree : sample preparation
The use of volumetric glassware that does not comply
with Class A requirements of the appropriate International
Standard of the International Organization for Standardization
(ISO) is acceptable if the validation or the system suitability
test of the method using such glassware have experimentally
demonstrated that the method is suitable for the intended
purpose.
CAUTION : when using high-pressure digestion vessels and
microwave laboratory equipment, the safety precautions and
operating instructions given by the manufacturer must be
followed.
General Notices (1) apply to all monographs and other texts
2.4.20. Determination of elemental impurities
MEASUREMENT
Method. The choice of the techniques depends mainly on
the sample matrix and the characteristics and specification
limits of the element(s) of interest. Analyse according to the
instructions of the manufacturer of the equipment regarding
program and wavelength.
System suitability. A system suitability test must be carried
out on the day of the analysis to ensure that the sample
preparation and measurement system are appropriate.
145
2.4.20. Determination of elemental impurities EUROPEAN PHARMACOPOEIA 10.0

Figure 2.4.20.-2. – Elemental impurities decision tree : measurement

Acceptance criterion for preparation of sample solution : a clear V1
solution is obtained.
Acceptance criterion for measurement system : the measured
concentration of a standard solution of the element at a
concentration within the range of the used calibration curve
does not differ from the actual concentration by more than
20 per cent.
Calculation. The blank value of reagents must be taken into
account for the calculation of the content. Upon completion
of the analysis, the concentration of a given element in
the sample is calculated by the software of the instrument
from the concentration of the element in the test solution.
If no calculation software is available or no indication for
calculation is given in the general chapter corresponding to
the method used, the concentration of a given element in
the sample can be calculated from the concentration of the
element in the solution using the following expression :
= volume of the initial sample preparation, in
millilitres ;
V2 = total volume of any dilution performed, in
millilitres ;
V3 = volume of initial sample preparation used in any
dilution performed, in millilitres.
VALIDATION REQUIREMENTS
Some validation requirements provided below may differ from
those provided in general chapters of the Ph. Eur. (e.g. 2.2.22
(AES), 2.2.23 (AAS), 2.2.57 (ICP-AES), 2.2.58 (ICP-MS)).
Before the initial use of the selected procedure, the analyst
must ensure that the sample preparation and measurement
method are appropriate for the element(s) of interest, sample
matrix and instrument used. This is accomplished by
following the validation procedure before the initial use and
the system suitability test on the day of the analysis.

V1 V2 For elemental impurities, validation of a limit test must

C=A´ ´ include specificity and limit of detection.
m V3
The following section defines the characteristics for the
C = concentration of element in the analysed sample, acceptability of a quantitative procedure. It must be
in micrograms per gram ; demonstrated experimentally that such a procedure complies
A with the validation requirements, with an appropriate system
= instrument reading of the concentration of the
suitability test using material spiked with a suitable reference
element in the sample solution, in micrograms per
material. The test materials must be spiked before any sample
millilitre ;
preparation steps. For example, if a test material is to be
m = mass of the sample in the initial sample solution, digested, the material must be spiked at the beginning of the
in grams ; digestion procedure.

146 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 10.0 2.4.22. Composition of fatty acids by GC

SPECIFICITY and 90 volumes of light petroleum R so that the plate dips

Specificity is the ability to ensure that the analytical procedure about 5 mm beneath the surface of the liquid. When the
(sample preparation and measurement) allows a reliable impregnation mixture has risen by at least 12 cm from the
determination of the element(s) of interest in the presence of lower edge of the plate, remove the plate and allow the solvent
components (e.g. carrier gas, impurities, matrix) that may be to evaporate for 5 min. Carry out the chromatography in the
expected to be present. same direction as the impregnation.
Acceptance criteria : the procedure must be able to assess Preparation of the mixture of fatty acids. Heat 2 g of the oil
unequivocally each elemental impurity to be determined with with 30 mL of 0.5 M alcoholic potassium hydroxide under a
this procedure in the presence of components that may be reflux condenser for 45 min. Add 50 mL of water R, allow
expected to be present, including other elemental impurities, to cool, transfer to a separating funnel and extract with
matrix components and other sources of interference ; three quantities, each of 50 mL, of ether R. Discard the ether
specificity is demonstrated by complying with the accuracy extracts, acidify the aqueous layer with hydrochloric acid R
requirement for the element(s) to be determined. and extract with three quantities, each of 50 mL, of ether R.
RANGE Combine the ether extracts and wash with three quantities,
Acceptance criterion : range is demonstrated by complying each of 10 mL, of water R ; discard the washings, dry the ether
with the recovery requirement. over anhydrous sodium sulfate R and filter. Evaporate the ether
on a water-bath. Use the residue to prepare the test solution.
ACCURACY The fatty acids may also be obtained from the soap solution
Verify the accuracy using a certified reference material prepared during the determination of the unsaponifiable
or by performing a test for recovery. Elemental impurity matter.
solutions CRS may be used. Test solution. Dissolve 40 mg of the mixture of fatty acids
The recovery may be determined on a sample of the substance obtained from the substance to be examined in 4 mL of
to be examined, spiked with a known quantity of a reference chloroform R.
standard of the element of interest (3 concentration levels in Reference solution. Dissolve 40 mg of the mixture of fatty acids
the range of 50-150 per cent of the intended specification limit, obtained from a mixture of 19 volumes of maize oil R and
even if the original concentration of the reference standard is 1 volume of rapeseed oil R in 4 mL of chloroform R.
at the specified value), in triplicate. Apply to the plate 3 μL of each solution. Develop over a path of
Acceptance criterion : spike recovery is within 70 per cent and 8 cm using a mixture of 10 volumes of water R and 90 volumes
150 per cent for the mean of 3 replicates at each concentration. of glacial acetic acid R. Dry the plate at 110 °C for 10 min.
REPEATABILITY Allow to cool and, unless otherwise prescribed, place the plate
Test samples : either 6 independent samples of the substance in a chromatographic chamber, with a tightly fitting lid, that
to be examined spiked with a suitable reference standard at has previously been saturated with iodine vapour by placing
the specified concentration level, or 3 concentration levels iodine R in an evaporating dish at the bottom of the chamber.
prepared in triplicate. After some time brown or yellowish-brown spots become
Acceptance criterion : the relative standard deviation is in both visible. Remove the plate and allow to stand for a few minutes.
cases not more than 20 per cent. When the brown background colour has disappeared, spray
with starch solution R. Blue spots appear which may become
INTERMEDIATE PRECISION brown on drying and again become blue after spraying with
The effect of random events (intra-laboratory variations) on water R. The chromatogram obtained with the test solution
the analytical precision of the method must be established. always shows a spot with an RF of about 0.5 (oleic acid) and
Acceptable experiments for establishing intermediate a spot with an RF of about 0.65 (linoleic acid) corresponding
precision include performing the repeatability analysis on to the spots in the chromatogram obtained with the reference
different days, or with different instrumentation, or by solution. With some oils a spot with an RF of about 0.75 may
different analysts. Only 1 of the 3 experiments is required to be present (linolenic acid). By comparison with the spot in the
demonstrate intermediate precision. chromatogram obtained with the reference solution, verify the
Acceptance criterion : the relative standard deviation is not absence in the chromatogram obtained with the test solution
more than 25 per cent. of a spot with an RF of about 0.25 (erucic acid).
LIMIT OF QUANTIFICATION
Use the results from the accuracy study. Determine the lowest 07/2016:20422
concentration meeting the acceptance criterion.
Acceptance criterion : the limit of quantification is below the
specification limit.
LIMIT OF DETECTION (ONLY APPLICABLE TO LIMIT
TESTS) 2.4.22. COMPOSITION OF FATTY
Determine the lowest concentration giving a signal clearly ACIDS BY GAS CHROMATOGRAPHY
distinct from that obtained with a blank solution.
Acceptance criterion : the limit of detection is not more than The test for foreign oils is carried out on the methyl esters
0.5 times the concentration of the specification limit. of the fatty acids contained in the oil to be examined by gas
chromatography (2.2.28).
01/2008:20421 METHOD A
This method is not applicable to oils that contain glycerides
of fatty acids with an epoxy-, hydroepoxy-, hydroperoxy-,
cyclopropyl or cyclopropenyl group, or those that contain a
large proportion of fatty acids of chain length less than 8 carbon
2.4.21. FOREIGN OILS IN FATTY OILS atoms or to oils with an acid value greater than 2.0.
BY THIN-LAYER CHROMATOGRAPHY Test solution. When prescribed in the monograph, dry the oil
to be examined before the methylation step. Weigh 1.0 g of
Examine by thin-layer chromatography (2.2.27) using the oil into a 25 mL round-bottomed flask with a ground-glass
kieselguhr G R as the coating substance. Impregnate a plate by neck fitted with a reflux condenser and a gas port into the flask.
placing it in a chromatographic tank containing the necessary Add 10 mL of anhydrous methanol R and 0.2 mL of a 60 g/L
quantity of a mixture of 10 volumes of liquid paraffin R solution of potassium hydroxide R in methanol R. Attach the

General Notices (1) apply to all monographs and other texts 147