Quantitation of neonicotinoid metabolites in human urine using gas chromatography-mass spectrometry

Abstract

Neonicotinoid insecticides (NEOs) are widely used in agriculture as foliar and seed treatments, for indoor and outdoor insect control, home gardening and pet products. The aim of this study is to develop and validate a simple method for quantitation of urinary NEO metabolites, 6-chloronicotinic acid (6CN), 2-chloro-1,3-thiazole-5-carboxylic acid (2CTCA) and 3-furoic acid (3FA) for biomonitoring of NEO exposure. The sensitivity and reliability of our method were examined in this study. Two mL urine was pipetted into a test tube, and 50 ?L H2SO4 (5 mol/L) and I.S. solution (10 mg/L isotope labeled 3PBA) were added for deconjugation procedure. After incubation at 86 oC for 2 hours, the solution was loaded into preconditioned SPE cartridge (Bond Elut Plexa PCX, Agilent Technologies). After the wash by 500 ?L formic acid solution (2%), the SPE cartridge was dried under vacuum for 3 min and eluted with 500 ?L methanol. The eluate was divided into two equal volumes in test tubes. These eluates were dried up with N2 gass, and the residues were resolved with 250 ?L acetonitrile in one test tube for 6CN and 2CTCA or with 250 ?L toluene in another test tube for 3FA. After derivatization using 40 ?L N,O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, each of 1 ?L sample was separately analyzed by gas chromatography-mass spectrometry. The regression equations ranging from 0.6 to 10 ?g/L were y=0.013x+0.0015 (r2=0.999) for 6CN, y=0.0054x+0.0002 (r2=0.999) for 2CTCA and y=0.0048x+0.0013 (r2=0.997) for 3FA. Our present method is simple, sensitive and reliable for the determination of urinary 6CN, 2CTCA and 3FA, i.e. lower limit of detection (0.1 ?g/L for each metabolites) compared with previous reports with satisfactory intra- and inter-day accuracy and precision (variability less than 12.3 %, R.S.D). This is the first report about determination of 2CTCA and 3FA in urine to the best of our knowledge.