Analyst, December, 1966, Vol. 91, pp. 779-782 779 The Analysis of the Organophosphorus Pesticide, Fenitrothion, an Infrared Method BY R. B. DELVES ( Woodstock Agricultural Research Centre, “Shell” Research Limited, Sittingbourne, Kent) AND v. P. WILLIAMS (Milstead Laboratory, “.Shell” Research Limited, Sittingbourne, Kent) An infrared method is described for determining the fenitrothion [00-di- methyl-0-( 3-methyl-4-nitrophenyl) -phosphorothioate] content of technical material after chromatography over silica gel. The fenitrothion is eluted with mcthylene chloride, and after evaporating the eluate to dryness the residue is dissolved in carbon disulphide to give a concentration of fenitrothion in the range 0.8 to 1 . 1 per cent. w/v. Measurements taken a t three absorption peaks in conjunction with related minima are used to calculate the fenitro- thion content from previously prepared calibration graphs that relate absorbance to fenitrothion content.The standard deviation of this method, based on ten samples of technical material, is 0.53 per cent. THE increasing use of organophosphorus pesticides necessitates the development of more specific methods of analysis. Non-specific methods, such as those based on the reduction of the nitro group, for example the method of O’Keeffe and Averelll for technical parathion, tend to give erroneously high results owing to the presence of “related impurities.” A search of the literature has revealed some general papers on fenitrothion [00-dimethyl-0-(3-methyl-4-nitro- phenyl)-phosph~rothioate]~~~~~~~ and two concerned with its analytical determinatiom6 p 7 One of these analytical methods involves polarography after separation of technical material by thin-layer chromatography, and the other utilises gas - liquid chromatography for residue analysis.Neither method was considered suitable for our purposes, and the object of our work has been to develop a rapid, specific infrared method for determining the fenitrothion content of technical and formulated materials (Notes 1, 2 and 3). EXPERIMENTAL In an attempt to isolate fenitrothion from associated impurities, preliminary separations were carried out by loose-layer chromatography. Examination of several solvent systems on the adsorbents, alumina, silica gel and Florisil, showed that a good separation could be obtained on silica gel by using methylene chloride as developing solvent.Chromatography over a column of silica gel (50 g) with methylene chloride as developing solvent isolated the major component from technical material (0.5 6). After elution of this material, which represented about 95 per cent. w/w recovery, a clear fraction was obtained NOTES- 1. This method of determination can also be applied to liquid formulations as follows- Make a slurry of 60 g of silica gel with petroleum spirit (boiling range 40” to 60” C) and methylene chloride (1 + 1 viv) and place it in the chromatographic column; drain off exress solvcnt. Take a sufficient formulation to contain 0-2 g of fenitrothion and transfer i t quantitatively with a minimum amount of petroleum spirit - methylene chloride t o the top of the silica gel column.Place a beaker under the column and allow the sollition to percolate into the adsorbent. -4dd small amounts of solvent t o ensure complctc adsorption of the fenitrothion into thc silica gel. Continue to elute with petroleum spirit - methylcne chloride (about 1.50 ml) until the formulation solvent is removed. Elute with 250ml o f methylene chloride and continue from this point as directed under Chromato- graphic Separation of Penitrothion. 2, Normally, formulation ingredients and decomposition products (if any) that are formed on storage will be retained on the adsorbent. 3. Possible interference will be detected if the conccntration of fenitrothion with any one of the peaks differs by more than & 3 per cent.(relativc) of thc mean. If this occurs, the particular value should be discarded and the remaining concentrations averaged.780 DELVES AND WILLIAMS: ANALYSIS OF THE ORGANOPHOSPHORUS [Analyst, Vol. 91 before the first impurity was eluted. The other impurities remained on the column as four separate zones. PROOF OF STRUCTURE OF THE MAJOR COMPONENT- The major component isolated by chromatography was examined by (a) mass spectro- metry with an A.E.I. Ltd. M.S. 2H mass spectrometer, (b) infrared spectrometry with a Grubb Parsons spectrometer and (c) thin-layer chromatography, by using a thin layer (275 p) of silica gel G and chloroform or methylene chloride - benzene (1 + 1, v/v) as develop- ing solvents. The chromatoplate was sprayed with 2,6-dichloro-~-benzoquinone-4-chlorimine, which appears to be specific for the P + S group by forming a red coloured derivative.* The mass spectrometer showed a parent peak at m/e 277 (fenitrothion) and intense fragment ions at m/e 260, m/e 125 and m/e 109.The fragment ion at m/e 260 was attributed to the elimination of OH from the 3-methyl-4-nitro substituents of fenitrothion. Beynon et aL9 have reported that o-nitrotoluene eliminates OH and this is accompanied by ring closure. This evidence confirms that the methyl and nitro groups are ortho to one another in the aromatic ring of fenitrothion. The fragment ion at m/e 125 was formed by the cleavage of the P-0 bond in the P-0-aromatic group to give a substituted phenoxy ion. The intensity of the ion a t m/e 109 was comparable to that of the ion a t m/e 125.It appears that there is a re-arrangement within the mass spectrometer followed by elimination of the phosphorus moiety to produce an ion at m/e 109. The infrared spectrum showed the presence of P-0-aromatic, P-0-methyl and probably P -+ S groupings. P -+ 0 and P-S-aromatic groupings were absent. The P -+ S group was confirmed by thin-layer chromatography, which showed one component only to be present. Infrared analysis of the phenol isolated after hydrolysis of the major component showed this to be identical with 4-nitro-m-cresol (Sadtler infrared spectrogram No. 23685). This confirmed that the methyl and nitro groups are in the 3 and 4 positions, respectively. Thus, the analytical evidence obtained by mass spectrometry, infrared spectrometry and thin-layer chromatography shows that the major component is fenitrothion, the structure of which is- APPARATUS- NO, I I I CH,O-P+ S OCH, METHOD Infrared s#ectrophotomcter-An instrument capable of quantitative analysis in the 2 to Sealed liquid absorption cell-0.4-mm path length.Hypodermic syringe-Glass, Luer type, 2-ml capacity. Chromatographic columns-15 x 500 mm, fitted with a glass tap and a solvent reservoir 15-p region is required. of approximately 500 ml. REAGENTS- Silica geLWhatman Chromedia SG3 1. Benzene, general-purpose reagent grade. Methylene chloride, general-purpose reagent grade. Carbon disulphide, R.D. H . Ltd. general-purpose reagent grade or equivalent. Fenitrothion-Analytical standard material of purity greater than 99 per cent.Prepare material suitable for analytical calibration purposes by using the chromatographic separation method.December, 19661 PESTICIDE FENITROTHION BY AN INFRARED METHOD 781 CHROMATOGRAPHIC SEPARATION OF FENITROTHION- Prepare a slurry of 50 g of silica gel with methylene chloride and transfer it to a chromato- graphic column with a cotton-wool plug to retain the adsorbent. Allow the solvent to pass through the column until the meniscus reaches the top of the adsorbent. Weigh, to the nearest 0.1 mg, an amount of sample that contains approximately 0.2 g of fenitrothion. Dissolve the fenitrothion in a minimum of benzene (3 ml) and transfer the solution quantitatively to the top of the column by using methylene chloride. Allow the solvent to percolate into the silica gel, then wash the inside of the chromatographic column with three 5-ml portions of solvent, allowing each portion to penetrate the adsorbent independently.Elute with 250 ml of methylene chloride and collect the eluate in a tared 400-ml beaker. When the solvent reaches the top of the adsorbent, stop the flow of solvent, wash the tip of the column with solvent and replace the 400-ml beaker with a 100-ml beaker. Evaporate the solvent contained in the 400-ml beaker at room temperature by using a forced draught. This is the fenitrothion residue. Collect a further 50-ml fraction and evaporate to dryness in a forced draught to confirm the complete elution of fenitrothion by the absence of residue. In the unlikely event of a residue being present, examine it by infrared spectroscopy as it may be a related impurity.Calculate the percentage extract (to give a guide to the fenitrothion content) and submit the extract to quantitative infrared analysis. Re-weigh the tared 400-ml beaker and obtain the weight of the extract. INFRARED ANALYSIS CALIBRATION OF APPARATUS- Into each of several 20-ml calibrated flasks, weigh (to the nearest 0.1 mg) 160, 180, 200 and 220-mg amounts of the standard sample of fenitrothion. Dissolve each in carbon di- sulphide, dilute to the marks, and mix thoroughly. The strengths of these solutions will therefore be 0.8, 0.9, 1.0 and 1.1 g per 100 ml. Fill the 0.4-ml cell with the most dilute of these standard solutions by means of the hypodermic syringe. Adjust the spectrophotometer to the optimum instrument settings with respect to gain, slit width, balance, response, chart speed and wavelength-scanning speed.Make duplicate scans over the 7.0 to 9.0-p region. Flush out the cell with carbon disulphide, dry, re-fill, in turn, with each of the remaining calibration solutions and, without changing instrument conditions, repeat the duplicate scans over the 7-0 to 9-0-p region. For each of the scans of the calibration solutions measure the transmitted radiant power, as a proportion of the incident radiant power, at the following wavelengths- (;) From the absorption peak at about 7-45 p to the minimum, i.e., reference point at (ii) From the absorption peak at about 8.05 p to the minimum, i.e., reference point at (iii) From the absorption peak at about 8.05 ,U to the minimum, i.e., reference point at (iv) From the absorption peak at about 8-55 p to the minimum, i.e., reference point at Construct a calibration graph for each absorption peak by plotting the percentage transmission on a logarithmic ordinate zleysus the corresponding concentration (g per 100 ml) on a linear abscissa.about 7.60 mp. about 7-95 p. about 8.30 p. about 8-50 p. Construct the line of best fit through each set of four points. PROCEDURE- Dissolve the material obtained from the chromatographic procedure in a volume of carbon disulphide sufficient to give a concentration of fenitrothion in the range 0-8 to 1-1 per cent. w/v. Fill the same 0.4-mm path-length cell that was used in calibrating the instrument with each sample solution in turn, and make duplicate scans over the 7.0 to 9-0-p region.The same instrument conditions must be used as for the calibration. Calculate the percentage transmission of the three specified peaks for each sample.782 DELVES AND WILLIAMS CALCULATION- fenitrothion in each solution. differ by more than 4 per cent. of the mean. Read from the appropriate calibration graph the concentration, in g per 100m1, of Take the average of the four concentrations thus obtained. Concentrations should not Calculate the fenitrothion content by using the following equation- c x v w Fenitrothion content per cent. by weight = ~ where C is the average concentration as read from calibration graphs, g per 100 ml; V is the volume of sample solution, ml; and W is the original weight of sample taken for chromatographic “clean-up,” g.RESLJLTS AKD CONCLUSIONS The results obtained by using the method described are shown in Table I. TABLE I FENITROTHION CONTENT OF TECHNICAL MATERIAL Technical Fenitrothion content, per cent. w/\v Standard fenitrothion, -h- deviation, sample number Individual Mean per cent. 1 92.4, 93.4, 92.6, 93.4, 92.5, 93.1 0.53 2 96.9, 97.5 97.2 - 3 96-4, 97.3 96.8 - 4 96-9, 97.3 97-1 - 92.8, 92.7, 93-8, 93.8, 93.1 The method described is specific and is used routinely for determining the fenitrothion content of technical material and its formulations. I t has proved to be satisfactory. The standard deviation of the method, which includes adsorption chromatography and infrared spectroscopy, is 0-53 per cent. We thank Mr. F. Wirtz-Peitz of Cologne University who, as an overseas student under IAESTE (U.K.), carried out much of the experimental work reported in this paper. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES O’Keeffe, K., and Averell, P. R., Analyt. Chem., 1951, 23, 1167. Nishizawa, Y., Fujii, I<., Kadota, T., Miyamoto, J., and Sakamoto, H., Agric. & Biol. Chem., Nishizawa, Y., Nakagawa, M., Suzuki, Y . , Sakamoto, H., and Mizutani, T., Ibid., 1961, 25, 597. Schrader, G., “Die Entwirklaing neuer inseklizider Phosphorsaure-Ester, ” J‘erlag Chcmie GmbH, LTchijama, M., and Okui, S., J . Fd Hyg. Soc. Japuw, 1962, 3, 277; Chrm. -4bsti/., 58, 3839b. Kovac, J., J . Chvomat., 1963, 11, 412. Dawson, J . A,, Dvriegan, L., and Thain, E. M., r2nalysf, 1964, 89, 495. Braithwaite, n. P., h’utzrr~, 1963, 200, 1011. Beynon, J . H., Saundcrs, R. A., and li~illiams, A . E., Tiidiistrie Chifl2. Belge, 1964, 4, 311. 1961, 25, 605. WeinheimiBergstr., 1963, p. 292. Received November loth, 1965