U.S. PHARMACOPEIA

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TEST FOR PESTICIDES
Unless otherwise specified in the individual monograph, the following methods may be used for the analysis of pesticides. Depending on the substance being examined, it may be necessary to modify, sometimes extensively, the procedure described hereafter. Additionally, it may be necessary to perform another method with another column having a different polarity, another detection method (e.g., mass spectrometry), or a different method (e.g., immunochemical method) to confirm the results.
Extraction— [NOTE—Use the following procedure for the analysis of samples of articles having a water content of less than 15%. Samples having a higher water content may be dried, provided that the drying procedure does not significantly affect the pesticide content.] To 10 g of the coarsely powdered substance under test, add 100 mL of acetone, and allow to stand for 20 minutes. Add 1 mL of a solution in toluene containing 1.8 g of carbophenothion per mL. Mix in a high-speed blender for 3 minutes. Filter this solution, and wash the residue with two 25-mL portions of acetone. Combine the filtrate and the washings, and heat, in a rotary evaporator, maintaining the temperature of the bath below 40 until the solvent has almost completely evaporated. To the residue add a few mL of toluene, and heat again until the acetone is completely removed. Dissolve the residue in 8 mL of toluene. Pass through a membrane filter having a 45-µm porosity, rinse the flask and the filter with toluene, dilute with toluene to 10.0 mL (Solution A), and mix.
Purification—
Organochlorine, Organophosphorus, and Pyrethroid Insecticides— The size-exclusion chromatograph is equipped with a 7.8-mm × 30-cm stainless steel column containing 5-µm packing L21. Toluene is used as the mobile phase at a flow rate of about 1 mL per minute.
Performance of the Column— Inject 100 µL of a solution in toluene containing, in each mL, 0.5 mg of methyl red and 0.5 mg of oracet blue. The column is not suitable unless the color 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 in toluene containing suitable concentrations of the pesticide of interest having the lowest molecular weight (for example, dichlorvos) and that having the highest molecular weight (for example, deltamethrin). Determine which fraction of the eluate contains both pesticides.
Purification of the Test Solution— Inject a suitable volume (100 to 500 µL) of Solution A into the chromatograph. Collect the fraction (Solution B) as determined above under Performance of the Column. Organophosphorus pesticides elute between 8.8 and 10.9 mL. Organochlorine and pyrethroid pesticides elute between 8.5 and 10.3 mL.
Organochlorine and Pyrethroid Insecticides— Into a 5-mm × 10-cm chromatographic column, introduce a piece of fat-free cotton and 0.5 g of silica gel treated as follows. Heat chromatographic silica gel in an oven at 150 for at least 4 hours. Allow to cool, and add dropwise a quantity of water corresponding to 1.5% of the weight of silica gel used. Shake vigorously until agglomerates have disappeared, and continue shaking by mechanical means for 2 hours. Condition the column with 1.5 mL of hexane. [NOTE—Prepacked columns containing about 0.50 g of a suitable silica gel may also be used, provided they have been previously validated.] Concentrate Solution B almost to dryness, with the aid of a stream of helium or oxygen-free nitrogen, and dilute with toluene to a suitable volume (200 µL to 1 mL, according to the volume injected in the preparation of Solution B). Quantitatively transfer this solution to the column, and proceed with the chromatography, using 1.8 mL of toluene as the mobile phase. Collect the eluate (Solution C).
Quantitative Analysis of Organophosphorus Insecticides—
Test Solution— Concentrate Solution B almost to dryness, with the aid of a stream of helium, dilute with toluene to 100 µL, and mix.
Standard Solution— Prepare at least three solutions in toluene containing each of the pesticides of interest and carbophenothion at concentrations suitable for plotting a calibration curve.
Chromatographic System— The gas chromatograph is equipped with an alkali flame-ionization detector or a flame-photometric detector and a 0.32-mm × 30-m fused silica column coated with a 0.25-µm layer of phase G1. Hydrogen is used as the carrier gas. Other gases, such as helium or nitrogen, may also be used. The injection port temperature is maintained at 250, and the detector is maintained at 275. The column temperature is maintained at 80 for 1 minute, then increased to 150 at a rate of 30 per minute, maintained at 150 for 3 minutes, then increased to 280 at a rate of 4 per minute, and maintained at this temperature for 1 minute. Use carbophenothion as the internal standard. [Note—If necessary, use a second internal standard to identify any possible interference with the peak corresponding to carbophenothion.] Inject the chosen volume of each solution, record the chromatograms, and measure the peak responses: the relative retention times are approximately those listed in Table 5. Calculate the content of each pesticide from the peak areas and the concentrations of the solution.
Table 5
Substance Relative Retention Time
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
Phosalone 1.18
Quantitative Analysis of Organochlorine and Pyrethroid Insecticides—
Test Solution— Concentrate Solution C almost to dryness, with the aid of a stream of helium or oxygen-free nitrogen, dilute with toluene to 500 µL, and mix.
Standard Solution— Prepare at least three solutions in toluene containing each of the pesticides of interest and carbophenothion at concentrations suitable for plotting a calibration curve.
Chromatographic System— The gas chromatograph is equipped with an electron-capture detector, a device allowing direct on-column cold injection, and a 0.32-mm × 30-m fused silica column coated with a 0.25-µm layer of phase G1. Hydrogen is used as the carrier gas. Other gases, such as helium or nitrogen, may also be used. The injection port temperature is maintained at 275, and the detector is maintained at 300. The column temperature is maintained at 80 for 1 minute, then increased to 150 at a rate of 30 per minute, maintained at 150 for 3 minutes, then increased to 280 at a rate of 4 per minute, and maintained at this temperature for 1 minute. Use carbophenothion as the internal standard. [NOTE—If necessary, use a second internal standard to identify any possible interference with the peak corresponding to carbophenothion.] Inject the chosen volume of each solution, record the chromatograms, and measure the peak responses: the relative retention times are approximately those listed in Table 6. Calculate the content of each pesticide from the peak areas and the concentrations of the solutions.
Table 6
Substance Relative Retention Time
-Hexachlorocyclohexane 0.44
Hexachlorobenzene 0.45
-Hexachlorocyclohexane 0.49
Lindane 0.49
-Hexachlorocyclohexane 0.54
-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
-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
1.49
Deltamethrin 1.54
*  The substance shows several peaks.