A method based on liquid chromatography (LC) with electrospray ionization tandem mass spectrometry (MS) for the analysis of Alachlor and its metabolites. 2chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) and 2.6-diethylaniline (DEA) in rat plasma and urine has been developed. The 13 C-labeled Alachlor and its metabolites were used as the internal standards for quantitation. The mass-spectrometer was set at ESI MS-SIM mode with a programming procedure. The LC flow rate was constant at 0.3 mL/min. The mobile phase gradient program started at a ratio of 30% acetonitrile and 70% water containing 1% acetic acid for 3 min and then increased to 100% acetonitrile within the next 4 min. An initial sample volume of 500 microl was loaded onto a 96-well Waters Oasis HLB solid phase extraction plate and concentrated into 50 microl acetonitrile, of which a 5-ul aliquot was injected into the LC-MS system. The retention times for Alachlor. CDEPA and DEA on the Xterta MS C8 column (2.5 11m, 2.1 x 50 mm) were 1.61 ,2.72 and 3.98 min, respectively with complete peak separation. The sensitivities of the MS detection for Alachlor, CDEPA and DEA were found to be 1 ng, 0.5 ng and 0.2 ng (perinjection), respectively with a signal/noise threshold of 2. The detection linearity range of at least 40 pg to 25 ng was observed with the three target compounds. Reproducibility of the sample handling and LC-MS analysis was good with a CV% of < / = 10%. Based on six duplicate samples per concentration within the concentration range specified, the average recoveries for Alachlor, CDEPA and DEA in rat plasma were found to be 80, 87 and 90%, respectively. Similar preliminaty results were also obtained with rat urine. Therefore, this sensitive, specific, reproducible and cost-effective LC-MS method may be used to identify and quantitate Alachlor and its major metabolites in bio-samples obtained from in vivo studies.
The Toxicologist. Society of Toxicology 40th Annual Meeting, March 25-29, 2001, San Francisco, California