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2.8.13.

Pesticide residues

EUROPEAN PHARMACOPOEIA 5.0

If the drug is intended for the preparation of extracts,


tinctures or other pharmaceutical forms whose preparation
method modifies the content of pesticides in the finished
product, the limits are calculated using the following
expression :

Figure 2.8.12.-2
Introduce into the flask the prescribed quantity of the drug
and continue the distillation as described above for the
time and at the rate prescribed. Stop the heating and after
10 min read the volume of liquid collected in the graduated
tube and subtract the volume of xylene previously noted.
The difference represents the quantity of essential oil in the
mass of the drug taken. Calculate the result as millilitres
per kilogram of drug.
When the essential oil is to be used for other analytical
purposes, the water-free mixture of xylene and essential
oil may be recovered as follows : remove the stopper K
and introduce 0.1 ml of a 1 g/l solution of sodium
fluoresceinate R and 0.5 ml of water R. Lower the mixture
of xylene and essential oil into the bulb-shaped swelling L
by means of the three-way tap, allow to stand for 5 min and
lower the mixture slowly until it just reaches the level of the
tap M. Open the tap anti-clockwise so that the water flows out
of the connecting tube BM. Wash the tube with acetone R
and with a little toluene R introduced through the filling
funnel N. Turn the tap anti-clockwise in order to recover the
mixture of xylene and essential oil in an appropriate flask.

extraction factor of the method of preparation,


determined experimentally.

Higher limits can also be authorised, in exceptional cases,


especially when a plant requires a particular cultivation
method or has a metabolism or a structure that gives rise to
a higher than normal content of pesticides.
The competent authority may grant total or partial
exemption from the test when the complete history (nature
and quantity of the pesticides used, date of each treatment
during cultivation and after the harvest) of the treatment of
the batch is known and can be checked precisely.
Sampling
Method. For containers up to 1 kg, take one sample from
the total content, thoroughly mixed, sufficient for the tests.
For containers between 1 kg and 5 kg, take three samples,
equal in volume, from the upper, middle and lower parts of
the container, each being sufficient to carry out the tests.
Thoroughly mix the samples and take from the mixture an
amount sufficient to carry out the tests. For containers of
more than 5 kg, take three samples, each of at least 250 g
from the upper, middle and lower parts of the container.
Thoroughly mix the samples and take from the mixture an
amount sufficient to carry out the tests.
Size of sampling. If the number (n) of containers is three or
fewer, take samples from each container as indicated above
01/2005:20813 under Method. If the number of containers is more than
three, take
samples from containers as indicated
under Method, rounding up to the nearest unit if necessary.
2.8.13. PESTICIDE RESIDUES
The samples are to be analysed immediately to avoid possible
degradation of the residues. If this is not possible, the
Definition. For the purposes of the Pharmacopoeia,
samples are stored in airtight containers suitable for food
a pesticide is any substance or mixture of substances
intended for preventing, destroying or controlling any pest, contact, at a temperature below 0 C, protected from light.
unwanted species of plants or animals causing harm during Reagents. All reagents and solvents are free from any
or otherwise interfering with the production, processing,
contaminants, especially pesticides, that might interfere
storage, transport or marketing of vegetable drugs. The item with the analysis. It is often necessary to use special quality
includes substances intended for use as growth-regulators,
solvents or, if this is not possible, solvents that have recently
defoliants or desiccants and any substance applied to crops been re-distilled in an apparatus made entirely of glass. In
either before or after harvest to protect the commodity from any case, suitable blank tests must be carried out.
deterioration during storage and transport.
Apparatus. Clean the apparatus and especially glassware to
ensure that they are free from pesticides, for example, soak
Limits. Unless otherwise indicated in the monograph,
for at least 16 h in a solution of phosphate-free detergent,
the drug to be examined at least complies with the limits
indicated in Table 2.8.13.-1. The limits applying to pesticides rinse with large quantities of distilled water R and wash with
acetone and hexane or heptane.
that are not listed in the table and whose presence is
suspected for any reason comply with the limits set by
Qualitative and quantitative analysis of pesticide residues.
European Community directives 76/895 and 90/642,
The analytical procedures used are validated according
including their annexes and successive updates. Limits for
to the regulations in force. In particular, they satisfy the
pesticides that are not listed in Table 2.8.13.-1 nor in EC
following criteria :
directives are calculated using the following expression :
the chosen method, especially the purification steps, are
suitable for the combination pesticide residue/substance
to be analysed, and not susceptible to interference from
co-extractives ; the limits of detection and quantification
are measured for each pesticide-matrix combination to
ADI = acceptable daily intake, as published by
be analysed,
FAO-WHO, in milligrams per kilogram of body
between 70 per cent to 110 per cent of each pesticide is
mass,
recovered,
M
= body mass in kilograms (60 kg),
the repeatability of the method is not less than the values
MDD = daily dose of the drug, in kilograms.
indicated in Table 2.8.13.-2,
218

See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0

the reproducibility of the method is not less than the


values indicated in Table 2.8.13.-2,

2.8.13. Pesticide residues

The following section is published for information.

Test for pesticides


the concentration of test and reference solutions and the
setting of the apparatus are such that a linear response is
ORGANOCHLORINE, ORGANOPHOSPHORUS AND
obtained from the analytical detector.
PYRETHROID INSECTICIDES
The following methods may be used, in connection with
Table 2.8.13.-1
the general method above. Depending on the substance
Limit
Substance
being examined, it may be necessary to modify, sometimes
(mg/kg)
extensively, the procedure described hereafter. In any case,
0.02
Alachlor
it may be necessary to use, in addition, another column
with a different polarity or another detection method (mass
0.05
Aldrin and Dieldrin (sum of)
spectrometry...) or a different method (immunochemical
1.0
Azinphos-methyl
methods...) to confirm the results obtained.
3.0
Bromopropylate
This procedure is valid only for the analysis of samples of
vegetable drugs containing less than 15 per cent of water.
0.05
Chlordane (sum of cis-, trans - and Oxythlordane)
Samples with a higher content of water may be dried,
0.5
Chlorfenvinphos
provided it has been shown that the drying procedure does
not affect significantly the pesticide content.
0.2
Chlorpyrifos
1. EXTRACTION
0.1
Chlorpyrifos-methyl
To 10 g of the substance being examined, coarsely
1.0
Cypermethrin (and isomers)
powdered, add 100 ml of acetone R and allow to stand
1.0
DDT (sum of p,p-DDT, o,p-DDT, p,p-DDE and p,p-TDE)
for 20 min. Add 1 ml of a solution containing 1.8 g/ml
of carbophenothion R in toluene R. Homogenise using a
0.5
Deltamethrin
high-speed blender for 3 min. Filter and wash the filter cake
Diazinon
0.5
with two quantities, each of 25 ml, of acetone R. Combine
the filtrate and the washings and heat using a rotary
1.0
Dichlorvos
evaporator at a temperature not exceeding 40 C until the
2.0
Dithiocarbamates (as CS2)
solvent has almost completely evaporated. To the residue
add a few millilitres of toluene R and heat again until the
3.0
Endosulfan (sum of isomers and Endosulfan sulphate)
acetone is completely removed. Dissolve the residue in 8 ml
0.05
Endrin
of toluene R. Filter through a membrane filter (45 m), rinse
2.0
Ethion
the flask and the filter with toluene R and dilute to 10.0 ml
with the same solvent (solution A).
0.5
Fenitrothion
2. PURIFICATION
1.5
Fenvalerate
2.1. Organochlorine, organophosphorus and
0.05
Fonofos
pyrethroid insecticides. Examine by size-exclusion
0.05
Heptachlor (sum of Heptachlor and Heptachlorepoxide)
chromatography (2.2.30).
The chromatographic procedure may be carried out using :
0.1
Hexachlorobenzene
a stainless steel column 0.30 m long and 7.8 mm in
0.3
Hexachlorocyclohexane isomers (other than )
internal diameter packed with styrene-divinylbenzene
0.6
Lindane (-Hexachlorocyclohexane)
copolymer R (5 m),
1.0
Malathion
as mobile phase toluene R at a flow rate of 1 ml/min.
Performance of the column. Inject 100 l of a solution
0.2
Methidathion
containing 0.5 g/l of methyl red R and 0.5 g/l of oracet blue
0.5
Parathion
2R R in toluene R and proceed with the chromatography.
0.2
Parathion-methyl
The column is not suitable unless the colour of the eluate
changes from orange to blue at an elution volume of about
1.0
Permethrin
10.3 ml. If necessary calibrate the column, using a solution
0.1
Phosalone
containing, in toluene R, at a suitable concentration, the
insecticide to be analysed with the lowest molecular mass
3.0
Piperonyl butoxide
(for example, dichlorvos) and that with the highest molecular
4.0
Pirimiphos-methyl
mass (for example, deltamethrin). Determine which fraction
of the eluate contains both insecticides.
3.0
Pyrethrins (sum of)
Purification of the test solution. Inject a suitable volume
1.0
Quintozene (sum of quintozene, pentachloroaniline and
of solution A (100 l to 500 l) and proceed with the
methyl pentachlorophenyl sulphide)
chromatography. Collect the fraction as determined above
(solution B). Organophosphorus insecticides are usually
Table 2.8.13.-2
eluted between 8.8 ml and 10.9 ml. Organochlorine and
Concentration of
Reproducibility
Repeatability
pyrethroid insecticides are usually eluted between 8.5 ml
the pesticide
(difference,
(difference,
and 10.3 ml.
(mg/kg)
mg/kg)
mg/kg)
2.2. Organochlorine and pyrethroid insecticides. In
0.010
0.005
0.01
a chromatography column, 0.10 m long and 5 mm in
internal diameter, introduce a piece of defatted cotton and
0.100
0.025
0.05
0.5 g of silica gel treated as follows : heat silica gel for
1.000
0.125
0.25
chromatography R in an oven at 150 C for at least 4 h.
General Notices (1) apply to all monographs and other texts

219

2.8.13. Pesticide residues

EUROPEAN PHARMACOPOEIA 5.0

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 0.50 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 to
dryness and dilute to a suitable volume with toluene R
(200 l 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).
3. QUANTITATIVE ANALYSIS
3.1. Organophosphorus insecticides. Examine by gas
chromatography (2.2.28), using carbophenothion R as
internal standard. It may be necessary to use a second
internal stan-dard to identify possible interference with the
peak corresponding to carbophenothion.

Table 2.8.13.-3
Substance

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

Test solution. Concentrate solution B in a current of helium The chromatographic procedure may be carried out using :
for chromatography R almost to dryness and dilute to 100 l a fused silica column 30 m long and 0.32 mm in internal
diameter the internal wall of which is covered with a layer
with toluene R.
0.25 m thick of poly(dimethyl)(diphenyl)siloxane R,
Reference solution. Prepare at least three solutions in
hydrogen for chromatography R as the carrier gas. Other
toluene R containing the insecticides to be determined and
gases such as helium for chromatography R or nitrogen
carbophenothion at concentrations suitable for plotting a
for chromatography R may also be used, provided the
calibration curve.
chromatography is suitably validated,

an
electron-capture detector,
The chromatographic procedure may be carried out using :
a device allowing direct cold on-column injection,
a fused-silica column 30 m long and 0.32 mm in internal maintaining the temperature of the column at 80 C for
diameter the internal wall of which is covered with a layer 1 min, then raising it at a rate of 30 C/min to 150 C,
0.25 m thick of poly(dimethyl)siloxane R,
maintaining at 150 C for 3 min, then raising the temperature
hydrogen for chromatography R as the carrier gas. Other at a rate of 4 C/min to 280 C and maintaining at this
gases such as helium for chromatography R or nitrogen temperature for 1 min, and maintaining the temperature
of the injector port at 250 C and that of the detector at
for chromatography R may also be used provided the
275 C. Inject the chosen volume of each solution. When the
chromatography is suitably validated.
chromatograms are recorded in the prescribed conditions,
the relative retention times are approximately those listed
a phosphorus-nitrogen flame-ionisation detector or a
in Table 2.8.13.-4. Calculate the content of each insecticide
atomic emission spectrometry detector,
from the peak areas and the concentrations of the solutions.
maintaining the temperature of the column at 80 C for
Table 2.8.13.-4
1 min, then raising it at a rate of 30 C/min to 150 C,
Relative rentention times
maintaining at 150 C for 3 min, then raising the temperature Substance
at a rate of 4 C/min to 280 C and maintaining at this
0.44
-Hexachlorocyclohexane
temperature for 1 min, and maintaining the temperature
0.45
Hexachlorobenzene
of the injector port at 250 C and that of the detector at
275 C. Inject the chosen volume of each solution. When the -Hexachlorocyclohexane
0.49
chromatograms are recorded in the prescribed conditions,
0.49
Lindane
the relative retention times are approximately those listed
in Table 2.8.13.-3. Calculate the content of each insecticide
0.54
-Hexachlorocyclohexane
from the peak areas and the concentrations of the solutions.
3.2. Organochlorine and pyrethroid insecticides. Examine
by gas chromatography (2.2.28), 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 l 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.
220

-Hexachlorocyclohexane

0.56

Heptachlor

0.61

Aldrin

0.68

cis-Heptachlor-epoxide

0.76

o,p-DDE

0.81

-Endosulfan

0.82

Dieldrin

0.87

p,p-DDE

0.87

o,p-DDD

0.89

Endrin

0.91

See the information section on general monographs (cover pages)

2.8.15. Bitterness value

EUROPEAN PHARMACOPOEIA 5.0

Substance

01/2005:20815

Relative rentention times

-Endosulfan

0.92

o,p-DDT

0.95

2.8.15. BITTERNESS VALUE

1.00

The bitterness value is the reciprocal of the dilution of a


compound, a liquid or an extract that still has a bitter taste.
p,p-DDT
1.02
It is determined by comparison with quinine hydrochloride,
1.29
cis-Permethrin
the bitterness value of which is set at 200 000.
1.31
trans-Permethrin
Determination of the correction factor
1.40
Cypermethrin*
A taste panel comprising at least 6 persons is recommended.
The mouth must be rinsed with water R before tasting.
1.47 and 1.49
Fenvalerate*
To correct for individual differences in tasting bitterness
1.54
Deltamethrin
amongst the panel members it is necessary to determine a
* The substance shows several peaks.
correction factor for each panel member.
Stock solution. Dissolve 0.100 g of quinine hydrochloride R
01/2005:20814 in water R and dilute to 100.0 ml with the same solvent.
Dilute 1.0 ml of this solution to 100.0 ml with water R.
Reference
solutions. Prepare a series of dilutions by placing
2.8.14. DETERMINATION OF TANNINS
in a first tube 3.6 ml of the stock solution and increasing the
IN HERBAL DRUGS
volume by 0.2 ml in each subsequent tube to a total of 5.8 ml ;
dilute the contents of each tube to 10.0 ml with water R.
Carry out all the extraction and dilution operations
protected from light.
Determine as follows the dilution with the lowest
concentration that still has a bitter taste. Take 10.0 ml of the
In the case of a herbal drug or a dry extract, to the stated
weakest solution into the mouth and pass it from side to side
amount of the powdered drug (180) or the extract in a
over the back of the tongue for 30 s. If the solution is not
250 ml round-bottomed flask add 150 ml of water R. Heat
found to be bitter, spit it out and wait for 1 min. Rinse the
on a water-bath for 30 min. Cool under running water and
mouth with water R. After 10 min, use the next dilution in
transfer quantitatively to a 250 ml volumetric flask. Rinse
order of increasing concentration.
the round-bottomed flask and collect the washings in the
volumetric flask, then dilute to 250.0 ml with water R. Allow Calculate the correction factor k for each panel member
the solids to settle and filter the liquid through a filter paper from the expression :
125 mm in diameter. Discard the first 50 ml of the filtrate.
In the case of a liquid extract or a tincture, dilute the stated
amount of the liquid extract or tincture to 250.0 ml with
water R. Filter the mixture through a filter paper 125 mm in n
= number of millilitres of the stock solution in the
diameter. Discard the first 50 ml of the filtrate.
dilution of lowest concentration that is judged to
Total polyphenols. Dilute 5.0 ml of the filtrate to 25.0 ml
be bitter.
with water R. Mix 2.0 ml of this solution with 1.0 ml of
phosphomolybdotungstic reagent R and 10.0 ml of water R Persons who are unable to taste any bitterness when using
and dilute to 25.0 ml with a 290 g/l solution of sodium
the reference solution prepared from 5.8 ml of stock solution
carbonate R. After 30 min measure the absorbance (2.2.25) have to be excluded from the panel.
at 760 nm (A1), using water R as the compensation liquid.
Sample preparation
Polyphenols not adsorbed by hide powder. To 10.0 ml of the
If necessary, reduce the sample to a powder (710). To 1.0 g of
filtrate, add 0.10 g of hide powder CRS and shake vigorously
sample add 100 ml of boiling water R. Heat on a water-bath
for 60 min. Filter and dilute 5.0 ml of the filtrate to 25.0 ml
for 30 min, stirring continuously. Allow to cool and dilute to
with water R. Mix 2.0 ml of this solution with 1.0 ml of
100 ml with water R. Shake vigorously and filter, discarding
phosphomolybdotungstic reagent R and 10.0 ml of water R
the first 2 ml of the filtrate. The filtrate is labelled C-1 and
and dilute to 25.0 ml with a 290 g/l solution of sodium
has a dilution factor (DF) of 100.
carbonate R. After 30 min measure the absorbance (2.2.25)
If liquids have to be examined, 1 ml of the liquid is diluted
at 760 nm (A2), using water R as the compensation liquid.
with a suitable solvent to 100 ml and designated C-1.
Standard. Dissolve immediately before use 50.0 mg of
pyrogallol R in water R and dilute to 100.0 ml with the
Determination of the bitterness value
same solvent. Dilute 5.0 ml of the solution to 100.0 ml
Test solutions :
with water R. Mix 2.0 ml of this solution with 1.0 ml of
(DF = 1000)
phosphomolybdotungstic reagent R and 10.0 ml of water R 10.0 ml of C-1 is diluted with water R to
100 ml : C-2
and dilute to 25.0 ml with a 290 g/l solution of sodium
carbonate R. After 30 min measure the absorbance (2.2.25) 10.0 ml of C-2 is diluted with water R to
(DF = 10 000)
at 760 nm (A3), using water R as the compensation liquid.
100 ml : C-3
Calculate the percentage content of tannins expressed as
20.0 ml of C-3 is diluted with water R to
(DF = 50 000)
pyrogallol from the expression :
100 ml : C-3A
Carbophenothion

10.0 ml of C-3 is diluted with water R to


100 ml : C-4
m1

mass of the sample to be examined, in grams,

m2

mass of pyrogallol, in grams.

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

(DF = 100 000)

Starting with dilution C-4 each panel member determines


the dilution which still has a bitter taste. This solution is
designated D. Note the DF of solution D is Y.
221

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