Determination of Carbofuran and its metabolites in plant tissues by electron capture gas chromatography

PRACE Tom NAUKOWE. XXII, zeszyt | INSTYTUTU s OCHRONY ROŚLIN 2 1980 Jerzy Dec Edward Czapliński: DETERMINATION OF CARBOFURAN AND ITS - METABOLITES IN PLANT TISSUES BY ELECTRON CAPTURE GAS CHROMATOGRAPHY I. INTRODUCTION Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methyl carba- mate) is an effective insecticide-nematocide with both contact and systemic activity. Residues of carbofuran in soil and plants are accompanied by residues of its metabolites formed through oxidation and hydrolysis reactions. The oxidation results in the production of 3-hydroxycarbofuran which can be further oxidized to 3- ketocarbofuran. Both carbofuran and its oxidation products hydrolyze forming respective phenols that in plant tissues can occur as conjugated glucosides. The conjugates are also frequent for 3-hydroxycarbofuran (Cook, Stanovick and Cassil, 1969). The subject of carbofuran and its metabolites determination in plant tissues was undertaken by (1969) as the first developed applied many authors. the method to release the conjugated Cook, in which Stanovick and acid hydrolysis 3-hydroxycarbofuran into Cassil was its aglycone form. This acid hydrolysis followed by extraction with organic solvents and column cleanup was then introduced into the methods developed by Turner and Caro (1973), Williams and Brown (1973), Getz (1977) and Nelsen and Cook (1978). The method of Nelsen and Cook (1978) deserves particular attention due determining carbofuran various crops. The to its generality. It proved to be effective in and 3-hydroxycarbofuran residues in sixteen methods of other authors mentioned above were developed rather for individual crops. Nelsen and Cook based on the fact that carbofuran unlike 3-hydroxycarbofuran was not found as a conjugated residue, therefore it could be determined separately without hydrolysis step. In the opinion of the authors the generality of their method was effected by separating the analysis for the compounds of interest. | Prace Naukowe 118 ń — XXII Pozna IOR Nelsen and Cook elaborated their mionic nitrogen-phosphorous detector (2) method intending to use a therfor final quantitative determina- tion and in this connexion the method can be used when such a detector _ is available. In the method presented in this work detection of carbofuran and its metabolites was accomplished by gas chromatography using an electron capture detector (EC) which is of wide distribution. This detector is sufficiently sensitive for carbamates if they are previously chemically converted to respective derivatives; in the case of this method to 2,4-dinitrophenyl ethers obtained in the reaction with 1-fluoro-2,4-dinitrobenzene (FDNB) (Holden, 1973). EC detector as a non- -selective one is sensitive for impurities extracted with compounds to be determined, so its use requires the method ensuring high degree of sample purity. There are two ways to carry out acid hydrolysis. The first one consists in boiling of ground plant material in water solution of strong acid and the second one in boiling of acidified extract obtained after mechanical homogenization of plant material with aqueous methanol (Getz, 1977). In this method the second approach was chosen. First because of fewer coextractives involved and then because of the fact that there were no tendency to form emulsions during extraction of methanol solution with methylene chloride what eliminated the necessity of antifoam agents. Methanol extracts were acidified with orthophosphoric acid as was proposed by Getz (1977). Other authors mentioned above performed the hydrolysis in the presence of hydrochloric acid. To clean samples well enough so that EC detector could be used additional acetonitrile-hexane partition, first described by Cook et al. (1969), was inserted into the method. Column cleanup was performed according to Getz (1977). The next step was ethoxylation. It was carried out in order to convert 3-hydroxycarbofuran to 3-ethoxycarbofuran (Nelsen and Cook, 1978) for ether derivatives of 3-hydroxycarbofuran itself were not sensitive to electron capture detection. Ether derivatives of 3-ethoxycarbofuran obtained in the reacttion with FDNB according to Holden (1973) were then determined by EC gas chromatography along with carbofuran and 3-ketocarbofuran ether derivatives. Presented method has been tested on nine various plant materials. li. METHODS 1. Reagents — solvents: acetone, hexane, methylene chloride, isooctane, methanol, acetonitrile (all redistilled), absolute ethanol, benzene (purified with sulphuric acid and distilled); J. Dec, E. Czaplicki — Carbofuran in plant tissues — potassium hydroxide — 0,5 N aqueous solution; — 1-fluoro-2,4-dinitrobenzene (FDNB) — distilled acetone (1.5 cm3/25 cm3); — borax — saturated aqueous solution; and 119 dissolved in analysis acc. Pye Series 104 Chromatograph with ®Ni electron capture | tector; — Food chopper, Hobart Model 84181 DG; -— Laboratory homogenizer ,,Mechanika Precyzyjna” type 324; — Isomantle Isopad LTD; — Condux mill type LS 10M. de- —- orthophosphoric acid — 10% aqueous solution; — hydrochloric acid — — aluminium oxide — 0.25 N, and concentrated; — sodium sulphate — anhydrous, dried at 600°C; to Brockmann 2. Apparatus for chromatographic II (neutral). adsorption | _ — 3. Sample preparation 5 Chopped plant material was packed into plastic bags and stored in a refrigerator at —-20°C. The samples of orchard grass, lupine, alfalfa, tobacco and carrot leaves were cut with knife into small pieces. The samples of carrot roots and potato tubers were chopped in Hobard food. — chopper, and barley (grain and straw) was ground using Condux mill type LS 10M. 4, Procedure © | а) Hydrolysis and extraction - _ Расе 25 g of macerated crop into a 1000 cm! container for homogenization. Add 250 cm? of methanol and 75 cm? of distilled water. Blend for 5 minutes. Filter with suction through 1 cm layer of Celit 545 in a Biichner funnel. Rinse the container and filter cake with two 100 cm3 portions of aqueous methanol Transfer the combined methanol to a definit (150 cm? methanol and 50 cm? water). filtrates to a 1000 cm® graduated cylinder, volume and take one half of the sample a 500 cm? round bottom flask. Add and reflux for one hour. Cool 15 cm® of 10% orthophosphoric to room temperature and transfer add into acid to 120 Prace Naukowe IOR Poznań — XXII (2) a 500 cm? separatory funnel rinsing the flask twice with 20 cm® of distilled water. Extract three times with 100 cm? of methylene chloride. Dry methylene chloride extracts with anhydrous sodium sulphate and combine in a 500 cm? round bottom flask. Evaporate the dried extract to about 30 cm? in rotary evaporator at 40°C. Add 50 cm? of acetcnitrile and evaporate to about 10 cm? at the same temperature. Transfer the acetonitrile concentrate to a 250 cm? separatory funnel rinsing the flask with additional aceto (...truncated)