518 J. Food Hyg. Soc. Vol. 19, No. 6

Studies on Analysis of Pesticide Residues in Foods (XXVIII)

Determination of 33 Organophosphorus Pesticides in Agricultural Crops

(Received January 25, 1978)

Youko KAWAMURA, Mitsuharu TAKEDA and Mitsuru UCHIYAMA

(National Institute of Hygienic Sciences 18-1 Kamiyoga 1-chome, Setagaya-ku, Tokyo)

A modified method for the simultaneous determination of 33 organophosphorus pesticides in agricultural crops was presented. After extraction of samples with acetonitrile, the pesticides are transfered into 20% dichloromethane in benzene, cleaned up by charcoal column chromatography, and then determined simultaneously by the gas liquid chromatography with a flame photometric detector using the column of 4% OV-101 and 10% QF-1. The efficiency of the method was evaluated by analyzing water and 7 kinds of crops fortified with the pesticides. The average recoveries of the pesticides from crops were ranged from 72.0% (fenthion) to 108.0% (tetrachlorvinphos) and those from water were quite satisfactory for all pesticides.

Attention has been widely drawn for low PF (Shimadzu Seisakusho Ltd., Kyoto, Japan) persistence of organophosphorus pesticides in with a flame photometric detector. crops and the environment. In Japan, over 2) Liquid chromatographic column: forty organophosphorus pesticides are registered 15mm id.×300mm with stopcock. as insecticides and fungicides.1) Those are 3) Adsorbent mixture for charcoal column: very similar in chemical and physical charac- A mixture of 1 part of activated charcoal teristics and, furthermore, several kinds of (Darco G-60) and 10 parts of michrocrystalline pesticides are applied simultaneously to a crop cellulose (E. Merck, Darmstadt, Germany). for the protection against several harmful 4) Solvents: All were redistilled by use insects and fungi until harvest. of all glass with a Widmer column. Many papers have reported the simulta- 5) Pesticide standards: All were obtained neous determination of the multicomponent from the Society of Agricultural Chemical residues of the limited organophosphorus pesti- Industry, Tokyo, Japan. Stock solutions of cides,2)～14) but the systematic determination each pesticides (0.1ng/ml) were prepared with method has not been established. n-hexane or acetone, and kept under refri- Authors have published a series of the gerated. reports on the simultaneous method in order 6) Pesticide standard solutions: The to develop the convenient methods.4),6),7),11)～14) pesticides detectable separately in GLC were On the basis of these results, a simplified grouped and mixed to make up the working determination method by gas liquid chromato- standard. Suitable dilution was made with graphy (GLC) was designed and the recoveries acetone or n-hexane when necessary. from 7 kinds of crops and water fortified 33 7) Crops: Cabbages, Chinese cabbages , organophosphorus pesticides were examined. tomatos, carrots, apples, Japanese pears and Mandarine oranges were purchased from Method markets. Apparatus and reagents Extraction 1) Gas liquid chromatograph: Model 4BM- In 1L Omni-mixer cup, 100g of chopped December 1978 Studies on Analysis of Pesticide Residues in Foods (XXVIII) 519 sample, 100ml of acetonitrile and ca. 20g of Table 1. Pesticide Groups for Fortification and Celite 545 were placed and blended for 10min at GLC Conditions high speed. The mixture was filtrated with suc- tion through Celite layer 545 (5～10mm thick- ness) placed on filter paper into 500ml suction flask. The residue on the filter paper was added into 50ml of water and extracted again with 100ml of acetonitrile. The filtrate was combined, transferred into 1L separatory fun- nel containing 300ml of 10% sodium chloride solution and extracted with two 100ml portions of 20% dichloromethane in benzene by vigorous shaking. The extract was washed with two 100ml portions of water, dried over anhydrous sodium sulfate for 1hr and concentrated to 5ml in vacuum rotary evaporator below 40℃. Charcoal cleanup Some glass wool and 5g of anhydrous sodium sulfate were placed at the bottom of a column, 5g of adsorbent mixture suspended in ca. 10ml of benzene was poured into the column, after packing 5g of anhydrous sodium sulfate was carefully added on top. The column was prewashed with 50ml of sample was placed. To the cup, 2ml of benzene. The concentrate described above was pesticide standard solution in acetone was transferred quantitatively to the column. When added and stirred well. The extraction was last of the sample reached top of anhydrous initiated 1hr after fortification. sodium sulfate layer, inside of the column was rinsed with 3～5ml of benzene. The column Results and Discussion was eluated with benzene, at the moderate Extraction flow rate (ca. 2ml/min). 150ml of eluate was In order to examine the more suitable solvents collected, concentrated to 5ml on vacuum rotary for extracting organophosphorus pesticides evaporator below 40°, and the solution remained simultaneously, the recoveries from homo- in a flaskwas evaporatedagain just to dryness by air jet. The residue was dissolvedin 2ml genized mandarine orange fortified with each pesticidewere compared by use of acetonitrile10) of acetone. and a mixture of acetone and benzene (1:2).15) Gas-liquid chromatography In the former, the recoveries were diverse column; 3mm id.×150cm glass column packed from 77.7% (disulfoton) to 103.5% (DAEP), with 4% OV-101 and 3mm id.×200cm glass and in the latter, from 68.5% (IPSP) to 96.5% column packed with 10% GF-1 on Gas-Chrom (isoxathion). Not so much difference was Q (60～80 mesh). recognized, but the former was a little better Gas flow rate; carrier gas nitrogen 1.0kg/ in the recoveries than the latter. These cmcm2, hydrogen 180ml/min, air 90ml/min. results suggest that the both are applicable Sample inlet and detector temperature; 250℃ as extracting solvents for the determination Sample size injected; 2μl of the pesticide residues in crops. Column temperature was corrected appro- Although the low solubility of color materials priately by reference to the previous paper,14) from samples into the latter seems to be more and risted in Table 1. preferable for the further cleanup, the universal Fortification procedure extractability of the former should be the In 1L Omni-mixer cup, 100g of homogenized unexceptionable advantage for the simultaneous 520 J. Food Hyg. Soc. Vol. 19, No. 6

Table 2. Recovery of Organophosphorus Pesticides from Mandarine Orange by Different Extraction Solvents

extraction of organophosphorus pesticides. improved cleanup method for the multicompo- Furthermore, color materials from samples nent pesticides. In fact, the adaptability of may not cause a big trouble, when the detectors the charcoal column to the simultaneous deter- which are highly specific to phosphorus, such mination of the pesticide residues was sup- as a flame photometric or an alkaline thermionic ported by Watts.17) detectors, are employed. It was found in the previous paper12) that From these factors the extraction with 50% ethylacetate in benzene gave generally acetonitrile was adopted in the present study. the best recoveries of organophosphorus pesti- Charcoal cleanup cides from the charcoal column among 5 eluting It was shown by Pardue3) and Beckman16) solvents (50% and 30% ethylacetate in benzene, that Florisil column chromatography used in acetone, 50% and 30% acetone in n-hexane). the method of AOAC10) is unapplicable to the However, in cases of the samples such as analysis of most of organophosphorus pesticides spinach, carrot, tomato and mandarine orange, and is not suitable for the simultaneous deter- the eluate from the column were colored (pale mination of the multicomponent residues of yellow). the pesticides. Thus in order to find better eluting solvents The utilization of a flame photometric or an for charcoal column chromatography, the reco- alkaline thermionic detectors made the cleanup veries of 30 organophosphorus pesticides from procedure simplified. Thus, authors have the column were examined by use of 3 different investigated the cleanup of the multicomponent solvents, benzene, 50% ethylacetate in benzene residues and reported a charcoal column and 50% acetonitrile in benzene.10) The results chromatography.4),6),7),12),13) The results of were shown in Table 2. Almost the same the previous reports reveal that the charcoal recoveries (87.6～105%). The preliminary test column chromatography is applicabl as an with the extracts of spinach and mandarine December 1978 Studies on Analysis of Pesticide Residues in Foods (XXVIII) 521

Table 3. Recovery of Organophosphorus Pesticides in Activated Charcoal Column Chlomatographic Step

Scheme 1. Determination Procedure for organophosphorus pesticides in agricultural crops. 522 J. Food Hyg. Soc. Vol. 19, No. 6 orange showed that the column chromatography shown in Scheme 1. The operation of GLC with the above solvent systems could not com- for the complete separation of 38 kinds of pletely, take off the colored materials, but organophosphorus pesticides under some dif- benzene gave the best results among them. ferent temperature conditions reported in the Recovery from crops previous paper.14) On the basis of the present results, the Finally, the evaluation of the proposed method chosen for the simultaneous deter- method was carried out by determining 33 mination of organophosphorus pesticides was organophosphorus pesticides added to water

Table 4. Recovery of Organophosphorus Pesticides Added to Water and Crops December 1978 Studies on Analysis of Pesticide Residues in Foods (XXVIII) 523 and 7 crops (fortification level: 0.025～0.12ppm). 2) Bowman, M.C., Beroza,M., Hill,K.R.: J. Table 3 shows that the recoveries of the pesti- Assoc.Offic. Anal. Chem., 54, 346 (1971). cides were ranged from 72.0% (fenthion) to 3) Pardue,J.R.: ibid.,54, 359 (1971). 108% (tetrachlorvinphos) in crops. And those 4) Fukuhara,k., Takeda, M., Uchiyama,M.: J. Food Hyg. Soc.of Japan,14, 524 (1974). from water were quite satisfactory. Dichlo- 5) Repley,B.P., Wilkinson, R.J., Chau, A.S.Y.: rvos, fenthion and thiometon gave relatively J. Assoc.Offic. Anal, Chem., 57, 1033 (1974). low recoveries. 6) Inoue,Y., Fukuhara, K., Takeda, M., Uchiyama, In sample solutions, no peaks interferring M.: J. Food Hyg. Soc. of Japan, 15, 337 with pesticides were found on gas chromato- (1974). grams. But only in cabbage extract, 3 big 7) Aoki,Y., Takeda, M., Uchiyama,M.: J. Assoc. peaks were recognized. Offic.Anal. Chem., 58, 1286 (1975). Some recovery data of isoxathion, methidathion 8) Leoni,V., Puccetti,G., Grella,A.: J. Chro- and propaphos are lacking, because they could matogr.,106, 119 (1975). come to our hands after the initiation of the 9) McLeod, H.A., Butterfield,A.G., Lewis,D., Phillips,W.E., Coffin,D.E.: Anal. Chem., present study. However, the satisfactory 47, 674 (1975). recoveries of these pesticides could be expected 10) Horwitz, W.: "Official method of analysis, from the preliminary test. 12th" Chapter 29 (1975), Assoc. Offic. Anal. The results presented here reveal that the Chem., Washington, D.C. method established in this study is applicable 11) Aoki, Y., Takeda, M., Uchiyama, M.: J. Hyg. to the simultaneous determination of the Chem. 22, 81 (1976). multicomponent residues of the pesticides in 12) Fukuhara, K., Takeda, M., Uchiyama, M.: water and crops. Bull. Inst. Hyg. Sci., 94, 14 (1976). 13) Fukuhara, K., Takeda, M., Uchiyama, M.: Acknowledgement ibid., 94, 18 (1976). Thanks are gratefully extended to the Society 14) Kawamura, Y., Takeda, M., Uchiyama, M.: J. Food Hyg. Soc. of Japan, 19, 511 (1978). of Agricultural Chemical Industry (Japan) for 15) "The Food Sanitation Law, The Test Method kindly supplying organophosphorus pesticides. of Food Sanitation", Chapter 1, p. 116～129

(1973), Ministry of Health and Wealfare, Japan. References 16) Beckman, H., Gae, D.: J. Assoc. Offic. Anal.

1) Fukunaga, K.: "The Handbook of pesticides" Chem., 52, 286 (1969). (1976), Japan Plant Protection Association, 17) Watts, R., Storherr, R.W., Pardue, J.R., Tokyo. Osgood, T.: ibid., 52, 522 (1969).