Analyst, January, 1973, Vol. 98, PP. 19-24 19 The Determination of Malathion and Dichlorvos Residues in Grain REPORT BY THE PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN OF THE COMMITTEE FOR ANALYTICAL METHODS FOR RESIDUES OF PESTICIDES AND VETERINARY PRODUCTS IN FOODSTUFFS OF THE MINISTRY OF AGRICULTURE, FISHERIES AND FOOD THE Panel was formed in 1969 by the Committee for Analytical Methods for Residues of Pesticides and Veterinary Products in Foodstuffs to study gas - liquid chromatographic methods for the determination of malathion and dichlorvos residues in stored grain. At that time, the Codex Committee on Pesticide Residues had under review a colorimetric method for the determination of malathion residues, which was evaluated by a United Kingdom panel some years ag0.l The Committee for Analytical Methods considered it advisable to establish a gas - liquid chromatographic method that would be more specific and up to date and might effectively replace the colorimetric method.As dichlorvos is being increasingly used in a similar way to malathion on stored grain, it was included in the study. Members of the Committee themselves, or deputed staff from their laboratories, acted as a nucleus of the Panel while representation was invited, and accepted, from the British Agrochemicals Association, the Association of Public Analysts, the Society for Analytical Chemistry and the (then) Pest Infestation Laboratory so as to co-ordinate the work of various interested laboratories in the United Kingdom. The members of the panel are listed in Appendix 111.The terms of reference of the Panel were- “To establish by collaborative study a gas - liquid chromatographic method for residues of malathion and dichlorvos in grain, bearing in mind its application to organophosphorus compounds in other commodities.” The residues limits under consideration for raw grain by the Codex Committee were 8 p.p.m. for malathion and 2 p.p.m. for dichlorvos. Some relevant literature on residue analysis was reviewed by the Panel. Two methods (at that time unpublished) particularly interested the Panel: those of Elgar, Marlow and Mathews2 and Crisp and Tarrant.3 In the method of Elgar et aZ., the powdered grain, moistened with water, is macerated with dichloromethane. The filtered extract is then steam distilled and the distillate extracted and examined by gas - liquid chromatography by using, for preference, a flame-photometric detector (clean-up is not so critical with grain when using this detector).In the method of Crisp and Tarrant, the grain is macerated with three successive portions of methanol and the extract is concentrated. After chromato- graphy on activated charcoal, the eluate is examined by gas - liquid chromatography by use of a phosphorus-sensitive thermionic detector (the initial chromatography serves to remove spurious peaks from the methanol used in extracting the grain). Initial individual investi- gation of these two methods by laboratories showed neither to be completely satisfactory in the hands of workers not already familiar with the methods. The main investigations of the Panel concerned the choice of solvent for extracting malathion and dichlorvos from grain.When spiking grain for this purpose, the Panel con- sidered that, in order to test the method or solvent, after the addition of small amounts of a solution of pesticide to the grain, the flask containing the ground grain and pesticide should be stoppered and allowed to stand for at least 24 hours in a deep-freeze. In this way, the pesticide would be more likely to be absorbed into the grain and therefore correspond more closely to a treated commercial grain than if the sample were analysed immediately after the addition of the pesticide. Ethyl a ~ e t a t e , ~ acetonitrile, dichloromethane - water and acetone were all tested collaboratively as extraction solvents for spiked samples (see Appendix 11) by blending, filtration and gas - liquid chromatography (i.e., without clean-up).However, usually none of these procedures was as effective as methanolic extraction without clean-up (see Table I). Although the recovery of malathion was usually satisfactory, that of dichlorvos 0 SAC; Crown Copyright Reserved.20 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN : [Analyst, Vol. 98 TABLE I RECOVERY OF MALATHION AND DICHLORVOS FROM WHEAT WHEN USING SOLVENTS OTHER THAN METHANOL Level of pesticides on Solvent grain, p.p.m. Ethyl acetate . . 0.1 to 0.5 Acetonitrile , . 0.1 to 0-5 Dichloromethane - water, 2 + 1 . . 0-2 to 1-0 Acetone . . . . 0.2 to 1.0 Mean recovery of Recovery of dichlorvos individual determinations relative to methanolic (spiked samples) per cent.extraction (range arid M a e v o s determinations) per cent. mean of individual 90 34 - 71 32 - - 33 to 123, mean 69 83 60 25 to 97, mean 56 - was inadequate in solvents other than methanol. Moisture content affects the extent of recovery of dichlorvos. Comparisons of the extraction of dichlorvos, from a reference sample of wheat treated in a manner similar to that used commercially, by dichloromethane - water, acetone and methanol axe given in Table 11. TABLE I1 COMPARISON OF RECOVERY OF DICHLORVOS FROM TREATED WHEAT BY USING DICHLOROMETHANE - WATER, ACETONE AND METHANOL Laboratory 1 2 3 4 5 6 7 8 9 Mean recovery of pesticide compared with methanolic extraction, per cent. r > Dichloromethane - water Acetone 84 16 65 38 70 30 75 41 79 29 35 39 33 64 57 One determination 97 84) only 14 A Absolute value of methanolic extraction, p.p.m.0.22 0.48 0.19 0.36 0.49 0.82 0.56 0.57 - The actual level of dichlorvos found by methanolic extraction varied with the time of storage before analysis but the ratio of dichloromethane - water or acetone extraction to methanolic extraction would be expected to be independent of the actual level present. Values for this ratio are variable but show clearly that methanol is the best solvent. A collaborative study was undertaken so as to obtain results for the reproducibility of the method with dichlorvos, in which duplicate analyses were made on five 100-g sub-samples on a reference sample of wheat treated in the normal commercial fashion; results are given in Table 111.Again, the actual value varied with the time between exposure and analysis. The standard deviation of individual determinations on a 100-g sub-sample in the range 0.07 to 0.30 pep.". was 0.013 p.p.m. The lowest level of detection, by a single determination, would therefore not be less than 0.02 p.p.m. The recovery of malathion from wheat that was spiked and stored for 24 hours in a closed container is shown in Table IV. Members used their own discretion in choosing the column and detector for the gas- liquid chromatography. A limited study by members showed that the method, which was used successfully on wheat, was also applicable to the determination of residues of these pesticides in barley, sorghum, maize and flour. Very low levels of pesticide (less than 0.06 p.p.m.) may require clean-up.Other organophosphorus pesticides are unlikely to interfere in the determination of dichlorvos during the gas - liquid chromatographic stage. Columns on which the pesticides indicated may interfere in the determination of malathion are: 1.3 per cent. Apiezon L onJanuary, 19731 THE DETERMINATION OF MALATHION AND DICHLORVOS RESIDUES IN GRAIN 21 TABLE I11 DETERMINATIONS OF DICHLORVOS ON TREATED WHEAT Duplicate determinations on 20 g of wheat taken from five 100-g amounts from a common sample Laboratory 1 2 3* 4 5 6 7 8 9 First extract . . Duplicate . . Mean .. .. First extract . . Duplicate , . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean .... First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. First extract . . Duplicate . . Mean . . .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. * . .. .. .. .. .. .. Dichlorvos found, p.p.m. r A 0-27 0.28 0.28 0.17 0.14 0.16 0.072 0.072 0.072 0.20 0.21 0.21 0.2 1 0.24 0.23 0.30 0.30 0.30 0.15 0-17 0.16 0.24 0.25 0-25 0.18 0.16 0-17 0.079 0.078 0.079 0.23 0.23 0.23 0.24 0.26 0.25 0-30 0.29 0.30 0.18 0-17 0-18 0.23 0.25 0.24 0.14 0.16 0.15 0-067 0.069 0.068 0.21 0.22 0.22 0.27 0.28 0.28 0-30 0.30 0.30 0.16 0.16 0.16 0.26 0.25 0.26 0.18 0.14 0.16 0.078 0.073 0.076 0.20 0.19 0.20 0.25 0.27 0.26 0.29 0.30 0-30 0.18 0.19 0.19 0.29 0.30 0.28 0.28 0.28 0.37 0.36 0.35 0.29 0.34 0.32 0.32 0-29 0.29 0.29 0.29 0.27 0.28 0.28 0.28 0.28 0.29 0.29 0.29 1 0.24 0.24 0.24 0.14 0.16 0.15 0.069 0.07 1 0.070 0.26 0.22 0.24 0.26 0.27 0.27 0.3 1 0.3 1 0.3 1 0.16 0.19 0.18 0.28 0.3 1 0.30 0.29 0.29 0.29 * Maintained a t room temperature for 1 week before analysis instead of in a deep-freeze.Chromosorb G, formothion (Rm 99), aspon (Rm 103) and paraoxon (Rm 108); 1.3 per cent. butane-1,4-diol succinate on Chromosorb G, schradan (Rm 98), bromophos (Rm 100) and malaoxon (Rm 108); and 20 per cent. DC 200 on Gas-Chrom Q, paraoxon (Rm 94) and fenthion (Rm 107) [Rm represents the position of a peak from the time of injection relative to that of malathion (malathion = loo)]. Malaoxon is not an important metabolite of malathion on grain as the degradation of malathion proceeds by hydrolysis rather than by oxidation.Analytical procedures for dichloroacetaldehyde, a decomposition product of dichlorvos, are so markedly different from those for dichlorvos itself that they could not be included in the work reported here. TABLE I V RECOVERY OF MALATHION FROM SPIKED WHEAT Theore tical Laboratory Recovery, per cent. concentration, p.p.m. 1 87 0.7 76 0.1 2 98 0.1 87 0.5 3 82, 82, 87 0.1 85, 82, 72 0.5 0.1 0.5 The Panel therefore recommends the method given in Appendix I for the determination of malathion and dichlorvos residues in grain.22 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN: [Analyst, Vol. 98 Appendix I RECOMMENDED METHOD FOR DETERMINING RESIDUES OF MALATHION AND DICHLORVOS IN GRAIN APPARATUS- Low-speed grinder.High-speed homogeniser. Buchner funnel with a sintered-glass plate-The plate was 6 to 10 cm in internal diameter. Buchner $ask, 250 or 500 ml capacity. Suction pump. Standard flasks. Gas - liquid chromatographic apparatus with detector suitable for determining organophos- phorus compounds. REAGENTS- Methanol-Analytical-reagent grade material, screened for its blank value, was used. A cetone-Analytical-reagent grade. Dichlorvos and malathion-Material of known purity, preferably of analytical-standard grade, was used. METHOD When received, store the samples in a deep-freeze (at -20 "C). EXTRACTION- Grind the undried grain to a powder (care must be taken not to overheat the grain, otherwise losses may occur). Take a 20-g sample for analysis and homogenise it with 40 ml of methanol for 2 minutes.Filter the homogenate through the sintered-glass Buchner funnel by use of a partial vacuum. Transfer the deposit back into the homogeniser and wash it with two 30-ml portions of methanol for 30 s each, refiltering each time. Evaporate the combined filtrates to about 2 ml a t a temperature below 36 "C (otherwise dichlorvos may be lost) and under a partial vacuum. Dilute the concentrate to a suitable volume with acetone, maintaining the same ratio of acetone to methanol in all solutions and in standards so as to avoid inaccurate results. [A dilute solution of dichlorvos in acetone (1 pg ml-1) can be stored for at least 1 week in a refrigerator without any detectable change in its concentration.] Examine the solutions by gas - liquid chromatography.GAS - LIQUID CHROMATOGRAPHY- Most of the work reported above required the use of a phosphorus-sensitive thermionic detector but a flame-photometric detector was also used. The following columns and conditions have been used and found to be satisfactory. (i) Phenyldiethanolamine succinate (3 per cent.) on 100 to 120-mesh Diatomite CQ in a 0.9 m x 6 mm 0.d. glass column operated at 175 "C for dichlorvos and 200 "C for malathion. Retention times were about 3 and 5 minutes, respectively, with a nitrogen gas flow-rate of 30 rnl min-l, a hydrogen flow-rate of 20 ml min-l and an air flow-rate of 350 ml min-l. (ii) Phenyldiethanolamine succinate (10 per cent.) on Gas-Chrom Q in a 1.5 m x 6 mm 0.d. glass column operated at 180 "C. Retention times were 2 minutes for dichlorvos and about 30 minutes for malathion with a nitrogen gas flow-rate of 27 ml min-l, a hydrogen flow-rate of 30 ml min-1 and an air flow-rate of 500 ml min-l.(iii) Phenyldiethanolamine succinate (4 per cent.) on Chromosorb G in a 1.5 m x 6 mm 0.d. glass column operated at 180 "C. The retention time of dichlorvos was 3 minutes with a nitrogen gas flow-rate of 40 ml min-l, a hydrogen flow-rate of 200 ml min-l, an air flow-rate of 32 ml min-l and an oxygen flow-rate of 22 ml min-l. (iv) Yhenyldiethanolamine succinate (10 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 1 m x 3 mm i.d. glass column operated at 140 "C. The retention time for dichlorvos was 2 minutes with a nitrogen gas flow-rate of lOOmlmin-l, a hydrogen flow-rate of 800 ml min-1 and an oxygen flow-rate of 100 ml min-l.With a 4 per cent. phenyldiethanol- amine succinate stationary phase at a temperature of 190 "C, malathion can be conveniently determined.January, 19731 THE DETERMINATION OF MALATHION AND DICHLORVOS RESIDUES IN GRAIN 23 ( v ) Butane-1,4-diol succinate (1.3 per cent.) on acid-washed silanised 100 to 120-mesh Chromosorb G in a 1.5 m x 3 mm i.d. glass column operated at 175 "C for dichlorvos and 220 "C for malathion. (vi) Butane-l,4-diol succinate (1.3 per cent.) on Chromosorb G in a 1.5 m x 6 mm 0.d. glass column operated at 200 "C. The retention time for malathion was 4 minutes with a nitrogen gas flow-rate of 28 ml min-1, a hydrogen flow-rate of 30 ml min-l and an air flow-rate of 500 ml min-1.(vii) Butane-1,4-diol succinate (1.3 per cent.) on silanised 60 to 80-mesh Chromosorb G in a 1.2 m x 4 mm i.d. stainless-steel column operated a t 170 "C for dichlorvos. The retention time for dichlorvos was 1.2 minutes with a nitrogen gas flow-rate of 70 ml min-l, a hydrogen flow-rate of 28 ml min-1 and an air flow-rate of 340 ml min-l. (viii) Diethyleneglycol succinate (6.4 per cent.) on 100 to 120-mesh Aeropak 30 in a 0-4 m x 3 mm 0.d. glass column operated at 135 "C for dichlorvos and 215 "C for malathion. Retention times were 1.7 minutes and 2.1 minutes, respectively, with a nitrogen gas flow-rate of about 15mlmin-1, a hydrogen flow-rate of 15mlmin-l and an air flow-rate of about 170 ml min-l. ( i x ) Diethyleneglycol succinate (5 per cent.) on 100 to 120-mesh Chromosorb W in a 1.6 m x 4 mm id.glass column operated at 160 "C for dichlorvos and 230 "C for malathion. Retention times were 6.3 and 9.5 minutes, respectively, with a nitrogen gas flow-rate of 30 ml min-l, a hydrogen flow-rate of 15 to 25 ml min-1 and an air flow-rate of 400 ml min-l. (x) OV-1 (2 per cent.) on 100 to 120-mesh Diatomite CQ in a 0.9 m x 6 mm 0.d. glass column operated at 120 "C for dichlorvos and 150 "C for malathion. Retention times were 3 and 4 minutes, respectively, with a nitrogen gas flow-rate of 30mlmin-1, a hydrogen flow-rate of 20mlmin-l and an air flow-rate of 350mlmin-l. ( x i ) GE XE-60 (2 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 0.9 m x 6 mm 0.d. glass column operated at 120 "C for dichlorvos and 180 "C for malathion.The nitrogen gas flow-rate was 30 ml min-1, the hydrogen flow-rate 110 ml min-l and the oxygen flow-rate 30 ml min-l. (xii) GE XE-60 (3 per cent.) on 100 to 120-mesh Gas-Chrom Q in a 1.5 m x 4 mm i.d. glass column operated at 140 "C for dichlorvos and 195 "C for malathion. The nitrogen gas flow-rate was 30mlmin-1, the hydrogen flow-rate 15 to 25ml min-l and the air flow-rate 400 ml min-l. (xiii) GE XE-60 (2.5 per cent.) with 0.25 per cent. Epikote 1001 on 80 to 100-mesh Gas-Chrom Q in a 1.5 m x 6 mm 0.d. glass column operated a t 178 "C for dichlorvos and 200 "C for malathion. Retention times were 0.6 minutes and 3 minutes, respectively, with a nitrogen gas flow-rate of 25mlmin-1, a hydrogen flow-rate of 24mlmin-l and an air flow-rate of 400 ml min-l.(xiv) GE XE-60 (2 per cent.) and 0-2 per cent. Epikote 1001 on 100 to 120-mesh Aero- pak 30 in a 1.5 m x 3 mm 0.d. glass column at 135 "C for dichlorvos and 215 "C for malathion. Retention times were 1.4 minutes for dichlorvos and 2.1 minutes for malathion, with a nitrogen gas flow-rate of 15 ml min-l, a hydrogen flow-rate of 15 ml min-l and an air flow-rate of 170 ml min-l. (xv) DC 200 (5 per cent.) on 70 to SO-mesh Aeropak 30 in a 1.5 m x 3 mm 0.d. stainless- steel column at 165 "C for dichlorvos and 230 "C for malathion. The nitrogen gas flow-rate was 25 ml min-l, the hydrogen flow-rate 15 ml min-l and the air flow-rate 200 ml min-l. The retention time for dichlorvos was 2.8 minutes. (mi) SE-30 (5 per cent.) on 70 to 80-mesh Aeropak 30 in a 1.5 m x 3 mm 0.d.stainless- steel column at 115 "C for dichlorvos and 157 "C for malathion. Retention times were 2 minutes and 12 minutes, respectively, with a nitrogen gas flow-rate of 20 ml min-l, a hydrogen flow-rate of 18 mlmin-l and an air flow-rate of 220mlmin-l. (xvii) SE-30 (5 per cent.) on 100 to 120-mesh Embacel in a 1.2 m x 6 mm 0.d. stainless- steel column at 150 "C for dichlorvos and 190 "C for malathion. Retention times were 2-3 minutes and 3.8 minutes, respectively, with a nitrogen gas flow-rate of 60 ml min-l, a hydrogen flow-rate of 28 ml min-l and an air flow-rate of 400 ml min-1. (xviii) QF-1 (2-5 per cent.) and 0.25 per cent. Epikote 1001 on 100 to 120-mesh Celite in a 1.5 m x 4 mm i.d. glass column operating at 150 "C for dichlorvos and 190 "C for malathion.Retention times were 3-0 minutes and 9.5 minutes, respectively, with a nitrogen24 PANEL ON MALATHION AND DICHLORVOS RESIDUES IN GRAIN gas flow-rate of 30 ml min-1, a hydrogen flow-rate of 15 to 26 ml min-l and an air flow-rate of 400 ml min-l. (xix) E 301 (2.5 per cent.) and 0.25 per cent. Epikote 1001 on 100 to 120-mesh Gas- Chrom Q in a 1.5 m x 6 mrn 0.d. stainless-steel column operating at 150 "C for dichlorvos and 190 "C for malathion. Retention times were 3.3 minutes and 5.2 minutes, respectively, with a nitrogen gas flow-rate of 60 ml min-l. It is suggested that one of the succinate ester types of stationary phase be used unless another suitable column is readily available. Recorder signals for the pesticides should normally be 40 per cent. or more of full-scale deflection.Appendix I1 METHOD OF FORTIFICATION OF SAMPLES OF GRAIN FOR RECOVERY EXPERIMENTS Circulate 20 g of ground grain in a 250-ml ground glass necked flask (Quickfit and Quartz) by using a wrist-action shaker. Then add slowly the required amount of pesticide in 1 ml of acetone at a rate of about one drop every 2 s and mix for 6 minutes by further shaking. Remove the solvent by using an intermittent, light stream of air. Stopper the flask and store it in a deep-freeze overnight to allow penetration into the grain. This sample should undergo extraction on the following day. Appendix I11 MEMBERSHIP OF THE PANEL The membership of the Panel was: Mr. W. Cassidy (Chairman), Mr. S. Bailey, Mr. P. M. Brown (from October, 1970), Mr. G. J. Dickes (from March, 1970), Mr. L. Donegan, Mr. J. W. Edmunds, Mr. F. B. Fishwick, Mr. D. C. Holmes, Mr. A. Holder (from October, 1970), Dr. D. F. Horler, Mr. F. R. Johnson (from October, 1970), Mr. A. F. Machin (until March, 1970), Mr. B. L. Mathews, Mr. M. P. Quick (from March, 1970), Mr. K. Rawlings (until October, 1970), Mr. R. C. Spalding (until June, 1970) and Dr. N. A. Smart (Secretary). REFERENCES 1. 2. 3. 4. Report by the Malathion Panel, Analyst, 1960, 85, 915. Elgar, K. E., Marlow, R. G., and Mathews, B. L., Ibid., 1970, 95, 875. Crisp, S., and Tarrant, K. R., Ibid., 1971, 96, 310. Watts, R. R., and Storherr, R. W., J . Ass. OH. A g r i ~ . Chem., 1965, 48, 1158. Received July 20th, 1972 Accepted August 24th, 1972 COMMITTEE FOR ANALYTICAL METHODS FOR RESIDUES OF PESTICIDES AND VETERINARY PRODUCTS IN FOODSTUFFS (DR. N. A. SMART, SECRETARY), MINISTRY OF AGRICULTURE, FISHERIES AND FOOD, PLANT PATHOLOGY LABORATORY, HATCHING GREEN, HARPENDEN , HERTFORDSHIRE.