Quality Control Methods For Medicinal Plant Materials OMS

Working document QAS/05.131/Rev.
1
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WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE
QUALITY CONTROL METHODS FOR MEDICINAL PLANT MATERIALS

Revised DRAFT UPDATE
September 2005
The need for a revision of the general methods included in Quality control methods for medicinal
plant materials, Geneva, World Health Organization, 1998, was expressed on various occasions
by experts collaborating in the Traditional Medicines Programme.
The attached draft document summarizes the above-mentioned group's discussions. It was
further discussed at a WHO consultation on 11-13 July 2005, and is now being mailed outside
the Organization for comment. We should appreciate receiving any comments you may have
thereon by 30 November 2005.
Title: Quality control methods for medicinal plant materials - Revised Draft Update, September 2005
© World Health Organization 2005
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Please send any request for permission to:

Ms Yukiko Maruyama, Scientist, Traditional Medicine (TRM), Technical Cooperation for Essential Drugs and Traditional
Medicine (TCM), World Health Organization, CH-1211 Geneva 27, Switzerland.
Fax: (41-22) 791 4730; e-mail: [email protected]
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Working document QAS/05.131/Rev.1
page 2
SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/05.131/Rev.1:

QUALITY CONTROL METHODS FOR MEDICINAL PLANT MATERIALS
REVISED DRAFT UPDATE
Deadline
First working draft prepared June 2003
Discussion at WHO working group meeting July 2003
First draft November 2003
First global review November 2003-January 2004
Revised draft May 2004
Second global review May-June 2004
Second revised draft July 2004
Discussion at WHO consultation July 2005
Revision of draft document June-July 2005
First update draft July 2005
Revised update draft September 2005
Third global review: deadline for receipt of comments 30 November 2005
Presentation to Fortieth WHO Expert Committee on 24-28 October 2005

Specifications for Pharmaceutical Preparations
page 3
Recommended analytical methods

Introduction
The test methods described in this document are presented as examples of suitable methods in the
detection of selected contaminants and residues in medicinal plant materials. Where available, test
methods applicable to different products and stages of herbal medicines, such as extracts and finished
herbal products, are also described. In addition to the test methods, some suggestions regarding
general limits for contaminants are included, where applicable. Both should be considered as the basis
for establishing national and regional requirements for limits and methodologies. WHO is currently
not able to recommend limits for contaminants since these are too diverse and there is a lack of
consensus. Also the test procedures cannot take into account all possible impurities but where such
impurities occur, validated methods should be developed and appropriate toxicological consideration
should be applied.
The analysis of medicinal plant materials is not restricted to those methods discussed or recommended
here and other techniques are also available. Details of analytical methods, such as volumetric
analysis, are described in international pharmacopoeias. (4, 5, 6, 7, 8)
When considering the choice of one of these methods the level of detection and the plant product
matrix used for the testing e.g. seeds containing oils, finished products, etc., must be taken into
account and modifications of the method made if required. The method of the determination has to be
validated for the respective matrix.
Sections of methods proposed for the update:
Sections Page
Determination of pesticide residues (currently Chapter 16) ……………………………... 3
Determination of arsenic and toxic metals (currently Chapter 17) ……………………………... 19
Determination of microorganisms (currently Chapter 18)……………………………… 27
Determination of aflatoxins (currently included in Chapter 18)…………………. 38
Determination of radioactive substances (currently Chapter 19)……………………………… 41
[Subsequently, Annex 1 (List of culture media and strains used for microbiological analysis) and
Annex 2 (List of reagents and solutions) will be updated by WHO accordingly.]
page 4
Determination of Pesticide Residues

Table of contents
General.......................................................................................................................3
A. Determination of total chlorine and phosphorus..................................................5
B. Determination of chlorides..................................................................................8
C. Determination of phosphates..............................................................................8
D. Qualitative and quantitative determination of organochlorine pesticides............9
E. Analysis of esters of organophosphorus compounds............................................11
F. Determination of specific pesticide residues in medicinal plant materials..........11
G. Determination of desmetryn, prometryn, and simazine residues..........................12
H. Determination of specific organochroline, organophosphorus, and pyrethroid pesticide
residues…………………………………………………………………………………..14
General
Methods for the determination of pesticide residues
Chromatography (mostly column and gas) is recommended as the principal method for the
determination of pesticide residues. These methods may be coupled with MS. Samples are extracted
by a standard procedure, impurities are removed by partition and/or adsorption, and the presence of a
moderately broad spectrum of pesticides is measured in a single determination. However, these
techniques are not universally applicable. Some pesticides are satisfactorily carried through the
extraction and clean-up procedures, others are recovered with a poor yield, and some are lost entirely.
In chromatography, the separations may not always be complete, pesticides may decompose or
metabolise, and many of the metabolic products are still unknown. Consequently, as a result of
limitations in the analytical technique and incomplete knowledge of pesticide interaction with the
environment, it is not yet possible to apply an integrated set of methods that will be satisfactory in all
situations.
Generally the methodology should be adapted to the type of herbal material being tested and
modifications may be necessary for different samples including seeds, leaves, oils, extracts, finished
products and for samples with different quantities of moisture present. Also the spectrum of
pesticides to be tested for is dependent on the specific pesticides used on the plant material and the
historical use of persistent pesticides in the regional or national area.
It is, therefore, desirable to test plant materials of unknown history for broad groups of compounds
rather than for individual pesticides. A variety of methods meet these requirements. Pesticides
containing chlorine in the molecule, for example, can be detected by the measurement of total organic
chlorine; insecticides containing phosphate can be measured by analysis for total organic phosphorus,
while pesticides containing arsenic and lead can be detected by measurement of total arsenic or total
lead, respectively. Similarly, the measurement of total bound carbon disulfide in a sample will provide
information on whether residues of the dithiocarbamate family of fungicides are present.
Importantly, where such general methods are employed care must be taken to ensure that results are
not adversely affected by contributions from certain plant constituents containing these targeted
elements.
If the pesticide to which the plant material has been exposed is known or can be identified by suitable
means, an established method for the determination of that particular pesticide residue should be
employed.
page 5
General aspects of analytical methodology
The samples should be tested as quickly as possible after collection, before any physical or chemical
changes occur. If prolonged storage is envisaged, the samples should preferably be stored in airtight
containers under refrigeration.
Water content of samples can also be problematic and in the USP 27 method (USP 27/NFD 22-2004)
and the EP Fifth Edition, content is limited to 15% and below for the determination of organochlorine
and pyrethroid insecticides.
Light can cause degradation of many pesticides, and it is therefore advisable to protect the samples
and any extracts or solutions from undue exposure.
The type of container or wrapping material used should not interfere with the sample or affect the
analytical results.
Solvents and reagents used in the analytical method should be free from substances that may interfere
with the reaction, alter the results or provoke degradation of the pesticide residue in the sample. It is
usually necessary to employ specially purified solvents or to distil them freshly in an all-glass
apparatus. Blank determinations with the solvents should be carried out, concentrating and testing
them as specified in the test procedure for the plant material concerned.
The simplest and quickest procedure should be used to separate unwanted material from the sample
(clean-up procedure) in order to save time when many samples have to be tested.
The process of concentrating solutions should be undertaken with great care, especially during the
evaporation of the last traces of solvent, in order to avoid losses of pesticide residues. For this reason,
it is often not advisable to remove the last traces of solvent. Agents, such as mineral oil or other oils
of low volatility that may help to preserve the solution could be added to retard the loss of the
relatively volatile pesticides, especially when the last traces of solvent are being evaporated.
However, these agents, while satisfactory in colorimetric procedures, are usually not desirable in gas
chromatographic methods. It may be necessary to evaporate heat-labile compounds using a rotary
vacuum apparatus.
Some examples of national and regional limits set for various types of pesticide residues are shown in
Table 1, below.
page 6
Table 1. Examples of national limits set for various pesticide residues

Limit
Substances EP1 Japan2
(ppm)
alachlor 0.02
aldrin and dieldrin (sum of) 0.05
azinphos-methyl 1.0
bromopropylate 3.0
chlordane (sum of cis-, trans, and oxythlordane) 0.05
chlorfenvinphos 0.5
chlorpyrifos 0.2
chlorpyrifos-methyl 0.1
cypermethrin (and isomers) 1.0
DDT (sum of p,p'- DDT o,p'-DDT p,p'- DDE p,p'- TDE) 1.0
DDT (sum of p,p'- DDT o,p'-DDT p,p'- DDD p,p'- DDE) 0.2
deltamethrin 0.5
diazine phosphorus
diazinon 0.5
dichlorvos 1.0
dimethoate
dimethoate oxide
dithiocarbamate (as CS2) 2.0
endosulfan (sum of isomers and endosulfan sulphate) 3.0
endrin 0.05
ethion 2.0
fenitrothion 0.5
fenvalerate 1.5
fonofos 0.05
heptachlor (sum of heptachlor and heptachlorepoxide) 0.05
hexachlorobenyene 0.1
hexachlorocyclohexane isomers (other than γ) 0.3
hexachlorocyclohexane isomers (sum of α,β,γ,and δ ) 0.2
lindane ( γ-Hexachlorocyclohexane) 0.6
malathion 1.0
methamidophos
methamidophos-acetyl
methidathion 0.2
parathion 0.5
parathion-methyl 0.2
permethrin 1.0
phosalone 0.1
piperonyl butoxide 3.0
pirimiphos-methyl 4.0
pyrethrins (sum of) 3.0
quintozene (sum of quintozene, pentachloroaniline and methyl 1.0
pentachlorophenyl sulphide)
1
: Applicable to medicinal plant materials included in the European pharmacopoeia, 5th Edition, unless otherwise
indicated in their respective monograph. Same values in USP 27 and Argentina pharmacopoeia F.A. Vol 1
2
: Currently applicable only to 5 medicinal plant materials (Ginseng Root, Powdered Ginseng Root, Red Ginseng Root,
Senna Leaf, and Powdered Senna Leaf) included in Japanese Pharmacopoeia, XIV.
A. Determination of total chlorine and phosphorus

Most pesticides contain organically bound chlorine or phosphorus.
Recommended procedure
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Preparation of samples
Reduce the plant material to a fine powder, and extract with a mixture of water and acetonitrile R.
Most pesticides are soluble in this mixture, while most cellular constituents (e.g. cellulose, proteins,
amino acids, starch, fats and related compounds) are sparingly soluble and are thus removed. A
number of polar and moderately polar compounds may also be dissolved; it is therefore necessary to
transfer the pesticides to light petroleum R. For pesticides containing chlorine, further purification is
seldom required, but for those containing phosphorus, further purification by column chromatography
may be necessary, eluting with mixtures of light petroleum R and ether R.
Preparation of the column

Use Florisil R grade 60/100 PR (or equivalent), activated at 650 °C, as the support. If this material is
obtained in bulk, transfer it immediately after opening to a 500-ml glass jar or bottle with a glass
stopper or foil-lined, screw-top lid. Store in the dark. Before use, heat at not less than 130 °C, cool in
a desiccator to room temperature and heat once again to 130 °C after 2 days.
Prepare a Florisil column (external diameter, 22 mm), which contains, after settling, 10 cm of
activated Florisil topped with about 1 cm of anhydrous sodium sulfate R. Pre-wet the column with 40-
50 ml of light petroleum R. Place a graduated flask under the column to receive the eluate.
Method
Grind the material to pass through a sieve no. 710 or 840 and mix thoroughly. Place 20-50 g of the
ground sample into a blender, add 350 ml of acetonitrile R with a water content of 35 % (to 350 ml of
water add sufficient acetonitrile R to produce 1000 ml). Blend for 5 minutes at a high speed. Filter
under vacuum through an appropriate funnel, diameter 12 cm, fitted with filter paper, into a 500-ml
suction flask.
Transfer the filtrate to a 250-ml measuring cylinder and record the volume. Transfer the measured
filtrate to a 1-litre separating funnel and carefully add 100 ml of light petroleum R. Shake vigorously
for 1-2 minutes, add 10 ml of sodium chloride (400 g/l) TS and 600 ml of water. Hold the separating
funnel in a horizontal position and mix vigorously for 30-45 seconds. Allow to separate, discard the
aqueous layer and gently wash the solvent layer with two 100-ml portions of water. Discard the
washings, transfer the solvent layer to a 100-ml glass-stoppered cylinder, and record the volume. Add
about 15 g of anhydrous sodium sulfate R and shake vigorously. The extract must not remain in
contact with this reagent for longer than 1 hour. Transfer the extract directly to a Florisil column; if
necessary, reduce the volume first to 5-10 ml. Allow it to pass through the column at a rate of not
more than 5 ml per minute. Carefully rinse the cylinder with two portions, each of 5 ml, of light
petroleum R, transfer them to the column, rinse with further small portions of light petroleum R if
necessary, and then elute at the same rate with 200 ml of ether/light petroleum TS1. Change the
receiver and elute with 200 ml of ether/light petroleum TS2. Again change the receiver and elute with
200 ml of ether/light petroleum TS3. Evaporate each eluate to a suitable volume, as required, for
further testing.
- The first eluate contains chlorinated pesticides (aldrin, DDE, TDE (DDD), o,p'-and p,p'-DDT,
HCH, heptachlor, heptachlor epoxide, lindane, methoxychlor), polychlorinated biphenyls (PCB),
and phosphated pesticides (carbophenothion, ethion, and fenchlorphos).
- The second eluate contains chlorinated pesticides (dieldrin and endrin) and phosphated pesticides
(methyl parathion and parathion).
- The third eluate contains phosphated pesticide (malathion).

page 8
Combustion of the organic matter

Combustion of the organic matter in oxygen is the preparatory step for the determination of chlorine
and phosphorus. The pesticide is extracted from the sample and purified, if necessary. The extract is
concentrated, evaporated to dryness, transferred to a sample holder, and burned in a suitable conical
flask flushed with oxygen. The gases produced during combustion are then absorbed in a suitable
solution. The absorbed chlorine is determined as chloride and the absorbed phosphorus as
orthophosphate, both colorimetrically.
Apparatus
The combustion is carried out in a 1-litre conical flask made of borosilicate glass, into the stopper of
which is fused one end of a piece of platinum wire about 1 mm in diameter. To the free end of the
wire is attached a piece of platinum gauze measuring about 1.5 x 2 cm to provide a means of holding
the sample clear of the absorbing liquid during combustion.
Sample holder for chlorine-containing residues

For a small quantity of solid material, use a sample holder made from a piece of halide-free filter-
paper about 5 cm long and 3 cm wide; for a small volume of liquid, preferably use a sample holder in
the form of a cone made from cellulose acetate film. Prepare the cone as follows: wearing cloth
gloves and using a suitable cardboard template cut the film in a circle of 4 cm radius.
Manually pin the two edges together to form a cone. Seal the joined edges using heat to form a seam
about 5 mm wide. Immerse the seam in acetone R to about one half of its width for 10 seconds.
Remove and dry it immediately in a stream of hot air. Using forceps, wash the cone by dipping in a 1-
litre beaker containing warm sodium hydroxide (~240 g/l) TS for 10 seconds at a temperature of about
60 °C. Rinse the cone thoroughly with water and allow to drain dry on a piece of aluminium foil.
Place each cone in a clean funnel (diameter 65 mm).
Sample holder for phosphorus-containing residues

Use a piece of halide-free filter-paper about 4 cm square as the sample holder.
Combustion of chlorine-containing residues

Transfer an aliquot of the extract as prepared above onto the sample holder, which is placed in a
funnel using a solvent that will not dissolve the sample holder. Allow the solvent to evaporate.
Wearing rubber gloves, remove the sample holder and its dry contents from the funnel, and fold it
over and up to form a small packet, about 1 cm2 in area, and secure it in the centre of the platinum
gauze. Insert a narrow strip of filter paper, about 1 x 3 cm, as a fuse into the top of the holder,
between the folds of the packet. Add 30 ml of water to the combustion flask. Moisten the neck of the
flask with water. Fill the flask thoroughly with oxygen by means of a tube with its end just above the
liquid. Ignite the free end of the paper strip and immediately insert the stopper. Hold the stopper
firmly in place. When vigorous burning has begun, tilt the flask to prevent incompletely burned
material from falling into the liquid. Immediately after combustion is completed, shake the flask
vigorously for 10 minutes to dissolve the combustion products. Place a small quantity of water around
the rim of the flask, and carefully withdraw the stopper. Rinse the stopper, platinum wire, platinum
gauze and sides of the flask with water. Transfer the liquid and liquids used for rinsing to a 50-ml
volumetric flask and dilute to volume with water.
Combustion of phosphorus-containing residues

Dip the sample holder made from filter paper into methanolic sodium hydroxide TS, and then suspend
it in a current of heated air. Immediately transfer about 0.2 ml of an aliquot of the extract as prepared
above to the sample holder with the aid of 0.2-ml portions of chloroform R using a micropipette.
Allow the solvent to evaporate from the paper, fold it to form a small packet about 1 cm2 in area and
page 9
place it in the centre of the platinum gauze. Insert a strip of filter paper, about 1 x 3 cm, as a fuse into
the top of the holder, between the folds of the packet. Add 10 ml of sulfuric acid (~37 g/l) TS to the
combustion flask and continue with the combustion as described above. Transfer the solution and the
liquid used for rinsing to a 25-ml volumetric flask and dilute to volume with water.
B. Determination of chlorides
Apparatus
The determination is made with a spectrophotometer capable of measuring absorbance at 460 nm
using absorption cells with path-lengths of 2 cm and 10 cm.
Method
Place 15 ml of the solution obtained after combustion in a 50-ml conical flask together with 1 ml of
ferric ammonium sulfate (0.25 mol/l) VS and 3 ml of mercuric thiocyanate TS. Swirl the contents of
the flask and allow to stand for 10 minutes. Transfer a portion of the solution to a 2-cm cell and
measure the absorbance at 460 nm using water in the reference cell. The reading should be made
promptly to minimize absorption of chloride from the air.
Prepare a standard solution of sodium chloride R containing 5 µg of chloride per ml. Transfer aliquots
of this solution (0 ml, 2 ml, 4 ml, 6 ml, 8 ml, and 10 ml) into a series of 50-ml conical flasks and
dilute to 15 ml with water. Develop the colour and measure the absorbances as described above. Plot
the absorbances against the chloride content of the dilutions in µg per ml and interpolate the chloride
content of the solutions of the material tested.
C. Determination of phosphates
The phosphomolybdate method is based on the reaction of phosphate ions with ammonium molybdate
to form a molybdophosphate complex, which is subsequently reduced to form a strongly blue-
coloured molybdenum complex. The intensity of the blue colour is measured spectrophotometrically.
This method is applicable for the determination of any phosphates that have undergone a prior
separation procedure.
Naturally occurring phosphates are present in most samples, and are often not removed during the
clean-up procedure. In order to obtain background values, therefore, it is necessary to proceed with
the determination for all samples, even those with no phosphate-containing pesticides. These
background values should be subtracted from the results obtained on testing pesticide residues.
Extracts of most uncontaminated materials contain about 0.05-0.1 mg/kg of phosphorus. Therefore,
no contamination with organophosphate pesticides can be assumed for results in this range.
Apparatus
The determination is made with a spectrophotometer capable of measuring absorbance at 820 nm
using an absorption cell with a path-length of 1 cm.
Method
Place 7 ml of the solution obtained after combustion in a calibrated 10-ml test-tube. Add 2.2 ml of
sulfuric acid (300 g/l) TS and mix the solution well. Add 0.4 ml of ammonium molybdate (40 g/l) TS
and swirl the mixture. Then add 0.4 ml of aminonaphtholsulfonic acid TS and swirl again. Heat the
solution to 100°C for 12 minutes (±2 minutes), cool, and transfer a portion to a 1-cm cell. Measure the
absorbance at 820 nm using water in the reference cell.
page 10
Prepare standard dilutions with a known content of phosphate and measure the absorbance as
described above. Plot the absorbances against the phosphate content of the dilutions in µg per ml and
interpolate the phosphate content of the solutions of the material tested.
D. Qualitative and quantitative determination of organochlorine pesticides
Preparation of sample
Place 20 g of powdered plant material (sieve no. 180), accurately weighed, in a 500-ml beaker (tall
form), mix with 98 ml of water and allow to macerate for at least 30 minutes. Add 200 ml of acetone
R; the resulting volume of extraction solvent will be 295 ml. Extract for 5 minutes, while cooling and
using a high-speed mixer. Filter the homogenized mixture through a porcelain filter (Büchner funnel,
diameter 70 mm) fitted with a filter paper, using a slight vacuum, into a 250-ml graduated cylinder,
allowing the process to last no longer than 1 minute, and then measure the volume (V) of the filtrate in
ml.
Method
Transfer the filtrate prepared as above to a 500-ml separating funnel. Add a quantity of sodium
chloride R equivalent in grams to one-tenth of the volume of the filtrate, then add 100 ml of
dichloromethane R. Shake vigorously for 5 minutes, allow the phases to separate and discard the
lower (aqueous) layer. Dry the acetone-dichloromethane phase, transfer it to a 500-ml conical flask,
add 25 g of anhydrous sodium sulfate R and swirl occasionally. Next, filter the solution into a 500-ml
flask with a ground-glass stopper using a glass funnel (diameter 100 mm) containing purified glass-
wool and anhydrous sodium sulfate R. Rinse the separating funnel, the conical flask and the glass
funnel twice with 10 ml of ethyl acetate R. Add 5 ml of 2,2,4-trimethylpentane R, and concentrate the
crude extract to about 2 ml in a rotary vacuum evaporator in a water-bath at 30-40 °C. Expel the
remaining solvent in a gentle stream of air.
To purify by gel chromatography, macerate 50 g of suitable beads (e.g. S-X3 bio-beads) in an elution
mixture of cyclohexane R and ethyl acetate R (1:1) and pour them into a chromatographic column
(length 600 mm, diameter 25 mm) adapted for use with a vacuum pump. Rinse the gel bed with the
elution mixture under air-free conditions. Dissolve the extract in the flask with 5 ml of ethyl acetate R.
Add 2 g of anhydrous sodium sulfate R, swirl gently and add 5 ml of cyclohexane R. Filter the
completely dissolved crude extract through a rapid filter into a 10-ml test-tube with a ground-glass
stopper and close the tube immediately. Then transfer 5 ml of the filtrate onto the gel column. Elute
with the elution mixture at an average rate of 5 ml/minute. Plant material components leave the gel
column first, followed by the active ingredients of pesticides. Fractionation must be determined for
each column, using appropriate reference substances.
Discard the first fraction (about 100 ml) containing the impurities. Collect the organochlorine
pesticides appearing in the next eluate (about 70 ml) in a flask with a ground-glass stopper. Add l0 ml
of 2,2,4-trimethylpentane R and concentrate the solution to about 5 ml in a rotary vacuum evaporator
and a water-bath at 30-40 °C. Pipette another 5 ml of 2,2,4-trimethylpentane R into the flask and
carefully evaporate the solution to about 1 ml (do not allow to become completely dry).
Calculate the amount of plant material in grams in the purified extract using the following formula:
V x Sample weight in g
590
where V = volume of filtrate.
page 11
To purify further, transfer 1 g of previously deactivated silica gel for column chromatography (70-230
mesh) containing 1.5 % of water, to a chromatographic column (length 25 cm, internal diameter 7
mm). Put 10 mm of anhydrous sodium sulfate R on top of the content of the column and cover with
purified glass wool. Before use rinse the column with 5 ml of hexane R. Allow the solvent to reach
the surface of the column filling, then transfer quantitatively, by means of a pipette, the purified
extract obtained by gel chromatography from the flask to the prepared silica gel column and rinse
with 1 ml of hexane R. Set the flask aside for subsequent elutions.
Using a 10-ml volumetric flask as the receiver, elute any residues of polychlorinated biphenyls from
the column with 10 ml of hexane R (eluate 0). Add 2 ml of an elution mixture composed of toluene
R/hexane R (35:65) to the flask and swirl. Quantitatively transfer the solution to the column. Using
another 10-ml volumetric flask as the receiver, elute the majority of the organochlorine pesticides
from the silica gel column using 6 ml of the same elution mixture. Dilute the contents of the flasks to
volume with the elution mixture (eluate 1).
Rinse the flask with 2 ml of toluene R and transfer it quantitatively to the column. Collect the eluate
in a third 10-ml volumetric flask. Add 8 ml of toluene R to the flask, swirl and transfer the solution to
the silica gel column; elute the remaining organochlorine pesticides using the same receiver. Dilute
the contents of the flask to volume with toluene R (eluate 2).
Evaluate the test solutions by capillary gas chromatography using an electron capture detector (ECD).
Confirm the findings obtained for the main column (first separation system) with a second capillary
column of different polarity (second separation system).
Determination by gas chromatography

A capillary gas chromatograph with an ECD is used for the measurement. Helium R is used as the
carrier gas and a mixture of argon R and methane R (95:5) as an auxiliary gas for the detection.
First separation system

Use a vitreous silica column, 30 m long with an internal diameter of 0.25 mm, packed with a
chemically bound phase of 5 % phenyl, 95% methyl-polysiloxane. Use the following temperature
programme:
- heat at 60 °C for 0.5 minutes;
- increase the temperature at a rate of 30 °C per minute to 160 °C and maintain this temperature for
2 minutes;
- increase the temperature at a rate of 2 °C per minute to 250 °C and maintain this temperature for 5
minutes.
Use a "split/split-free" injector to inject the sample solution and maintain the injection port at a
temperature of 240 °C. Inject a volume of 1 µl at a rate of 30 seconds ("split-free"). The detector
temperature should be 300 °C.
Second separation system

Use a vitreous silica column, 15 m long with an internal diameter of 0.25 mm, packed with a
chemically bound phase of 7 % cyanopropyl, 7 % phenyl, 86 % methyl-polysiloxane. Use the
following temperature programme:
- heat at 60 °C for 0.2 minutes;
- increase the temperature at a rate of 30 °C per minute to 180 °C and maintain this temperature for
1 minute;
- increase the temperature at a rate of 2 °C per minute to 250 °C and maintain this temperature for 5
minutes.
page 12
Use an on-column injector to inject a volume of 1 µl of the sample solution. The detector temperature
should be 300 °C.
Use the "external standard" method for the qualitative and quantitative evaluation of the
organochlorine pesticides in the test solutions with reference solutions of the following pesticides: α-,
β-, γ- and δ-hexachlorocyclohexane (HCH); hexachlorobenzene; quintozene; aldrin; dieldrin; endrin;
α- and β-endosulfan; endosulfan sulfate; heptachlor, heptachlorepoxide; camphechlor; TDE, DDE and
DDT (both o,p'- and p,p'-isomers); methoxychlor.
Measure the peak height of the pesticides obtained in the chromatograms and calculate the
concentration of the residues in mg/kg using the following formula:
ht x 10 wr
x
w hr
where ht = peak height obtained for the test solution in mm,

w = quantity of sample in the purified extract (g),
wr = quantity of pesticide in ng in the reference solution injected,
hr = peak height obtained for the reference solution in mm.
E. Analysis of esters of organophosphorus compounds

Despite the fact that most organophosphorus compounds undergo rapid decomposition, Member
States may elect to test for them because of their harmful nature if present in significant
concentrations. Testing may be more relevant in the case of herbal medicines used in high
concentrations and frequency.
The extraction and the clean-up procedures can be performed as described above, but the detection
requires a phosphorus flame ionization detector (P-FID).
F. Determination of specific pesticide residues in medicinal plant material

General recommendations
For the total determination, mix thoroughly 1 kg of plant material.
In order to obtain reliable chromatographic results, do one or more of the following:
- repeat the separation using another column;
- use a different separation system;
- use a different detector system;
- apply a coupling technique;
- prepare a derivative;
- perform chromatography with a mixture of the sample and a reference substance;
- change the sample preparation;
- use a fractionated elution during the column-chromatography clean-up procedure of the plant
extract and test every fraction by chromatography; and
- compare the distribution coefficient of the material with that of a reference substance.
Prior to the quantitative determination of the material to be tested, check whether there is a linear
relationship between the values obtained for the reference substance and its concentration over the
range 0.1-2 times the standard concentration. Otherwise, prepare another concentration range or
page 13
evaluate the results using a reference curve. Use any suitable mechanical or manual technique for the
chromatographic determination.
Store the reference solutions protected from light to prevent decomposition. Use glass vessels closed
with glass stoppers and keep them in a container saturated with the solvent employed to avoid any
increase in concentration due to evaporation. Check the loss by evaporation by interim weighing of
the vessels.
Rate of recovery
The rate of recovery (R) is the percentage of the reference material, originally added to the plant
material that is determined using the method described below.
G. Determination of desmetryn, prometryn, and simazine residues

(footnote: In future development of this method, elimination of chloroform by other appropriate solvents is
recommended)
Preparation of the plant material extract

Place 10 g of powdered plant material in a 500-ml conical flask and add 125 ml of chloroform R.
Shake the mixture for 60 minutes and filter under reduced pressure through a filter paper (medium
grade) into a round-bottomed flask. Wash the residue with 3 successive volumes each of 25 ml of
chloroform R.
Method
Concentrate the combined filtrates to a volume of 3-5 ml using a rotary vacuum evaporator and a
water-bath at 40 °C. Transfer the extract to a chromatographic column as prepared below, rinsing the
round-bottomed flask twice with 5 ml of chloroform R.
Preparation of chromatographic column

Use a glass tube (internal diameter 20-22 mm) with a restricted orifice and protected with a sintered-
glass plate (e.g. P10 or P16, glass filter G4; or P40, glass filter G3). Fill the column with chloroform
R, and then pour purified aluminium oxide R into it to form a 100-mm thick layer. The support
material should remain covered with chloroform R. After transferring the extract and the rinsing
liquids to the column, elute with 150 ml of chloroform R, at a rate of 1-2 drops per second, collecting
the eluate in a round-bottomed flask. The first purifying process is completed when no further eluate
drips from the column.
Evaporate the eluate to dryness using a rotary vacuum evaporator and a water-bath at 40 °C. To the
residue add 10 ml of light petroleum R and transfer the mixture to a chromatographic column
containing a layer of purified aluminium oxide R, 50 mm thick, in light petroleum R. Elute the
mixture with 90 ml of light petroleum R, using this to rinse the round-bottomed flask, at a rate of 1-2
drops per second. Discard the eluate. Dissolve any remaining residue, which has not dissolved in light
petroleum R in 10 ml of a mixture composed of 60 volumes of chloroform R, and 40 volumes of light
petroleum R and transfer the solution to the column. Rinse the round-bottomed flask twice more with
10 ml of the solvent mixture. Transfer the liquid used for rinsing to the column. Elute with 120 ml of
the same solvent mixture, at a rate of 1-2 drops per second and collect the eluate in a round-bottomed
flask. The second purifying process is completed when no further eluate drips from the column.
Evaporate the eluate to dryness using a rotary vacuum evaporator and a water-bath at 40 °C. To
prepare a purified extract for the determination by gas chromatography, dissolve the residue in
sufficient acetone R to produce a volume of 10 ml. If an especially purified extract is required,
proceed as described below.
page 14
To the residue add 10 ml of light petroleum R and 10 ml of dimethyl sulfoxide R. Shake the mixture
and transfer it to a separating funnel. Extract the dimethyl sulfoxide layer twice with 10 ml of light
petroleum R. Discard the petroleum ether extract. Then add 100 ml of water to the dimethyl sulfoxide
layer and extract 3 times, each with 20 ml of chloroform R. Extract the combined chloroform extracts
twice with 20 ml of water and evaporate them to dryness using a rotary vacuum evaporator and a
water-bath at 40 °C. Transfer the residue along with a mixture of 10 ml of light petroleum R and 10
ml of hydrochloric acid (1 mol/l) VS to a separating funnel and extract the mixture first with 10 ml
and then with 5 ml of hydrochloric acid (1 mol/l) VS. Discard the petroleum ether layer and adjust the
pH of the combined aqueous solutions to a value between 7 and 8 using sodium hydroxide (1 mol/l)
VS. Extract the solution 3 times, each with 20 ml of chloroform R. Dry the combined chloroform
extracts with anhydrous sodium sulfate R and filter into a round-bottomed flask, rinsing the funnel 3
times with 10.0-ml portions of chloroform R. Evaporate the filtrate to dryness using a rotary vacuum
evaporator and a water-bath at 40 °C. Dissolve the residue in sufficient acetone R to produce 10 ml of
especially purified extract to be used for the determination by gas chromatography.
Use the extracts as indicated below for the following plant materials:
No. Material No. Material

1 Flores Calendulae 10 Fructus Foeniculi
2 Flores Chamomillae 11 Herba Millefolii
3 Folia Melissae 12 Herba Plantaginis lanceolatae
4 Folia Menthae piperitae 13 Radix Althaeae
5 Folia Salviae 14 Radix Angelicae
6 Folia Thymi 15 Radix Levistici
7 Fructus Carvi 16 Radix Petroselini
8 Fructus Coriandri 17 Radix Valerianae
9 Fructus Cynobasti
For materials no. 1 and 2, use an especially purified extract (see above pages); for materials no. 3-17,
use a purified extract (see above pages).
Determination of the rate of recovery
Prepare five individual samples using each of the following procedures:
1. To prepare solution S2, first dissolve separately 0.04 g of each of the reference substances,
desmetryn R, prometryn R and simazine R, in sufficient acetone R to produce 100 ml. Then place 5
ml of each solution into a 100-ml volumetric flask and dilute the mixture to volume with acetone R
(S2). Place 10 g of powdered plant material into a 500-ml conical flask and add 1 ml of solution S2.
Shake this mixture mechanically for 60 minutes; if necessary, repeat the operation manually and then
proceed as described under "Preparation of the plant material extract". Use either the purified or
especially purified extract for the determination by gas chromatography, as specified in the test
procedure for the plant material concerned.
2. Treat 10 g of powdered plant material as described under "Preparation of the plant material
extract". Use either the purified extract or the especially purified extract for the determination by gas
chromatography, as specified in the test procedure for each individual plant material.
Calculate the rate of recovery (R) in % using the following formula:

page 15
2(a-b)
c
where a = average quantity in mg/kg of the 5 residues obtained using procedure 1,
b = average quantity in mg/kg of the 5 residues obtained using procedure 2,
c = quantity of reference substances in mg contained in solution S2 during procedure 1.
The rate should be within the range 70-120 %. It is specific for each drug.
Determination by gas chromatography

Perform the determination as described in the International Pharmacopoeia.
Apparatus
The equipment consists of:
- a glass column 1.2 m long, internal diameter 2 mm;
- a suitable stationary liquid phase;
- a suitable diatomaceous support.
Use nitrogen R as the carrier gas with a flow rate of 30 ml/min. The sample injection block should be
maintained at 230 °C, the column at 190 °C and the detector, which should be nitrogen-selective, at
300 °C. In addition:
- volume of sample solution to be injected: 2.0 µl;
- separation characteristics: h. 1.2 x 10-3 for desmetryn R; RS . 1.2 for prometryn R and simazine R;
- relative standard deviation (precision of chromatographic system): sr. 0.05 for desmetryn R,
prometryn R and simazine R.
Method
Chromatogram T. To determine the separation characteristics, inject solution S2 (for the preparation of
solution S2 see "Determination of the rate of recovery" above). Chromatograms A1-A5. To determine
the relative standard deviation, inject solution S2 and repeat the determination 5 times.
Chromatogram S2. Inject 1 ml of solution S2 for the determination of the rate of recovery. Dilute 1 ml
of solution S2 to 10 ml with acetone R and inject it for the chromatographic determination. On the
chromatogram the peaks occur in the following sequence: prometryn, simazine, desmetryn.
Chromatogram P2. Inject the purified extract or the especially purified extract. Determine using an
external standard: a = 0.0005. To convert the values obtained to percentage by weight, multiply the
concentration in mg/kg by 104.
The total maximum permissible amount of residues due to desmetryn, prometryn and simazine is 2
mg/kg of plant material.
H. Determination of specific organochlorine, organophosphorus and pyrethroid

insecticide residues
Depending on the substance being examined, it may be necessary to modify, sometimes extensively,
the procedure described hereafter. In any case, it may be necessary to use, in addition, another column
with a different polarity or another detection method (e.g. mass spectrometry) or a different method
(e.g. immunochemical methods) to confirm the results obtained.
This procedure is valid only for the analysis of samples of medicinal plant materials containing less
page 16
than 15 per cent of water. Samples with a higher content of water may be dried, provided it has been
shown that the drying procedure does not affect significantly the pesticide content.
1. Extraction.
To 10 g of the substance being examined, coarsely powdered, add 100 ml of acetone R and allow to
stand for 20 min. Add 1 ml of a solution containing 1.8 µg/ml of carbophenothion R in toluene R.
Homogenise using a high-speed blender for 3 min. Filter and wash the filter cake with two quantities,
each of 25 ml, of acetone R. Combine the filtrate and the washings and heat using a rotary evaporator
at a temperature not exceeding 40 °C until the solvent has almost completely evaporated. To the
residue add a few millilitres of toluene R and heat again until the acetone is completely removed.
Dissolve the residue in 8 ml of toluene R. Filter through a membrane filter (45 µm), rinse the flask
and the filter with toluene R and dilute to 10.0 ml with the same solvent (solution A).
2. Purification
2.1. Organochlorine, organophosphorus and pyrethroid insecticides.

Examine by size-exclusion chromatography.
The chromatographic procedure may be carried out using:
— a stainless steel column 0.30 m long and 7.8 mm in internal diameter packed with styrene-
divinylbenzene copolymer R (5 µm),
— as mobile phase toluene R at a flow rate of 1 ml/min.
Performance of the column. Inject 100 µl of a solution containing 0.5 g/l of methyl red R and 0.5 g/l
of oracet blue 2R R in toluene R and proceed with the chromatography. The column is not suitable
unless the colour of the eluate changes from orange to blue at an elution volume of about 10.3 ml. If
necessary calibrate the column, using a solution containing, in toluene R, at a suitable concentration,
the insecticide to be analysed with the lowest molecular mass (for example, dichlorvos) and that with
the highest molecular mass (for example, deltamethrin). Determine which fraction of the eluate
contains both insecticides.
Purification of the test solution. Inject a suitable volume of solution A (100 µl to 500 µl) and proceed
with the chromatography. Collect the fraction as determined above (solution B). Organophosphorus
insecticides are usually eluted between 8.8 ml and 10.9 ml. Organochlorine and pyrethroid
insecticides are usually eluted between 8.5 ml and 10.3 ml.
2.2. Organochlorine and pyrethroid insecticides.

In a chromatography column, 0.10 m long and 5 mm in internal diameter, introduce a piece of
defatted cotton and 0.5 g of silica gel treated as follows: heat silica gel for chromatography R in an
oven at 150 °C for at least 4 h. Allow to cool and add dropwise a quantity of water R corresponding to
1.5 per cent of the mass of silica gel used; shake vigorously until agglomerates have disappeared and
continue shaking for 2 h using a mechanical shaker. Condition the column using 1.5 ml of hexane R.
Prepacked columns containing about 0.5 g of a suitable silica gel may also be used provided they are
previously validated.
Concentrate solution B in a current of helium for chromatography R or oxygen-free nitrogen R almost

page 17
to dryness and dilute to a suitable volume with toluene R (200 µl to 1 ml according to the volume
injected in the preparation of solution B). Transfer quantitatively onto the column and proceed with
the chromatography using 1.8 ml of toluene R as the mobile phase. Collect the eluate (solution C).
Quantitative analysis
1. Organophosphorus insecticides.
Examine by gas chromatography, using carbophenothion R as internal standard. It may be necessary
to use a second internal standard to identify possible interference with the peak corresponding to
carbophenothion.
Test solution. Concentrate solution B in a current of helium for chromatography R almost to dryness
and dilute to 100 µl with toluene R.
Reference solution. Prepare at least three solutions in toluene R containing the insecticides to be
determined and carbophenothion at concentrations suitable for plotting a calibration curve.
— a fused-silica column 30 m long and 0.32 mm in internal diameter the internal wall of which is
covered with a layer 0.25 µm thick of poly(dimethyl)siloxane R,
— hydrogen for chromatography R as the carrier gas. Other gases such as helium for
chromatography R or nitrogen for chromatography R may also be used provided the
chromatography is suitably validated.
— a phosphorus-nitrogen flame-ionisation detector or a atomic emission spectrometry detector,
maintaining the temperature of the column at 80 °C for 1 min, then raising it at a rate of 30 °C/min to
150 °C, maintaining at 150 °C for 3 min, then raising the temperature at a rate of 4 °C/min to 280 °C
and maintaining at this temperature for 1 min, and maintaining the temperature of the injector port at
250 °C and that of the detector at 275 °C. Inject the chosen volume of each solution. When the
chromatograms are recorded in the prescribed conditions, the relative retention times are
approximately those listed in Table 2. Calculate the content of each insecticide from the peak areas
and the concentrations of the solutions.
page 18
Table 2
Substances Relative retention times
dichlorvos 0.20
fonofos 0.50
diazinon 0.52
parathion-methyl 0.59
chlorpyrifos-methyl 0.60
pirimiphos-methyl 0.66
malathion 0.67
parathion 0.69
chlorpyrifos 0.70
methidathion 0.78
ethion 0.96
carbophenothion 1.00
azinphos-methyl 1.17
phosalon 1.18
[Note: Relative retention times are very close. If distinction needed further development of a method
is needed]
2. Organochlorine and pyrethroid insecticides.

Examine by gas chromatography, using carbophenothion as the internal standard. It may be necessary
to use a second internal standard to identify possible interference with the peak corresponding to
carbophenothion.
Test solution. Concentrate solution C in a current of helium for chromatography R or oxygen-free

nitrogen R almost to dryness and dilute to 500 µl with toluene R.
Reference solution. Prepare at least three solutions in toluene R containing the insecticides to be
determined and carbophenothion at concentrations suitable for plotting a calibration curve.
— a fused silica column 30 m long and 0.32 mm in internal diameter the internal wall of which is
covered with a layer 0.25 µm thick of poly(dimethyl)(diphenyl)siloxane R,
— hydrogen for chromatography R as the carrier gas. Other gases such as helium for
chromatography R or nitrogen for chromatography R may also be used, provided the
chromatography is suitably validated,
— an electron-capture detector,
— a device allowing direct cold on-column injection,
maintaining the temperature of the column at 80 °C for 1 min, then raising it at a rate of 30 °C/min to
150 °C, maintaining at 150 °C for 3 min, then raising the temperature at a rate of 4 °C/min to 280 °C
and maintaining at this temperature for 1 min, and maintaining the temperature of the injector port at
250 °C and that of the detector at 275 °C. Inject the chosen volume of each solution. When the
chromatograms are recorded in the prescribed conditions, the relative retention times are
approximately those listed in Table 3. Calculate the content of each insecticide from the peak areas
and the concentrations of the solutions.
page 19
Table 3
Substances Relative retention times

α- hexachlorocyclohexane 0.44
hexachlorobenzene 0.45
β- hexachlorocyclohexane 0.49
lindane 0 49
δ- hexachlorocyclohexane 0.54
ε- hexachlorocyclohexane 0.56
heptachlor 0.61
aldrin 0.68
Cis-heptachlor-epoxide 0.76
o,p'-DDE 0.81
α-endosulfan 0.82
dieldrin 0.87
p,p'-DDE 0.87
o,p'-DDD 0.89
endrin 0.91
Β-endosulfan 0.92
o,p'-DDT 0.95
carbophenothion 1.00
p,p'- DDT 1.02
Cis-permethrin 1.29
trans-permethrin 1.31
cypermethrin* 1.40
fenvalerate* 1.47 and 1.49
deltamethrin 1.54
* The substance shows several peaks

page 20
Determination of arsenic and toxic metals

Table of contents
A. Limit tests..................................................................................................19
A.1. Limit test for arsenic..................................................................20
A.2. Limit test for cadmium and lead................................................22
A.3 Limit test for total toxic metals as lead......................................23
A.4 Limit test for total toxic metals as lead in extracts.................................. 23
B. Determination of specific toxic metals.................................................... 24
B.1. Detection of cadmium, copper, iron, lead, nickel and zinc...... 25
B.2. Detection of arsenic and mercury..............................................25
A. Introduction/background
Quantitative Tests and Limits Tests in general accurately determine the concentrations of toxic metals
as impurities and contaminants. The latter are unavoidably present in these samples being tested i.e.
herbals and their herbal products. Member States can elect to use either of these types of tests and
their choices would be influenced by the nature of the sample and the contaminants or residue, on a
case-by-case basis. Another factor would be that the identified and chosen method(s) that can be
applied to control heavy metals should be relevant and acceptable to the requirements at a regional
and national level.
Some examples of proposed national limits for arsenic and toxic metals in various types of herbal
products are shown in Table 4, below. Country figures were based on information provided by
respective national health authorities.
TABLE 4. EXAMPLES OF NATIONAL LIMITS FOR ARSENIC AND TOXIC METALS IN HERBAL MEDICINES AND PRODUCTS
Arsenic Lead Cadmium Chromium Mercury Copper Total Toxic

Metals as
(As) (Pb) (Cd) (Cr) (Hg) (Cu) Lead
For herbal medicines
raw medicinal plant 5 ppm 10 ppm 0.3 ppm 2 ppm 0.2 ppm
Canada materials
finished herbal products 0.01mg/day 0.02mg/day 0.006mg/day 0.02mg/day 0.02mg/day
China to be medicinal plant materials 2 ppm 10 ppm 1 ppm 0.5 ppm 20 ppm
changed..
Malaysia finished herbal products 5 mg/kg 10 mg/kg 0.5 mg/kg
Republic of Korea medicinal plant materials 30 ppm
Singapore finished herbal products 5 ppm 20 ppm 0.5 ppm 150 ppm
Thailand medicinal plant material, 4 ppm 10 ppm 0.3 ppm

finished herbal products
WHO recommendations (2) 10 mg/kg 0.3 mg/kg
For other herbal products
National Sanitation Foundation draft proposal

(Raw Dietary supplement)* 5 ppm 10 ppm 0.3 ppm 2 ppm
National Sanitation Foundation draft proposal
(Finished Dietary Supplement) * 0.01mg/day 0.02mg/day 0.006mg/day 0.02mg/day 0.02mg/day
*Dietary supplements - NSF International Draft Standard (Draft Standard NSF 173-2001), National Sanitation Foundation International, Ann Arbor,
Michigan, USA, 2001
page 21
The metals are widely distributed throughout nature and occur freely in soil, water and as mentioned
before are often components of certain pesticides.
In general, during analyses of the metals one should always aim to use the best and latest methods
whenever possible. However, it is crucial to ensure that all methods are fully validated, not forgetting
the need to validate the integrity of the starting matrix of plant product. This imperative should apply
to both governments and companies/applicants submitting these methods as part of their applications
to the country’s regulatory authority for market authorization.
Limit Tests find wide application in the world of pharmaceutical medicines where it is common to test
for substances such as chlorides, sulfates, arsenic and heavy metals. Thus they will be very useful in
the testing of herbals and their products. Limit Tests can also be modified in many instances to
function as true limits test where the actual amount of toxic metal can be estimated with great
accuracy.
The need for inclusion of tests for toxic metals and acceptance criteria should be studied during
development and based on knowledge of the plant species, its growth and or cultivation and the
manufacturing process. The choice of procedures for control should take account of this information:
the use of a limit test versus a specific quantitative method will depend on the level of control
required on a particular material, e.g. starting material, etc., and should be justified.
In general, where the burden of the plant material with heavy metals is unknown it is suggested that it
be identified qualitatively and quantitatively on several batches collected preferably over several
years. These data should be used to establish acceptance limits that should be checked by appropriate
limit tests.
A. 1 Limit test for arsenic

Arsenic is abundantly found in nature and its presence in herbals and herbal medicines should be no
different to its wide occurrence in foods.
A popular method relies on the digestion of the plant matrix and then subjection of the digestate to a
comparative colorimetric test in a special apparatus.
Procedure
Preparation of the sample by acid digestion

Place 35-70 g of coarsely ground material, accurately weighed, in a Kjeldahl flask, capacity 800-1000
ml. Add 10-25 ml of water and 25-50 ml of nitric acid (~1000 g/l) TS and then carefully add 20 ml of
sulphuric acid (~1760 g/l) TS. Heat cautiously so that no excessive foaming takes place. Gradually
add nitric acid (~1000 g/l) TS, drop by drop, until all the organic matter is destroyed. This is achieved
when no further darkening of the solution is observed with continued heating, and a clear solution
with copious vapours of sulfur trioxide is obtained. Cool, and add 75 ml of water and 25 ml of
ammonium oxalate (25 g/l) TS. Heat again until sulfur trioxide vapours develop. Cool, transfer with
the help of water to a 250-ml volumetric flask, and dilute to volume with water.
Apparatus
A suitable type of apparatus is constructed as follows. A wide-mouthed bottle of about 120-ml
capacity is fitted with a rubber bung through which passes a glass tube. The tube, made from ordinary
glass tubing, has a total length of about 200 mm and an internal diameter of exactly 6.5 mm (external
page 22
diameter about 8 mm). The lower end of the tube is drawn out to an internal diameter of about 1 mm,
and there is a hole not less than 2 mm in diameter blown in the side of the tube, near the constricted
part. The tube is positioned so that when the bottle contains 70 ml of liquid the constricted end is
above the surface of the liquid and the hole in the side is below the bottom of the bung. The upper
end of the tube has a flat, ground surface at a right angle to the axis of the tube, with slightly rounded-
off edges.
One of two rubber bungs (about 25 mm x 25 mm), each with a central hole of exactly 6.5 mm
diameter, is fitted onto the upper end of the tube. The other bung is fitted with a piece of glass tube
about 3 mm long and with an internal diameter of exactly 6.5 mm and with a similar ground surface.
One end of each of the tubes is flush with the larger end of the bungs, so that when these ends are held
tightly together with a rubber band or a spring clip, the openings of the two tubes meet to form a true
tube. Alternatively, the two bungs may be replaced by any suitable construction satisfying the
conditions described in the test.
Method
Moisten some cotton-wool with lead acetate (80 g/l) TS, allow to dry, and lightly pack into the tube
which fits into the wide-mouthed bottle to not less than 25 mm from the top. Between the flat
surfaces of the tubes, place a piece of mercuric bromide paper AsR that is large enough to cover their
openings (15 mm x 15 mm). The mercuric bromide paper AsR can be fitted by any other means
provided that:
- the whole of the evolved gas passes through the paper;
- the portion of the paper in contact with the gas is a circle 6.5 mm in diameter; and
- the paper is protected from sunlight during the test.
Place an aliquot (25-50 ml) of the solution being tested, prepared as described above, in the wide-
mouthed bottle, add 1 g of potassium iodide AsR and l0 g of granulated zinc AsR, and place the
prepared glass tube assembly quickly in position. Allow the reaction to proceed for 40 minutes.
Compare any yellow stain that is produced on the mercuric bromide paper AsR with a standard stain
produced in a similar manner with a known quantity of dilute arsenic AsTS. Examine the test and
standard stains without delay in daylight; the stains fade with time.
The most suitable temperature for carrying out the test is generally about 40 °C but, as the rate of
evolution of the gas varies somewhat with different batches of granulated zinc AsR, the temperature
may have to be adjusted to obtain an even evolution of gas. Placing the apparatus on a warm surface,
care being taken to ensure that the mercuric bromide paper AsR remains dry throughout may
accelerate the reaction.
Between successive tests, the tube must be washed with hydrochloric acid (~250 g/l) AsTS, rinsed
with water and dried.
Preparation of standard stain

Add 10 ml of stannated hydrochloric acid (~250 g/l) AsTS and 1 ml of dilute arsenic AsTS to 50 ml
of water. The resulting solution, when treated as described in the general test, yields a stain on
mercuric bromide paper AsR referred to as the standard stain (10 µg of As).
The amount of arsenic in the medicinal plant material is estimated by matching the depth of the colour
on the sample paper with that of the standard stain.
page 23
A. 2 Limit test for cadmium and lead

Procedure
Apparatus
The equipment consists of a digestion vessel, consisting of a vitreous silica crucible (DIN 12904),
"tall form", height 62 mm, diameter 50 mm, capacity 75 ml, with a vitreous silica cover.
Materials used are:

- digestion mixture: 2 parts by weight of nitric acid (~1000 g/l) TS and 1 part by weight of
perchloric acid (~1170 g/l) TS.
- reference materials: olive leaves (Olea europaea)1 and hay powder2.
Clean scrupulously with nitric acid (~1000 g/l) TS the digestion vessel and all other equipment to be
used for the determination rinse thoroughly several times with water and dry at 120 °C.
Preparation of the sample

For the wet digestion method in an open system, place 200-250 mg of air-dried medicinal plant
material, accurately weighed, finely cut and homogeneously mixed, into a cleaned silica crucible. Add
1.0ml of the digestion mixture, cover the crucible without exerting pressure and place it in an oven
with a controlled temperature and time regulator (computer-controlled, if available).
Heat slowly to 100 °C and maintain at this temperature for up to 3 hours; then heat to 120 °C and
maintain at this temperature for 2 hours. Raise the temperature very slowly to 240 °C, avoiding losses
due to possible violent reactions especially in the temperature range of 160-200 °C, and maintain at
this temperature for 4 hours. Dissolve the remaining dry inorganic residue in 2.5 ml of nitric acid
(~1000 g/l) TS and use for the determination of heavy metals.
Every sample should be tested in parallel with a blank.
Method
The contents of lead and cadmium may be determined by inverse voltametry or by atomic absorption
spectrophotometry.
A. 3 Limit tests for total toxic metals as lead

In this method, the heavy metals are the metallic inclusions that are darkened with sodium sulphide
TS in acidic solution, as their quantity is expressed in terms of the quantity of lead (Pb).
Preparation of sample solution and blank solution

Place an amount of the sample, directed in the monograph, in a quartz or porcelain crucible, cover
loosely with a lid, and carbonise by gentle ignition. After cooling, add 2 ml of nitric acid and 5 drops
of sulphuric acid, heat cautiously until white fumes are evolved, and incinerate by ignition between
500 °C and 600 °C. Cool, add 2 ml of hydrochloric acid, evaporate to dryness on a water-bath,
moisten the residue with 3 drops of hydrochloric acid, add 10 ml of hot water, and warm for 2
1
BCR reference material CRM No. 62 Community Bureau of Reference, obtainable from BCR, Directorate-General X11,
Commission of the European Communities, 200 rue de la Loi, B-1049 Brussels, Belgium.
2
Obtainable from IAEA/V-10, International Atomic Energy Agency, Analytical Quality Control Services, Laboratory
Geibersdorf, P.O. Box 100, A-Vienna, Austria.
page 24
minutes. Then add 1 drop of phenolphthalein TS, add ammonia TS drop-wise until the solution
develops a pale red colour, add 2 ml of dilute acetic acid, filter, if necessary, and wash with 10 ml of
water. Transfer the filtrate and washings to a Nessler tube, and add water to make 50 ml. Designate it
as the test solution.
The control solution is prepared as follows: Evaporate a mixture of 2 ml of nitric acid, 5 drops of
sulphuric acid, and 2 ml of hydrochloric acid on a water-bath, further evaporate to dryness on a sand-
bath, and moisten the residue with 3 drops of hydrochloric acid. Hereinafter, proceed as directed in
the test solution, and then add the volume of standard lead solution directed in the monograph and
water to make 50 ml.
Procedure
Add 1 drop of sodium sulfide TS to each of the test solution and the control solution, mix thoroughly,
and allow to stand for 5 minutes. Then compare the colours of both solutions by viewing the tubes
downward or transversely against a white background. The test solution has no more colour than the
control solution.
A. 4 Limit test for total toxic metals as lead in extracts

Ignite 0.3 g of extracts to ash, warm with 3 ml of dilute hydrochloric acid, and filter. Wash the residue
with two 5 ml portions of water. Neutralize the combined filtrate and washings by adding ammonia
TS, filter, if necessary, and add 2 ml of dilute acetic acid and water to make 50 ml. Perform the Heavy
Metals Limit Test using this solution as the test solution. Control solution: Proceed with 3 ml of dilute
hydrochloric acid in the same manner as directed in the preparation of the test solution, and add 3 ml
of standard lead solution 1 ppm and water to make 50 ml.
Procedure
Add 1 drop of sodium sulfide TS to each of the test solution and the control solution, mix thoroughly,
and allow to stand for 5 minutes. Then compare the colours of both solutions by viewing the tubes
downward or transversely against a white background. The test solution has no more colour than the
control solution.
B. Determination of specific toxic metals

Atomic absorption spectrometry (AA) is used for the determination of the amount or concentration
specific heavy metals. AA uses the phenomenon that atoms being in the ground state absorb the light
of specific wavelength, characteristic of the respective atom, when the light passes through an atomic
vapour layer of element to be determined (Japanese Pharmacopoeia, 14th ed.).
Caution must be exercised when using the recommended closed high-pressure digestion vessels and
microwave laboratory equipment, and there should be full familiarity with the safety and operating
instructions given by the manufacturer.
Procedure
Apparatus
The apparatus consists of the following:
page 25
Usually the apparatus consists of a light source, a sample atomiser, a spectroscope, a photometer and a
recording system.
ii. a digestion flask, polytetrafluoroethylene flask with a volume of about 120 ml, fitted with an
airtight closure, a valve to adjust the pressure inside the container and a
polytetrafluoroethylene tube to allow release of gas(1), A good example is the Digestion Vessel
Assembly P/N ZZ 1000
iii. a system to make flasks airtight, using the same torsional force(2), an example is the CEM
Capping station calibration
iv. a microwave oven, with a magnetron frequency of 2450 MHz, with a selectable output from 0
to 630 ± 70 W in 1 per cent increments, a programmable digital computer, a
polytetrafluoroethylene-coated microwave cavity with a variable speed exhaust fan, a rotating
turntable drive system and exhaust tubing to vent fumes
v. an atomic absorption spectrometer, equipped with an appropriate lamp for each element as
source of radiation and a deuterium lamp as background corrector; the system is fitted with a
sample atomiser of which there are three types with: the flame type, the electrochemical type
and the cold-vapour type.
(a) a graphite furnace (in the electrochemical type) as atomisation device for cadmium, copper,
iron, lead, nickel, zinc. An example is the Vapour Generation Accessory
(b) an automated continuous-flow hydride vapour generation system for arsenic and mercury(3).
(1)
Digestion Vessel Assembly P/N ZZ 1000 is suitable.
(2)
CEM Capping station calibration is suitable.
(3)
Vapour Generation Accessory is suitable.
Method
Clean all the glassware and laboratory equipment with a 10 g/l solution of nitric acid R in water R
before use.
Test solution. In a digestion flask place the prescribed quantity of the substance to be examined (about
0.5 of powdered drug or 0.5 g of fatty oil). Add 3 ml of nitric acid R, 1 ml hydrogen peroxide R and
1 ml of hydrochloric acid R. Seal the flask so that it is 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 and to each add 4
ml of sulphuric acid R. Repeat the digestion programme. After cooling in air, 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 water R and collect the rinsings in the volumetric
flask. Add 1 ml of 10 g/l solution of magnesium nitrate R and 1 ml of 100 g/l solution of ammonium
dihydrogen phosphate R and dilute to 50 ml with water R.
Blank solution. Mix 3 ml of nitric acid R 1 ml hydrogen peroxide R (30%) and 1ml of hydrochloric
acid R in a digestion flask. Carry out the digestion in the same manner as for the test solution.
page 26
B. 1 Detection of cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), nickel (Ni)
and zinc (Zn)
Measure the content of cadmium, copper, iron, lead, nickel and zinc by the standard additions method
using reference solutions of each heavy metal and the instrumental parameters that may be used are
described in Table 5.
The absorbance value of the compensation liquid (blank solution) is subtracted from the value
obtained with the test solution.
Table 5
Cd Cu Fe Ni Pb Zn
Wavelength nm 228.8 324.8 248.3 232 283.5 213.9
Slit width nm 0.5 0.5 0.2 0.2 0.5 0.5
hollow-cathodeLamp mA 6 7 5 10 5 7
current
Ignition temperature αC 800 800 800 800 800 800
Atomisation temperature αC 1800 2300 2300 2500 2200 2000
Background corrector On On On On On On
Nitrogen flow Litre/min 3 3 3 3 3 3
B. 2 Detection of arsenic (As) and mercury (Hg)

Measure the content of arsenic and mercury in comparison with the reference solutions of arsenic or
mercury at a known concentration by direct calibration using an automated continuous-flow hydride
vapour generation system.
The absorbance value of the compensation liquid (blank solution) is automatically subtracted from the
value obtained with the test solution.
Arsenic
Sample solution. To 19 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 possibility to stabilize adding ascorbic acid. Allow the test
solution to stand at room temperature for about 50 min or at 70 °C for about 4 min.
Acid reagent. Hydrochloric acid R.
Reducing reagent. A 6 g/l solution of sodium tetrahydroborate R in a 5 g/l solution of sodium
hydroxide R.
The instrumental parameters in Table 6 may be used.
Mercury
Sample solution. Test solution or blank solution, as prescribe above.
Acid reagent. A 515 g/l solution of hydrochloric acid R.
Reducing reagent. A 10 g/l solution of stannous chloride R or sodium tetrahydroborate in dilute
hydrochloric acid R.
The instrumental parameters in Table 6 may be used.
page 27
Table 6
As Hg
Wavelength nm 193.7 253.7
Slit width nm 0.2 0.5
Hollow-cathode 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
Sample solution flow rate ml/min 7.0 7.0
Absorption cell Quartz (heated) Quartz (unheated)
Background corrector on on
Nitrogen flow rate litre/min 0.1 0.1
Heating 800C 100C

page 28
Determination of Microbiological contaminants

Table of contents
A. Total viable aerobic count (TVC)............................................................27
A. 1. Pre-treatment of test plant materials .......................................... 27
A. 2. Test procedures........................................................................................ 28
A. 3. Effectiveness of the culture medium, confirmation of antimicrobial
substances and validity of the counting methods ……………………… 30
B. Test for specific microorganisms .......................................................... 31
B. 1. Pretreatment of the materials being examined.......................... 31
B. 2. Enterobacteria and certain other gram-negative bacteria.......... 31
Detection of bacteria............................................................. 31
Quantitative evaluation......................................................... 31
Escherichia coli..................................................................... 32
Salmonella spp...................................................................... 32
Pseudomonas aeruginosa...................................................... 33
Staphylococcus aureus.......................................................... 33
Clostridia.............................................................................. 34
Shigella................................................................................. 34
B.3. Validation of tests for specific microorganisms................... 35
C. Microbial contamination limits in herbal medicines......................... 35
A. Total viable aerobic count (TVC)
The total viable aerobic count (TVC) of the plant material being examined is determined, as specified
in the test procedure, using one of the following methods: membrane-filtration, plate count or serial
dilution. Aerobic bacteria and fungi (moulds and yeasts) are determined by this TVC method.
Usually a maximum permitted level is set for certain products but whenever the TVC exceeds this
level then it is unnecessary to proceed with determination of specific organisms; the material should
then be rejected without being subjected to further testing.
Procedure
A. 1 Pretreatment of the test plant material
Depending on the nature of the crude medicinal plant material, grind, dissolve, dilute, suspend or
emulsify it using a suitable method and then eliminate any antimicrobial properties by dilution,
neutralization or filtration. Either Phosphate Buffer pH 7.2, Buffered Sodium Chloride-Peptone
Solution. pH 7.0 or fluid medium, used for the test, is used to suspend or dilute this test specimen.
Different materials have special requirements, which have to be met for acceptable pre-treatment to be
performed. Some examples follow:
i. Materials containing tannins, antimicrobial substances

Some herbal preparations present difficulties in determining microbial levels e.g. those
containing high tannin, essential oils. When test specimens have antimicrobial activity or
contain antimicrobial substances, any such antimicrobial properties are removed as mentioned
before. For the test, use a mixture of several portions selected at random from the bulk or from
the contents of a sufficient number of containers. If the test specimens are diluted with fluid
medium, the test should be performed quickly..
page 29
ii. Water-soluble materials

Dissolve or dilute 10 g or 10 ml of plant material, unless otherwise specified in the test
procedure for the material concerned, in lactose broth or another suitable medium proven to
have no antimicrobial activity under the conditions of the test. Adjust the volume to 100 ml
with the same medium. (Noting that some materials may require the use of larger volumes.) If
necessary, adjust the pH of the suspension to about 7.
iii. Non-fatty materials insoluble in water

Suspend 10 g or 10 ml of the plant material, unless otherwise specified in the test procedure
for the material concerned, in lactose broth or another suitable medium proven to have no
antimicrobial activity under the conditions of the test; dilute to 100 ml with the same medium.
(Noting that some materials may require the use of a larger volume.) If necessary, divide the
material and homogenize the suspension mechanically. A suitable surfactant, such as a
solution of Polysorbate 20R or 80 R containing 1 mg per ml may be added to aid dissolution.
If necessary, adjust the pH of the suspension to about 7.
iv. Fatty materials

Homogenize 10 g or 10 ml of material, unless otherwise specified in the test procedure for the
material concerned, with 5 g of Polysorbate 20R or 80R. If necessary, heat to a temperature
not exceeding 40 °C. (Occasionally, it may be necessary to heat to a temperature of up to 45
°C, for the shortest possible time). Mix carefully while maintaining the temperature in a water-
bath or oven. Add 85 ml of lactose broth or another suitable medium proven to have no
antimicrobial activity in the conditions of the test, if necessary heated to a temperature not
exceeding 40 °C. Maintain this temperature for the shortest time necessary until an emulsion is
formed and, in any case, for not more than 30 minutes. If necessary, adjust the pH of the
emulsion to about 7.
A.2 Test procedures
Plate count
For bacteria use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 1 ml of the pre-
treated plant material and about 15 ml of liquefied casein-soybean digest agar at a temperature not
exceeding 45 °C. Alternatively, spread the material on the surface of the solidified medium in a Petri
dish. If necessary, dilute the material to obtain an expected colony count of not more than 300.
Prepare at least two dishes using the same dilution, invert them and incubate them at 30-35 °C for 48-
72 hours, unless a more reliable count is obtained in a shorter period of time. Count the number of
colonies formed and calculate the results using the plate with the largest number of colonies, up to a
maximum of 300.
For fungi use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 1 ml of the pre-treated
material and about 15 ml of liquefied Sabouraud glucose agar with antibiotics (also used is Potato
Dextrose Agar with antibiotics) at a temperature not exceeding 45 °C. Alternatively, spread the
pretreated material on the surface of the solidified medium in a Petri dish. If necessary, dilute the
material as described above to obtain an expected colony count of not more than 100. Prepare at least
two dishes using the same dilution and incubate them upright at 20-25 °C for 5 days, unless a more
reliable count is obtained in a shorter period of time. Count the number of colonies formed and
calculate the results using the dish with not more than 100 colonies.
page 30
Membrane filtration
Use membrane filters with a nominal pore size of not greater than 0.45 µm, and with a proven
effectiveness of retaining bacteria, e.g. cellulose nitrate filters are used for aqueous, oily and weakly
alcoholic solutions, while cellulose acetate filters are better for strongly alcoholic solutions. The
technique below uses filter discs of about 50 mm in diameter. Where filters of a different diameter are
used, adjust the volumes of the dilutions and washings accordingly. Sterilize, by appropriate means,
the filtration apparatus and the membrane as the solution is introduced, filtered and examined under
aseptic conditions, and the membrane then transferred to the culture medium.
The detailed method

Transfer 10 ml or a solution containing 1g of the material to each of two membrane filter
apparati and filter immediately. If necessary, dilute the pretreated material to obtain an
expected colony count of 10-100. Wash each membrane, filtering three or more successive
quantities of approximately 100 ml of a suitable liquid such as buffered sodium chloride-
peptone solution of pH 7.0. For fatty materials, a suitable surfactant may be added, such as
Polysorbate 20R or 80R. Transfer one of the membrane filters, intended primarily for the
enumeration of bacteria, to the surface of a plate with casein-soybean digest agar and the
other, intended primarily for the enumeration of fungi, to the surface of a plate with Sabouraud
glucose agar with antibiotics. Incubate the plates for 5 days, unless a more reliable count can
be obtained otherwise, at 30-35 °C for the detection of bacteria and at 20-25 °C for the
detection of fungi. Count the number of colonies formed. Calculate the number of
microorganisms per gram or per ml of the material tested, if necessary counting bacteria and
fungi separately.
Serial dilution
Prepare a series of 12 tubes each containing 9-10 ml of soybean-casein digest medium. To each of
the:
a. first group of three tubes add 1 ml of the 1:10 dilution of dissolved, homogenized material
(containing 0.1 g or 0.1 ml of specimen) prepared as described later in the guideline
b. second group of three tubes add 1 ml of a 1:100 dilution of the material and to the
c. third group of three tubes add 1 ml of a 1:1000 dilution of the material
d. last three tubes add 1 ml of the diluent.
Incubate the tubes at 30-35 °C for at least 5 days. No microbial growth should appear in the last three
tubes. If the reading of the results is difficult or gives an uncertain reading, owing to the nature of the
material being examined, prepare a subculture in a liquid or a solid medium, and evaluate the results
after a further period of incubation. Determine the most probable number of microorganisms per g or
ml of the material using Table 7.
If, for the first column, the number of tubes showing microbial growth is two or less, the most
probable number of microorganisms per g or per ml is less than 100.
page 31
Table 7. Determination of total viable aerobic count
a
Number of tubes with microbial Growth Most probable number of
100mg or 10mg or 1mg or microorganisms per g or ml
0.1 ml per tube 0.01 ml per tube 0.001 ml per tube
3 3 3 >1100
3 3 2 1100
3 3 1 500
3 3 0 200
3 2 3 290
3 2 2 210
3 2 1 150
3 2 0 90
3 1 3 160
3 1 2 120
3 1 1 70
3 1 0 40
3 0 3 95
3 0 2 60
3 0 1 40
3 0 0 23
a
Amounts in mg or ml are quantities of original plant material.
A. 3. Effectiveness of the culture medium, confirmation of antimicrobial substances and

validity of the counting method
The following strains are normally used (see also Annex 2):
Staphylococcus aureus NCIMB 8625 (ATCC 6538-P, CIP 53.156) or NCIMB 9518 (ATCC
6538, CIP 4.83, IFO 13276)
Bacillus subtilis NCIMB 8054 (ATCC 6633, CIP 52.62, IFO 3134)
Escherichia coli NCIMB 8545 (ATCC 8739, CIP 53.126, IFO 3972)
Candida albicans ATCC 2091 (CIP 1180.79, IFO 1393) or ATCC 10 231 (NCPF 3179,
CIP 48.72, IFO 1594)
Allow the test strains to grow separately in tubes containing Soybean-Casein Digest Medium at 30-35
°C for 18-24 hours for aerobic bacteria and between 20-25 °C for Candida albicans, for 48 hours.
[Antibiotics are often added to the culture medium to attain a certain selectivity]
Dilute portions of each of the cultures using Buffered Sodium Chloride-Peptone Solution pH 7.0 or
Phosphate Buffer, pH 7.2 to prepare test suspensions containing about 50 to 200 viable cfu
(microorganisms) per ml. Growth-promoting qualities are tested by inoculating 1 ml of each
microorganism into each medium. The test media are satisfactory if clear evidence of growth appears
in all the inoculated media after incubation at the indicated temperature for 5 days. When a count of
test organisms with a test specimen is less than one-fifth of that without the test specimen, any such
effect must be eliminated by dilution, filtration, neutralization or inactivation.
page 32
To confirm the sterility of the medium and of the diluent and the aseptic performance of the test, carry
out the TVC method using sterile Buffered Sodium Chloride-Peptone Solution pH 7.0 or Phosphate
Buffer, pH 7.2 as a control. There should be no growth of microorganisms.
To validate the method, a count for the test organism should be obtained differing by not more than a
factor of 10 from the calculated value for the inoculum.
B. Tests for specific microorganisms

Microbial tests should be applied to starting plant materials, intermediate and finished products where
necessary. Enterobacteria and certain other gram-negative bacteria Escherichia coli, Salmonella and
Staphylococcus aureus, are included as target strains of the test.
The conditions of the tests for microbial contamination are designed to minimize accidental
contamination of the materials being examined and thus precautions must be taken to ensure
conditions do not adversely affect any microorganisms that are to be measured.
Procedure
B. 1 Pretreatment of the material being examined

Refer to the Sampling and Preparation of the Test solution in TVC, including the elimination of any
antimicrobial substances, which may be present.
B. 2 Test Procedure for the Enterobacteriaceae and certain other Gram-negative

bacteria
Detection of bacteria
Homogenize the pretreated material appropriately and incubate at 30-37 °C for a length of time
sufficient for revivification of the bacteria but not sufficient for multiplication of the organisms
(usually 2-5 hours). Shake the container, transfer aliquots equivalent to 1g or 1ml of the homogenized
material to 100ml of Enterobacteriaceae enrichment broth-Mossel and incubate at 35-37 °C for 18-48
hours. Prepare a subculture on a plate with violet-red bile agar with glucose and lactose. Incubate at
35-37 °C for 18-48 hours. The material passes the test if no growth of colonies of Gram-negative
bacteria is detected on the plate.
Quantitative evaluation
Inoculate a suitable amount of Enterobacteriaceae enrichment broth-Mossel with quantities of
homogenized material prepared as described under "Detection of bacteria" above, appropriately
diluted as necessary, containing 1g, 0.1 g and 10 µg, or 1 ml, 0.1 ml and 10 µl, of the material being
examined. Incubate at 35-37 °C for 24-48 hours. Prepare a subculture of each of the cultures on a
plate with violet-red bile agar with glucose and lactose in order to obtain selective isolation. Incubate
at 35-37 °C for 18-24 hours. The growth of well-developed colonies, generally red or reddish in
colour, of Gram-negative bacteria constitutes a positive result. Note the smallest quantity of material
that gives a positive result. Determine the probable number of bacteria using Table 8.
page 33
Table 8. Determination of Enterobacteriaceae and certain other Gram-negative bacteria
Result for each quantity or volume Probable number of

1.0 g or 1.0 ml 0.1g or 0.1 ml 0.01 g or 0.01 ml bacteria per g of material
+ + + More than 102
+ + . Less than 102 but more than 10
+ . . Less than 10 but more than 1
. . . Less than 1
Escherichia coli
Transfer a quantity of the homogenized material in lactose broth, prepared and incubated as described
above, and containing 1 g or 1 ml of the material being examined, to 100 ml of MacConkey broth and
incubate at 43-45 °C for 18-24 hours.
Prepare a subculture on a plate with MacConkey agar and incubate at 43-45 °C for 18-24 hours.
Growth of red, generally non-mucoid colonies of Gram-negative rods, sometimes surrounded by a
reddish zone of precipitation, indicates the possible presence of E. coli. This may be confirmed by the
formation of indole at 43.5-44.5 °C or by other biochemical reactions. The material passes the test if
no such colonies are detected or if the confirmatory biochemical reactions are negative.
Salmonella spp.
Incubate the solution, suspension or emulsion of the pretreated material prepared as described above
at 35-37 °C for 5-24 hours, as appropriate for enrichment.
Primary test
Transfer 10 ml of the enrichment culture to 100 ml of tetrathionate bile brilliant green broth and
incubate at 42-43 °C for 18-24 hours. Prepare subcultures on at least two of the following three agar
media: deoxycholate citrate agar; xylose, lysine, deoxycholate agar; and brilliant green agar. Incubate
at 35-37 °C for 24-48 hours. Carry out the secondary test if any colonies are produced that conform to
the description given in Table 9.
Table 9. Description of Salmonella colonies appearing on different culture media

Medium Description of colony
Deoxycholate citrate agar Well developed, colourless
Xylose, lysine, deoxycholate agar Well developed, red, with or without black centres
deoxycholate agar
Brilliant green agar Small, transparent and colourless, or opaque, pink or
white (frequently surrounded by a pink to red zone)
Secondary test
Prepare a subculture of any colonies showing the characteristics described in Table 9 on the surface of
triple sugar iron agar using the deep inoculation technique. The latter is done by first, inoculating the
inclined surface of the culture medium, followed by a stab culture with the same inoculating needle
and then, incubating at 35-37 °C for 18-24 hours. The test is positive for the presence of Salmonella
species. if a change of colour from red to yellow is observed in the deep culture (but not in the surface
culture), usually with the formation of gas with or without production of hydrogen sulfide in the agar.
Confirmation is obtained by appropriate biochemical and serological tests.
page 34
The material being examined passes the test if cultures of the type described do not appear in the
primary test, or if the confirmatory biochemical and serological tests in the secondary test are
negative.
Pseudomonas aeruginosa
Pretreat the material being examined as described under A.1 but using buffered sodium chloride-
peptone solution, pH 7.0, or another suitable medium shown not to have antimicrobial activity under
the conditions of the test, in place of lactose broth. Inoculate 100 ml of soybean-casein digest medium
with a quantity of the solution, suspension or emulsion thus obtained containing 1 g or 1 ml of the
material being examined. Mix and incubate at 35-37 °C for 24-48 hours. Prepare a subculture on a
plate of cetrimide agar and incubate at 35-37 °C for 24-48 hours. If no growth of microorganisms is
detected, the material passes the test. If growth of colonies of Gram-negative rods occurs, usually with
a greenish fluorescence, apply an oxidase test and test the growth in soybean-casein digest medium at
42 °C. The following method may be used. Place 2 or 3 drops of a freshly prepared 0.01 g/ml solution
of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride R on filter-paper and apply a smear of
the suspected colony; the test is positive if a purple colour is produced within 5-10 seconds. The
material passes the test if cultures of the type described do not appear or if the confirmatory
biochemical test is negative.
Staphylococcus aureus
Prepare an enrichment culture as described for Pseudomonas aeruginosa. Prepare a subculture on a
suitable medium such as Baird-Parker agar. Incubate at 35-37 °C for 24-48 hours. The material passes
the test if no growth of microorganisms is detected. Black colonies of Gram-positive cocci often
surrounded by clear zones may indicate the presence of Staphylococcus aureus. For catalase-positive
cocci, confirmation may be obtained, for example, by coagulase and deoxyribonuclease tests. The
material passes the test if cultures of the type described do not appear or if the confirmatory
biochemical test is negative.
Clostridia
Add 10 g (10 ml) of the herbal materials, preparation or product to be examined into two suitable
vessels, each containing 100 ml of Cooked-Meat Medium, previously heated just prior to use, to 100
°C for a few minutes and cooled to 37 °C. To distinguish between sporing and non-sporing organisms,
immediately seal one vessel with a layer of sterile paraffin or agar, heat the other vessel at 65 °C for
30 minutes, and then similarly seal.
Incubate both vessels at 35 °C to 37 °C and examine every 24-hours up to 4 days. Growth of sporing
organisms alone will take place in the vessel which was heated after inoculation.
Table 10. Characteristics of Clostridium species on Cooked-Meat Medium
Clostridium botulinum Clostridium perfringens Clostridium tetani

No digestion of meat; No digestion of meat; No digestion of meat;
much gas, white sediment meat turns pink colour burnt organic smell
If no growth occurs in both vessels, the sample passes the test for absence of Clostridia and other
anaerobic bacteria.
If sporing anaerobic organisms (Table 10) are found inoculate the cultures, each in duplicate, on one
half the surface of 5% defibrinated Sheep Blood Agar Medium plates. Incubate at 37 °C for 48 hours,
page 35
one plate anaerobically and the other aerobically, to check that the organisms will not grow under
aerobic condition.
After 24 and 48 hours examine the appearance of the colonies together with the type and extent of
hemolysis, and also examine microscopically for spore formation using Gram stain or spore stain
techniques. Match the result with the description in Table 11, and proceed for further identification of
specified clostridia.
Table 11. Characteristics of clostridium species on 5% defibrinated Sheep Blood Agar Medium
Clostridium botulinum Clostridium perfringens Clostridium tetani
Colonies Irregular, translucent with a Large, circular, convex, Transparent with long
granular surface and semitranslucent, smooth feathery spreading
indefinite fimbriated with an entire edge. projections.
spreading edge
Hemolysis + Double zone +
Spores Oval, central, subterminal Absent Spherical and terminal
distend bacilli (drum stick)
Shigella
The method described below is adopted from WHO guidelines for the control of epidemics due to
Shigella dysenteriae type 1.
Direct inoculation of agar plates

Use 2 or 3 loopfuls of herbal materials, preparations, products. Incubate plates at 35-37 °C for 18-24
hours.
Inoculate a general purpose plating medium of low selectivity and one of moderate or high selectivity.
MacConkey agar is recommended as a medium of low selectivity. MacConkey agar with 1 mcg/ml of
potassium tellurite has been reported to be particularly useful for S. dysenteriae type 1 (Sd1). Use a
small inoculum. Incubate at 35-37 °C for 18-24 hours.
Xylose-lysine-desoxycholate (XLD) agar is recommended as a medium of moderate or high

selectivity for isolation of Shigella. Desoxycholate citrate agar (DCA) is a suitable alternative.
Do not use salmonella-shigella (SS) agar, as it often inhibits growth of Sd1.
Each new batch of medium should be controlled for quality before routine use by inoculating it with
known reference strains and observing their growth and colony characteristics.
Identification of colonies on plating media

Colonies suspicious for Shigella will appear as follows:
MacConkey agar: Convex, colourless, 2-3 mm
XLD agar: Red, smooth, 1-2 mm
DCA agar: Colourless, translucent, 2-3 mm
Identify well-separated colonies of typical appearance to be transferred from each of the plating media
for further testing by making a mark on the bottom of the Petri plate.
Whenever possible a person experienced with the identification of Shigella should train laboratory
workers who are unfamiliar with its identification.
page 36
Inoculation of Kligler iron agar (KIA)

Pick three characteristic colonies from the plating media and inoculate into KIA as follows: stab the
butt and then streak the slant with a zigzag configuration. Pay attention to proper labelling of the
tubes. If screw-cap KIA tubes are used, make sure that the caps are loose. Incubate overnight. On the
following morning, examine the reactions in the KIA tubes. Tubes suspicious for Shigella will have
an acid (yellow) butt and an alkaline (red) slant. They will not produce gas (no bubbles or cracks in
the agar) and will not produce hydrogen sulfide (no black along the stab line).
Triple sugar iron agar (TSI) can also be used for the identification of Shigella. It will give the same
reactions as KIA.
B. 3 Validation of the tests for specific microorganisms
If necessary, grow separately the test strains listed in Table 12 on the culture media indicated at
30-35 °C for 18-24 hours. Dilute portions of each of the cultures using buffered sodium chloride-
peptone solution pH 7.0 so that the test suspensions contain about 103; microorganisms per ml. Mix
equal volumes of each suspension and use 0.4 ml (approximately 102 microorganisms of each strain)
as an inoculum in tests for Escherichia coli, Salmonella spp., Pseudomonas aeruginosa and
Staphylococcus aureus, in the presence of the material being examined, if necessary. The test method
should give a positive result for the respective strain of microorganism.
Table 12. Validation of tests for detection of specific microorganisms in the herbal sample
Microorganism Strain numbera Medium
Escherichia coli e.g. NCIMB 8545 lactose broth
(ATCC 8739, CIP 53.126, IFO 3972)
Pseudomonas aeruginosa e.g. NCIMB 8626 soybean-casein digest
(ATCC 9027, CIP 82.118) medium
Salmonella typhimurium No strain number is recommended. Species not lactose broth
pathogenic for humans, such as Salmonella abony
(NCTC 6017, CIP 80.39), may be used
Add from Thailand
Pharmacopoeia
Staphylococcus aureus e.g. NCIMB 8625 soybean-casein digest

(ATCC 6538 P, CIP 53.156) or NCIMB 9518 medium
(ATCC 6538, CIP 4.83, IFO 13276)
a
See Annex 2
For other microorganism see overview under A3
C. Microbial contamination limits in herbal materials, preparations and

products
Different limits are set according to the use of the herbal material and the material itself. Some
examples are given here:
page 37
a. For contamination of raw medicinal plant, and medicinal plant materials intended for further
processing (including additional decontamination by a physical or chemical process) the limits,
adapted from the provisional guidelines established by an international consultative group (26), are
given for untreated medicinal plant material harvested under acceptable hygienic conditions:
- Escherichia coli, maximum 104 per gram;
- mould propagules, maximum 105 per gram
- shigella, absence per gram or ml.
b. For medicinal plant materials that have been pretreated (e.g. with boiling water as used for
herbal teas and infusions) or that are used as topical dosage forms:
- aerobic bacteria, maximum 107 per gram;
- yeasts and moulds, maximum 104 per gram;
- other enterobacteria, maximum 104 per gram;
- clostridia, absence per 1 gram;
- salmonellae, absence per 1 gram;
- shigella, absence per 1 gram .
c. For other medicinal plant materials for internal use:

- other enterobacteria, maximum 103, per gram;
- salmonellae, absence per 1 gram;
d. For herbal medicines to which boiling water is added before use:

- salmonellae, absence per 1 gram ;
e. For other herbal medicines:

- Escherichia coli, absence per 1 gram ;
- salmonellae, absence per 1 gram ;
- shigella, absence per 1 gram ;
page 38
Determination of Aflatoxins
Whenever testing for aflatoxins is required they should be determined after using a suitable clean-up
procedure, during which great care should be taken not to be exposed or to expose the working or
general environment to these dangerous and toxic substances. Thus Member States should adapt their
GLP and GMP accordingly. Only products that have a history of aflatoxin contamination need to be
tested.
Test for aflatoxins

These tests are designed to detect the possible presence of aflatoxins B1, B2, G1 and G2, which are
highly toxic contaminants in any material of plant origin.
Some examples of proposed national limits for Aflatoxin in various types of herbal products are
described as below.
Herbal materials, preparations and products

Argentine
(Determination method: HPLC based technique using a monoclonal antibody immunoaffinity column)
• For herbs, herbal materials, herbal preparations used for herbal tea infusions
20µg/kg for aflatoxins B1 + B2 + G1 + G2 with the condition that aflatoxin B1≤5 µg/kg
• For finished herbal products for internal or topical use

Absence per 1 gram
Germany
• Any materials used in manufacture of medicinal products (including medicinal herbal products)
2 µg/kg* for aflatoxin B1 or 4 µg/kg* for total sum of aflatoxins B1, B2, G1 and G2
(the determination of the level of aflatoxin content has to be based on sampling procedures taking into
account a potential heterogenic distribution in the material)
* (calculated on at least 88% of dry weight)
Procedure
Test Method
The method described below does not require the use of toxic solvents, such as chloroform and
dichloromethane. It uses a multifunctional column, which contains lipophilic and charged active sites,
and HPLC using fluorescence detection to determine aflatoxins B1, B2, G1 and G2. The advantages
of employing a multifunctional column are: (1) high total recoveries of aflatoxins B1, B2, G1 and G2
(more than 85%) and (2) the column can be kept (stocked) at room temperature and for a fairly long
time prior to use.
Standard solutions of Aflatoxin B1, B2, G1 and G2 (2.5 ng/ml)

Stock standard solution: weigh exactly 1.0 mg of crystalline material of each aflatoxins B1, B2, G1
and G2 and dissolve in 50 ml of toluene-acetonitrile (9:1) solution by shaking vigorously in a glass
flask and obtain standard stock solution (20 µg/ml). Stock standard solution should be kept in a tight
container, sealed/covered with Aluminium filament, and kept at 4 °C in dark (keep in refrigerator).
Working standard solution: 0.5 ml of stock standard solution was added with toluene-acetonitrile
(9:1) solution to 200 ml [Working standard solution (50 ng/ml)].
page 39
Standard solution: Take 1.0 ml of Working standard solution was added with toluene-acetonitrile
(9:1) solution to 20 ml [Final standard solution (2.5 ng/ml)].
Standard solution for Liquid Chromatography analysis:

Take 0.25 ml of the final standard solution (described as above) into a centrifuge glass tube and
evaporated to dryness at 40 °C or by using nitrogen air stream. To derivatize3 aflatoxins B1 and G1
(precolumn derivatization), add 0.1 ml of trifluoro acetic Acid (TFA) solution to the residue in the
tube, tightly close the tube and shake strongly. Allow the tube to stand at room temperature for 15
min in the dark. Add 0.4 ml of acetonitrile: water (1:9) solution to the tube. A 20 µl portion of the
sample solution in the tube was subjected to liquid chromatography analysis.
A medicinal plant material for test was ground to uniform consistency using the coffee mill, and 50g
test sample was extracted with 400 ml of acetonitrile-water (9:1) by shaking vigorously in a glass
flask fitted with a stopper for 30 min or by using a mechanical blender for 5 min. The solution was
filtered through a filter paper or centrifuged. A 5 ml portion of the filtrate or the top clean layer was
transferred to a multifunctional column [such as a MultiSep #228 cartridge column (Romer Labs) or
an Autoprep MF-A (Showa-denko)] and passed through at a flow rate of 1 ml/min. The aflatoxins
existing in a sample passed through the column as the first elute. Obtain the first 1ml elute as the test
solution.
0.5 ml of the test solution in a centrifuge glass tube was evaporated to dryness at 40 °C or by using
nitrogen air stream to exclude solvent.
To derivatize aflatoxins B1 and G1 (precolumn derivatization), add 0.1 ml of trifluoro acetic acid
(TFA) solution to the residue in the tube, tightly close the tube and shake strongly. Allow the tube to
stand at room temperature for 15 min in the dark. Add 0.4 ml of acetonitrile-water (1:9) solution to
the tube. A 20 µl portion of the sample solution in the tube was subjected to liquid chromatography
analysis.
Method
Liquid Chromatography (LC) conditions:
The mobile phase was acetonitrile-methanol-water (1:3:6)4. The mobile phase was degassed by
sonication. An Octadesyl-Sylica gel (ODS) column (4.6 mm I.D. X 250 mm, 3 –5 µm), such as
Inertsil ODS-3 (4.6 mm I.D. X 250 mm, 3 µm) was connected as the liquid chromatography column.
The column was maintained at 40 °C with a flow rate of 1 ml/min. The aflatoxin and derivatives were
detected at the excitation and emission wavelengths of 365 nm and 450 nm, respectively. The
injection volume was 20µl.
If an impurity peak overlaps the peaks corresponding to aflatoxins, the second liquid chromatography
conditions described below are recommended.
Alternative LC conditions:
The mobile phase was methanol-water (3:7). The mobile phase was degassed by sonication. A
fluorocarbonated column, such as Wako-pack Fluofix 120E (4.6 mm I.D. X 250 mm, 5 µm) was
connected as the liquid chromatograph column. The column was maintained at 40 °C with a flow rate
3
.
4
If the sample solution contains a lot of impurity, the column should be washed by acetonitrile for 5-10 min and
reconditioned with the mobile phase for 10 min before the next analysis.
page 40
of 1 ml/min. The aflatoxin and its derivatives were detected at the excitation and emission
wavelengths of 365 nm and 450 nm, respectively. The injection volume was 20 µl.
Interpretation of the results
Comparison of retention time of peak area or peak heights of respective aflatoxin in chromatograms:
if they are bigger or higher than one obtained in respective standard aflatoxin solution, it should be
regarded positive for respective aflatoxin in the sample solution.
page 41
Determination of Radioactive Contaminants
Method of measurement
Plant materials may be contaminated for a long period of time with airborne radioactive materials.for
example following a severe nuclear accident. These materials may deposit on leaves of vegetables
and medicinal plants as well. Their activity concentration and type of radioactive contamination can
be measured by Radiation monitoring laboratories of most of the WHO Member States. The activity
concentration of radioisotopes in herbs should be assessed by the competent national radiohygiene
laboratories accounting for the relevant recommendations of the international organization, such as
Codex Alimentarius - International Atomic Energy Agency (IAEA), FAO and WHO) .
Since radionuclides from accidental discharges vary with the type of facility involved, a generalized
method of measurement is so far not available. However, should such contamination be of concern,
suspect samples can be analysed by a competent laboratory. Details of laboratory techniques are
available from the International Atomic Energy Agency (IAEA).5
5
International Atomic Energy Agency (IAEA), Analytical Quality Control Services, Laboratory Seibersdorf, PO Box 100,
Vienna, Austria.
page 42
8. Bibliography
(1) WHO Traditional Medicine Strategy: 2002–2005. Geneva, World Health Organization, 2002 (document
WHO/EDM/TRM/2002.1).
http://www.who.int/medicines/library/trm/strategytrm.shtml
(2) Quality control methods for medicinal plant materials. Geneva, World Health Organization, 1998.
http://www.who.int/medicines/library/trm/medicinalplants/qualcontrolmethods.shtml
(3) WHO guidelines on good agricultural and field collection practices(GACP) for medicinal plants. Geneva,
World Health Organization, 2003
http://www.who.int/medicines/library/trm/medicinalplants/agricultural.shtml
(4) International pharmacopoeia, Third edition, Volume 1. Geneva, World Health Organization, 1979 - update
to 4th edition…
(5) International pharmacopoeia, Third edition, Volume 2. Geneva, World Health Organization, 1981
(9) Good manufacturing practices: main principles for pharmaceutical products. Annex 4 of WHO Expert
Committee on Specifications for Pharmaceutical Preparations, Report of the thirty-seventh session.
Geneva, World Health Organization, 2003 (WHO Technical Report Series, No. 908). (These
guidelines are also included in Quality assurance of pharmaceuticals: a compendium of guidelines
and related materials, Vol. 2, Updated edition: good manufacturing practices and inspection. Geneva,
World Health Organization, 2004).
(10) Good manufacturing practices supplementary guidelines for manufacture of herbal medicinal
products. Annex 8 of WHO Expert Committee on Specifications for Pharmaceutical Preparations.
Thirty-fourth report. Geneva, World Health Organization, 1996 (WHO Technical Report Series, No.
863), (These guidelines are also included in Quality assurance of pharmaceuticals: a compendium
of guidelines and related materials, Vol. 2, Updated edition: good manufacturing practices and
inspection. Geneva, World Health Organization, 2004). [These supplementary guidelines are being
updated]
(11) Good storage practices of pharmaceutical products. Annex 9 of WHO Expert Committee on
Specifications for Pharmaceutical Preparations, Report of the thirty-seventh session, Geneva,
World Health Organization, 2003 (WHO Technical Report Series, No. 908) .
(12) Good trade and distribution practices (GTDP) of pharmaceutical starting materials. Annex 2 of WHO
Expert Committee on Specifications for Pharmaceutical Preparations. Report of the thirty-eighth
session. Geneva, World Health Organization, 2004 (WHO Technical Report Series, NO. 917).
(13) General guidelines for methodologies on research and evaluation of traditional medicine.
Geneva, World Health Organization, 2000 (document WHO/EDM/TRM/2000.1).
http://www.who.int/medicines/library/trm/who-edm-trm-2000-1/who-edm-trm-2000-1en.shtml
(14) Guidelines for assessment of herbal medicines. Annex 11 of WHO Expert Committee on
Specifications for Pharmaceutical Preparations. Thirty-fourth report. Geneva, World Health
Organization, 1996 (WHO Technical Report Series, No. 863), (These guidelines are also
page 43
included in Quality assurance of pharmaceuticals: a compendium of guidelines and related

materials, Vol. 1. Geneva, World Health Organization, 1997).
(15) WHO monographs on selected medicinal plants, Vol. 1. Geneva, World Health Organization, 1999.
http://www.who.int/medicines/library/trm/medicinalplants/monograph_volume_one.shtml
(16) WHO monographs on selected medicinal plants, Vol. 2. Geneva, World Health Organization,
2002.
http://www.who.int/medicines/library/trm/medicinalplants/monograph_volume_two.shtml
(17) Codex Alimentarius Code of Practice, General Principles of Food Hygiene, 2nd ed. Rome, Joint
FAO/WHO Food Standards Programme, 2001 (document Codex Alimentarius GL 33).
(18) Codex Alimentarius Guidelines for the production, processing, labelling and marketing of
organically produced food. Rome, Joint FAO/WHO Food Standards Programme, 2001 (document
Codex Alimentarius GL 32, Rev. 1).
(19) Codex Alimentarius Code for hygienic practice for spices and dried aromatic plants. Rome, Joint
FAO/WHO Food Standards Programme, 1995 (document Codex Alimentarius CAC/RCP 42).
(20) Pesticide Residues in Food – Methods of Analysis and Sampling. Codex Alimentarius. Vol. 2A, Part
1, 2nd Edition, Rome, Joint FAO/WHO Food Standards Programme, 2000
(21) Facts about low-level radiation. Vienna, International Atomic Energy Agency, 1986.
(22) Derived intervention levels for radionuclides in food. Guidelines for application after widespread
radioactive contamination resulting from a major radiation accident. Geneva, World Health
Organization, 1988.
(23) Codex Alimentarius Commission. Guide. Codex maximum limits for pesticide residues, Part 2, April
1992. Rome, Food and Agriculture Organization of the United Nations, 1992 (unpublished FAO
document CX/PR2-1992; available in hard copy and electronic format from FAO).
(24) Public health impact of pesticides used in agriculture. Geneva, World Health Organization, 1990.
(25) Official herbicide recommendations for vegetable crops, herbs and medicinal plants. The Hague,
International Society for Horticultural Science, 1981.
(26) International Consultative Group on Food Irradiation. Consultation on microbiological criteria for
foods to be further processed including by irradiation. Geneva, World Health Organization, 1989: 21
(unpublished WHO document WHO/ EHE/FOS/89.5; available on request from Office of Global and
Integrated Environmental Health, World Health Organization, 1211 Geneva 27, Switzerland).
(27) Quality control methods. In: Remington: the science and practice of pharmacy, 19th ed. Easton, PA,
MACK, 1995: 118-119.
(28) Lachman L et al. Quality control charts. In: The theory and practice of industrial pharmacy.
Philadelphia, Lea & Febiger, 1986: 817-824.
(29) Sampling procedures for industrially manufactured pharmaceuticals. In: WHO Expert Committee on
Specifications for Pharmaceutical Preparations. Thirty-first report. Geneva, World Health
Organization, 1990 (WHO Technical Report Series, No. 790) Annex 2, pp. 34-47. [These guidelines
are being updated]
page 44
(30) Good manufacturing practices: guidelines on the validation of manufacturing processes. In: WHO
Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-fourth report. Geneva,
World Health Organization, 1996 (WHO Technical Report Series, No. 863): 80-96. (These guidelines
are also included in Quality assurance of pharmaceuticals: a compendium of guidelines and related
materials, Vol. 2, Updated edition: good manufacturing practices and inspection. Geneva, World
Health Organization, 2004).
(31) Lowe DA. Guide to international recommendations on names and symbols for quantities and on units
of measurement. Geneva, World Health Organization, 1975.
(32) The SI for the health professions. Geneva, World Health Organization, 1977
National and Regional pharmacopoeias

European Pharmacopoeia, 5th edition, Strasbourg, Council of Europe, 2004.
Farmacopea Argentina, Volume 1, 7th edition, Buenos Aires, Ministry of Health, 2003
The Japanese pharmacopoeia, 14th ed.(English edition.). Tokyo, Ministry of Health, Labour and Welfare,
2001.
The Korean Pharmacopoeia, 7th edition. (English edition.). Seoul, Korea Food and Drug Administration, and
Korean Association of Official Compendium for Public Health, 1998.
The Korean Pharmacopoeia, 8th edition. (English edition.). Seoul, Korea Food and Drug Administration, and
Korean Association of Official Compendium for Public Health, 2004.
Pharmacopoeia of the People’s Republic of China. Volume I (English edition). Beijing, Chemical Industry
Press, 2000.
Pharmacopoeia of the People’s Republic of China. Volume 2 (English edition). Beijing, Chemical Industry
Press, 2000
Thai herbal pharmacopoeia, Volume 1, Bangkok, Department of Medical Sciences, Ministry of Public Health,
1998.
Thai herbal pharmacopoeia, Volume 2, Bangkok, Department of Medical Sciences, Ministry of Public Health,
2000.
Thai pharmacopoeia, Volume 1, Bangkok, Department of Medical Sciences, Ministry of Public Health, 1987
The United States pharmacopoeia 27 and the National Formulary 22. Rockville, MD, The United States
Pharmacopoeia Convention, Inc., 2003.
page 45
Annex 1. List of Culture Media and Strains used for Microbiological Analysis
Culture media
The following media are satisfactory, but other media may be used if they have similar nutritive and selective
properties for the microorganisms to be tested.
Baird-Parker agar
Procedure. Dissolve 10 g of pancreatic digest of casein R, 5 g of beef extract R, 1g of water-soluble yeast
extract R, 5 g of lithium chloride R, 20 g of agar R, 12 g of glycine R and 10 g of sodium pyruvate R in
sufficient water to produce 950 ml. Heat to boiling for 1 minute, shaking frequently and adjust the pH to 6.6-
7.0 using sodium hydroxide (0.5 mol/l) VS. Sterilize in an autoclave at 121 °C for 15 minutes, cool to 45-50 °C
and add 10 ml of a sterile 0.01 g/ml solution of potassium tellurite R and 50 ml of egg-yolk emulsion.
Brilliant green agar

Procedure. Dissolve 10 g of dried peptone R (meat and casein), 3 g of water-soluble yeast extract R, 5 g of
sodium chloride R, 10 g of lactose R, 10 g of sucrose R, 20 g of agar R, 0.08 g of phenol red R and 12.5 mg of
brilliant green R in sufficient water to produce 1000 ml. Heat to boiling for 1 minute. Using sodium hydroxide
(0.05 mol/l) VS adjust the pH to 6.7-7.1. Immediately before use, sterilize in an autoclave at 121 °C for 15
minutes, cool to 50 °C and pour into Petri dishes.
Buffered sodium chloride-peptone solution pH 7.0

Procedure. Dissolve 3.56 g of potassium dihydrogen phosphate R, 7.23 g of disodium hydrogen phosphate R,
4.30 g of sodium chloride R and 1 g of dried peptone R (meat and casein) in sufficient water to produce 1000
ml. Polysorbate 20 R or polysorbate 80 R may be added, 0.001-0.01 g /ml. Sterilize in an autoclave at 121 °C
for 15 minutes.
Casein-soybean digest agar

Procedure. Dissolve 15 g of pancreatic digest of casein R, 3 g of papaic digest of soybean meal R, 5 g of
sodium chloride R and 15 g of agar R in sufficient water to produce 1000 ml. Using sodium hydroxide (0.05
mol/l) VS adjust the pH to 7.1-7.5. Sterilize in an autoclave at 121 °C for 15 minutes.
Cetrimide agar
Procedure. Dissolve 20 g of pancreatic digest of gelatin R, 1.4 g of magnesium chloride R, 10 g of potassium
sulfate R, 0.3 g of cetrimide R, 13.6 g of agar R and 10 ml of glycerol R insufficient water to produce 1000 ml.
Heat to boiling for 1 minute with shaking. Using sodium hydroxide (0.05 mol/l) VS adjust the pH to 7.0-7.4.
Sterilize in an autoclave at 121 °C for 15 minutes.
Cooked-meat medium (WHO will add)
Defibrinated Sheep Blood Agar medium (WHO will add)
Deoxycholate citrate agar

Procedure. Dissolve 10 g of beef extract R, 10 g of dried peptone R (meat), 10 g of lactose R, 20 g of sodium
citrate R, 1 g of iron (III) citrate R, 5 g of sodium deoxycholate, 13.5 g of agar R and 20 mg of neutral red R in
sufficient water to produce 1000 ml. Heat gently to boiling for 1 minute, cool to 50 °C and adjust the pH to 7.1-
7.5 using sodium hydroxide (0.05 mol/l) VS. Pour into Petri dishes. Do not heat in an autoclave.
Enterobacteriaceae enrichment broth-Mossel

Procedure. Dissolve 10 g of pancreatic digest of gelatin R, 5 g of glucose hydrate R, 20 g of dehydrated ox bile
R, 2 g of potassium dihydrogen phosphate R, 8 g of disodium hydrogen phosphate R and 15 mg of brilliant
green R in sufficient water to produce 1000 ml. Heat to boiling for 30 minutes and cool immediately. Using
sodium hydroxide (0.05 mol/l) VS adjust the pH to 7.0-7.4.
page 46
Kligler's iron agar (KIA)

Procedure. Dissolve the agar in the meat infusion broth, or alternatively in meat extract broth, by heating in a
boiling water-bath or in steam at 100° C. Bring the molten nutrient agar to 80 °C in a water-bath. Add and
dissolve the lactose, peptone, proteose peptone, NaCl, glucose, ferrous sulfate, and sodium thiosulfate and mix
well. Adjust the pH to 7.4. Add 6 ml of 0.5% solution of phenol red and mix well. Distribute in screw-cap
tubes (15 x 150 or 16 x 160 mm) in 5-6 ml amounts and sterilize by autoclaving at 121 °C for 15 minutes.
Allow the medium to cool and set with a slant of 2.5 cm and a butt 2.5 cm deep. Record batch number and date
on the label and then store at room temperature not exceeding 25 °C.
An additional 10 g of peptone, 3 g of beef extract, 3 g of yeast extract, and one litre of distilled water may be
used in place of meat infusion broth.
Lactose broth
Procedure. Dissolve 3 g of beef extract R, 5 g of pancreatic digest of gelatin R and 5 g of lactose R in sufficient
water to produce 1000 ml. Using sodium hydroxide (0.05 mol/l) VS adjust the pH to 6.7-7.1. Sterilize in an
autoclave at 121 °C for 15 minutes.
MacConkey agar
Procedure. Dissolve 17 g of pancreatic digest of gelatin R, 3 g of dried peptone R (meat and casein), 10 g of
lactose R, 5 g of sodium chloride R, 1.5 g of bile salts R, 13.5 g of agar R, 30 mg of neutral red R and 1 mg of
crystal violet R in sufficient water to produce 1000 ml. Using sodium hydroxide (0.05 mol/l) VS adjust the pH
to 6.9-7.3. Heat to boiling for 1 minute with constant shaking then sterilize in an autoclave at 121 °C for 15
minutes.
MacConkey broth
Procedure. Dissolve 20 g of pancreatic digest of gelatin R, 10 g of lactose R, 5 g of dehydrated ox bile R and
10 mg of bromocresol purple R in sufficient water to produce 1000 ml. Using sodium hydroxide (0.05 mol/l)
VS adjust the pH to 7.1-7.5. Sterilize in an autoclave at 121 °C for 15 minutes.
Sabouraud glucose agar with antibiotics

Procedure. Dissolve 10 g of dried peptone R (meat and casein), 40 g of glucose hydrate R and 15 g of agar R in
sufficient water to produce 1000 ml. Using acetic acid (~60 g/l) TS adjust the pH to 5.4-5.8. Sterilize in an
autoclave at 121 °C for 15 minutes. Immediately before use, add sterile solutions of 0.10 g of benzylpenicillin
sodium R and 0.1 g of tetracycline R per litre of medium or alternatively, before sterilization, add 0.05 g of
chloramphenicol R per litre of medium.
Soybean-casein digest medium

Procedure. Dissolve 17 g of pancreatic digest of casein R, 3 g of papaic digest of soybean meal R, 5 g of
sodium chloride R, 2.5 g of dipotassium hydrogen phosphate R and 2.5 g of glucose hydrate R in sufficient
water to produce 1000 ml. Using sodium hydroxide (0.05 mol/l) VS adjust the pH to 7.1-7.5. Sterilize in an
autoclave at 121 °C for 15 minutes.
Tetrathionate bile brilliant green broth

Procedure. Dissolve 8.6 g of dried peptone R, 8 g of dehydrated ox bile R, 6.4 g of sodium chloride R, 20 g of
calcium carbonate RI, 20 g of potassium tetrathionate R and 0.07 g of brilliant green R in sufficient water to
produce 1000 ml. Using sodium hydroxide (0.05 mol/l) VS adjust the pH to 6.8-7.2. Heat just to boiling; do not
reheat.
Triple sugar iron agar

Procedure. Dissolve 3 g of beef extract R, 3 g of water-soluble yeast extract, 20 g of dried peptone R (casein
and beef), 5 g of sodium chloride R, 10 g of lactose R, 10 g of sucrose R, 1 g of glucose hydrate R, 0.3 g of
brown ammonium iron(III) citrate R, 0.3 g of sodium thiosulfate R, 25 mg of phenol red R and 12 g of agar R
in sufficient water to produce 1000 ml. Heat to boiling for 1 minute with shaking. Using sodium hydroxide
(0.05 mol/l) VS adjust the pH to 7.2-7.6. Distribute in tubes and sterilize in an autoclave at 121 °C for 15
minutes. Allow to set in an inclined position covered with a butt.
page 47
Violet-red bile agar with glucose and lactose

Procedure. Dissolve 3.0 g of water-soluble yeast extract R, 7.0 g of pancreatic digest of gelatin R, 1.5 g of bile
salts R, 10.0 g of lactose R, 5.0 g of sodium chloride R, 10.0g of glucose hydrate R, 15.0 g of agar R, 30 mg of
neutral red R and 2.0 mg of crystal violet R in sufficient water to produce 1000 ml. Heat to boiling and adjust
the pH to 7.2-7.6 using sodium hydroxide (0.05 mol/l) VS. Do not heat in an autoclave.
Xylose, lysine, deoxycholate agar

Procedure. Dissolve 3.5 g of xylose R, 5 g of L-lysine R, 7.5 g of lactose R, 7.5 g of sucrose R, 5 g of sodium
chloride R, 3 g of water-soluble yeast extract R, 0.08 g of phenol red R, 13.5 g of agar R, 2.5 g of sodium
deoxycholate R, 6.8 g of sodium thiosulfate R and 0.8 g of brown ammonium iron(III) citrate R in sufficient
water to produce 1000 ml. Using sodium hydroxide (0.05 mol/l) VS, adjust the pH to 7.2-7.6. Heat just to
boiling, cool to 50 °C and pour into Petri dishes. Do not heat in an autoclave.
Strains of microorganisms
The strains of microorganism referred to throughout the text are suitable, but others may be used if they have
similar properties. The designations of the strains and the addresses from which they may be obtained are as
follows:
ATCC American Type Culture Collection, 12301 Park Lawn Drive, Rockville, MD 20852, USA.
CIP Collection de l'Institut Pasteur, Service de la Collection Nationale de Cultures de

Microorganismes (CNCM), 25 rue du Docteur Roux, F 75015 Paris, France.
IFO Institute for Fermentaion, Osaka (IFO) microorganism strains, The Culture Collection
Division, The NITE Biological Resource Center (NBRC), Department of Biotechnology,
National Institute of Technology and Evaluation (NITE), 2-5-8, Kazusa-kamatari, Kisarazu
City, Chiba 292-0818, Japan
NCIMB National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen AB24
3RY, Scotland, United Kingdom.
NCPF National Collection of Pathogenic Fungi, PHLS Mycology Reference Laboratory, Public
Health Laboratory, Kingsdown, Bristol BS2 8EL, England, United Kingdom.
NCTC National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue,
London NW9 5HT, England, United Kingdom.
page 48
Annex 2. List of reagents and solutions

[To be updated by WHO in accordance with changes.]
The reagents, test solutions and volumetric solutions mentioned in this publication are described blow.
Reagents are denoted by the abbreviation R, test solutions by the abbreviation TS, and volumetric
solutions by the abbreviations VS. The concentration of the reagents solutions is expressed in g/l, that
is, grams of anhydrous substance per litre of water or solvent, as indicated. Where no solvent is
indicated, demineralized water should be used. The procedures for the preparation f test solutions that
20
require special attention are given in detail. The designation of d denotes the relative density d 20 :,
i.e. measured in air at 20 °C in relation to water at 20 °C. Colour Index (C.I.) numbers are provided
for strains.
Acetic acid, glacial, R. C2H4O2; d ~1.048.

A suitable commercially available reagent.
Acetic acid (~300 g/l) TS. A solution of glacial acetic acid R containing about 300g of C2H4O2 per litre
(approximately 5 mol/l); d ~ 1.037.
Acetic acid (~60g/l) TS. Acetic acid (~300g/l) TS, diluted to contain about 60g of C2H4O2 per litre
(approximately 1 mol/l); d ~ 1.008.
Acetic acid, dilute.

Dilute 6g of glacial acetic acid with water to make 100ml (1 mol/l).
Acetone R. C3H6O.
Acetonitrile R. Methyl cyanide, C2H3N.

Description. A clear, colourless liquid.
Miscibility. Freely soluble with water.
Aflatoxin mixture TS.

Procedure. Prepare a mixed working standard in a mixture of 98 volumes of chloroform R and 2 volumes of
acetonitrile R, containing 0.5 µg of each of aflatoxins B1 and G1 per ml, and 0.1 µg of each of aflatoxins
B2 and G2 per ml.
Note. Aflatoxins are highly toxic and should be handled with care. National legal requirements should be
followed.
Suitable commercially available working standards.
Agar R.
Aluminium chloride R. AIC13, 6H2O.

Aluminium oxide, purified, R. Al2O3.

A suitable commercially available reagent for column chromatography.
1,2,4-Aminonaphtholsulfonic acid R. C10H9NO4S.

page 49
Description. A white to slightly brownish pink powder.

Solubility. Sparingly soluble in water.
Aminonaphtholsulfonic acid TS.

Procedure. Add 0.25g of 1,2,4-aminonaphtholsulfonic acid R to 100ml of freshly prepared sodium
metabisulfite (150g/l) TS with mechanical stirring. After stirring for 15 minutes, add 0.5 g of anhydrous
sodium sulfite R. After stirring for an additional 5 minutes, filter the mixture.
Storage. Keep in a brown bottle.
Note. This reagent should be prepared freshly every week.
Ammonia solution NH4OH (R or TS, check all)

Ammonia Water, A suitable commercially available reagent, Specific gravity: about 0.90, Density:
0.908 g/ml, Content: 28-30%.
Ammonia TS.
To 400 ml of ammonia solution add water to make 1000 ml (10%).
Ammonia (~100g/l) TS. Ammonia (~260g/l) TS, diluted to contain about 100g of NH3 per litre (approximately
6 mol/l); d 0.956.
Ammonium dihydrogen phosphate R. (NH4)H2PO4.

Monobasic ammonium phosphate. A white, crystalline powder or colourless crystals, freely soluble in water.
Ammonium iron(III) citrate, brown, R. Ferric ammonium citrate, brown; soluble ferric citrate.
Contains about 9% of NH3, 16.5-18.5% of Fe, and about 65% of hydrated citric acid.
Description. Reddish brown granules, garnet-red transparent scales, or brownish yellow powder; odourless or
slight odour of NH3. Very deliquescent.
Solubility. Very soluble in water; practically insoluble in ethanol (~750g/l) TS.
Storage. Store in a well closed container, protected from light.
Ammonium molybdate R. (NH4)6Mo7O24,4H2O.

Ammonium molybdate (40g/l) TS. A solution of ammonium molybdate R containing about 40g of (NH4)
6
Mo7O24,4H2O per litre.
Ammonium oxalate R. C2H8N2O4,H2O.

Ammonium oxalate (25 g/l) TS. A solution of ammonium oxalate R containing about 27g of C2H8N2O4 per
litre.
Ammonium thiocyanate R. CH4N2S.

Ammonium thiocyanate (75 g/l) TS. A solution of ammonium thiocyanate R containing about 75 g of
CH4N2S per litre (approximately 1 mol/l).
Argon R. Ar. Contains not less than 999.95 ml of Ar per litre.

Arsenic, dilute, AsTS. One millilitre contains 10 µg of arsenic.

page 50
Procedure. Dilute 1 ml of strong arsenic AsTS with sufficient water to produce 100 ml.
Note. Dilute arsenic AsTS must be freshly prepared.
Arsenic, strong, AsTS.

Procedure. Dissolve 0.132 g of arsenic trioxide R in 6 ml of sodium hydroxide (80g/l) TS, by gentle heating.
Dilute the cooled solution with 20ml of water, and add 50 ml of hydrochloric acid (250g/l) TS and
sufficient water to produce 100 ml.
Arsenic trioxide R. As2O3.

Beef extract R. A residue from beef broth obtained by extracting fresh, sound, lean beef by cooking with water
and evaporating the resulting broth at a low temperature, usually under reduced pressure until a thick
pasty residue is obtained.
Benzylpenicillin sodium R. C16H17N2NaO4S. Quality conforms to the monograph in The international

pharmacopoeia. Vol. 2, p. 51.
Bile salts R.
Description. A concentrate of beef bile, the principal constituent of which is sodium desoxycholate, determined
as cholic acid.
Solubility. Soluble in water and in ethanol (750g/l) TS.
Acidity. pH of a 0.02 g/ml solution 5.8-6.2.
Brilliant green R. Malachite green G; basic green 1; C.I. 42040; C27H34N2O4S.

Description. Small, glistening golden crystals.
Solubility. Soluble in water and ethanol (750 g/l) TS.
Bromine AsTS.
Procedure. Dissolve 30g of potassium bromide R in 40 ml of water, add 30g of bromine R and dilute with
sufficient water to produce 100 ml. The solution complies with the following test: Evaporate 10 ml
nearly to dryness on a water-bath, add 50 ml of water, 10ml of hydrochloric acid (250g/l) AsTS, and
sufficient stannous chloride AsTS to reduce the remaining bromine, and apply the general test for
arsenic. The colour of the stain produced is not more intense than that produced from a 1-ml standard
stain, showing that the amount of arsenic does not exceed 1 µg/ml.
Bromocresol purple R. C21H16Br2O5S.

Calcium carbonate R1. CaCO3.

Carbophenothion. C11H16ClO2PS3. O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl]- phosphorodi- thionate.

Yellowish liquid, practically insoluble in water, miscible with organic solvents.
d 425 : about 1.27.
Cetrimide R. Contains not less than 96.0% and not more than 101.0% of alkyltrimethylammonium bromide,
calculated as C17H38BrN with reference to the dried substance.
Description. A white or almost white, voluminous, free-flowing powder; slight characteristic odour.
page 51
Solubility. Soluble in two parts of water; freely soluble in ethanol (750g/l) TS.
Chloramphenicol R. C11H12Cl2N2O5. Quality conforms to the monograph in The international

pharmacopoeia, Vol. 2, p. 64.
Chloroform R. CHCl3.
Crystal violet R. C25H30ClN3.

Cyclohexane R. C6H12.
Desmetryn R. C9H17N5S. 2-methylmercapto-4-methylamino-6-isopropylamino-S-triazine.

A commercially available reagent suitable for use as a reference material.
Dichloromethane R. Methylene chloride, CH2Cl2.

Description. A clear colourless, mobile liquid.
Miscibility. Freely miscible with ethanol (750g/l) TS and ether R.
Boiling range. Not less than 95% distils between 39 and 41°C.
Residue on evaporation. After evaporation on a water-bath and drying at 105°C, leaves not more than 0.5
mg/ml.
Dimethyl sulfoxide R. C2H6OS.

Description. A colourless liquid; odourless or with a slight unpleasant odour.
Mass density (ρ20). 1.10kg/l.
Dimethyl yellow R. C.I. 11020; 4-dimethylaminoazobenzene; C14H15N3.

Caution. Dimethyl yellow R is carcinogenic.
Description. Produces a red colour in moderately acidic alcoholic solutions and yellow colour in weakly acidic
and alkaline solutions.
Homogeneity. Carry out the method for thin-layer chromatography, using silica gel G as the coating substance
and dichloromethane R as the mobile phase. Apply 10 µl of a 0.1 mg/ml solution in dichloromethane R.
After removing the plate from the chromatographic chamber allow it to dry in air. Only one spot appears
on the chromatogram.
Dipotassium hydrogen phosphate R. K2HPO4.

Disodium edetate R. C10H14N2Na2O8,2H2O.

Disodium hydrogen phosphate R. Na2HPO4,12H2O.

Ethanol R.
page 52
Ethanol (750 g/l) TS.

Ether R. C4H10O.
Ether/light petroleum TS1.

Procedure. Dilute 60 ml of ether R with sufficient distilled light petroleum R to produce 1000 ml.

Procedure. Dilute 150ml of ether R with sufficient distilled light petroleum R to produce 1000 ml.

Procedure. Dilute 500 ml of ether R with sufficient distilled light petroleum R to produce 1000 ml.
Ethyl acetate R. C4H8O2.

Ferric ammonium sulfate R. FeH4NO8S2,12H2O.

This reagent should be free of chlorides.
Ferric ammonium sulfate (0.25 mol/l) VS. Ferric ammonium sulfate R, dissolved in nitric acid (750 g/l) TS
to contain 120.5 g of . FeH4NO8S2,12H2O in 1000 ml.
Procedure. Dissolve 120.5 g of ferric ammonium sulfate R in a sufficient quantity of nitric acid (750g/l) TS
to produce 1000ml. The reagent should be free of chlorides.
Ferrous sulfate. FeSO4,7 H2O

Florisil R.
A suitable commercially available material for column chromatography.
Glucose hydrate R. Monohydrate of α-D-glucopyranose, C6H12O6, H2O. Contains not less than 99.0% and not
more than 101.5% of C6H12O6, calculated with reference to the dried substance.
Description. Colourless crystals or a white crystalline or granular powder; odourless.
Solubility. Soluble in about 1 part of water and in about 60 parts of ethanol (750 g/l) TS; more soluble in
boiling water and in boiling ethanol (750g/l) TS. Acidity. Dissolve 5g in 50ml of carbon-dioxide-free
water R. Neutralization requires not more than 0.5ml of carbonate-free sodium hydroxide (0.02mol/l)
VS, phenolphthalein/ethanol TS being used as indicator.
Specific optical rotation. Dissolve 100 mg, previously dried to constant weight, in 1 ml of water, and add a few
drops of ammonia (100g/l) TS;
= +52 to +53°.
Soluble starch or sulfites. Dissolve 1 g in 10 ml of water and add 1 drop of iodine TS; the liquid is coloured
yellow.
Loss on drying. Dry to constant weight at 105°C; loses not less than 80mg/g and not more than 100mg/g.
Sulfated ash. Not more than 1.0mg/g.
Assay. Dissolve about 0.1 g, accurately weighed, in 50 ml of water, add 30 ml of iodine (0.1 mol/l) VS and 10
ml of sodium carbonate (50g/l) TS, and allow to stand for 20 minutes. Add 15 ml of hydrochloric acid
(70g/l) TS and titrate the excess of iodine with sodium thiosulfate (0.1 mol/l) VS, using starch TS as
page 53
indicator. Perform a blank determination and make any necessary corrections. Each ml of iodine (0.1
mol/l) VS is equivalent to 9.008 mg of C6H12O6.
Glycerol R. Propane- 1,2,3-triol with small amounts of water, C3H8O3. Contains not less than 970g/kg of
C3H8O3.
Description. A clear, almost colourless, syrupy and hygroscopic liquid; odourless.
Miscibility. Miscible with water and ethanol (750g/l) TS; practically immiscible with ether R and chloroform
R.
Mass density (ρ20). Not less than 1.256kg/l.
Refractive index ( n D20 : ). Not less than 1.469.
Acrolein and other reducing substances. Mix 1 ml with 1 ml of ammonia (100g/l) TS and heat in a water-
bath at 60°C for 5 minutes; the liquid is not coloured yellow. Remove from the water-bath and add 3
drops of silver nitrate (40g/l) TS; the liquid does not become coloured within 5 minutes.
Sulfated ash. Not more than 0.5 mg/ml.
Glycine R. Aminoacetic acid, C2H5NO2.

Helium R. He. Contains not less than 999.95 ml of He per litre.

Helium for chromatography. He.

Contains not less than 99.995 per cent V/V of He.
Hexane. C6H14.
A colourless, flammable liquid, practically insoluble in water, miscible with ethanol and with ether.
d 2020 : 0.659 to 0.663.
n D20 : 1.375 to 1.376
Distillation range. Not less than 95 per cent distils between 67 °C and 69 °C.
Hexane R. n-Hexane, C6H14.

Description. A colourless, mobile, highly inflammable liquid.
Boiling range. Distils completely over a range of 1°C between 67.5 and 69.5°C.
Mass density (ρ20). 0.658-0.659kg/l.
Refractive index ( n D20 : ). 1.374-1.375.
Hydrochloric acid R. HCl

Hydrochloric acid (1 mol/l) VS. Hydrochloric acid (250g/l) TS, diluted with water to contain 36.47g of HCl
in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 1 mol/l solution in the following manner:
Dissolve about 1.5g, accurately weighed, of anhydrous sodium carbonate R (previously dried at 270°C
for 1 hour) in 50 ml of water and titrate with the hydrochloric acid solution, using methyl orange/ethanol
TS as indicator. Each 52.99 mg of anhydrous sodium carbonate R is equivalent to 1 ml of hydrochloric
acid (1 mol/l) VS.
page 54
Hydrochloric acid (~420 g/l) TS. d 1.18.

Hydrochloric acid (~250 g/l) AsTS. Hydrochloric acid (250g/l) TS that complies with the following tests A
and B:
A. Dilute 10ml with sufficient water to produce 50ml, add 5ml of ammonium thiocyanate (75g/l) TS and stir
immediately; no colour is produced.
B. To 50 ml add 0.2 ml of bromine AsTS, evaporate on a water-bath until reduced to 16 ml, adding more
bromine AsTS if necessary to ensure that an excess, as indicated by the colour, is present throughout the
evaporation. Add 50 ml of water and 5 drops of stannous chloride AsTS and apply the general test for
arsenic. The colour of the stain produced is not more intense than that produced from a 0.2-ml standard
stain, showing that the amount of arsenic does not exceed 0.05 µg/ml.
Hydrochloric acid (~250 g/l) TS. A solution of hydrochloric acid (420g/l) TS in water, containing
approximately 250g of HCl per litre; d 1.12.
Hydrochloric acid (~250 g/l), stannated, AsTS.

Procedure. Dilute 1 ml of stannous chloride AsTS with sufficient hydrochloric acid ( 250g/l) AsTS to
produce 100ml.
Hydrochloric acid (~70g/l) TS.

Procedure. Dilute 260ml of hydrochloric acid ( 250g/l) TS with sufficient water to produce 1000ml
(approximately 2mol/l); d 1.035.
Hydrochloric acid, dilute, R

Dilute 23.6 ml of hydrochloric acid with water to make 100 ml (10%).
Hydrogen for chromatography. H2.

Contains not less than 99.95 per cent V/V of H2.
Indophenol blue R. C.I. 49700; C18H26N2O.

Description. A violet-black powder.
Solubility. Practically insoluble in water; soluble in chloroform R.
Homogeneity. Carry out the method for thin-layer chromatography, using silica gel G as the coating substance
and dichloromethane R as the mobile phase. Apply 10 µl of a 0.1 mg/ml solution in dichloromethane R
and develop. After removing the plate from the chromatographic chamber allow it to dry in air. Only one
spot appears on the chromatogram.
Iodine R. I2.
Iodine TS.
Procedure. Dissolve 2.6 g of iodine R and 3 g of potassium iodide R in sufficient water to produce 100 ml
(approximately 0.1 mol/l).
Iodine (0.1 mol/l) VS. Iodine R and potassium iodide R, dissolved in water to contain 25,38g of I2 and 36.0 g
of KI in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 0.1 mol/l solution by titrating 25.0 ml with
sodium thiosulfate (0.1 mol/l) VS, using starch TS as indicator.
Iron(III) citrate R. Ferric citrate, C6H5FeO7, H2O.

page 55
Lactose R. C12H22O11.
Lead acetate R. C4H6O4Pb,3H2O.

Lead acetate (80 g/l) TS. A solution of lead acetate R in freshly boiled water containing about 80g/l of .
C4H6O4Pb (approximately 0.25mol/l).
Lithium chloride R. LiCl.

Description. White, deliquescent crystals or granules.
Solubility. Freely soluble in water; soluble in acetone R and ethanol (~750g/l) TS. Storage. Store in a tightly
closed container.
L-Lysine R. C6H14N2O2.
Description. Crystalline needles or hexagonal plates.
Solubility. Soluble in water; very slightly soluble in ethanol (~750g/l) TS; insoluble in ether R.
Melting point. About 213°C with decomposition.
Specific optical rotation. Dissolve 0.2g in 10ml of hydrochloric acid (~250g/l) TS;
= about +21.5°.
Magnesium chloride R. MgCl2,6H2O.

Magnesium nitrate R. Mg(NO3)2

see Magnesium nitrate hexahydrate.
Magnesium nitrate hexahydrate R. Mg(NO3)2,6 H2O

Mercuric bromide R. HgBr2.

Mercuric bromide AsTS.

Procedure. Dissolve 5 g of mercuric bromide R in sufficient ethanol (~750 g/l) TS to produce 100 ml.
Mercuric bromide paper AsR.

2
Procedure. Use smooth, white filter-paper weighing 65-120 g/m . The thickness of the paper in mm should be
approximately equal numerically to the weight expressed as above, divided by 400. Soak pieces of filter-
paper, not less than 25 mm in width, in mercuric bromide AsTS, decant the superfluous liquid, suspend
the paper over a non-metallic thread and allow it to dry, protected from light.
Storage. Store the mercuric bromide paper AsR in stoppered bottles in the dark.
Note. Paper that has been exposed to sunlight or to vapours of ammonia must not be used as it produces only a
pale stain or no stain at all.
Mercuric thiocyanate R. C2HgN2S2.

Mercuric thiocyanate TS. A saturated solution of mercuric thiocyanate R in ethanol (~750g/l) TS.
page 56
Mercury R. Hg.
Methane R. CH4.
Methanol R. CH4O.
Methyl orange R. Sodium salt of 4'-dimethylaminoazobenzene-4-sulfonic acid, C14H14N3NaO3S.

Methyl orange/ethanol TS.

Procedure. Dissolve 0.04g of methyl orange R in sufficient ethanol (~1508/l) TS to produce 100ml.
Methyl red. C.I. 13020. 2-(4-Dimethylamino-phenylazo)benzoic acid. C15H15N3O2.

A dark-red powder or violet crystals, practically insoluble in water, soluble in alcohol.
Neutral red R. C.I. 50040; C.I. Basic red; C15H17ClN4.

Nitric acid (~750 g/l) TS.

Procedure. Dilute 750ml of nitric acid ( 1000g/l) TS with sufficient water to produce 1000ml (approximately
12mol/l).
Nitric acid (~1000 g/l) TS. d ~ 1.41.

Nitric acid R. HNO3 (Concentration: 69-70%, Density: about 1.42 g / ml)

Nitrogen. N2.
Nitrogen, washed and dried.
Nitrogen for chromatography.

Contains not less than 99.95 per cent V/V of N2.
Nitrogen, oxygen-free.
Nitrogen R which has been freed from oxygen by passing it through alkaline pyrogallol solution R.
Nitrogen R. N2.
Oracet blue 2R. CI 61110. 1-Amino-4-(phenylamino)anthracene-9,10-dione. C20H14N2O2.

mp: about 194 °C.
Ox bile, dehydrated, R. Dehydrated, purified fresh bile.

page 57
Pancreatic digest of casein R.

Pancreatic digest of gelatin R.

Papaic digest of soybean meal R.

Peptone, dried, R. A variety of peptones are available from casein, meat, beef or a mixture of these.
Perchloric acid (~1170 g/l) TS. d ~ 1.67.

Petroleum, light, R.
Phenolphthalein R. C20H14O4.
Phenolphthalein TS.
Dissolve 1g of phenophthalein in 100ml of ethanol R.
Phenolphthalein/ethanol TS.
Procedure. Dissolve 1.0g of phenolphthalein R in sufficient ethanol (~750g/l) TS to produce 100 ml.
Phenol red R. Phenolsulfonphthalein, C19HI4O5S.

Phosphate buffer solution, pH 7.2

Mix 50 ml of 0.2 mol/l potassium dihydrogenphosphate TS for buffer solution and 34.7 ml of 0.2 mol/l sodium
hydroxide VS, and add water to make 200 ml.
Poly(dimethyl)(diphenyl)siloxane.
Contains 95 per cent of methyl groups and 5 per cent of phenyl groups. DB-5, SE52. Stationary phase for gas
chromatography.
Poly(dimethyl)siloxane.
Silicone gum rubber (methyl). Organosilicon polymer with the appearance of a semi-liquid, colourless gum.
Polysorbate 20 R. Quality conforms to the monograph in The international pharmacopoeia, Vol. 4, p. 202.
Polysorbate 80 R. Quality conforms to the monograph in The international pharmacopoeia, Vol. 4, p. 202.
Potassium bromide R. KBr.

Potassium dihydrogen phosphate R. KH2PO4.

Potassium dihydrogen phosphate TS (0.2 mol/l)

Dissolve 27.22g of potassium dihydrogenphosphate in water to make 1000 ml.
page 58
Potassium hydrogen phthalate R. C8H5KO4.

Potassium hydroxide.. KOH

Potassium iodide R. KI.

Potassium iodide AsR. Potassium iodide R that complies with the following test: Dissolve 10g of potassium
iodide R in 25 ml of hydrochloric acid (~250g/l) As TS and 35 ml of water, add 2 drops of stannous
chloride As TS and apply the general test for arsenic; no visible stain is produced.
Potassium sulfate R. K2SO4.

Potassium tellurite R. K2TeO3 (approx.)

Potassium tetrathionate R. K2S4O6.

Prometryn R. C10H19N5S.
Propylene glycol R. Propane diol, C3H8O2.

2-Propanol R. Isopropyl alcohol; C3H8O.

Pyrogallol R. C6H6O3. Benzene-1,2,3-triol.

White crystals, becoming brownish on exposure to air and light, very soluble in water, in alcohol and in ether,
slightly soluble in carbon disulphide. On exposure to air, aqueous solutions, and more rapidly alkaline
solutions, becomebrown owing to the absorption of oxygen.
mp: about 131 °C.
Store protected from light.
Pyrogallol solution, alkaline.

Dissolve 0.5 g pyrogallol R in 2 ml of carbon dioxide-free water R. Dissolve 12 g of potassium hydroxide R in
8 ml of carbon dioxide-free water R. Mix the two solutions immediately before use.
Silica gel G
Description. A fine, white, homogeneous powder with an average particle size of between 10 and 44 µm
containing about 130g of calcium sulfate, hemihydrate per kg.
Content of calcium sulfate. Place about 0.25 g, accurately weighed, in a flask with a ground-glass stopper, add
3 ml of hydrochloric acid (~70g/l) TS and 100 ml of water and shake vigorously for 30 minutes. Filter
through a sintered-glass filter and wash the residue. Using the combined filtrate and washings, carry out
the assay for calcium by complexometry (The international pharmacopoeia, Vol. 1, page 128). Each ml
of disodium edetate (0.05 mol/l) VS is equivalent to 7.26 mg of CaSO4,1/2H2O (MW 145.1).
page 59
pH. Shake 1 g with 10 ml of carbon-dioxide-free water R for 5 minutes. Measure the pH potentiometrically
(The international pharmacopoeia, Vol. 1, page 96); pH is about 7.
Preparation. Suspend 30g in 60 ml of water, shaking vigorously for 30 seconds. Carefully coat the cleaned
plates with a layer 0.25 mm thick using a spreading device. Allow the coated plates to dry in air.
Separating power. Apply separately to the adsorbent layer 10 µl of 0.10 mg/ml solutions containing
respectively indophenol blue R, sudan red G R and dimethyl yellow R in toluene R. Develop the plate
with the same solvent over a distance of 10 cm. The chromatogram shows three clearly separated spots,
the spot of indophenol blue near the points of application, that of dimethyl yellow in the middle of the
chromatogram, and that of sudan red G between the two.
Silica gel R.
Silver nitrate R. AgNO3.

Silver nitrate (40 g/l) TS. A solution of silver nitrate R containing about 42.5g of AgNO3 per litre
Simazine R. C7H12CIN5.
Soda lime R.
Sodium carbonate R. Na2CO3,10H2O.

Sodium carbonate, anhydrous, R. Na2CO3.

Sodium carbonate (50 g/l) TS. A solution of sodium carbonate R containing about 50g of Na2CO3 per litre
Sodium chloride R. NaCl.

Sodium chloride (400 g/l) TS. A solution of sodium chloride R containing about 400g of NaCl per litre.
Sodium citrate R. C6H5Na3O7,2H2O.

Quality of substance conforms to the mono-graph in The international pharmacopoeia, vol. 3, p. 192.
Sodium deoxycholate R. C23H39NaO4. Containing not less than 90% of C23H39NaO4.

Sodium hydroxide R. NaOH.

Sodium hydroxide (1 mol/l) VS. Sodium hydroxide R dissolved in water to produce a solution containing
40.01g of NaOH in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 1 mol/l solution in the following manner:
Dry about 5g of potassium hydrogen phthalate R at 105°C for 3 hours and weigh accurately. If the
page 60
potassium hydrogen phthalate is in the form of large crystals, they should be crushed before drying.
Dissolve in 75ml of carbon-dioxide-free water R and titrate with the sodium hydroxide solution, using
phenolphthalein/ethanol TS as indicator. Each 0.2042g of potassium hydrogen phthalate is equivalent to
1 ml of sodium hydroxide (1 mol/l) VS. Standard solutions of sodium hydroxide should be
restandardized frequently.
Storage. Solutions of alkali hydroxides absorb carbon dioxide when exposed to air. They should therefore be
stored in bottles with suitable non-glass, tightly-fitting stoppers, provided with a tube filled with soda
lime R.
Sodium hydroxide (0.5 mol/l) VS. Sodium hydroxide R dissolved in water to produce a solution containing
20.00g of NaOH in 1000 ml.
Method of standardization. Ascertain the exact concentration of the solution following the method described
under sodium hydroxide (1 mol/l) VS.
Sodium hydroxide (0.05 mol/l) VS. Sodium hydroxide R, dissolved in water to produce a solution containing
2.000g of NaOH in 1000ml.
Sodium hydroxide (0.02 mol/l), carbonate-free, VS. Sodium hydroxide R, dissolved in water to produce a
solution containing 0.8001 g of NaOH in 1000 ml.
Sodium hydroxide (~240 g/l) TS. A solution of sodium hydroxide R containing about 240g of NaOH per litre
of carbon-dioxide-free water R.
Sodium hydroxide, methanolic TS.

Procedure. Dissolve 2.5 g of sodium hydroxide R in 10ml of carbon-dioxide-free water R. Add 1 ml of
propylene glycol R and dilute to 100 ml with methanol R.
Sodium metabisulfite R. Na2O5S2.

Sodium metabisulfite (150 g/l) TS.

A solution of sodium metabisulfite R containing about 150g of Na2O5S2 per litre.
Sodium pyruvate R. C3H3NaO2.

Description. An almost white to white powder or a crystalline powder. Solubility. Soluble in water.
Sodium sulfate, anhydrous, R. Na2SO3. A suitable commercially available reagent.
Sodium sulfide TS. Na2S

Dissolve 5 g of sodium sulfide ennaehydrate in a mixture of 10 ml of water and 30 ml of glycerin. Or dissolve
5g of sodium hydroxide in a mixture of 30 ml of water and 90 ml of glycerin, saturate half volume of this
solution with hydrogen sulfide, while cooling, and mix with the remaining half. Preserve in well-filled,
light-resistant bottles. Use within 3 months.
Sodium sulfide ennaehydrate R. Na2S,9H2O

Sodium sulfite, anhydrous, R. Na2SO3

page 61
Sodium tetrahydroborate R. NaBH4.

Hygroscopic cristals, freely soluble in water, soluble in ethanol.
Sodium thiosulfate R. Na2S2O3,5H2O.

Sodium thiosulfate (0.1 mol/l) VS. Sodium thiosulfate R, dissolved in water to produce a solution containing
15.82g of Na2S2O3 in 1000 ml.
Method of standardization. Ascertain the exact concentration of the 0.1 mol/l solution in the following manner:
transfer 30.0ml of potassium dichromate (0.0167 mol/l) VS to a glass-stoppered flask and dilute with
50ml of water. Add 2g of potassium iodide R and 5ml of hydrochloric acid (~250g/l) TS, stopper and
allow to stand for 10 minutes. Dilute with 100 ml of water and titrate the liberated iodine with the sodium
thiosulfate solution, using starch TS as indicator. Sodium thiosulfate solutions should be restandardized
frequently.
Stannous chloride R. SnCl2,2H2O.

Stannous chloride AsTS.

Procedure. Prepare from stannous chloride TS by adding an equal volume of hydrochloric acid ( 250g/l) TS,
boiling down to the original volume, and filtering through a fine-grained filter-paper.
Test for arsenic. To 10 ml add 6 ml of water and 10 ml of hydrochloric acid (∼ 250 g/l) AsTS, and distil 16ml.
To the distillate add 50ml of water and 2 drops of stannous chloride AsTS and apply the general test for
arsenic. The colour of the stain produced is not more intense than that produced from a 1-ml standard
stain, showing that the amount of arsenic does not exceed 1 µg/ml.
Starch R.
Starch, soluble, R.
Starch TS.
Procedure. Mix 0.5 g of starch R or of soluble starch R with 5 ml of water, and add this solution, with constant
stirring, to sufficient water to produce about 100ml; boil for a few minutes, cool, and filter.
Note. Starch TS should be freshly prepared.
Styrene-divinylbenzene copolymer.
Porous, rigid, cross-linked polymer beads. Several grades are available with different sizes of beads. The size
range of the beads is specified after the name of the reagent in the tests where it is used.
A suitable commercially available material.
Sucrose R. C12H22O11.
Sudan red G R. 1-(4-Phenylazophenylazo)-2-naphthol; sudan III; solvent red 23; C.I. 26100; C22H16N4O.
Description. A reddish brown powder.
Solubility. Practically insoluble in water; soluble in chloroform R.
Sulfuric acid R. H2SO4

Sulfuric acid (~1760 g/l) TS. d 1.84.

page 62
Sulfuric acid (~300 g/l) TS.

Procedure. Add 171 ml of sulfuric acid (~1760g/l) TS to sufficient water to produce 1000 ml (approximately
3mol/l).
Sulfuric acid (~37g/l) TS.

Procedure. Add 21.5 ml of sulfuric acid (~1760g/l) TS to sufficient water to produce 1000ml (approximately
0.375mol/l).
Tetracycline R. C22H24N2O8.
N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride R. C10H16N2, 2HCl.

Description. Whitish grey crystals.
Toluene R. C7H8. Methylbenzene.

A clear, colourless, flammable liquid, very slightly soluble in water, miscible with alcohol.
d 2020 : 0.865 to 0.870.
bp: about 110 °C.
Trifluoroacetic acid (TFA). CF3COOH.

2,2,4-Trimethylpentane R. C8H18.
Water, carbon-dioxide-free, R. Water that has been boiled vigorously for a few minutes and protected from
the atmosphere during cooling and storage.
Yeast extract, water-soluble, R.

Zinc R, Zn; granulate, powder, or dust.

Zinc, AsR, granulated. Granulated zinc R that complies with the following tests:
Limit of arsenic. Add 10ml of stannated hydrochloric acid (~250g/l) AsTS to 50ml of water, and apply the
general test for arsenic; use 10 g of granulated zinc R and allow the reaction to continue for 1 hour; no
visible stain is produced.
Test for sensitivity. Repeat the test for arsenic with the addition of 0.1 ml of dilute arsenic AsTS; a faint, but
distinct yellow stain is produced.
Zinc acetate R. C4H6O4Zn,2H2O.

Zinc acetate/aluminium chloride TS.

Procedure. Dissolve 200g of zinc acetate R and 5g of aluminium chloride R in sufficient water to produce 1000
ml.
***

Quality Control Methods For Medicinal Plant Materials OMS

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Working document QAS/05.131/Rev.

WORLD HEALTH ORGANIZATION

QUALITY CONTROL METHODS FOR MEDICINAL PLANT MATERIALS

Please send any request for permission to:

SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/05.131/Rev.1:

First working draft prepared June 2003

Discussion at WHO working group meeting July 2003

First draft November 2003

First global review November 2003-January 2004

Revised draft May 2004

Second global review May-June 2004

Second revised draft July 2004

Discussion at WHO consultation July 2005

Revision of draft document June-July 2005

First update draft July 2005

Revised update draft September 2005

Third global review: deadline for receipt of comments 30 November 2005

Presentation to Fortieth WHO Expert Committee on 24-28 October 2005

Recommended analytical methods

Sections of methods proposed for the update:

Determination of Pesticide Residues

Methods for the determination of pesticide residues

General aspects of analytical methodology

Table 1. Examples of national limits set for various pesticide residues

A. Determination of total chlorine and phosphorus

Preparation of the column

- The third eluate contains phosphated pesticide (malathion).

Combustion of the organic matter

Sample holder for chlorine-containing residues

Sample holder for phosphorus-containing residues

Combustion of chlorine-containing residues

Combustion of phosphorus-containing residues

D. Qualitative and quantitative determination of organochlorine pesticides

Determination by gas chromatography

First separation system

Second separation system

where ht = peak height obtained for the test solution in mm,

E. Analysis of esters of organophosphorus compounds

F. Determination of specific pesticide residues in medicinal plant material

G. Determination of desmetryn, prometryn, and simazine residues

Preparation of the plant material extract

Preparation of chromatographic column

No. Material No. Material

Determination of the rate of recovery

Prepare five individual samples using each of the following procedures:

Calculate the rate of recovery (R) in % using the following formula:

Determination by gas chromatography

H. Determination of specific organochlorine, organophosphorus and pyrethroid

2.1. Organochlorine, organophosphorus and pyrethroid insecticides.

The chromatographic procedure may be carried out using:

— as mobile phase toluene R at a flow rate of 1 ml/min.

2.2. Organochlorine and pyrethroid insecticides.

Concentrate solution B in a current of helium for chromatography R or oxygen-free nitrogen R almost

The chromatographic procedure may be carried out using:

— a phosphorus-nitrogen flame-ionisation detector or a atomic emission spectrometry detector,

2. Organochlorine and pyrethroid insecticides.

Test solution. Concentrate solution C in a current of helium for chromatography R or oxygen-free

The chromatographic procedure may be carried out using:

— a device allowing direct cold on-column injection,