Appendix VII Limit Tests 3 - British Pharmacopoeia

8/6/2021 Appendix VII Limit Tests - British Pharmacopoeia
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Appendix VII Limit Tests

Nessler Cylinders
(No Ph. Eur. method)
Where the use of Nessler cylinders is prescribed in a test of the Pharmacopoeia, Nessler cylinders complying with the following requirements should be used.
Nessler cylinders comply with British Standard 612:1966 (Specification for Nessler cylinders). They are of clear glass with a nominal capacity of 50 mL; the overall height is about 15
cm, the external height to the 50-mL mark 11.0 to 12.4 cm, the thickness of the wall 1.0 to 1.5 mm and the thickness of the base 1.0 to 3.0 mm. The external heights to the 50-mL
mark of cylinders used for a test must not differ by more than 1 mm.
Tubes for Comparative Tests
(Ph. Eur. method 2.1.5)
Tubes used for comparative tests are matched tubes of colourless glass with a uniform internal diameter. The base is transparent and flat.
A column of the liquid is examined down the vertical axis of the tube against a white background, or if necessary, against a black background. The examination is carried out in
diffused light.
It is assumed that tubes with an internal diameter of 16 mm will be used. Tubes with a larger internal diameter may be used instead but the volume of liquid examined must then be
increased so that the depth of liquid in the tubes is not less than where the prescribed volume of liquid and tubes 16 mm in internal diameter are used.
Limit Test for Aluminium
Place the prescribed solution in a separating funnel and shake with 2 quantities, each of 20 mL, and then with one 10 mL quantity of a 5 g/L solution of hydroxyquinoline R in
chloroform R. Dilute the combined chloroform solutions to 50.0 mL with chloroform R (test solution).
Prepare a standard in the same manner using the prescribed reference solution.
Prepare a blank in the same manner using the prescribed blank solution.
Measure the intensity of the fluorescence (2.2.21) of the test solution (I1), of the standard (I2) and of the blank (I3) using an excitant beam at 392 nm and a secondary filter with a
transmission band centred on 518 nm or a monochromator set to transmit at this wavelength.
The fluorescence (I1-I3) of the test solution is not greater than that of the standard (I2-I3).
Limit Test for Ammonium
Unless otherwise prescribed, use method A.
METHOD A
Introduce the prescribed solution into a test-tube or dissolve the prescribed quantity of the substance to be examined in 14 mL of water R in a test-tube. Make the solution alkaline if
necessary by the addition of dilute sodium hydroxide solution R, dilute to 15 mL with water R and add 0.3 mL of alkaline potassium tetraiodomercurate solution R. Prepare a
standard by mixing 10 mL of ammonium standard solution (1 ppm NH4) R, 5 mL of water R and 0.3 mL of alkaline potassium tetraiodomercurate solution R. Stopper the test-tubes.
After 5 min, any yellow colour in the test solution is not more intense than that in the standard.
METHOD B
In a 25 mL jar fitted with a cap, place the prescribed quantity of the finely powdered substance to be examined and dissolve or suspend in 1 mL of water R. Add 0.30 g of heavy
magnesium oxide R. Close immediately after placing a piece of silver manganese paper R 5 mm square, wetted with a few drops of water R, under the polyethylene cap. Swirl,
avoiding projections of liquid, and allow to stand at 40 °C for 30 min. If the silver manganese paper shows a grey colour, it is not more intense than that of a standard prepared at the
same time and in the same manner using the prescribed volume of ammonium standard solution (1 ppm NH4) R, 1 mL of water R and 0.30 g of heavy magnesium oxide R.
Limit Test for Arsenic
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A. Lead acetate paper/cotton
Figure 2.4.2.-1. – Apparatus for the limit test for arsenic (method A)
Dimensions in millimetres
METHOD A
The apparatus (see Figure 2.4.2.-1) consists of a 100 mL conical flask closed with a ground-glass stopper through which passes a glass tube about 200 mm long and about 5 mm in
internal diameter. The lower part of the tube tapers to an internal diameter of 1 mm, and about 20 mm from its tip is a lateral orifice 2-3 mm in diameter. When the tube is in position
in the stopper, the lateral orifice is at least 3 mm below the lower surface of the stopper. A second glass tube of the same internal diameter is connected to the first tube. The second
tube is bent twice at right angles and the free end of the tube tapers to an internal diameter of 1 mm. This end is immersed in a test-tube containing 3.0 mL of silver
diethyldithiocarbamate solution R. Other suitable equipment may be used. Into the first tube insert 50-60 mg of lead acetate cotton R, loosely packed, or a small plug of cotton and a
rolled piece of lead acetate paper R weighing 50-60 mg.
In the conical flask, dissolve the prescribed quantity of the substance to be examined in 25 mL of water R, or in the case of a solution adjust the prescribed volume to 25 mL with
water R. Add 15 mL of hydrochloric acid R, 0.1 mL of stannous chloride solution R and 5 mL of potassium iodide solution R, allow to stand for 15 min and introduce 5 g of activated
zinc R. Assemble the 2 parts of the apparatus immediately and immerse the flask in a water-bath at a temperature such that a uniform evolution of gas is maintained. Prepare a
standard in the same manner, using 1 mL of arsenic standard solution (1 ppm As) R, diluted to 25 mL with water R. If foaming occurs, 1 mL of 2-propanol R may be added to the
flask.
After at least 2 h, the colour obtained in the test-tube with the test solution is not more intense than that obtained with the standard.
Suitability test The colour obtained in the test-tube with the standard is at least as intensely coloured as 3 mL of a mixture of 3.0 mL of yellow primary solution, 0.6 mL of red
primary solution and 11.40 mL of a solution of hydrochloric acid R (10 g/L HCl) (2.2.2, Method I).
METHOD B
Introduce the prescribed quantity of the substance to be examined into a test-tube containing 4 mL of hydrochloric acid R and about 5 mg of potassium iodide R and add 3 mL of
hypophosphorous reagent R. Heat the mixture on a water-bath for 15 min, shaking occasionally. Prepare a standard in the same manner, using 0.5 mL of arsenic standard solution
(10 ppm As) R.
After heating on the water-bath, the colour obtained in the test-tube with the test solution is not more intense than that obtained with the standard.
Limit Test for Calcium
All solutions used for this test are prepared with distilled water R.
To 0.2 mL of alcoholic calcium standard solution (100 ppm Ca) R add 1 mL of ammonium oxalate solution R. After 1 min add a mixture of 1 mL of dilute acetic acid R and 15 mL of
the prescribed solution or of a solution containing the prescribed quantity of the substance to be examined, and shake. Prepare a standard in the same manner using a mixture of
10 mL of aqueous calcium standard solution (10 ppm Ca) R, 1 mL of dilute acetic acid R and 5 mL of distilled water R.
After 15 min, any opalescence in the test solution is not more intense than that in the standard.
Limit Test for Chlorides
To 15 mL of the prescribed solution add 1 mL of dilute nitric acid R and pour the mixture as a single addition into a test-tube containing 1 mL of silver nitrate solution R2. Prepare a
standard in the same manner using 10 mL of chloride standard solution (5 ppm Cl) R and 5 mL of water R. Examine the tubes laterally against a black background.
After standing for 5 min protected from light, any opalescence in the test solution is not more intense than that in the standard.
Limit Test for Fluorides
Introduce into the inner tube of the apparatus (see Figure 2.4.5.-1) the prescribed quantity of the substance to be examined, 0.1 g of acid-washed sand R and 20 mL of a mixture of
equal volumes of sulfuric acid R and water R. Heat the jacket containing tetrachloroethane R maintained at its boiling point (146 °C). Heat the steam generator and distil, collecting
the distillate in a 100 mL volumetric flask containing 0.3 mL of 0.1 M sodium hydroxide and 0.1 mL of phenolphthalein solution R. Maintain a constant volume (20 mL) in the tube
during distillation and ensure that the distillate remains alkaline, adding 0.1 M sodium hydroxide if necessary. Dilute the distillate to 100 mL with water R (test solution). Prepare a
standard in the same manner by distillation, using 5 mL of fluoride standard solution (10 ppm F) R instead of the substance to be examined. Into two glass-stoppered cylinders
introduce 20 mL of the test solution and 20 mL of the standard and 5 mL of aminomethylalizarindiacetic acid reagent R.
After 20 min, any blue colour in the test solution (originally red) is not more intense than that in the standard.
Figure 2.4.5.-1. – Apparatus for limit test for fluorides
Limit Test for Heavy Metals
The methods described below require the use of thioacetamide reagent R. As an alternative, sodium sulfide solution R1 (0.1 mL) is usually suitable. Since tests prescribed in
monographs have been developed using thioacetamide reagent R, if sodium sulfide solution R1 is used instead, it is necessary to include also for methods A, B and H a monitor
solution, prepared from the quantity of the substance to be examined prescribed for the test, to which has been added the volume of lead standard solution prescribed for
preparation of the reference solution. The test is invalid if the monitor solution is not at least as intense as the reference solution.
METHOD A
Test solution 12 mL of the prescribed aqueous solution of the substance to be examined.
Reference solution (standard) A mixture of 10 mL of lead standard solution (1 ppm Pb) R or lead standard solution (2 ppm Pb) R, as prescribed, and 2 mL of the prescribed
aqueous solution of the substance to be examined.
Blank solution A mixture of 10 mL of water R and 2 mL of the prescribed aqueous solution of the substance to be examined.
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately. Examine the solutions after 2 min.
System suitability The reference solution shows a slight brown colour compared to the blank solution.
Result Any brown colour in the test solution is not more intense than that in the reference solution.
If the result is difficult to judge, filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying moderate and
constant pressure to the piston. Compare the spots on the filters obtained with the different solutions.
METHOD B
Test solution 12 mL of the prescribed solution of the substance to be examined prepared using an organic solvent containing a minimum percentage of water (for example,
dioxan containing 15 per cent of water or acetone containing 15 per cent of water).
Reference solution (standard) A mixture of 10 mL of lead standard solution (1 or 2 ppm Pb), as prescribed, and 2 mL of the prescribed solution of the substance to be examined
in an organic solvent. Prepare the lead standard solution (1 or 2 ppm Pb) by dilution of lead standard solution (100 ppm Pb) R with the solvent used for the substance to be
examined.
Blank solution A mixture of 10 mL of the solvent used for the substance to be examined and 2 mL of the prescribed solution of the substance to be examined in an organic
solvent.
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately. Examine the solutions after 2 min.
System suitability The reference solution shows a slight brown colour compared to the blank solution.
METHOD C
Test solution Place the prescribed quantity (not more than 2 g) of the substance to be examined in a silica crucible with 4 mL of a 250 g/L solution of magnesium sulfate R in
dilute sulfuric acid R. Mix using a fine glass rod. Heat cautiously. If the mixture is liquid, evaporate gently to dryness on a water-bath. Progressively heat to ignition and continue
heating until an almost white or at most greyish residue is obtained. Carry out the ignition at a temperature not exceeding 800 °C. Allow to cool. Moisten the residue with a few drops
of dilute sulfuric acid R. Evaporate, ignite again and allow to cool. The total period of ignition must not exceed 2 h. Take up the residue in 2 quantities, each of 5 mL, of dilute
hydrochloric acid R. Add 0.1 mL of phenolphthalein solution R, then concentrated ammonia R until a pink colour is obtained. Cool, add glacial acetic acid R until the solution is
decolorised and add 0.5 mL in excess. Filter if necessary and wash the filter. Dilute to 20 mL with water R.
Reference solution (standard) Prepare as described for the test solution, using the prescribed volume of lead standard solution (10 ppm Pb) R instead of the substance to be
examined. To 10 mL of the solution obtained add 2 mL of the test solution.
Monitor solution Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) R prescribed for
preparation of the reference solution. To 10 mL of the solution obtained add 2 mL of the test solution.
Blank solution A mixture of 10 mL of water R and 2 mL of the test solution.
To 12 mL of each solution, add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately. Examine the solutions after 2 min.
System suitability:
— the reference solution shows a slight brown colour compared to the blank solution,
— the monitor solution is at least as intense as the reference solution.
METHOD D
Test solution In a silica crucible, mix thoroughly the prescribed quantity of the substance to be examined with 0.5 g of magnesium oxide R1. Ignite to dull redness until a
homogeneous white or greyish-white mass is obtained. If after 30 min of ignition the mixture remains coloured, allow to cool, mix using a fine glass rod and repeat the ignition. If
necessary repeat the operation. Heat at 800 °C for about 1 h. Take up the residue in 2 quantities, each of 5 mL, of a mixture of equal volumes of hydrochloric acid R1 and water R.
Add 0.1 mL of phenolphthalein solution R and then concentrated ammonia R until a pink colour is obtained. Cool, add glacial acetic acid R until the solution is decolorised and add
0.5 mL in excess. Filter if necessary and wash the filter. Dilute to 20 mL with water R.
Reference solution (standard) Prepare as described for the test solution using the prescribed volume of lead standard solution (10 ppm Pb) R instead of the substance to be
examined and drying in an oven at 100-105 °C. To 10 mL of the solution obtained add 2 mL of the test solution.
Monitor solution Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) R prescribed for
preparation of the reference solution and drying in an oven at 100-105 °C. To 10 mL of the solution obtained add 2 mL of the test solution.
Blank solution A mixture of 10 mL of water R and 2 mL of the test solution.
To 12 mL of each solution, add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately. Examine the solutions after 2 min.
System suitability:
— the reference solution shows a slight brown colour compared to the blank solution,
METHOD E
Test solution Dissolve the prescribed quantity of the substance to be examined in 30 mL of water R or the prescribed volume.
Figure 2.4.8.-1. – Apparatus for the test for heavy metals
Reference solution (standard) Unless otherwise prescribed, dilute the prescribed volume of lead standard solution (1 ppm Pb) R to the same volume as the test solution.
Prepare the filtration apparatus by adapting the barrel of a 50 mL syringe without its piston to a support containing, on the plate, a membrane filter (nominal pore size 3 µm) and
above it a prefilter (Figure 2.4.8.-1).
Transfer the test solution into the syringe barrel, put the piston in place and then apply an even pressure on it until the whole of the liquid has been filtered. In opening the support
and removing the prefilter, check that the membrane filter remains uncontaminated with impurities. If this is not the case replace it with another membrane filter and repeat the
operation under the same conditions.
To the prefiltrate or to the prescribed volume of the prefiltrate add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately and allow to
stand for 10 min and again filter as described above, but inverting the order of the filters, the liquid passing first through the membrane filter before passing through the prefilter
(Figure 2.4.8.-1). The filtration must be carried out slowly and uniformly by applying moderate and constant pressure to the piston of the syringe. After complete filtration, open the
support, remove the membrane filter, and dry using filter paper.
In parallel, treat the reference solution in the same manner as the test solution.
Result The colour of the spot obtained with the test solution is not more intense than that obtained with the reference solution.
METHOD F
Test solution Place the prescribed quantity or volume of the substance to be examined in a clean, dry, 100 mL long-necked combustion flask (a 300 mL flask may be used if the
reaction foams excessively). Clamp the flask at an angle of 45°. If the substance to be examined is a solid, add a sufficient volume of a mixture of 8 mL of sulfuric acid R and 10 mL
of nitric acid R to moisten the substance thoroughly; if the substance to be examined is a liquid, add a few millilitres of a mixture of 8 mL of sulfuric acid R and 10 mL of nitric acid R.
Warm gently until the reaction commences, allow the reaction to subside and add additional portions of the same acid mixture, heating after each addition, until a total of 18 mL of
the acid mixture has been added. Increase the amount of heat and boil gently until the solution darkens. Cool, add 2 mL of nitric acid R and heat again until the solution darkens.
Continue the heating, followed by the addition of nitric acid R until no further darkening occurs, then heat strongly until dense, white fumes are produced. Cool, cautiously add 5 mL
of water R, boil gently until dense, white fumes are produced and continue heating to reduce to 2-3 mL. Cool, cautiously add 5 mL of water R and examine the colour of the solution.
If the colour is yellow, cautiously add 1 mL of strong hydrogen peroxide solution R and again evaporate until dense, white fumes are produced and reduce to a volume of 2-3 mL. If
the solution is still yellow in colour, repeat the addition of 5 mL of water R and 1 mL of strong hydrogen peroxide solution R until the solution is colourless. Cool, dilute cautiously with
water R and rinse into a 50 mL colour comparison tube, ensuring that the total volume does not exceed 25 mL. Adjust the solution to pH 3.0-4.0, using short range pH indicator
paper as external indicator, with concentrated ammonia R1 (dilute ammonia R1 may be used, if desired, as the specified range is approached), dilute with water R to 40 mL and mix.
Add 2 mL of buffer solution pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. Mix immediately. Dilute to 50 mL with water R and mix.
Reference solution (standard) Prepare at the same time and in the same manner as the test solution, using the prescribed volume of lead standard solution (10 ppm Pb) R.
Monitor solution Prepare as described for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) R prescribed for the
preparation of the reference solution.
Blank solution Prepare as described for the test solution, omitting the substance to be examined.
Examine the solutions vertically against a white background after 2 min.
System suitability:
— the reference solution shows a brown colour compared to the blank solution,
METHOD G
CAUTION: when using high-pressure digestion vessels the safety precautions and operating instructions given by the manufacturer must be followed. The digestion cycles have to
be elaborated depending on the type of microwave oven to be used (for example, energy-controlled microwave ovens, temperature-controlled microwave ovens or high-pressure
ovens). The cycle must conform to the manufacturer's instructions. The digestion cycle is suitable if a clear solution is obtained.
Test solution Place the prescribed amount of the substance to be examined (not more than 0.5 g) in a suitable, clean beaker. Add successively 2.7 mL of sulfuric acid R, 3.3 mL
of nitric acid R and 2.0 mL of strong hydrogen peroxide solution R using a magnetic stirrer. Allow the substance to react with a reagent before adding the next one. Transfer the
mixture to a dry high-pressure-resistant digestion vessel (fluoropolymer or quartz glass).
Reference solution (standard) Prepare as described for the test solution, using the prescribed volume of lead standard solution (10 ppm Pb) R instead of the substance to be
examined.
Monitor solution Prepare as prescribed for the test solution, adding to the substance to be examined the volume of lead standard solution (10 ppm Pb) R prescribed for the
preparation of the reference solution.
Blank solution Prepare as described for the test solution, omitting the substance to be examined.
Close the vessels and place in a laboratory microwave oven. Digest using a sequence of 2 separate suitable programmes. Design the programmes in several steps in order to
control the reaction, monitoring pressure, temperature or energy depending on the type of microwave oven available. After the first programme allow the digestion vessels to cool
before opening. Add to each vessel 2.0 mL of strong hydrogen peroxide solution R and digest using the second programme. After the second programme allow the digestion vessels
to cool before opening. If necessary to obtain a clear solution, repeat the addition of strong hydrogen peroxide solution R and the second digestion programme.
Cool, dilute cautiously with water R and rinse into a flask, ensuring that the total volume does not exceed 25 mL.
Using short-range pH indicator paper as external indicator, adjust the solutions to pH 3.0-4.0 with concentrated ammonia R1 (dilute ammonia R1 may be used as the specified range
is approached). To avoid heating of the solutions use an ice-bath and a magnetic stirrer. Dilute to 40 mL with water R and mix. Add 2 mL of buffer solution pH 3.5 R. Mix and add to
1.2 mL of thioacetamide reagent R. Mix immediately. Dilute to 50 mL with water R, mix and allow to stand for 2 min.
Filter the solutions through a suitable membrane filter (nominal pore size 0.45 µm). Carry out the filtration slowly and uniformly, applying moderate and constant pressure to the
piston. Compare the spots on the filters obtained with the different solutions.
System suitability:
— the spot obtained with the reference solution shows a brown colour compared to the spot obtained with the blank solution,
— the spot obtained with the monitor solution is at least as intense as the spot obtained with the reference solution.
Result The brown colour of the spot obtained with the test solution is not more intense than that of the spot obtained with the reference solution.
METHOD H
Test solution Dissolve the prescribed quantity of the substance to be examined in 20 mL of the solvent or solvent mixture prescribed.
Reference solution Dilute the prescribed volume of lead standard solution (10 ppm Pb) R to 20 mL with the solvent or solvent mixture prescribed.
Blank solution 20 mL of the solvent or solvent mixture prescribed.
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix. (In some cases precipitation occurs, in which case the specific monograph would describe re-dissolution in a defined
volume of a given solvent.) Add to 1.2 mL of thioacetamide reagent R. Mix immediately and allow to stand for 2 min. Filter the solutions through a suitable membrane filter (nominal
pore size 0.45 µm). Compare the spots on the filters obtained with the different solutions.
System suitability The spot obtained with the reference solution shows a brownish-black colour compared to the spot obtained with the blank solution.
Result The brownish-black colour of the spot obtained with the test solution is not more intense than that of the spot obtained with the reference solution.
Limit Test for Iron
Dissolve the prescribed quantity of the substance to be examined in water R and dilute to 10 mL with the same solvent or use 10 mL of the prescribed solution. Add 2 mL of a
200 g/L solution of citric acid monohydrate R and 0.1 mL of thioglycollic acid R. Mix, make alkaline with ammonia R and dilute to 20 mL with water R. Prepare a standard in the same
manner, using 10 mL of iron standard solution (1 ppm Fe) R.
After 5 min, any pink colour in the test solution is not more intense than that in the standard.
Limit Test for Lead in Sugars
Determine the lead by atomic absorption spectrometry (2.2.23, Method II).
Test solution Dissolve 20.0 g of the substance to be examined in a mixture of equal volumes of dilute acetic acid R and water R and dilute to 100.0 mL with the same mixture of
solvents. Add 2.0 mL of a clear 10 g/L solution of ammonium pyrrolidinedithiocarbamate R and 10.0 mL of methyl isobutyl ketone R and then shake for 30 s protected from bright
light. Allow the layers to separate and use the methyl isobutyl ketone layer.
Reference solutions Prepare 3 reference solutions in the same manner as the test solution but adding 0.5 mL, 1.0 mL and 1.5 mL respectively of lead standard solution
(10 ppm Pb) R in addition to the 20.0 g of the substance to be examined.
Set the zero of the instrument using methyl isobutyl ketone R treated as described for the test solution without the substance to be examined. Measure the absorbance at 283.3 nm
using a lead hollow-cathode lamp as source of radiation and an air-acetylene flame.
The substance to be examined contains not more than 0.5 ppm of lead, unless otherwise prescribed.
Limit Test for Magnesium
To 10 mL of the prescribed solution add 0.1 g of disodium tetraborate R. Adjust the solution, if necessary, to pH 8.8 to pH 9.2 using dilute hydrochloric acid R or dilute sodium
hydroxide solution R. Shake with 2 quantities, each of 5 mL, of a 1 g/L solution of hydroxyquinoline R in chloroform R, for 1 min each time. Allow to stand. Separate and discard the
organic layer. To the aqueous solution add 0.4 mL of butylamine R and 0.1 mL of triethanolamine R. Adjust the solution, if necessary, to pH 10.5 to pH 11.5. Add 4 mL of the solution
of hydroxyquinoline in chloroform, shake for 1 min, allow to stand and separate. Use the lower layer for comparison. Prepare a standard in the same manner using a mixture of 1 mL
of magnesium standard solution (10 ppm Mg) R and 9 mL of water R.
Any colour in the solution obtained from the substance to be examined is not more intense than that in the standard.
Limit Test for Magnesium and Alkaline-earth Metals
To 200 mL of water R add 0.1 g of hydroxylamine hydrochloride R, 10 mL of ammonium chloride buffer solution pH 10.0 R, 1 mL of 0.1 M zinc sulfate and about 15 mg of mordant
black 11 triturate R. Heat to about 40 °C. Titrate with 0.01 M sodium edetate until the violet colour changes to full blue. To the solution add the prescribed quantity of the substance to
be examined dissolved in 100 mL of water R or use the prescribed solution. If the colour of the solution changes to violet, titrate with 0.01 M sodium edetate until the full blue colour
is again obtained.
The volume of 0.01 M sodium edetate used in the second titration does not exceed the prescribed quantity.
Limit Test for Heavy Metals in Herbal Drugs and Herbal Drug Preparations
CAUTION: when using closed high-pressure digestion vessels and microwave laboratory equipment, be familiar with the safety and operating instructions given by the manufacturer.
APPARATUS
The apparatus typically consists of the following:
— as digestion flasks, polytetrafluoroethylene, perfluoroalkoxy polymer, quartz or glass flasks with a volume of 20-150 mL, fitted with an airtight closure, a valve to adjust the
pressure inside the container and a polytetrafluoroethylene tube to allow release of gas;
— a system to make flasks airtight, using the same torsional force for each of them;
— a programmable microwave oven (e.g. with a magnetron frequency of 2450 MHz, with a selectable output from 0 to 1500 ± 70 W in 1 per cent increments), a
polytetrafluoroethylene-coated microwave cavity with a variable speed exhaust fan, a rotating turntable drive system and exhaust tubing to vent fumes;
— an atomic absorption spectrometer (2.2.23), an inductively coupled plasma-atomic emission spectrometer (2.2.57), or an inductively coupled plasma-mass spectrometer
(2.2.58).
METHOD
Examine by atomic absorption spectrometry (AAS) (2.2.23), inductively coupled plasma-atomic emission spectrometry (ICP-AES) (2.2.57), or inductively coupled plasma-mass
spectrometry (ICP-MS) (2.2.58).
Deviations from the experimental parameters of the sample preparation procedure and the method description below are acceptable provided that the validation requirements are
met and the system suitability test is fulfilled on the day of the analysis.
Sample preparation
Clean all the glassware and laboratory equipment with a 10 g/L solution of nitric acid R before use.
Test solution In a digestion flask, place the prescribed quantity of the substance to be examined (about 0.50 g of powdered herbal drug (1400) (2.9.12)). Add 4 mL of heavy
metal-free hydrochloric acid R and 6 mL of heavy metal-free nitric acid R. Make the flask airtight.
Place the digestion flasks in the microwave oven. Carry out the digestion in 3 steps according to the following programme, used for 7 flasks each containing the test solution: 80 per
cent power for 15 min, 100 per cent power for 5 min, 80 per cent power for 20 min.
At the end of the cycle, allow the flasks to cool in air or water. After cooling, open each digestion flask and introduce the clear, colourless solution obtained into a 50 mL volumetric
flask. Rinse each digestion flask with 2 quantities, each of 15 mL, of heavy metal-free dilute nitric acid R, collect the rinsings in the volumetric flask and dilute to 50.0 mL with
water R. Modifiers (e.g. in the case of AAS with electrothermal atomisation, 1.0 mL of a 10 g/L solution of magnesium nitrate R and 1.0 mL of a 100 g/L solution of ammonium
dihydrogen phosphate R) and stabilising agents may be used, if necessary.
Blank solution Mix 4 mL of heavy metal-free hydrochloric acid R and 6 mL of heavy metal-free nitric acid R in a digestion flask. Carry out the digestion in the same manner as for
the test solution.
DETERMINATION OF ARSENIC, CADMIUM, COPPER, NICKEL AND LEAD USING AAS (2.2.23) WITH
ELECTROTHERMAL ATOMISATION
Measure the content of arsenic, cadmium, copper, nickel and lead by direct calibration (2.2.23, Method I) or by the standard additions method (2.2.23, Method II), using reference
solutions of each heavy metal and the instrumental parameters recommended in Table 2.4.27.-1.
The absorbance value of the blank solution is subtracted from the value obtained with the test solution.
Table 2.4.27.-1. – Instrumental parameters for AAS with electrothermal atomisation
As Cd Cu Ni Pb
Wavelength nm 193.7 228.8 324.8 232 283.5
Slit width nm 0.5 0.5 0.5 0.2 0.5
Lamp current mA 10 6 7 10 5
Ignition temperature °C 1400 800 800 800 800
Atomisation temperature °C 2600 1800 2300 2500 2200
Gas flow rate L/min 3 3 3 3 3
DETERMINATION OF ARSENIC AND MERCURY USING AAS (2.2.23) WITH COLD-VAPOUR OR HYDRIDE ATOMISATION
Measure the content of arsenic and mercury by direct calibration (2.2.23, Method I) or by the standard additions method (2.2.23, Method II), using reference solutions of arsenic or
mercury and an automated continuous-flow hydride vapour generation system.
The absorbance value of the blank solution is subtracted from the value obtained with the test solution.
Arsenic
Sample solution To 19.0 mL of the test solution or of the blank solution as prescribed above, add 1 mL of a 200 g/L solution of potassium iodide R. Allow the test solution to stand
at room temperature for about 50 min or at 70 °C for about 4 min.
Acid reagent Heavy metal-free hydrochloric acid R.
Reducing reagent 6 g/L solution of sodium tetrahydroborate R in a 5 g/L solution of sodium hydroxide R.
The recommended instrumental parameters in Table 2.4.27.-2 may be used.
Mercury
Sample solution Test solution or blank solution, as prescribed above.
Acid reagent 515 g/L solution of heavy metal-free hydrochloric acid R.
Reducing reagent 10 g/L solution of stannous chloride R in heavy metal-free dilute hydrochloric acid R.
The recommended instrumental parameters in Table 2.4.27.-2 may be used.
Table 2.4.27.-2. – Instrumental parameters for AAS with cold-vapour or hydride atomisation
As Hg
Wavelength nm 193.7 253.7
Slit width nm 0.2 0.5
Lamp current mA 10 4
Acid reagent flow rate mL/min 1.0 1.0
Reducing reagent flow rate mL/min 1.0 1.0
As Hg
Sample solution flow rate mL/min 7.0 7.0
Absorption cell Quartz (heated) Quartz (unheated)
Nitrogen flow rate L/min 0.1 0.1
DETERMINATION OF ARSENIC, CADMIUM, COPPER, MERCURY, NICKEL AND LEAD USING ICP-AES (2.2.57)
Measure the content of arsenic, cadmium, copper, mercury, nickel and lead by direct calibration (2.2.23, Method I), using reference solutions of each heavy metal or a mixture of all
measured metals, and the instrumental parameters recommended in Table 2.4.27.-3.
The emission value of the blank solution is subtracted from the value obtained with the test solution.
Table 2.4.27.-3. – Instrumental parameters for ICP-AES
As Cd Cu Hg Ni Pb
Wavelength nm 193.696/ 214.438/ 324.754/ 184.950/ 231.604/ 220.351/
197.197/ 226.502/ 327.396/ 253.652/ 231.997/ 283.306/
189.042 228.802 224.700 435.835 352.454 168.215
Ar, Monitorline nm 430.010 430.010 430.010 430.010 430.010 430.010
Plasma energy W 1200 1200 1200 1200 1200 1200
Peak algorithm with background

yes yes yes yes yes yes
correction
DETERMINATION OF ARSENIC, CADMIUM, COPPER, MERCURY, NICKEL AND LEAD USING ICP-MS (2.2.58)
Measure the content of arsenic, cadmium, copper, mercury, nickel and lead by direct calibration (2.2.23, Method I) using reference solutions of each heavy metal and the analytical
isotopes and additional masses recommended in Table 2.4.27.-4.
The signal intensity of the blank solution is subtracted from the value obtained with the test solution.
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.
Table 2.4.27.-4. – Recommended analytical isotopes and additional masses for ICP-MS
Isotope Element of Interest
75 Arsenic
106, 108, 111, 114 Cadmium
63, 65 Copper
202 Mercury
60, 62 Nickel
206, 207, 208 Lead
VALIDATION REQUIREMENTS
The analytical procedures used must be validated in accordance with the relevant general methods AAS (2.2.23), ICP-AES (2.2.57) and ICP-MS (2.2.58). Additionally, the following
criteria must be fulfilled.
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 heavy metal to be determined with this procedure in the presence of components that may be
expected to be present, including other heavy metals, matrix components and other sources of interference; specificity is demonstrated by complying with the accuracy requirement
for the metal(s) to be determined.
RANGE
The calibration range of each metal is within the linear range of the method; test solutions containing residues at a level outside the calibration range may be diluted to
concentrations within the calibration 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 greater than the value indicated in Table 2.4.27.-5.
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 with different analysts. Only 1 of the 3 experiments is required to
demonstrate intermediate precision.
Acceptance criterion The relative standard deviation is not greater than the value indicated in Table 2.4.27.-5.
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.
Table 2.4.27.-5
Concentration range of the metal (mg/kg) Repeatability (RSD) (per cent) Intermediate precision (RSD) (per cent)
0.01 - 1 20 32
>1 10 16
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 0.1 times the concentration of the specification limit.
Limit Test for Nickel in Polyols
Determine the nickel by atomic absorption spectrometry (2.2.23, Method II).
Test solution Dissolve 20.0 g of the substance to be examined in a mixture of equal volumes of dilute acetic acid R and water R and dilute to 100.0 mL with the same mixture of
solvents. Add 2.0 mL of a saturated solution of ammonium pyrrolidinedithiocarbamate R (about 10 g/L) and 10.0 mL of methyl isobutyl ketone R and then shake for 30 s protected
from bright light. Allow the layers to separate and use the methyl isobutyl ketone layer.
Reference solutions Prepare 3 reference solutions in the same manner as the test solution but adding 0.5 mL, 1.0 mL and 1.5 mL respectively of nickel standard solution
(10 ppm Ni) R in addition to the 20.0 g of the substance to be examined.
Set the zero of the instrument using methyl isobutyl ketone R treated as described for preparation of the test solution omitting the substance to be examined. Measure the
absorbance at 232.0 nm using a nickel hollow-cathode lamp as source of radiation and an air-acetylene flame.
The substance to be examined contains not more than 1 ppm of nickel, unless otherwise prescribed.
Limit Test for Phosphates
To 100 mL of the solution prepared and, if necessary, neutralised as prescribed add 4 mL of sulfomolybdic reagent R3. Shake and add 0.1 mL of stannous chloride solution R1.
Prepare a standard in the same manner using 2 mL of phosphate standard solution (5 ppm PO4) R and 98 mL of water R. After 10 min, compare the colours using 20 mL of each
solution.
Any colour in the test solution is not more intense than that in the standard.
Limit Test for Potassium
To 10 mL of the prescribed solution add 2 mL of a freshly prepared 10 g/L solution of sodium tetraphenylborate R. Prepare a standard in the same manner using a mixture of 5 mL of
potassium standard solution (20 ppm K) R and 5 mL of water R.
Limit Test for Sulfates
All solutions used for this test must be prepared with distilled water R.
Add 3 mL of a 250 g/L solution of barium chloride R to 4.5 mL of sulfate standard solution (10 ppm SO4) R1. Shake and allow to stand for 1 min. To 2.5 mL of this suspension add
15 mL of the prescribed solution and 0.5 mL of acetic acid R. Prepare a standard in the same manner using 15 mL of sulfate standard solution (10 ppm SO4) R instead of the
prescribed solution.
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Appendix VII Limit Tests 3 - British Pharmacopoeia

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Appendix VII Limit Tests

(No Ph. Eur. method)

Tubes for Comparative Tests

(Ph. Eur. method 2.1.5)

Limit Test for Aluminium

(Ph. Eur. method 2.4.17)

transmission band centred on 518 nm or a monochromator set to transmit at this wavelength.

Limit Test for Ammonium

(Ph. Eur. method 2.4.1)

Unless otherwise prescribed, use method A.

Limit Test for Arsenic

(Ph. Eur. method 2.4.2)

A. Lead acetate paper/cotton

Figure 2.4.2.-1. – Apparatus for the limit test for arsenic (method A)

rolled piece of lead acetate paper R weighing 50-60 mg.

Limit Test for Calcium

(Ph. Eur. method 2.4.3)

Limit Test for Chlorides

(Ph. Eur. method 2.4.4)

Limit Test for Fluorides

Figure 2.4.5.-1. – Apparatus for limit test for fluorides

Limit Test for Heavy Metals

(Ph. Eur. method 2.4.8)

— the monitor solution is at least as intense as the reference solution.

— the monitor solution is at least as intense as the reference solution.

Figure 2.4.8.-1. – Apparatus for the test for heavy metals

above it a prefilter (Figure 2.4.8.-1).

operation under the same conditions.

preparation of the reference solution.

Examine the solutions vertically against a white background after 2 min.

— the monitor solution is at least as intense as the reference solution.

Blank solution 20 mL of the solvent or solvent mixture prescribed.

Limit Test for Iron

(Ph. Eur. method 2.4.9)

(Ph. Eur. method 2.4.10)

Determine the lead by atomic absorption spectrometry (2.2.23, Method II).

Limit Test for Magnesium

(Ph. Eur. method 2.4.6)

Limit Test for Magnesium and Alkaline-earth Metals

(Ph. Eur. method 2.4.7)

(Ph. Eur. method 2.4.27)

The apparatus typically consists of the following:

Table 2.4.27.-1. – Instrumental parameters for AAS with electrothermal atomisation

Wavelength nm 193.7 228.8 324.8 232 283.5

Slit width nm 0.5 0.5 0.5 0.2 0.5

Ignition temperature °C 1400 800 800 800 800

Atomisation temperature °C 2600 1800 2300 2500 2200

Gas flow rate L/min 3 3 3 3 3

mercury and an automated continuous-flow hydride vapour generation system.

Acid reagent Heavy metal-free hydrochloric acid R.

The recommended instrumental parameters in Table 2.4.27.-2 may be used.

Sample solution Test solution or blank solution, as prescribed above.

Acid reagent 515 g/L solution of heavy metal-free hydrochloric acid R.

The recommended instrumental parameters in Table 2.4.27.-2 may be used.

Table 2.4.27.-2. – Instrumental parameters for AAS with cold-vapour or hydride atomisation

Wavelength nm 193.7 253.7

Slit width nm 0.2 0.5

Acid reagent flow rate mL/min 1.0 1.0

Reducing reagent flow rate mL/min 1.0 1.0

Sample solution flow rate mL/min 7.0 7.0

Absorption cell Quartz (heated) Quartz (unheated)