苯已被證實會導致白血病，國際癌症研究中心 (International Agency for Research on Cancer, IARC)已經將苯列為group1的致癌性物質。目前苯基硫醇酸 (S-phenylmercapturic acid, S-PMA)被證實是一個具有高特異性的苯暴露生物指標，然而以往缺乏敏感度與繁複前處理的分析方法較不適於評估苯暴露量。因此為了例行性樣本分析，發展一個高敏感度且能免除繁複樣本前處理的自動化分析方法是有必要的。本研究的目的在於發展自動化固相萃取技術並利用電灑離子化法串聯式質譜儀(electrospray ionization-tandem mass spectrometry, ESI-MS/MS)來分析定量尿中苯的生物指標S-PMA，此系統可以自動化分析並免除繁複的前處理。本實驗室已將自動化固相萃取技術與電灑離子化法串聯式質譜儀系統已架構完畢，所欲發展的分析方法之檢量線、精密度、回收率、準確度、方法偵測極限與樣本儲存穩定度都有被加以描述，檢量線利用13C6 S-PMA當作內標準品其相關係數達 (R2=0.998)，本方法在尿液基質內之偵測極限達2.09 μg/g creatinine，尿液樣本添加S-PMA濃度相當為12.5、25、50 μg/g creatinine經過重複分析變異係數各別為4.82 %、5.62 %、6.24 %。尿液樣本以4 ℃與-20 ℃儲存，其樣本儲存穩定度結果是令人滿意的，分析相當12.5與50 μg/g creatinine濃度尿液樣本儲存8週後回收率介於88.9 %~105.7 %之間。本研究亦有探討解凍次數對樣品儲存穩定度的影響，樣本在10次的解凍後仍有相當良好的回收率 (93.2 % ~101.5 %)。另外此分析方法的適用性亦藉由台灣國家衛生研究院實驗室來加以驗證。

Benzene exposure has been linked to the occurrence of various types of leukemia. International Agency for Research on Cancer (IARC) has classified benzene as a group 1 carcinogen. S-phenylmercapturic acid (S-PMA) is so far considered to provide best specificity for benzene exposure. However, traditional analytical methods employed for assessing benzene exposure have been considered either lacking necessary sensitivities or require tedious procedures in the sample preparation. This study aims to develop a sensitive and automatic analytical systems to eliminate tedious sample preparation for the routine analysis of urinary S-PMA.An on-line solid phase extraction and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) system was developed for the quantitative detection of benzene exposure biomarker, S-PMA, in urine. The system is easily automated and eliminates time-consuming sample preparation procedures.The calibration curve, precision, recovery, accuracy, detection limit, and stability of the system have been characterized. Good linear correlation (R2 = 0.998) was observed for calibration curves established for S-PMA-spiked urines with the use of stable isotope-labeled 13C6 S-PMA as internal standard. The detection limit of the method for S-PMA in urine matrix was determined to be 2.09 μg/g creatinine. The coefficients of variation (CV) generated by repeated analyses of urine samples containing 12.5, 25, and 50 μg/g creatinine were calculated to be 4.82 %, 5.62 %, 6.24 %, respectively. The stability of sample storage at 4 ℃ or -20 ℃ was assessed and the results were satisfactory. The analytical recovery of urine sample containing 12.5 and 50 μg/g of creatinine were very good (88.9 % ~ 105.7 %) after 8-week storage using two different conditions. The stability for frozen-and-thaw cycles was also described. The samples were stable up to 10 frozen-and-thaw cycles with good recovery (93.2 % ~ 101.5 %). The method was validated by another analytical laboratory located in the National Health Research institutes, Taiwan.

Boogaard PJ, van Sittert NJ. Suitability of S-phenyl mercapturic acid and trans-trans-muconic acid as biomarkers for exposure to low concentrations of benzene. Environmental Health Perspectives 104:1151-7(1996).

Bruins AP. Mechanistic aspects of electrospry ionization. Journal of Chromatography A 794:345-57(1998).

Dor F, Dab W, Empereur-Bissonnet P, Zmirou D. Validity of biomarkers in environmental health studies: the case of PAHs and benzene. Critical Reviews in Toxicology 29:129-68(1999).

Einig T, Dehnen W. Sensitive determination of the benzene metabolite S-phenylmercapturic acid in urine by high-performance liquid chromatography with fluorescence detection. Journal of Chromatography A 697:371-5(1995).

Einig T, Dunemann L, Dehnen W. Sensitive gas chromatographic method for determination of mercapturic acids in human urine. Journal of Chromatography-Biomedical Applications 687:379-85(1996).

Fang MZ, Shin MK, Park KW, Kim YS, Lee JW, Cho MH. Analysis of urinary S-phenylmercapturic acid and trans, trans-muconic acid as exposure biomarkers of benzene in petrochemical and industrial areas of Korea. Scandinavian Journal of Work Environment & Health 26:62-6(2000).

Ghittori S, Imbriani M, Maestri L, Capodaglio E, Cavalleri A. Determination of S-phenylmercapturic acid in urine as an indicator of exposure to benzene. Toxicology Letters 108:329-34(1999).

Ghittori S, Maestri L, Fiorentino ML, Imbriani M. Evaluation of occupational exposure to benzene by urinalysis. International Archives of Occupational & Environmental Health 67:195-200(1995).

Hayes RB, Yin SN, Dosemeci M, Li GL, Wacholder S, Travis LB, Li CY, Rothman N, Hoover RN, Linet MS. Benzene and the dose-related incidence of hematologic neoplasms in China. Journal of the National Cancer Institute 89:1065-71(1997).

Inoue O, Kanno E, Kakizaki M, Watanabe T, Higashikawa K, Ikeda M. Urinary phenylmercapturic acid as a marker of occupational exposure to benzene. Industrial Health 38:195-204(2000).

International Agency for Research on Cancer (IARC). Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. Chemicals, Industrial processes and industries associated with cancer in humans. Supplement7, Lyon, France, 1987.

Jongeneelen FJ, Dirven HA, Leijdekkers CM, Henderson PT, Brouns RM, Halm K. S-phenyl-N-acetylcysteine in urine of rats and workers after exposure to benzene. Journal of Analytical Toxicology 11:100-4(1987).

Melikian AA, O'Connor R, Prahalad AK, Hu P, Li H, Kagan M, Thompson SM. Determination of the urinary benzene metabolites S-phenylmercaturic acid and trans,trans-muconic acid by liquid chromatography-electrospray ionization-tandem mass spectrometry. Carcinogenesis 20:719-726(1999).

Ong CN, Kok PW, Ong HY, Shi CY, Lee BL, Phoon WH, Tan KT. Biomarkers of exposure to low concentrations of benzene: a field assessment. Occupational & Environmental Medicine 53:328-33(1996).

Ong CN, Lee BL. Determination of benzene and its metabolites: application in biological monitoring of environmental and occupational exposure to benzene. Journal of Chromatography-Biomedical Applications 660:1-22(1994).

Popp W, Rauscher D, Muller G, Angerer J, Norpoth K. Concentrations of benzene in blood and S-phenylmercapturic and t,t-muconic acid in urine in car mechanics. International Archives of Occupational & Environmental Health 66:1-6(1994).

Qu Q, Melikian AA, Li G, Shore R, Chen L, Cohen B, Yin S, Kagan MR, Li H, Meng M, Jin X, Winnik W, Li Y, Mu R, Li K. Validation of biomarkers in humans exposed to benzene: urine metabolites. American Journal of Industrial Medicine 37:522-31(2000).

Stanek W, Krenmayr P, Scherer G, Schmid ER. Quantitative determination of N-acetyl(-L-)cysteine derivatives in human urine by tandem mass spectrometry. Biological Mass Spectrometry 22:133-42(1993).

Stommel P, Muller G, Stucker W, Verkoyen C, Schobel S, Norpoth K. Determination of S-phenylmercapturic acid in the urine--an improvement in the biological monitoring of benzene exposure. Carcinogenesis 10:279-82(1989).

Van Sittert NJ, Boogaard PJ, Beulink GD. Application of the urinary S-phenylmercapturic acid test as a biomarker for low levels of exposure to benzene in industry. British Journal of Industrial Medicine 50:460-9(1993).

Wallace LA. Major sources of benzene exposure. Environmental Health Perspectives 82:165-9(1989).

Weaver VM, Buckley T, Groopman JD. Lack of specificity of trans,trans-muconic acid as a benzene biomarker after ingestion of sorbic acid-preserved foods. Cancer Epidemiology, Biomarkers & Prevention 9:749-55(2000).

World Health Organization (WHO). Biological Monitoring of Chemical Exposure in the Workplace Guidelines. Geneva, World Health Organization,1996.