2.4.19. Alkaline impurities in fatty oils
01/2008:20419 If no sample preparation and/or measurement method is
described in the specific 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.
04/2013:20420
2.4.20. DETERMINATION OF METAL
CATALYST OR METAL REAGENT
RESIDUES
INTRODUCTION
This chapter describes the general approach for the
determination of metal catalyst or metal reagent residues
in substances for pharmaceutical use. As the chemical
composition of the considered substances and the specification
limits for the metal(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 specific monograph or
by a system suitability test for methods 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 metal, 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 metal at the specified limit stated in the
specific 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 metal residues, if the method has been verified before the
initial use by a system suitability test or a validation procedure
according to this chapter.
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EUROPEAN PHARMACOPOEIA 9.
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
peroxide, at various concentrations, can be used to dissolve
the samples. The viscosity of sulfuric acid is greater than
that 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 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 of
metals, organic solvents of the highest purity possible with
regard to metal 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 metal(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 open- and 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 metal(s) of interest and the concentration
range of the metals to be quantified. Open-vessel digestion
is not recommended for the analysis of volatile metals.
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 metals 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
metals onto the surface of a vessel, particularly in ultra-trace
analyses. Contamination of sample solutions by metals and
ions present in the container can also lead to inaccurate results.
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
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 9.0
Figure 2.4.20.-1. – Metal residues decision tree : sample preparation
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 metal catalyst or metal reagent residues
MEASUREMENT
Method. The choice of the techniques depends mainly on
the sample matrix and the characteristics and specification
limits of the metal(s) of interest. Analyse according to the
instructions of the manufacturer of the apparatus regarding
programme and wavelength.
139
2.4.20. Determination of metal catalyst or metal reagent residues
Figure 2.4.20.-2. – Metal residues decision tree : measurement
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.
Acceptance criterion for preparation of sample solution : a clear
solution is obtained.
Acceptance criterion for measurement system : the measured
concentration of a standard solution of the metal 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 metal in the sample
is calculated by the software of the instrument from the
concentration of the metal in the test solution. If no calculation
software is available or no indication for calculation is given in
the corresponding general chapter in section 2.2. Physical and
physicochemical methods, the concentration of a given metal
in the sample can be calculated from the concentration of the
metal in the solution using the following expression :
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EUROPEAN PHARMACOPOEIA 9.0
C = concentration of metal in the analysed sample, in
micrograms per gram ;
A = instrument reading of the concentration of the
metal in the sample solution, in micrograms per
millilitre ;
m = mass of the sample in the initial sample solution,
in grams ;
V1 = 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 metal(s), sample matrix and
See the information section on general monographs (cover pages)
EUROPEAN PHARMACOPOEIA 9.0
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.
For metal residues, validation of a limit test must include
specificity and limit of detection.
The following section defines the characteristics for the
acceptability of a quantitative procedure. It must be
demonstrated experimentally that such a procedure complies
with the validation requirements, with an appropriate system
suitability test using material spiked with a suitable reference
material. The test materials must be spiked before any sample
preparation steps. For example, if a test material is to be
digested, the material must be spiked at the beginning of the
digestion procedure.
SPECIFICITY
Specificity is the ability to ensure that the analytical procedures
for sample preparation and measurement allow a reliable
determination of the metal(s) in the presence of components
(e.g. carrier gas, impurities, matrix) that may be expected to
be present.
Acceptance criteria : the procedure must be able to assess
unequivocally each metal residue to be determined with
this procedure in the presence of components that may be
expected to be present, including other metal residues, matrix
components, and other sources of interference ; specificity is
demonstrated by complying with the accuracy requirement
for the metal(s) to be determined.
RANGE
Acceptance criterion : range is demonstrated by complying
with the recovery requirement.
ACCURACY
Verify the accuracy using a certified reference material (CRM)
or by performing a test for recovery.
Recovery. Recovery may be determined on a sample of the
substance to be examined, spiked with a known quantity of a
reference standard of the metal (3 concentration levels in the
range of 50-150 per cent of the intended specification limit,
even if the original concentration of the reference standard is
at the specified value), in triplicate.
Acceptance criterion : spike recovery is within 70 per cent and
150 per cent for the mean of 3 replicates at each concentration.
REPEATABILITY
Test samples : either 6 independent samples of the substance
to be examined spiked with a suitable reference standard at
the specified concentration level, or 3 concentration levels
prepared in triplicate.
Acceptance criterion : the relative standard deviation is in both
cases not more than 20 per cent.
INTERMEDIATE PRECISION
The effect of random events (intra-laboratory variations) on
the analytical precision of the method must be established.
Acceptable experiments for establishing intermediate
precision include performing the repeatability analysis on
different days, or with different instrumentation, or by
different analysts. Only 1 of the 3 experiments is required to
demonstrate intermediate precision.
Acceptance criterion : the relative standard deviation is not
more than 25 per cent.
LIMIT OF QUANTIFICATION
Determine the lowest concentration meeting the acceptance
criterion. Use the results from the accuracy study.
Acceptance criterion : the limit of quantification is below the
specification limit.
LIMIT OF DETECTION (ONLY APPLICABLE TO LIMIT
TESTS)
Determine the lowest concentration giving a signal clearly
distinct from that obtained with a blank solution.
Acceptance criterion : the limit of detection is not more than
0.5 times the concentration of the specification limit.
General Notices (1) apply to all monographs and other texts
2.4.22. Composition of fatty acids by GC
01/2008:20421
2.4.21. FOREIGN OILS IN FATTY OILS
BY THIN-LAYER CHROMATOGRAPHY
Examine by thin-layer chromatography (2.2.27) using
kieselguhr G R as the coating substance. Impregnate a plate by
placing it in a chromatographic tank containing the necessary
quantity of a mixture of 10 volumes of liquid paraffin R
and 90 volumes of light petroleum R so that the plate dips
about 5 mm beneath the surface of the liquid. When the
impregnation mixture has risen by at least 12 cm from the
lower edge of the plate, remove the plate and allow the solvent
to evaporate for 5 min. Carry out the chromatography in the
same direction as the impregnation.
Preparation of the mixture of fatty acids. Heat 2 g of the oil
with 30 mL of 0.5 M alcoholic potassium hydroxide under a
reflux condenser for 45 min. Add 50 mL of water R, allow
to cool, transfer to a separating funnel and extract with
three quantities, each of 50 mL, of ether R. Discard the ether
extracts, acidify the aqueous layer with hydrochloric acid R
and extract with three quantities, each of 50 mL, of ether R.
Combine the ether extracts and wash with three quantities,
each of 10 mL, of water R ; discard the washings, dry the ether
over anhydrous sodium sulfate R and filter. Evaporate the ether
on a water-bath. Use the residue to prepare the test solution.
The fatty acids may also be obtained from the soap solution
prepared during the determination of the unsaponifiable
matter.
Test solution. Dissolve 40 mg of the mixture of fatty acids
obtained from the substance to be examined in 4 mL of
chloroform R.
Reference solution. Dissolve 40 mg of the mixture of fatty acids
obtained from a mixture of 19 volumes of maize oil R and
1 volume of rapeseed oil R in 4 mL of chloroform R.
Apply to the plate 3 μL of each solution. Develop over a path of
8 cm using a mixture of 10 volumes of water R and 90 volumes
of glacial acetic acid R. Dry the plate at 110 °C for 10 min.
Allow to cool and, unless otherwise prescribed, place the plate
in a chromatographic chamber, with a tightly fitting lid, that
has previously been saturated with iodine vapour by placing
iodine R in an evaporating dish at the bottom of the chamber.
After some time brown or yellowish-brown spots become
visible. Remove the plate and allow to stand for a few minutes.
When the brown background colour has disappeared, spray
with starch solution R. Blue spots appear which may become
brown on drying and again become blue after spraying with
water R. The chromatogram obtained with the test solution
always shows a spot with an RF of about 0.5 (oleic acid) and
a spot with an RF of about 0.65 (linoleic acid) corresponding
to the spots in the chromatogram obtained with the reference
solution. With some oils a spot with an RF of about 0.75 may
be present (linolenic acid). By comparison with the spot in the
chromatogram obtained with the reference solution, verify the
absence in the chromatogram obtained with the test solution
of a spot with an RF of about 0.25 (erucic acid).
07/2016:20422
2.4.22. COMPOSITION OF FATTY
ACIDS BY GAS CHROMATOGRAPHY
The test for foreign oils is carried out on the methyl esters
of the fatty acids contained in the oil to be examined by gas
chromatography (2.2.28).
141