496 Artalyst, August, 1967, Vol. 92, $9. 496-500 The Determination of Malathion in Formulations by a Method Based on Cleavage by Alkali BY A. C. HILL,* M. AKHTAR, M. MUMTAZ AND J. A. OSMANI (Department of Locust Warning and Plant Quarantine, Malir Halt, Karachi, Pakistan) A method for the determination of malathion in formulations is de- scribed. The malathion dissolved in methanol is hydrolysed a t room tem- perature by sodium hydroxide in the presence of phenol to give 00-dimethyl- phosphorodithioic acid. The latter is converted into its yellow copper complex by reaction with copper(I1) sulphate in acetate buffer solution, and the complex extracted with chloroform. The copper( 11) sulphate remain- ing in the aqueous layer is then determined iodimetrically. The free 00-di methylphosphorodithioic acid in the sample, and other materials that are likely to interfere, are accounted for in a parallel determination, which omits the hydrolysis stage.Hence the amount of malathion in the sample can be calculated. COLORIMETRIC methods for the determination of malathion that have been reported to date are all based on the following scheme of reactions- CHr COOCzHs I I I I Cu2+ ions (in dilute HCI) The malathion, I, in ethanol is cvnverted to sodium 00-dimethylphosphorodithioate, 11, by treatment with alkali. The latter is then reacted with copper(I1) ions in dilute hydrochloric acid to give a yellow-coloured complex, 111, which can be extracted into various organic solvents. In the original colorimetric method,l which is intended for the determination of malathion residues, the malathion (0.25 to 2.5 mg) is dissolved in a mixture of ethanol (25 ml) and carbon tetrachloride (100 ml), and is treated with 1 ml of 6 N sodium hydroxide for 1 minute.The solution is then shaken with 2 per cent. aqueous sodium chloride (75 ml) for 1 minute, the layers allowed to separate and the carbon tetrachloride layer discarded. The aqueous layer is washed with 25 ml of carbon tetrachloride, acidified with 1 ml of 7 N hydrochloric acid and washed once more with 25ml of carbon tetrachloride. Finally, the * On loan to the Department from “Shell” Research Limited, Woodstock Agricultural Research Centre, Sittingbourne, Kent, England, through the Colombo Plan.HILL, AKHTAR, MUMTAZ AND OSMANI 497 aqueous layer is shaken for 1 minute with exactly 25ml of carbon tetrachloride, together with 2 ml of 1 per cent. copper sulphate solution, and the optical density of the yellow copper complex in the carbon tetrachloride layer is measured at 418 mp.The amount of malathion in the sample is found by comparing the optical density measurement against a standard graph prepared with various known amounts of pure insecticide. Upham2 reported a modification of the original method that is intended for the deter- mination of malathion in formulations. In this modification the use of acetonitrile was introduced for the extraction of solid formulations, and the carbon tetrachloride replaced by cyclohexane as extractant for the yellow complex to improve colour stability. Ware,3$4 in a further modification, eliminated the standard graph by carrying a standard through the procedure with the sample, and also replaced the washing stages by a single “ferric- oxidation” step (first mentioned by Upham2) to remove materials likely to interfere by reducing copper(I1) to copper(1) ions.O r l o ~ k i , ~ on behalf of a further collaborative study group, reported a more restrictive version of the method reported by Ware. Despite the considerable amount of collaborative work that has been carried out on the colorimetric method, none of the versions reported to date appears to be entirely satis factory. The World Health Organisation Expert Committee on Insecticides, in its Fifteenth Report,6 noted that methods for the assay of malathion often give faulty results when applied to poor quality products and that, in particular, methods based on the cleavage by alkali of the molecule might be greatly improved if the mechanism of this cleavage were known.The Committee recommended that this mechanism should be investigated. Double7 noted that colorimetric methods of malathion determination were unsatisfactory for commercial dust formulations containing malathion, DDT and lindane ; reproducible results could not be obtained. In January 1964, a reliable method was urgently required to check the toxicant contents of malathion formulations being produced in Pakistan. The most recent version of the colorimetric rnethod3y4 available was tried, and gave erratic results. Neither the polaro- graphic8 nor the infrared spectrophotometric method2,g could be used owing to non-availability of the necessary equipment, and attempts were therefore made to improve the colorimetric method.A consideration of the colorimetric method, particularly in relation to the purpose for which it was required, suggested some modifications that might improve its reliability and, at the same time, would make it more economic in routine use. Firstly, it was thought that the use of methanol, instead of ethanol, as the medium in which to carry out the cleavage by alkali of the malathion molecule, would afford some degree of protection to the CH,O-P bonds in both malathion and 00-dimethylphosphorodithioic acid against cleavage by alkali. This protection would be expected to act through a concentration (mass action) effect.Secondly, at least 500 ml of ethanol, an expensive solvent in Pakistan, was required to process each sample through the procedure. Thirdly, the formation and extraction of the yellow complex was being carried out from hydrochloric acid solution, and thus under conditions that could be conducive to the observed instability of the yellow complex. In view of these considerations it was decided to adopt the use of methanol, instead of ethanol, as the medium for the cleavage by alkali and to use sodium acetate - acetic acid buffer solution to terminate the alkali treatment, so that the yellow complex would be formed and extracted under milder conditions of acidity. Initially, work was carried out with the intention of incorporating these features into the colorimetric method.However, this eventually led to the development of a method with a titrimetric finish, which is reported below. METHOD SCOPE OF THE METHOD- The method is intended for use in the determination of S-(1,2-dicarbethoxyethy1)- 00-dimet hylphosphorodit hioat e in malathion insecticide and its formulations. Compounds in these materials, which, like S-( 1,2-dicarbethoxyethyl)-OO-dimethylphos- phorodithioate are converted to 00-dimethylphosphorodithioic acid by the alkali treatment, will interfere with the method described. Known compounds of this type1 are 0000-tetra methylpyrophosphorotrithioate and bis(dimethoxyphosphorothiono) disulphide. Either one or both of the half-esters of S-(l,2-dicarboxyethyl)-00-dimethylphosphorodithioate, namely498 HILL et al.DETERMINATION OF MALATHION I N FORMULATIONS [Analyst, VOl. 92 S- [( 1-carboxy-2-carbethoxy)ethyl]-00-dimethylphosphorodithioate and S- [ (1-carbethoxy- 2-carboxy)ethyl]-00-dimethylphosphorodithioate, are also converted into 00-dimethyl- phosphorodithioic acid. The dicarboxylic acid, S-( 1,2-dicarboxyethyl)-00-dimethylphos- phorodithioate, is not converted into 00-dimetliylphosphorodithioic acid by alkali treatment and does not interfere. The method has not been applied to formulations that contain mixtures of malathion with other pesticides. Gusathion, Imidan, ekatin, ethion, dimethoate and other dialkyl- phosphorodithioate insecticides will certainly interfere, and so also will captan and dithio- carbamate fungicides. However, it is considered probable that the method will be applicable to malathion formulations containing DDT, methoxychlor, perthane, heptachlor, aldrin, dieldrin, endrin, BHC (technical), lindane, toxaphene or DDVP.APPARATUS- Measwing flasks-50 and 100-ml capacity. Sepayating f unnels-250-ml capacity, with close-fit ting stoppers. Pipettes-1, 10 and 25-ml capacity. Graduated measuriflg cylinders-25 and 50-ml capacity. Conical (or iodine) jasks-250-ml capacity, with glass stoppers. Burette-Of at least 25-ml capacity, calibrated in O-l-ml divisions. Sodium hydroxide (carbonate-free), 3 N. Afethanol-General-purpose reagent grade, containing not more than 0.25 per cent. Phenol solution, 30 per ceut. w / v iqz methanol. Chloro form-General-purpose reagent “A” grade. Potassium iodate soldon, 0.05 N. Sodium thiosulphate solution, 0.02 N-Dissolve 10-00 g of sodium thiosulphate penta- hydrate in 2 litres of water that has been freshly distilled from alkaline potassium perman- ganate.Keep in a dark bottle. Add 20 mg of mercury(I1) iodide to the solution to protect it from possible deterioration by Thiobacillus thioparus. These precautions10 are necessary to ensure good storage stability. Standardise against 0.05 N potassium iodate in the con- ventional manner. REAGENTS- w/w of water. CoPper(I1) sulphate solution, 0.02 N. Bufer solution-Dissolve 48 g of sodium acetate trihydrate and 12 ml of glacial acetic acid in distilled water and make up to 1 litre. Potassium iodide-Iodine-free, general-purpose reagent grade. Potassium thiocyanate-General-purpose reagent grade. Starch solution, 1 per cent.w/u i~ water. SAMPLE PREPARATION- Technical malathion and emulsiJiable concentrates-No sample preparation is necessary. Weigh accurately directly into a 50-ml measuring flask an amount of technical malathion or emulsifiable concentrate that contains 0.75 to 0.90g of pure malathion. Dissolve it in methanol and make up to the mark. Mix thoroughly. This is the sample solution. Wettable powders and dust concentrates (25 and 50 per cent.)-Weigh accurately into a 50-ml beaker an amount of the formulation that contains 0.75 to 0.90g of pure malathion. Disperse it in 20 ml of methanol and filter through a fine porosity paper, collecting the filtrate directly in a 50-ml measuring flask. Wash the beaker and filter-paper with 10-ml portions of methanol, allowing each wash to percolate through the filter before adding the next. Adjust the extract to the 50-ml mark and mix thoroughly.This is the sample solution. PROCEDURE- (a) Determination of malathion plus free 00-dimethylphosphorodithioic acid and any inter- fering materials-Transfer by pipette a 10-ml aliquot of the sample solution into a 250-ml separating funnel containing a mixture of 1 ml of 3 N sodium hydroxide and 1 ml of 30 per cent. phenol solution, each solution being added by pipette. Stopper the funnel, mix the solutions by tilting the funnel and by gentle swirling (not by shaking), and allow to stand for 45 minutes. Then add, with a graduated cylinder, 25 ml of buffer solution and 50 mlAugust, 19671 BY A METHOD BASED ON CLEAVAGE BY ALKALI 499 of chloroform, followed by 25 ml, added by pipette, of 0.02 N copper (11) sulphate solution.Stopper the funnel and shake it vigorozdy for at least 2 minutes (see Note 1). Allow the layers to separate and drain off the yellow chloroform layer into a 100-ml measuring flask. Extract the blue aqueous layer three times with 10-ml portions of chloroform, collecting these washings in turn in the 100-ml measuring flask. If the third wash with chloroform shows any yellow colour (as has been found with some emulsifiable concentrate formulations), carry out further washings with 10-ml portions of chloroform until the washings are colourless (see Note 2). Drain the blue aqueous layer into an iodine flask and wash out the separating funnel with two 10-ml portions of distilled water. Add 2-0 g of potassium iodide to the contents of the flask, dissolve it by swirling, and titrate the liberated iodine with sodium thiosulphate solution.Then add 1-Og of potassium thiocyanate and titrate until the yellow colour of the aqueous dispersion becomes faint. Finally, add 3 ml of 1 per cent. starch solution and titrate dropwise to the end-point (blue to colourless). (b) Determination of free 00-dimethylphosphorodit~ioic acid and afzy inter fering materials- Transfer by pipette a 10-ml aliquot of the sample solution into a separating funnel containing a mixture of 1 ml of 3 N sodium hydroxide, 1 ml of 30 per cent. phenol solution (each solution added by pipette) and 25 ml of buffer solution. Add 50 ml of chloroform, followed by 25 ml, added by pipette, of 0.02 N copper(I1) sulphate solution.Stopper the funnel and shake it vigoyously for at least 2 minutes (see Note 1). Allow the layers to separate and drain the slightly yellow chloroform layer three times with 10-ml portions of chloroform, collecting the chloroform washings in the 100-ml flask (see Note 2). Drain the blue aqueous layer into a 250-ml iodine flask and wash out the separating funnel with two 10-ml portions of distilled water. Proceed with the iodimetric titration of the copper(I1) sulphate remaining in the aqueous solution as described in section ( a ) above. Record the volume, Tb, of sodium thiosulphate used in the titration. Continue until the yellow colour fails to return. Record the volume, T , of sodium thiosulphate used in the titration.NOTES- phorodithioic acid is quantitatively reacted with the copper sulphate. desired). 10 g of calcium oxide for each litre of chloroform extract being distilled. 1. The instructions given for shaking should be followed to ensure that the 00-diniethylphos- 2 . The chloroform extracts may be kept for measurement of their optical densities (if this is The chloroform may be recovered (90 per cent.) by distillation from a flask containing CALCULATION OF RESULTS- formula- Calculate the percentage of malathion in the sample taken for analysis from the following B (Tb - T ) x 13.23 x 100 W Malathion, per cent. w/w = where B is the weight in grams of copper(I1) sulphate pentahydrate to which 1 ml of the sodium thiosulphate solution is equivalent ; Tb is the titre in millilitres of sodium thiosulphate solution required for the copper(I1) sulphate remaining after reaction with the unhydrolysed malathion sample ; T is the titre in millilitres of sodium thiosulphate solution required for the copper(I1) sulphate remaining after reaction with the hydrolysed malathion sample ; and W is the original weight in grams of malathion sample taken for analysis.The figure 13-23 takes into account that one molecule of copper(I1) sulphate pentahydrate reacts with two molecules of 00-dimethylphosphorodithioic acid derived from two molecules of malathion, and that only one-fifth of the malathion weighed is used in each determination. ACCURACY AND PRECISION OF RESULTS- cent. malathion showed- A statistical analysis of twelve consecutive results obtained with a fresh sample of 99.6 per Average result, 99.57 per cent.; range, 1.7 per cent.; standard deviation, 0.61 per cent.; “95 per cent.error,’’ 1-22 per cent.; and standard error, 0.18 per cent.500 HILL, AKHTAR, MUMTAZ AND OSMANI DIFFERENCES BETWEEN THE PRESENT METHOD AND FORMER METHODS- Although the present method is based on the same principle as the colorimetric method originally described by Norris, Vail and Averel1,l and later modified by Upham,2 Ware3y4 and Orlo~ki,~ it differs considerably in practice, particularly in the following respects. Conversion of the malathion into 00-dimethylphosphorodithioic acid is carried out by using equivalent amounts of sodium hydroxide and phenol in methanol for the hydrolysis stage, instead of using sodium hydroxide in ethanol.Conversion of the 00-dimethylphosphorodithioic acid into its copper complex and extraction of the complex from the aqueous layer are carried out at pH 4.8 to 5.0 in the presence of acetate buffer solution, instead of in hydrochloric acid s 0 1 u t i o n . l ~ ~ ~ ~ ~ ~ ~ Also, chloroform is used as extractant for the complex, instead of carbon tetrachloride1 or cy~lohexane.~ 93 94 y5 Compounds such as thiols and mercaptans, which are likely to interfere in the method by reducing copper(I1) to copper(1) ions, are accounted for in a parallel determination, which omits the hydrolysis stage, instead of being eliminated during the procedure by a “ferric-oxidation step.”2 7 3 9 4 9 5 The procedure is finished titrimetrically instead of colorimetrically.The yellow complex, as formed under the conditions of the method, is apparently much more stable than that obtained with former methods. Since mid-1964, the method has been successfully applied to the determination of S-(l,2-dicarbethoxyethyl)-00-dimethylphosphorodithioate in malathion imported from the U.S.A., as well as to 50 to 60 samples of malathion formulations produced in Pakistan. Toxicant contents determined by the method did not differ by more than 2 per cent. (absolute) from contents stated by producers in supply contracts. We thank Mr. H. M. Abbas, Director of the Department of Locust Warning and Plant Quarantine, for permission to publish this paper, and Mr. M. Rahman of the Department of Agriculture, Government of East Pakistan, who provided some technical assistance. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Norris, M. V., Vail, W. A., and Averell, P. R., J . Agvic. F d Chem., 1954, 2, 570. Upham, S. D., J . Ass. OH. Agric. Chem., 1960, 43, 360. Ware, J. H., Ibid., 1961, 44, 608. -, Ibid., 1962, 45, 529. Orloski, E. J., Ibid., 1964, 47, 248. “Specifications for Pesticides Used in Public Health,” World Health Organisation Expert Com- mittee on Insecticides, Fifteenth Report, Geneva, October 19th-25th, 1965, p. 31. Double, R. C., J . Ass. 08. Agric. Chem., 1964, 47, 693. Jura, W. H., A n a l y t . Chem., 1955, 27, 525. “Malathion Manual for Insecticide Formulators,” compiled by Cyanamid International. Vogel, A. I., “A Textbook of Quantitative Inorganic Analysis, Theory and Practice,” Second Edition, Longmans, Green & Co., London, New York and Toronto, 1951, p. 333. Received November 30th, 1966