Determination of the Insecticide Promecarb by Fluorogenic Labelling with Dansyl Chloride
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ANALYST, AUGUST 1991, VOL. 116 851 Determination of the Insecticide Promecarb by Fluorogenic Labelling With Dansyl Chloride F. Garcia Sanchez and C. Cruces Blanco Department of Analytical Chemistry, Faculty of Sciences, The University, 29071 Malaga, Spain The use of spectrofluorimetry to determine the fluorescent derivative of the insecticide promecarb, following hydrolysis to the corresponding phenol in basic media and subsequent coupling with the labelling agent dansyl chloride, is described and discussed. A study of media of different basicity and of different temperatures for both reactions gave optimum conditions of 20 min for the hydrolysis reaction and 10 min for the labelling reaction at 55 "C in 0.05 mol dm-3 sodium hydrogen carbonate solution with a reagent to insecticide ratio of 12: 1. The effect of the solvent on the formation of the dansyl derivative and on the extraction process was studied using nine and seven solvents, respectively. The use of a mixture of acetone and water (50 + 50, v/v) and an extraction into cyclohexane gave the best results. The minimum detectable concentration of promecarb in the experimental assays was 100 ng ml-1. The error and relative standard deviation at a concentration level of 0.6 pg ml-1 were 9.7 and 10.9%, respectively. Air samples containing promecarb at different concentration levels were analysed. Keywords: Promecarb insecticide determination; air samples; dansyl chloride labelling; spectrofluorimetry The N-methylcarbamate insecticide promecarb (3-isopropyl- Experimental 5-methylphenyl methylcarbamate) acts against many pests and is effective both as a contact and a stomach poison. The Apparatus risks associated with exposure to this type of insecticide are All spectrofluorimetric measurements were performed on a caused by the accumulation of endogenous acetylcholine in Perkin-Elmer Model MPF-43A fluorescence spectrophoto- human beings, due to the inhibition of cholinesterase. meter, equipped with an Osram XBO 150 W xenon lamp, For these reasons, and taking into account the paucity of excitation and emission grating monochromators, 1 X 1 cm analytical methods described in the literature for this insecti- quartz cells, a Hamamatsu R-777 photomultiplier and a cide,' simple and rapid assays are required. Carbamate Perkin-Elmer 023 recorder. Instrument sensitivity was adjus- pesticides have frequently been analysed by thin-layer2 or gas ted daily, using a Rhodamine B bar as a reference standard. A chromatography.3 These procedures, however, are not water-bath circulator (Frigiterm S-382) was used for tempera- entirely satisfactory in terms of the sensitivity normally ture control. An electric shaker (Selecta) was used for required for residue analysis. extraction procedures. Because promecarb does not show native fluorescence, the technique of spectrofluorimetry has not been previously Reagents applied to its determination. This difficulty has been over- come in the present work by introducing a highly fluorescent A stock solution (1 mg ml-1) of promecarb (>99% pure, moiety into the pesticide molecule. Such an approach, called Pestanal quality, Riedel-de Haen, Hannover, Germany) was fluorogenic labelling, has found much application in the field prepared in analytical-reagent grade acetone. A working of amino acid and peptide chemistry4 and also in pesticide standard solution of 60 pg ml-1 was prepared from this stock residue analysis.5-9 solution by dilution with acetone. The dansyl chloride used The reagent most often used in such labelling techniques is (Sigma, St. Louis, MO, USA) was prepared as solutions (1 dansyl chloride [5-(dimethylamino)naphthalene-l-sulphonyl mg ml-1 and 470 pg ml-1) in ACS spectrophotometric grade chloride], which reacts with primary and secondary amino acetone (Gold Label; Aldrich, Milwaukee, WI, USA). groups and phenolic hydroxy groups to form highly fluores- Sodium hydroxide, sodium carbonate and sodium hydrogen cent derivatives. The extensive application of dansyl chloride carbonate were of analytical-reagent grade (Merck, Darm- is due not only to its ability to react with compounds having an stadt, Germany); solutions of these salts were prepared in amine or phenol moiety but also to its ability to react with both de-ionized water. All of the solvents used were of analytical- the amine and phenol hydrolysis products of carbamates, reagent grade. All pesticides tested were of 99% purity or resulting in two derivatives suitable for the quantification of better and were used without further purification. carbamate residues in the low nanogram range. This proce- dure has permitted the determination of numerous carbamate Reaction Procedure insecticides, 1@12 organophosphate insecticides,*3,14 herbi- cides7.15 and fungicides.16 Similar procedures have been used Different volumes (30,21,15,9 and 3 pl) of the stock solution for determining amino acids,17?18 food additives such as of promecarb in acetone (1 mg ml-1) and 50, 40, 30, 20 and monosodium glutamatel9 or alkaloids in complex phar- 10 pl of the working standard solution of the same reagent in maceutical dosage forms.20.21 acetone (60 pg ml-1) were placed in 15 ml test-tubes. A 0.5 ml In the present work, the use of dansyl chloride as a volume of 0.1 mol dm-3 sodium hydrogen carbonate solution fluorogenic labelling reagent for the determination of the was added and the tubes were loosely stoppered and heated in carbamate insecticide promecarb is described. The reaction, a water-bath at 55 "C for 20 min. The tubes were then cooled based on the formation of a fluorescent derivative of the to room temperature and 0.5 ml of a solution of dansyl phenolic hydrolysis product of the carbamate, has been chloride in acetone (470 pg ml-1) was added. The tubes were studied in detail and can be carried out in less than 40 min. heated in the water-bath for a further 10 min at 55 "C and then 852 ANALYST, AUGUST 1991, VOL. 116 allowed to cool to room temperature. A 3 ml volume of cyclohexane was added and the tubes were shaken for 1 min. Immediately after sample preparation, when both layers were clearly separated, the fluorescence intensity of the organic layer was measured at an excitation wavelength of 340 nm and an emission wavelength of 485 nm against a reagent blank. Promecarb Hydrolysed Standard Procedure for Air Samples promecarb Air samples were collected in a workroom environment (dry A temperature, 29 "C; humidity temperature, 16 "C; relative humidity, 52%) of 40.5 m2 (4.5 x 9.0 m) with 37 mm three-body filter cassettes equipped with mixed cellulose ester OH membrane filters of 0.8 pm pore size [Mine Safety Applicans Co. (MSA), Pittsburgh, PA, USA]. Battery-powered per- sonal sampling pumps (Model Fixt-Flo, MSA) with the capacity to operate at between 1 and 2 1 min-1 were calibrated at flow rates of 1.00 1 min-1 over a period of 24 h. Each filter so2 I sample was treated with 25 ml of acetone. After the mixture 0 had been shaken for 30 min at 25 "C and 160 rev min-1, the Dansyl chloride I acetone extract was transferred into a round-bottomed glass funnel and evaporated to incipient dryness in a rotary evaporator at 45 "C. The dry residue was then dissolved in acetone and taken to a final volume of 10 ml with the same solvent. An aliquot of this solution (100 p1) was then treated as described under Reaction Procedure. Results and Discussion Unless indicated otherwise, the conditions employed in the S03H optimization experiments were the same as those employed in Sulphonic acid the Experimental section. From earlier work in the field of carbamate pesticide B analysis,7~"~14~*2~23it is known that the labelling reaction with Fig. 1 Over-all reaction scheme for A, the hydrolysis; and B, the dansyl chloride involves two distinct steps: (a) hydrolysis of dansylation of promecarb the carbamate; and (b) coupling of the hydrolysis product with dansyl chloride. The coupling reaction proceeds faster than either the hydrolysis of the carbamate or the hydrolysis of the reagent to It can be seen that solvents that are immiscible with water the corresponding sulphonic acid. As the rate of hydrolysis of (such as chloroform and isobutyl methyl ketone) give no dansyl chloride is constant at a constant pH or temperature, fluorescence, indicating that dansylation has not taken place. only the rate of hydrolysis of the carbamate will govern the Solvents with high relative permittivities and hydrogen yield of the dansyl derivative. A reaction scheme is shown in bonding capacity (such as water, ethanol and methanol) give Fig. 1 for the formation of the derivative of the N-methyl- high fluorescence both in the analyte and reagent blank, carbarnate, promecarb. because of the absence of hydrolysis of the excess of dansyl Several reaction procedures were attempted for the hydroly- chloride to the corresponding sulphonic acid. The fluor- sis of the insecticide to the corresponding phenol. Different escence emission occurs also at longer wavelengths in highly concentrations (0.02, 0.04, 0.06, 0.08 and 0.10 mol dm-3) of polar solvents as reported previously.26 As the extent of the sodium hydroxide, sodium hydrogen carbonate and sodium solute-solvent interaction increases, the emission is shifted to carbonate were tried using a heating time of 30 min (hydrolysis shorter wavelengths. reaction) and 20 min (dansylation reaction) at 45 "C. 1,4-Dioxane, despite having a low relative permittivity, is The fluorescence intensity of the organic layer (benzene) miscible with water and gives, together with acetonitrile and was compared with that of a blank signal. The results (Fig. 2) acetone, a good signal-to-noise ratio. It is deduced from this indicated that, by using sodium hydrogen carbonate solution study that a change in the polarity of the reaction medium for the hydrolysis process, stable analytical measurements and modifies, to a great extent, the characteristics of the labelling a good signal-to-noise ratio were obtained with a constant pH reaction.