| Summary: | 碩士 === 國立成功大學 === 環境醫學研究所 === 92 === Urinary metabolites S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t,t-MA) have been proposed as reliable biomarkers for benzene exposure. However, traditional analytical methods require tedious procedures in sample preparation such as solid phase extraction and chemical derivatization, and may be unsuitable in routine analyses. The aim of this study is to develop an on-line automatic sample cleanup system with electrospray ionization tandem mass spectrometry (ESI-MS/MS) in order to quantitate S-PMA and t,t-MA simultaneously. The urine samples were adjusted to pH 2, passed through 0.2 μm PVDF membranes, and injected into an analytical system developed in our laboratory. The analytical system was constructed by an autosampler, a reversed -phase C18 trap cartridge, a 200 μL loop, one 6-port and one 10-port two-position switching valves, a triple-stage quadrupole MS/MS instrument equipped with an ESI source, and controlling computer software for automation. The calibration curve, detection limit, recovery, precision, accuracy and the stability of sample storage for the system have been characterized. Calibration plots of S-PMA and t,t-MA standards spiked into urine blanks over a wide concentration range (1/32 ~8 fold BEIs, the BEIs for S-PMA and t,t-MA were 25 and 500 μg/g creatinine) showed good linearity (r2 = 0.9986, 0.9996). The detection limits for S-PMA and t,t-MA were 0.25 and 2.46 μg/g creatinine, respectively. The analytical recovery of urine samples spiked with S-PMA and t,t-MA standards at 1/2, 1 and 2 BEI levels were 106 %、101 %、96.5 % and 93.7 %、95.6 %、101 %. The pooled coefficients of variation of repeated measurements for both analytes at these levels were 2.7 % and 4.3 %. The stability of sample storage at 4℃ or -20℃ were assessed. The recovery of urine sample spiked with S-PMA and t,t-MA standards with 1/2 and 1 BEI levels at 4℃ were 95.1~114 % 、 95.3~102 % ; and at -20℃ were 87.5~ 110 %、 96.9~100 %, respectively. In addition, urine samples from different benzene exposure groups were collected and measured in this system. The relative errors of repeated measurements were all below 15%. Without tedious manual sample clean-up, the analytical system was able to quantify simultaneously S-PMA and t,t-MA in less than 20 minutes.
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