April, 19611 LAWS AND WEBLEY 249 The Determination of Organo-phosphorus Insecticides in Vegetables A General Method for Insecticide Residues BY E. Q. LAWS AND D. J. WEBLEY (Department of Scientific aizd Industrial Research, Laboratory of the Government Chemist, Clement's Inn Passage, Strand, London, W.C.2) A method is described for the extraction of insecticides from treated cabbages and other vegetables and for their separation into water-soluble and petroleum-soluble groups. The further separation of members of each group from associated plant material is described for eighteen organo-phos- phorus insecticides, and the quantitative separation of both groups is discussed, with particular reference to mixtures of (a) phorate with its oxygen-analogue sulphone and (b) parathion-methyl with Phosdrin.THE method described by Laws and Webleyl for extracting and determining demeton-methyl is applicable to a number of water-soluble insecticides. It is not applicable, as it stands, to insecticides preferentially soluble in light petroleum, since the solvent systems used fail to extract the hydrocarbon-soluble insecticides from the macerated plant material, and no attempt is made to utilise the light petroleum washings. Modifications have consequently been made to the extraction procedure, and these ensure that both types of compound are extracted from the plant tissue before they are separated by partition. EXPERIMENTAL EXTRACTION AND SEPARATION- The first extraction is carried out by macerating the sliced vegetable with dichloromethane.After filtration, washing and then evaporation of the solvent, partition is effected between light petroleum and a 15 per cent. solution of methanol in water. At this stage, the insecticide is distributed as shown in the diagram below (solutions A and B)- Sliced vegetable material I Macerate with dichloromethane (All common insecticides extracted) I I Partition between light petroleum and 15 per cent. methanol - water I Petroleum-soluble organb-phosphorus insecticides Water-soluble okgano-phosphorus insecti- (solution A) cides (solution B) I I Chromatography on alumina; light petroleum as eluting agent I 1 I I Chlorthion* Wash column successively Disyston* with light petroleum and a Diazinon 15 per cent. solution of diethyl Fenchlorphos ether in light petroleum Phenkapton Gusathion* Phorate (Thimet*) Malathion S1752 Parathion-m ethyl I * Trade name. Chromatography on carbon; chloroform as eluting agent I Demeton-methyl and metabolites Dime fox Morphothion Phorate oxygen-analogue sulphone Phosdrin" Phosphamidon Rogor* Trichlorphon From this point in the determination the two groups are treated separately by different methods.The petroleum-soluble portion (solution A) is chromatographed on a column of graded alumina, light petroleum being used to elute the insecticides, and the water-soluble250 LAWS AND WEBLEY: THE: DETERMINATION OF [Vol. 86 portion (solution B) is examined by Laws and Virebley's chromatographic method1 involving elution with chloroform from a carbon column. The final eluates from the columns in both groups contain the insecticides substantially free from organic phosphorus of plant origin.TABLE: I RECOVERIES OF PETROLEUM- AND WATER-SOLUBLE INSECTICIDES The amount of each insecticide present was 50 to 70 pg. Petroleum-soluble insecticides were eluted with light petroleum and water-soluble insecticides with chloroform Petroleum-soluble group A r Insecticide Chlorthion* .. .. Disyston" . . .. .. Diazinon . . .. .. Fenchlorphos . . .. Gusathion* .. .. Malathion . . .. .. Parathion-methyl . . .. Phenkapton . . . I Phorate (Thimet*) . . S1752 . . .. .. Recovery from alumina column, 64 72 76 90 90 66 95 91 75 100 % Water-soluble group - A I 1 Recovery Recovery of Recovery of from insecticide insecticide added to carbon added to cabbage, Insecticide column, cabbage, % % % 66 Demeton-meth yl 95 and metabolites .. 85 76 89 Dimefox .. . . . . 98 80 89 Morphothion . . .. 91 84 79 Phorate oxygen-analogue 75 sulphone , . .. 80 70 65 Phosdrin* . . .. . . 90 65 74 Phosphamidon . . .. 78 70 75 Rogor* . . .. .. 80 65 70 Trichlorphon . . . . 75 57 * Trade name. TABLE I1 RECOVERY OF ADDED PHORATE AND ITS OXYGEN-ANALOGUE SULPHONE FROM CABBAGE The insecticides were added to 50-g portions of cabbage (range 0.25 to 4-0 p.p.m.) Insecticide added & Oxygen- analogue Phorate, sulphone, r g 209.3 11 1.6 111.6 55.8 55.8 55.8 27.9 27.9 13.9 Nil 55.8 r g 200.0 106.6 55.3 106.6 53.3 26.7 53.3 26.7 13.4 53.3 Nil Insecticide recovered Recovery r Phorate, rg 146.5 83.7 74.8 27.9 47.4 55.8 24.0 17.9 11.7 Nil 37.4 (Oxygen- analogue sulphone, r g 128-0 64.0 19.7 90.6 53.3 21.6 38.4 17.4 8.7 33.0 Nil Average .. Phorate, 70 76 67 50 85 100 86 64 84 Nil 67 75 % Oxygen- analogue sulphone, % 64 60 37 85 100 81 72 65 65 62 Nil 69 As this procedure isolates the insecticides in a reasonably pure condition, the method used to determine the residue is a matter of choice; determination of phosphorus, cholin- esterase assay, infra-red spectrography, gas chromatography or specific chemical or colori- metric methods present themselves as possibilities. The spectrophotometric determination of phosphorus as the molybdenum-blue complex was used to obtain the results reported in this paper. The solubility of the insecticides in light petroleum or water was first investigated, and then the behaviour on the appropriate chromatographic column was studied.Quantita- tive recovery experiments were next carried out on solutions of the commercially pure insecticide, and, finally, the compounds were added to raw cabbage and the complete method was applied.April, 19611 ORGANO-PHOSPHORUS INSECTICIDES I N VEGETABLES 251 In all the determinations reported here, the two phases of the partitition were completely andysed to ensure that the observed losses were not due to incomplete separation at this stage. In considering the recoveries of the various insecticides, the difficulties of residue work must be borne in mind. There are systematic losses at various stages of the procedure, and these are well known to practitioners in this field. Extraction from the plant rarely exceeds 95 per cent., and recovery from the chromatographic column varies with the purity of the insecticide; commercially pure insecticides may contain from 90 to 100 per cent.of pure active ingredient. Further, some insecticides are volatile, and, although the proposed method includes precautions to avoid losses due to volatility, it is a factor to be reckoned with at all stages. It is generally accepted that, for this type of analysis, an over-all recovery of 70 to 80 per cent. is satisfactory. The results of the experiments are shown in Table I. APPLICATIONS OF METHOD- To illustrate the application of the method to mixtures of insecticides, two series of recovery experiments were carried out on cabbage; two insecticides were used in each series, one component of each pair being petroleum-soluble and the other water-soluble. The chosen mixtures were (a) phorate (Thimet) with its oxygen-analogue sulphone and (b) parathion-me t hyl with Phosdrin .TABLE I11 RECOVERY OF ADDED PARATHION-METHYL AND PHOSDRIN FROM CABBAGE The insecticides were added to 50-g portions of cabbage (range 0.5 to 2-0 p.p.m.) Insecticide added Insecticide recovered s Parathion- methyl, Phosdrin, Pg Pg 97.5 112-0 97.5 56-0 58.5 112.0 58.5 56.0 29.3 56.0 58-5 33.6 29-3 33.6 97.5 Nil Nil 112.0 97-5 112.0 58.5 Nil Nil 56.0 I 1 Parathion- methyl, Phosdrin, Pg Pg 84.8 75.0 60.5 38.1 49.3 80.6 37.4 42.6 17.9 26.9 33.9 21.5 25.5 20.5 59.5 Nil Nil 62.7 -* 73.9 30.4 Nil Nil 37-0 Average . . * Solution lost. Recovery methyl, Phosdrin, G O ? % % 87 62 84 64 61 58 87 60 Nil 52 Nil 65 -* 67 68 72 76 48 64 61 Nil 56 66 Nil 66 65 The compound 00-diethyl S(ethylthiomethy1) phosphorothiolothionate, I, is known as I t undergoes a series of oxidative changes, either by chemical or enzyme phorate (Thimet).systems, the main product of which is the oxygen-analogue sulphone, 11. Samples of compounds I and 11, provided by Cyanamid of Great Britain Ltd., had a purity of 98 per cent., based on phosphorus content. Suitable standard solutions were prepared, each containing approximately 15pg of insecticide per ml of the final dilution, and the amounts of insecticide added and recovered are shown in Table 11. Compound I is typical of the petroleum-soluble group and compound I1 of the water-soluble group.252 LAWS AND WEBLEY: THE DETERMINATION OF [Vol.86 The compounds 00-dimethyl O-@-nitrophenyl phosphorothionate, 111, and 2-methoxy- carbonyl-l-methylvinyl dimethyl phosphate, IV, are commercially known as parathion-methyl and Phosdrin, respectively ; the former is petroleum-soluble and the latter water-soluble. S CHa-0 A sample of compound I11 having a purity of :LOO per cent. (by phosphorus content) was provided by Fisons Pest Control Ltd., and one of compound IV (purity of 96 per cent. by phosphorus content and 88 per cent. by extraction with chloroform) by Shell Chemicals Ltd. Two standard solutions, one containing 19 pg of compound I11 per ml and the other 11 pg of compound IV per ml were prepared, and the results of recovery experiments with these solutions are shown in Table 111. To show that the method was capable of wider application, some experiments were carried out on other vegetables and some fruits; the blank values found are shown in Table IV.TABLE :[V BLANK VALUES FOUND FOR 50-g SAMPLES OF VARIOUS VEGETABLES AND FRUITS Elution with light petroleum from alumina column Elution with chloroform from carbon column f Sample Cabbage .. . . .. Apples . . . . . . Peas. . . . . . . . Plums . . .. . . Cherries . . .. .. Lettuce . . .. .. Tomatoes . . . . .. Potatoes (tubers) . . .. Brussels sprouts . . .. Carrots . . . . .. Optical-density blank value 0.040 0.070 0.010 0.015 0.017 0.025 0-055 0.017 0-018 0.044 - r - Equivalent amount of phosphorus, Pg 0.64 1.12 0.15 0.23 0.26 0.42 0-89 0.26 0.27 0.68 A Optical-density blank value 0.045 0.032 0.016 0.0 13 0.018 0.015 0.015 0.043 0.040 0.040 - Equivalent amount of phosphorus, Pg 0.72 0.50 0.23 0.22 0.29 0.23 0.23 0.69 0.64 0.64 METHOD The method consists in the extraction of the insecticides from the plant material, their separation into petroleum- and water-soluble groups , chromatography of the groups on alumina and activated carbon, respectively, anti then determination of the phosphorus by spectrophotometric measurement of the rnolybdenum-blue complex.APPARATUS- Unicam SP500 absorption spectrophotometer. Glass tubes for chromatography-Tubes 1.5 c:m in diameter and 14 cm long, fitted with a tap at the lower end and a B19 ground-glass' joint at the upper end for attachment to a reservoir of solvent. M.S.E. homogeniser, with 100-ml Vortex beakei.s-0btainable from Measuring and Scientific Equipment Ltd., London, S.W.l. REAGENTS- All reagents must be of recognised analytical grade and phosphate-free. Chloroform. Dichloromethane. Diethyl ether. Light petroleum, boiling range 40" to 60" C. Methanol, absolute.April, 19611 ORGANO-PHOSPHORUS INSECTICIDES I N VEGETABLES 253 Alumina-Heat chromatographic aluminium oxide at 500" C for 2 hours to ensure conversion to the y-form. Adjust the activity to Brockmann grade V by adding 15 per cent. w/w of water. Active carbon-Heat 14- to 22-mesh carbon (grade 207, type B, obtainable from Messrs. Sutcliffe Speakman, Leigh, Lancashire) at 600" C in closed crucibles for 2 hours to remove organic impurities, boil twice with concentrated hydrochloric acid for 30 minutes on each occasion, wash free from acid with water, and dry in an oven at 100" to 110" C.Perchloric acid, N. Sulphuric acid, 10 and 1 N. Nitric acid, sp.gr. 1.420. Hydrochloric acid, s9.g. 1.180. Ammonia solution, sp.gr. 0.880. Ammonium molybdate solution-Dissolve 50 g of ammonium molybdate in 400 ml of Stannous chloride solution, concentrated-Dissolve 10 g of stannous chloride dihydrate Stannous chloride solution, dihte-Dilute the concentrated solution 200-fold with 1 N Ethanolic sulphuric acid-Mix 5 ml of concentrated sulphuric acid with 245 ml of absolute Isobutyl alcohol - benzene mixture, (1 + 1, v/v). Standard insecticide solutions-Prepare from the pure active ingredients, and suitably dilute. Potassium dihydrogen orthophosphate. EXTRACTION AND SEPARATION- Shred a representative sample of the plant in a slicing-and-grating machine, and transfer 5 0 g of the shredded material to the homogeniser beaker.If an addition of insecticide is required, add an aliquot of standard insecticide solution, and set aside for 30 minutes. Add 100 ml of dichloromethane, macerate for 15 minutes, filter the mixture through a Buchner funnel, and wash the solid on the filter-pad with 60ml of dichloromethane. Transfer the combined filtrate and washings to a separating funnel, and run the lower organic layer into a 260-ml conical flask. Wash the small aqueous layer with two 15-ml portions of dichloro- methane, and add the washings to the main extract. Reject the aqueous layer, and heat the dichloromethane solution on a hot-plate in a current of air until all the solvent has evaporated, taking care not to heat the flask overmuch in removing the last traces.Im- mediately add 5ml of methanol to the residue, pour the methanol solution into a 100-ml separating funnel, wash the flask with 30 ml of light petroleum and 25 ml of water, and add the washings to the contents of the separating funnel. Shake the funnel vigorously, allow to settle, and run the lower aqueous layer back into the 250-ml conical flask. Re-extract the light petroleum with 5 to 10 ml of water, run the aqueous layer into the conical flask, and pour the light petroleum layer from the top of the funnel into a 100-ml flask. Return the aqueous layer to the funnel, rinsing the flask with 30 ml of light petroleum and 10 ml of water. Shake vigorously, and separate as before.Rinse the funnel with a little light petroleum, and add the rinsings to the petroleum extract. Again return the aqueous portion to the funnel, extract it with four 20-ml portions of chloroform, and combine the chloroform extracts. CHROMATOGRAPHY- Petroleum-soluble insecticides-Evaporate the light petroleum extract to about 10 ml by heating on a water bath in a current of air. Prepare a column 1.5 cm in diameter from 8 g of the Brockmann grade V alumina; use light petroleum as liquid phase. Transfer the extract to the column, and elute with 150 ml of light petroleum a t about 1.5 ml per minute. To recover Gusathion and malathion, elute with 150 ml of light petroleum - diethyl ether mixture (85 + 15 v/v). Water-soluble insecticides-Evaporate the chloroform extract to about 10 ml by heating on a hot-plate; blow a current of air across the surface of the solvent t o cool the liquid and so minimise loss of volatile insecticides.With chloroform as liquid phase, prepare a column 10 M sulphuric acid, and dilute to 1 litre with distilled water. in 26ml of hydrochloric acid, spgr. 1.180. sulphuric acid. Prepare a fresh solution daily. ethanol .254 LAWS AND WEBLEY: THE DETERMINATION OF vol. 86 from 4 g of active carbon in a tube similar to that used for the alumina. Transfer the extract to the column, and elute with 100 ml of chloroform at 1.5 ml per minute. TREATMENT OF ELUATES- Evaporate the light petroleum, the light petroleum - diethyl ether and the chloroform eluates from the columns in separate conical flasks fitted with ground-glass joints; heat the first two eluates on a water bath and the third on a hot-plate, and assist evaporation with a stream of air to avoid loss of volatile insecticides.The chloroform solution need not be taken completely to dryness, as the last traces cd chloroform will boil away from the mixed acids and water in the final stages of the determination. Wet combustion of separated insecticides-To each evaporated eluate add 10 ml of water, 4 ml of N perchloric acid, 5 ml of nitric acid, sp.gr. 1-420, and 1 ml of hydrochloric acid, sp.gr. 1-180, and heat the solution under reflux beneath a Liebig condenser for 40 minutes. Drain the cooling water from the condenser, and leave until brown fumes begin to appear. At this point, remove the condenser, and evaporate the solution until fumes of perchloric acid are evolved.Rinse the condenser with 6 to 7 mi of water, add the rinsings to the contents of the flask, and again evaporate until white fumes are evolved. Add 3ml of water and 4ml of ammonia solution, sp.gr. 0.880, and remove the excess of ammonia by boiling. If it is known that volatile insecticides are not present, the heating under reflux may be omitted; the solution is heated to fumes with the acids as described above and then with 6 ml of water, with 2 ml of nitric acid and again with 5 ml of water before the ammonia solution is added. Place the test solution containing the phosphorus in a 100-ml separating funnel, and bring the volume to 10 ml, including any washings. Add 3.5 ml of ammonium molybdate solution, and mix after adjusting the total volume to 14ml.If the molybdate solution is added before transfer to the separating funnel, the final volume should still be 14 ml. Add 10 ml of isobutyl alcohol - benzene mixture, and shake the funnel vigorously. Allow the layers to separate, and discard the lower layer. Wash the remaining organic layer with 5 to 10 ml of N sulphuric acid, and again discard the lower layer. Add 15 ml of the dilute stannous chloride solution, shake for 5 to 10 seconds, and allow the layers to separate. Discard the lower aqueous layer, and allow the organic layer to fill the bore of the tap, but do not allow any of this layer to run to waste. Remove any of the aqueous layer left in the stem of the separating funnel with a piece of cotton-wool, and run the organic layer, which is blue if phosphorus is present, into a 10-ml measuring cylinder fitted with a stopper.Wash the separating funnel with 1 to 2ml of the ethanolic sulphuric acid, add the washings to the contents of the cylinder, and adjust the volume to 10ml with ethanolic sulphuric acid. Insert the stopper, and mix thoroughly. Measure the optical derisity of the solution in 1-cm cells at 730 mp with a spectrophotometer; use as blank solution a mixture of 4 parts of isobutyl alcohol - benzene mixture and 1 part of ethanolic sulphuric acid. The volume at this stage is about 8ml. NOTES- For successful production of molybdenum blue from orthophosphate, the acidity should be between 0.5 and 1.5 N and the amount of neutral salts other than ammonium molybdate should not exceed 0.5 g in 10 ml of test solution.To fulfil these conditions, the initial phosphate solution, whether derived from a wet combustion during a deter- mination or from the standard solution in the preparation of a calibration graph, is made up to 14m1, including 3 5 m l of the ammonium molybdate solution, which is in 4 N sulphuric acid. The time of shaking with the stannous chloride solution is restricted to 10 seconds, because a longer time results in a decrease in the intensity of the final colour. I t is important that the procedure should be carried out as rapidly as possible from the addition of the stannous chloride solution to the production of the final solution. The final colour is stabilised by the addition of the ethanolic sulphuric acid and remains unchanged for a considerable time.The method is essentially that of Berenblum and Chain2 and was more recently described by Field and Laws3 Minor chan,pes in procedure have been included in the above.April, 19611 ORGANO-PHOSPHORUS INSECTICIDES IN VEGETABLES 265 DISCUSSION OF RESULTS A general picture of the application of the method to eighteen different insecticides is shown in the diagram on p. 249 and in Table I. Recoveries of mixtures of phorate with its oxygen-analogue sulphone a t various levels are reported in Table I1 and those of mixtures of parathion-methyl with Phosdrin in Table 111. Blank values obtained on vegetables and fruit are summarised in Table IV. From Table I it can be seen that recovery of insecticide is sometimes higher in the presence of the vegetable matter than in its absence.We believe that this is caused by displacement of the adsorbed insecticide by some part of the extracted plant material, which results in the insecticide leaving the column earlier than it would if no such substance were present. In general, the results are reasonable for the type of system studied. The plant material contains phosphorus many hundreds of times in excess of the amount contributed by the insecticide, and the greater part of this is removed by the column. It is very important that the stated rates of elution are not exceeded, as experiment has shown that the amount of interfering plant substance passing through the column grows rapidly as the rate of elution is increased.Most of the experiments were carried out on cabbage, as this material is readily available and is typical of the crops likely to be examined. Since fruit is also likely to be sprayed with insecticide and is often eaten without preparation, some results were obtained to illustrate the wider application of the method (Table IV). Gusathion and malathion are unlike the other petroleum-soluble organo-phosphorus insecticides studied in that they are not eluted from the alumina column by light petroleum alone. They are, however, eluted by a mixture of light petroleum and diethyl ether (85 + 15 v/v). Some of the malathion is sometimes eluted by the light petroleum before the application of the mixture, but the mixture removes it all. The “plant blank” is not excessive when the mixed solvent is used.Preliminary examination of the petroleum-soluble insecticides on the alumina column demonstrated that the various compounds were eluted by different amounts of solvent. The amount chosen (150 ml) is the minimum that will effectively elute all the insecticides studied except malathion and Gusathion. I t is possible that separation of the individual insecticides could be achieved by using a second column of alumina and a fraction collector, with the purified eluate from the first column as starting material. The-general utility of the method lies in the fact that it divides the insecticides into two distinct groups and is applicable as a general method of analysis to a t least eighteen well known insecticides; it is potentially useful for a far greater number of compounds. Although it is accepted that methods are available for determining chlorinated insecticides, such as DDT, BHC and dieldrin, it should be mentioned that these compounds will be found in the light petroleum solution obtained by the proposed method and that this solution could in fact be used as a starting point in the determination of such residues. With one exception, optical-density blank values obtained on the vegetables so far tested have not exceeded 0.050, which corresponds to about 0.10 p.p.m. of insecticide. It would be possible to deter- mine residues of the order of 0.10 p.p.m. in a 50-g sample. This paper is published by permission of the Government Chemist, Department of Scientific and Industrial Research. REFERENCES 1. 2. 3. Laws, E. Q., and Webley, D. J., Analyst, 1959, 84, 28. Berenblum, J., and Chain, E., Biochem. J., 1938, 32, 295. Field, K., and Laws, E. Q., Analyst, 1957, 82, 667. Received Octobev 13th, 1960