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1 理 勞 暴露 類 CNOS93-04 行 93 年 年 李 亮 參 行 年

2 勞 暴露 尿 勞 暴露 來 暴露 勞 尿 便 理 暴露 勞 尿 行 量 60 µm polydimethylsiloxane divinylbenzene (PDMS DVB) 尿 度 µg/ml µg/ml 度 11.6 µg/ml µg/ml 類 度 參數 尿 率 86 ~ 94 率 86% ~ 114% 異 數 4.9 % 3.75 % 尿 -20 度 令 µg/ml ng/ml 度尿 2 率 81.5 %~123.4 % 率 ~ 尿 0.43 ng/ml 0.22 ng/ml 尿 度 尿 Abstract Urinary phenol and cresol is analyzed widely to determine benzene and toluene exposure in human. Most methods utilize direct measurements of phenols and cresols after extraction from urine using gas chromatography or high-performance liquid chromatography. We describe a novel and simple method to measure phenol and cresol from urine exposed to benzene and toluene. A solid phase microextraction and liquid chromatography (SPME-HPLC) system is here applied to the determination of the benzene and toluene exposure biomarker in urine. The PDMS DVB fiber (60 µm) was evaluated. The method was optimized using spiked urine samples in a range from µg/ml to µg/ml for pheol and from µg/ml to µg/ml for cresol. The effects of equilibration time and ionic strength of samples on the adsorption step were studied. The parameters affecting the desorption process, such as desorption mode, solvent composition and desorption time, were optimized. The method is reproducible and repeatable with recoveries calculated from spiked urine samples, varying from about 86% to 94% for phenol and 86% to 114% for cresol. The coefficients of variation (CV) generated by repeated analyses of urine samples were 4.9 % and 3.75 %, respectively. The stability of sample storage at -20 was assessed and the results were satisfactory. The analytical recovery of urine samples were very good (81.5 %~123.4 % for phenol and ~ or cresol). The limits of detection for phenol and cresol were 0.43 ng/ml and 0.22 ng/ml respectively. The method enabled the determination of phenol and cresol at low levels in urine samples. Keywords: solid phase microextraction, urine, biological monitoring 勞 尿 了 勞 暴露 暴露 列 理 列 見 來 識 不 了更 見 (International Agency for Research on Cancer, IARC) 列 group1

3 Cytochrome P450 2E1 來 列 trans,transmuconaldehyde trans,trans-muconic acid(t,t-ma) catechol (CAT) hydroquinone (HQ) 1,2,4-benzenetriol (BT) glutathione S-phenylmercapturic acid (S-PMA) 來 [1-4] 尿 p- 尿 [5] 尿 ph 1 Sep-pak Cl8 濾 濾 / / 理 離 來 S-PMA 來 硫 - 硫 硫 HPLC [6-9] 理 來論 GC 都 理 都不 HPLC 不 糖 硫 [10] 尿 量 暴露 尿 理 行尿 流 GC [11-15] 不 更 浪 漏 率不 - 便 - 便 了例行 量 理 (solid- phase microextraction SPME) 了 便 靈 度 量 [16-20] 利 SPME 量 µg/l 度 SPME 量 3.1 料 Phenol (99 ) P-cresol(methylphenol 99 ) ChemService NaCl(sodium chloride 99.5 ) GR Merck 晴 TEDIA99.8 Fluka HPLC HITACHI L-7100 Pump SH- IMAPDZU SPD-10A UV-VIS DETECTOR SPME SPME-HPLC SUPELCO. SPME-HPLC 六 60 µl 離 ZORBAX Eclipse XDB-C18 LC (Agilent Techn-ologies) 4.6 mm 75 mm 3.5 µm 流 80( ):20( 晴 ):0.02( ) 流 1 L/min 切 六 INJECT 來 流 離 行 流 SUPELCO Solid Phase Microextraction

4 Holder 連 SUPELCO Solid Phase Microextraction Fiber 1250 r- pm 度 行 condition fiber fiber 15 切 Inject 流 15 離 留 行 行 切 Inject 流 行 論 類 SPME 率 類 CW-TPR 60 µm PDMS DVB 行 量 率 CW-TPR 量 PDMS DVB 了 立 力 率 數 尿 ph 2 2mL 4 ml PDMS DVB 尿 min 行 1 30 min 狀 fiber 30 min Area response 8e+5 6e+5 4e+5 2e Absorption time (min) Phenol Cresol 尿 SPME-HPLC 了 來 兩 了 立 HCl ph 2 ~ 3 不 例 / 晴 / 行 / 晴 / (80/20/0.02%) 行 ~ 94 6 ~8 留 HCl ph 2 ~ 3 行 5 行 不 1 3 5

5 不 不 類 尿 留 離 流 5 兩 留 若 不 留 省 留 留 類 類 留 (%) 留 (%) 流 流 類 離 度 度降 Na 2 SO 4 NaCl KCl NH 4 Cl 類 行 類 不 類 度不 類 2 Na 2 SO 4 NaCl KCl NH 4 Cl 類 Na 2 SO 4 離 度 NaCl 來 NaCl 來 類 Area resopnse 5e+5 4e+5 3e+5 2e+5 1e+5 Na 2 SO 4 NaCl Kcl NH 4 Cl No salt ph 類 ph 不 離 度 異 ph ph 行 60 µm P-DMS/DVB 30 SPME-HPLC 5 1 ph=2 量 不 ph 度 異 不 度 ph 2 ~ 3 3 Area resopnse 4e+5 3e+5 2e+5 1e+5 0 Phenol Cresol ph 尿 3.2 留 列 量 立 不 度 尿 行 度 量 度 ~ µg/ml ~ 1.16 µg/ml 量 度 度 ~ 7.18ug/mL 度 量 ph ph 2 ph 3 ph 4 ph 7 0 Phenol Cresol Salt

6 R 度 ~ 5.8 ug/ml 度 量 R 了 尿 matrix effect 尿 離 離 尿 1 1 度 ~ µg/ml ~ 1.16 µg/ml 量 不 量 率 若 尿 離 量 不 度 率 不 量 率 90 論 立 量 率 異不 不 率 勞 尿 ng/ml 348ng/mL 離 量 率 率 86 ~ 114 異 數 1 ~ 8 尿 不 立 量 度 (µg/ml) R 2 y= x ~ 尿 y= x : 尿 (1:1) y= x y= x ~ 1.16 尿 y= x : 尿 (1:1) y= x 不 量 ( 尿 : =1:1) 尿 率 量 尿 量 度 率 (%)±(n=3) 率 (%)±(n=3) (µg/ml) ( 尿 : 尿 ( 尿 : =1:1) =1:1) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.4

7 4.2.4 暴露 尿 不 度 ng/ml ng/ml-20 冷 冷 ng/ml 率 80 ~ 120 良 度 5.7 ng/ml 4.64 ng/ml 尿 (standard deviation SD) (LOD) 0.43 ng/ml 0.22 ng/ml 論 SPME 靈 度 來 尿 PDMS/DVB 30 類 NaCl 尿 ph 2 行 不論 尿 離 尿 離 1 1 量 尿 度 率 80 理 不 量 度 ~1.436µg/mL ~ 1.16µg/mL R 率 86 ~ ng/ml 0.22 ng/ml 異 數 1.63 ~ 7.75 良 SPME 理 HPLC 理 流 靈 度 ng/ml 尿 度 Medicine, 37: [2] Ong, C.N., Lee, B.L., 1994; Journal of Chromatography-Biomedical Applications, 660:1-22. [3] R.K. Boutwell and D.K. Bosch, Cancer Res.,19 (1959) 413 [4] K.E. Murray and R.F. Adams, J. Chromatogr., 413 (1998) 143 [5]Ong, C.N., Lee, B.L., Ong, H.Y., and Heng, L.E., 1988; Determination of Urinary Phenol [6] Dor, F., Dab, W., Empereur-Bissonnet, P., Zmirou, D., 1999; Critical Reviews in B Toxicology, 29: [7] Ong, C.N., Kok, P.W., Ong, H.Y., Shi, C.Y., Lee, B.L., Phoon, W.H., Tan, K.T., 1996; Occupational & Environmental Medicine, 53: [8] Popp, W., Rauscher, D., Muller, G., Angerer, J., Norpoth, K., 1994; International Archives of Occupational & Environmental Health, 66:1-6. [9] Ghittori, S., Maestri, L., Fiorentino, M.L., Imbriani, M., 1995; International Archives of Occupational & Environmental Health, 67: [10] Crispin H. Pierce, Yili Chen, Russell L. Dills, David A. Kalman Michael S. Morgan, Toxicology Letters 129 (2002) 65 [11] E. G. Schmidt, J. Biol. Chem., 179 (1949) 211 [12]C.N. Ong, B.L. Lee and L.E. Heng, J. Anal.Toxicol., 12 (1998) 159 [13] M. Balikova and J. Kohlicek, J. Chromatogr., 497 (1989) 159 [14] L. Weber, J. Chromatogr., 574 (1992) 349 [15] C. V. Eadsforth and P. C. Coveney, Analyst, 109 (1984) 175 [16] Arthur. C.-L., Pawliszyn, J. Anal. Chem. 62 (1990) 2145 [17] Zhang. Z, Pawliszyn, J. Anal. Chem. 65 (1993) 1843 [18] Zhang. Z, Yang. M.-J, Pawliszyn, J. Anal. Chem. 66 (1994) 844A [19] Tadeusz Gorecki, Xiaomei Yu, Janusz Pawliszyn Analyst 124 (1999) ] [20] Y.J. Wu, C. R. Ou, M.K. Lee, R.S. Chou, T. S. Shih, 2001 Occupational Health Conference 參 [1] Qu, Q., Melikian, A.A., Li, G., Shore, R., Chen, L., Cohen, B., Yin, S., Kagan, M.R., Li, H., Meng, M., Jin, X., Winnik, W., Li, Y., Mu, R., Li, K., 2000; American Journal of Industrial