成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	施政甫 Shih, Jeng-Fu
論文名稱：	發展自動化樣品淨化暨電灑離子化串聯式質譜儀裝置來同時量測尿液中苯暴露之生物指標S-PMA及t,t-MA An automatic clean-up and ESI-MS/MS system for simultaneous quantitation of urinary S-PMA and t,t-MA as biomarkers of benzene exposure
指導教授：	廖寶琦 Liao, Pao-Chi
學位類別：	碩士 Master
系所名稱：	醫學院 - 環境醫學研究所 Department of Environmental and Occupational Health
論文出版年：	2004
畢業學年度：	92
語文別：	中文
論文頁數：	90
中文關鍵詞：	苯、4-已二烯二酸、電灑離子化法串聯式質譜儀、尿液、2，苯基硫醇酸
外文關鍵詞：	ESI-MS/MS, t-MA, t, S-PMA, benzene, urine
相關次數：	點閱：62 下載：1
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

　　尿液中的苯基硫醇酸 (S-phenylmercapturic acid, S-PMA)及2,4-已二烯二酸 (trans, trans-muconic acid, t,t-MA)被認為是可反映苯暴露之良好的生物指標。然而傳統上之分析方法皆需要繁複的前處理步驟，如固相萃取或衍生化過程，並不利於例行性的樣本分析。因此，本研究目的為發展自動化樣品淨化裝置並利用電灑離子化法串聯式質譜儀 (electrospray ionization-tandem mass spectrometry, ESI-MS/MS)來達到同時量測尿液中的S-PMA及t,t-MA，此系統可以自動化分析並免除繁複的前處理。尿液樣品經pH調整及0.2 μm PVDF材質的濾紙過濾後，進入本研究所開發之系統進行分析。此分析系統包含了自動化取樣裝置、一個C18的管柱、一個200μL的loop、一個六孔閥、一個十孔閥、電灑離子化串聯式質譜儀及控制系統自動化的電腦所架構而成。此分析方法之檢量線、方法偵測極限、回收率、精密度、準確度及樣本儲存穩定度皆有描述及評估。在尿液空白中添加S-PMA、t,t-MA之標準品進行檢量線的配製，濃度範圍為1/32倍BEI至8倍BEI值（BEI分別是：S-PMA為25 μg/g creatinine、t,t-MA 為500 μg/g creatinine），其r2分別為0.9986及0.9996。方法偵測極限S-PMA及t,t-MA分別為0.25 及2.46 μg/g creatinine。於尿液基質中添加S-PMA及t,t-MA 相當於1/2、1及2倍之BEI值濃度，其回收率分別為106 %、101 %、96.5 %及93.7 %、95.6 %、101 %。重複性分析之共同變異係數分別為2.7 %及4.3 %。而將尿液基值中添加S-PMA及t,t-MA 相當於1/2、1倍BEI值濃度分別以4℃及-20℃儲存，其儲存8週後回收率於4℃分別介於95.1~114 %及95.3~102 %之間；於-20℃分別介於87.5~ 110 %及96.9~100 %之間。此外，本研究亦收集不同暴露族群之尿液樣本並以此分析方法進行量測，其重複分析之相對誤差均小於15%。由上述結果說明，排除了冗長的前處理，尿液樣本經由本系統即可在20分鐘內同時將S-PMA 及t,t-MA分析完成。

　　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.

第一章 序論..................................................................................................................1
1-1 研究背景..................................................................................................................1
1-2 研究目的..................................................................................................................2
第二章 文獻回顧..................................................................................................................3
2-1 苯暴露的來源..................................................................................................................3
2-2 苯的特性、毒性作用及健康危害..................................................................................................................3
2-3 苯在體內的代謝途徑..................................................................................................................6
2-4 苯暴露的生物指標..................................................................................................................8
2-4-1 生物偵測..................................................................................................................8
2-4-2 苯各種生物指標之優劣.................................................................................................................11
2-4-3 苯生物指標之相關規範.................................................................................................................14
2-4-4 同時分析S-PMA 與t,t-MA 之重要性.................................................................................................................14
2-5 過去分析尿液中S-PMA 及t,t-MA 之方法及優劣比較.................................................................................................................14
第三章 研究方法與材料.................................................................................................................17
3-1 研究架構.................................................................................................................17
3-2 分析儀器與設備.................................................................................................................19
3-3 樣本前處理.................................................................................................................20
3-4 質譜儀參數找尋............................................................................................................,....20
3-4-1 找尋分析物之前趨離子及產物離子.................................................................................................................23
3-4-2 電壓參數之調整.................................................................................................................23
3-4-3 離子源參數之調整.................................................................................................................23
3-5 自動化淨化系統架構及參數找尋.................................................................................................................26
3-5-1 自動化淨化系統架構.................................................................................................................26
3-5-2 t,t-MA 轉移時間參數找尋.................................................................................................................26
3-5-3 S-PMA 水沖提時間參數找尋.................................................................................................................28
3-6 內標準品之合成與測試.................................................................................................................28
3-7 空白分析.................................................................................................................31
3-7-1 試劑空白分析.................................................................................................................31
3-7-2 尿液基質空白分析.................................................................................................................31
3-8 方法驗證程序.................................................................................................................31
3-8-1 檢量線之製備標準.................................................................................................................31
3-8-2 方法偵測極限.................................................................................................................32
3-8-3 回收率.................................................................................................................33
3-8-4 精密度.................................................................................................................33
3-8-5 準確度.................................................................................................................33
3-8-6 樣品儲存穩定性.................................................................................................................34
第四章 結果與討論.................................................................................................................36
4-1 S-PMA 及t,t-MA 之前驅離子及產物離子.................................................................................................................36
4-2 自動化淨化系統參數找尋結果.................................................................................................................36
4-2-1 t,t-MA 轉移時間參數之最佳化.................................................................................................................36
4-2-2 S-PMA 水沖提時間參數之最佳化.................................................................................................................38
4-3 內標準品之適用性.................................................................................................................39
4-4 空白分析結果.................................................................................................................40
4-4-1 試劑空白分析.................................................................................................................40
4-4-2 尿液基質空白分析.................................................................................................................40
4-5 方法驗證程序之結果.................................................................................................................43
4-5-1 檢量線之製備標準.................................................................................................................43
4-5-2 方法偵測極限.................................................................................................................45
4-5-3 回收率.................................................................................................................45
4-5-4 精密度.................................................................................................................46
4-5-5 準確度.................................................................................................................47
4-5-6 樣品儲存穩定性.................................................................................................................47
4-6 不同暴露族群之分析結果.................................................................................................................50
第五章 結論.................................................................................................................53
第六章 參考文獻.................................................................................................................56
附錄一.................................................................................................................62

                                    

Aksoy M. Different types of malignancies due to occupational exposure to benzene: a review of recent observations in Turkey. Environ Res. 23: 181-190 (1980).

Baselt RC. Benzene in biological monitoring methods for industrial chemicals. Davis: Biomedical Publications. 37-42 (1980).

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. Environ Health Persp. 104: 1151-1157 (1996).

Brugnone F, Perbellini L, Maranelli G, Romeo L, Guglielmi G, Lombardini F. Reference values for blood benzene in the occupationally unexposed general population. Int Arch Occup Environ Health. 64: 179-184 (1992).

Bruins AP. Mechanistic aspects of electrospry ionization. J Chromatogr A. 794: 345-357 (1998).

Dor F, Dab W, Empereur-Bissonnet P, Zmirou D. Validity of biomarkers in environmental health studies: the case of PAHs and benzene. Crit Rev Toxicol. 29: 129-168 (1999).

Duarte-Davidson R, Courage C, Rushton L, Levy L. Benzene in the environment: an assessment of the potential risks to the health of the population. Occup Environ Med. 58: 2-13 (2001).

Einig T, Dehnen W. Sensitive determination of the benzene metabolite S-phenylmercapturic acid in urine by high-performance liquid chromatography with fluorescence detection. J Chromatogr A. 697: 371-375 (1995).

Einig T, Dunemann L, Dehnen W. Sensitive gas chromatographic method for determination of mercapturic acids in human urine. J Chromatogr B. 687: 379-385 (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. Scand J Work Environ Health. 26: 62-66 (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. Toxicol Lett. 108: 329-334 (1999).

Ghittori S, Maestri L, Fiorentino ML, Imbriani M. Evaluation of occupational exposure to benzene by urinalysis. Int Arch Occup Environ 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. J Natl Cancer Inst. 89: 1065-1071 (1997).

Inoue O, Kanno E, Kakizaki M, Watanabe T, Higashikawa K, Ikeda M. Urinary phenylmercapturic acid as a marker of occupational exposure to benzene. Ind 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. J Anal Toxicol. 11: 100-104 (1987).

Lauwerys RR, Hoet P. Industrial chemical exposure : guideline for biological monitoring. 2nd ed. Lewis publishers (1993).

Liao PC, Li CM, Lin LC, Hung CW. An online automatic sample cleanup system for the quantitative detection of the benzene exposure biomarker S-phenylmercapturic acid in human urine by electrospray ionization tandem mass spectrometry. J Anal Toxicol. 26: 205-210 (2002).

Lin FM, Wu HL, Kou HS, Lin SJ. Ultratrace analysis for trans,trans-muconic acid by electrophoric derivatization and capillary gas chromatography. Anal Chim Acta. 455: 111-116 (2002).

Linos A, Kyle RA, O'Fallon WM, Kurland LT, A case-control study of occupational exposures and leukaemia. Int J Epidemiol. 9: 131-135 (1980).

Marrubini G, Coccini T, Maestri L, Manzo L. Effect of sorbic acid administration on urinary trans,trans-muconic acid excretion in rats exposed to low levels of benzene. Food Chem Toxicol. 40: 1799-1806 (2002).

Marrubini G, Coccini T, Manzo L. Direct analysis of urinary trans,trans-muconic acid by coupled column liquid chromatography and spectrophotoetric ultraviolet detection: method applicability to human urine. J Chromatogr B. 758: 295-305 (2001).

Melikian AA, O'Connor R, Hu P, Thompson SM. Determination of the urinary benzene metabolites S-phenylmercapturic 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. Occup Environ Med. 53: 328-333 (1996).

Ong CN, Lee BL. Determination of benzene and its metabolites: application in biological monitoring of environmental and occupational exposure to benzene. J Chromatogr B. 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. Int Arch Occup Environ 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. Am J Ind Med. 37: 522-531 (2000).

Ruppert T, Scherer G, Tricker AR, Adlkofer F. trans,trnas-Muconic acid as a biomarker of non-occupational environmental exposure to benzene. Int Arch Occup Environ Health. 69: 247-251 (1997).

Rinsky RA, Smith AB, Hornung R. Benzene and leukemia. An epidemiologic risk assessment. New Engl J Med. 316: 1044-1050 (1987).

Sandmeyer EE. Aromatic hydrocarbons: benzene. Patty’s Industrial Hygiene and Toxicology. 3: 3253-3283 (1981).

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: 133-142 (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: 279-282 (1989).

Tunek A, Hogstedt B, Olofsson T. Mechanism of benzene toxicity. Effect of benzene and benzene metabolites on bone marrow cellularity, number of granulopoietic stem cells and frequency of micronuclei in mice. Chem Biol. 39: 129-138 (1982).

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. Br J Ind Med. 50: 460-469 (1993).

Wallace LA. Major sources of benzene exposure. Environ Health Persp. 82: 165-169 (1989).

Weisel C, Yu R, Roy A. Georgopoulos P. Biomarkers of environmental benzene exposure. Environ Health Persp. 104: 1141-1146 (1996)

Wong O. An industry-wide mortality study of chemical workers occupationally exposed to benzene. II: Dose response analyses. Br J Ind Med. 44: 382-395 (1987).

Yin SN, Hayes RB, Linet MS, Li GL, Dosemeci M, Travis LB, Li CY, Zhang ZN, Li DG, Chow WH, Wacholder S, Wang YZ, Jiang ZL, Dai TR, Zhang WY, Chao XJ, Ye PZ, Kou QR, Zhang XC, Lin XF, Meng JF, Ding CY, Zho JS, Blot WJ. A Cohort Study of Cancer Among Benzene-Exposed Workers in China: Overall Results. Am J Ind Med. 29: 227-235 (1996).

校內：立即公開
校外：2004-06-30公開

簡易檢索 / 詳目顯示

相關論文