Objective

1. SIMULTANEOUS QUANTIFICATION OF THREE MERCAPTURICACIDS OF ACRYLONITRILE AND COTININE IN URINE USING ISOTOPE-DILUTION ON-LINE LC-MS/MS Chia-Fang Wu1, Wei-Chung Shih1, Shi-Nian Uang2, Kuen-Yuh Wu11Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University, Taipei, Taiwan 2Insitute of Occupational Safety and Health, Taipei, Taiwan Figure 1. Acrylonitrile Metabolism Introduction Acrylonitrile (AN) is a widely-used industrial chemical in the production of various household articlesand has been classified as a probable human carcinogen (Group 2B) by IARC in 1999. It is also present in tobacco smoke, about 1-2 µg/cigarette. AN can be detoxified either by glutathione S-transferase (GST) to form N-acetyl-S-(2-cyanoethyl)cysteine (CEMA) , or metabolized by CYP2E1 and further detoxified by GST to form mercapturic acids N-acetyl-S-(2-hydroxyethyl)cysteine (HEMA) and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine (CHEMA) (Figure 1.). Objective The objective of this study was to develop an isotope-dilution on-line solid-phase-extraction (SPE) high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method to quantify urinary CEMA, HEMA, CHEMA, and cotinine to validate mercapturic acids (MAs) as proper biomarkers for AN exposure. Figure 2. Mass spectra of CEMA HEMA, CHEMA, and cotinine with the predicted structures of the fragments 86 128 CEMA HEMA Materials & Methods Sample Preparation Urine blanks were collected from 9 non-occupational exposed andnon-smoking populations. These 9 spot urines were then pooled together as the urine blanks (urinary creatinine: 99 mg/dL) and stored at -20℃ until used for analysis. Calibration curve ranged from 0.1 to 2000 µg/L and was prepared by spiking standard analytes to the matrices. The samples were then added with equal volume of 5 mM ammonium formate and centrifuged at 12,000 rpm for 10 min after vigoroulsy mixing. One hundred nano-grams of d3-CEMA, d4-HEMA, and d3-cotinine were spiked into I mL of the samples. Fifteen micro-liters were then injected into the on-line LC-MS/MS system for analysis. On-Line SPE LC-MS/MS Analysis The on-line system consisted of a C18 cleanup cartridge (Inertsil, ODS-3, 5µm, 4.6×33mm), an autosampler, and a switching valve. By switching the position of the valve, analytes could be extracted in the cleanup cartridge (position A) first, and then be eluted (position B) by the binary mobile phase system (A: 0.1% formic acid in acetonitrile, B: 0.1% formic acid + 5 mM ammonium acetate) to the analytical column (Atlantis T3, 3µm, 2.1x100mm) to perform the chromatography. LC-MS/MS system (Waters Micromass Quattro Premier XE Mass Spectrometer) was operated under negative ESI for CEMA, HEMA, CHEMA, and positive ESI for cotinine. 206 215 162 77 213 146 CHEMA Cotinine 80 84 231 177 102 98 Figure 3. Chromatogramsof CEMA, HEMA, CHEMA, and Cotinine in urine matrix CEMA 8.1 min HEMA 7.4 min Results & Discussions Multiple reaction monitoring (MRM) mode was operated to monitor the transitions of the 3 mercapturic acids and cotinine (Figure 2.). Retention time of each analyte is shown in the chromatograms (Figure 3.). Quantification was conducted on the basis of calibration curve in urine matrix (Figure 4.). It appeared that present method showed outstanding performance (Table 1.). The on-line SPE system minimized the time for sample preparation, and the run time for each sample was just 15 minutes in this method. With excellent sensitivity and specificity of this LC-MS/MS method, the MAs in urine samples collected from exposed workers will definitely be detectable. CHEMA 7.6 min Cotinine 7.2 min Figure 4. Calibration curves of CEMA, HEMA, CHEMA, and Cotinine in different matrices Table 1. The Analytical Result Conclusions This present method simultaneously analyzes urinary CEMA, HEMA, CHEMA, and cotinine with outstanding performance. It will be applied to analyze real samples of exposed workers to assess AN exposures and to validate these MAs as proper biomarkers for AN exposures in future works.