砷是人類已知的致癌物質，並被認為是導致烏腳病的一項可能因子。然而，砷對人體所造成之毒害，往往無法從總濃度看出，因此須進一步進行體液中砷之物理性或化學性的物種分析。本研究之目的係發展血清樣品中含砷物種之分離及鑑定方法，即利用Size exclusion chromatography-ICP-MS (SEC-ICP-MS) 及Reversed phase LC-ICP-MS (RPLC-ICP-MS) 進行含砷物種之分離，繼而利用electrospray ionization mass (ESI MS) 進行物種鑑定，期能提供砷在人體中傳輸與代謝機制之相關資訊。
本研究結合二種層析技術進行物種分離，因此稱之為二維液相層析(two-dimensional liquid chromatography)。血清樣品首先經由SEC管柱依照分子量大小之不同而將蛋白質分離，繼而導入ICP-MS進行砷的測定。由於血清中之成份過於複雜，SEC無法完全將含砷物種與其它基質分離，因此須再利用RPLC作進一步之純化。由實驗的結果顯示，此SEC-ICP-MS連線系統能將血清中之含砷物種分離開來，估計其分子量分佈約在1500 Da左右。上述分離所得之含砷物種續經RPLC分離純化，結果顯示可將原來的含砷物種再細分出一種含砷物種與二種不含砷之未知物種，唯從層析圖譜上看出，前者仍受到相當嚴重的溶劑訊號干擾。最後將初步純化所得之含砷物種利用ESI MS進行測定，由於受到過多的雜訊干擾以及含砷物種濃度過低，目前在質譜的鑑定上仍有困難。

Arsenic has been proved to be a toxic and cancerogenic element. Besides, the biological importance is dependent on the species in which arsenic appears. Therefore, the elucidation of the mechanisms governing the essentiality and toxicity of trace elements in living organisms is critically dependent up the possibility of the characterization and determination of physical or chemical forms of these elements involved in life processes. The purpose of this study is to develop a separation system allowing the identification of arsenic species in serum samples. The analytical system was based on a two dimensional liquid chromatography system consisting of size-exclusion chromatography (SEC) and reversed-phase liquid chromatography (RPLC) with on-line detection of arsenic by inductively coupled plasma mass spectrometry (ICP-MS) and then the arsenic-containing fraction was analysed by ES-MS. In the first-dimension separation, SEC was found to be particularly well suited for protein separation and it provides easily interpretable qualitative information: the molecular weight of the arsenic specie is about 1500 Da. After SEC separation, RPLC was emplyed for second dimensional separation of the fraction containing arsenic. The effects of spectral interferences for the determination of arsenic species in serum samples with the proposed analytical system were investigated, and the results indicated that the on-line ICP-MS determination can be completely free from interferences with the prior column separation step.

1. A. S. Medel, Spectrochim. Acta., 53, 197 (1998).
2. J. Szpunar, R. Lobinski, Pure Appl. Chem., 71, 899 (1999).
3. E. Nieboer, Analyst., 117, 549 (1992).
4. M. Bernhard, F. E. Brinckman, and P. J. Sadler, "The Importance of Chemical Speciation in Environmental Processes", Springer-Verlay,Berlin (1986).
5. R. Lobinski, and J. Szpunar, Analytica Chimica Acta., 400, 321 (1999).
6. G. Berthon, "Handbook Metal-Ligand Interactions in Biological Fluids",Marcel, Dekker, Inc, New York (1995).
7. R. Lobinski, Analusis., 26 (1998).
8. S. J. Lippard, and J. M. Berg, "Principles og Bioinorganic Chemistry"., Mill Valey, CA (1994).
9. W. Kaim, and B. Schwederski, "Bioinorganic Chemistry:Inorganic Elements in the Chemistry of Life"., Wiley, Chichester (1998).
10. W. Kaim, and B. Schwederski, "Bioinorganic Chemistry:Inorganic Elements in the Chemistry of Life"., Wiley, Chichester (1991).
11. M. J. Stillman, C. F. Shaw, and K. T. Suzuki, "Metallothioneins Synthesis,Structure and Properties of Metallothioneins, Phytochelatins and Metalthiolate Complexes"., VCH, New York (1992).
12. J. F. Riodan, and B. L. Valee, Metallobiochemistry Part B. Metallothionein and Related Molecules, Methods in Enzymology., 205, Academic Press, New York (1991).
13. L. Ebdon, S. Hill, and R. W. Ward, Analyst., 111,1113 (1986).
14. L. Ebdon, S. Hill, and R. W. Ward, Analyst., 112,1 (1987).
15. P. E. Gardiner, J. Anal. At. Spectrom., 3,163 (1988).
16. V. D. Lewis, S. H. Nam, and I. T. Urasa, J. Chromatogr., 27,468 (1989).
17. E. L. Giroux, Biochem. Med., 12, 258 (1975).
18. B. A. Bidlingmeyer, J. Chromatogr. Sci., 18, 525 (1980).
19. T. J. Bruno, "Chromatographic and Electrophoretic Methods"., Englewood Cliffs, New Jersey (1991).
20. C. T. Wehr, and R. E. Majors, Regeneration of Biopolymer Columns., 5, 942 (1987).
21. C. T. Mant, and R. S. Hodges, Requirement for Peptide Standards in Reversed-phase HPLC., 4, 250 (1986).
22. C. Horvath, W. Melander, I. Molnar, and P. Molnar, Anal. Chem., 125, 129 (1976).
23. C. T. Mant, "High-Performance Liquid Chromatography of Peptides and Proteins:Separation, Analysis, and Conformation"., Alberta, Canada (1991).
24. J. O. Gregory, and W. J. James, Anal. Chem., 69,2283 (1997).
25. R. Purohit and S. Devi, Anal. Chim. Acta., 259, 53 (1992).
26. X. P. Yan and F. Adams, J. Anal. At. Spectrom., 12, 459 (1997).
27. K. E. LaFreniere, V. A. Fassel, and D. E. Eckels, Anal. Chem., 59, 879 (1987).
28. G. K. C. Low, G. E. Batley, and S. J. Buchanan, Chromatographia., 22, 292 (1986).
29. M. Thompson, and J. N. Walsh, "Handbook of Indcvtively Coupled Plasma Spectrometry"., Chapman and Hall, New York (1989).
30. K. E. Jarvis, A. L. Gray, and R. S. Houk, "Handbook of Indcvtively Coupled Plasma Spectrometry"., Blackie, Glasgow (1992).
31. B. Gercken, and R. M. Barnes. Anal. Chem., 63, 283 (1991).
32. V. E. Negretti de Bratter, S. Recknagel, and D. Gawlik, Fresenius J. Anal. Chem., 353, 137 (1995).
33. K. T. Suzuki, S. Yoneda, M. Itoh, and M. Ohmichi, J. Chromatogr. Biomed. Appl., 670, 63 (1995).
34. K. T. Suzuki, M. Itoh, and M. Ohmichi, Toxicology., 103, 157 (1995).
35. K. T. Suzuki, M. Itoh, and M. Ohmichi, J. Chromatogr. Biomed. Appl., 666, 13 (1995).
36. A. Makarov, and J. Szpunar, Analusis., 26, M44 (1998).
37. L. M. Owen, H. M. Crews, and R. C. Hutton, A. Amino acid. Analyst., 117, 649 (1992).
38. C. K. Shum, and R. S. Houk, Anal. Chem., 65, 2972 (1993).
39. K. A. High , J. S. Blais, B. A. J. Methven, Analyst., 120, 629 (1992).
40. J. Wang, R. S. Houk, D. Dreessen, and D. R. Wiederin., J. Am. Chem. Soc., 120, 5793 (1998).
41. K. Takatera, N. Osaki, H. Yamaguchi, and T. Watanabe, Anal. Sci., 10, 567 (1994).
42. J. Szpunar, H. Chasaigne, O. Donard, J. Bettmer, R. Lobinski, "Applications of ICP-MS"., RSC, Cambridge (1997).
43. A. H. Pan, F. Tie, B. G. Ru, L. Y. Li, and T. Shen, Biomed. Chromatogr., 6, 205 (1992).
44. G. J. Opiteck, and J. W. Jorgenson, Anal. Chem., 69, 2283 (1997).
45. R. Lobinski, and J. Szpunar, Analytica Chimica Acta., 400, 321 (1999).
46. C. J. Chen, M.M. Wu, S. S. Lee, J. D. Wang, S. H. Cheng, and H. Y. Wu, Arteriosclerosis., 8, 452 (1988).
47. 黃雅莉，國防醫學院公共衛生研究所碩士論文 (1995).
48. X. C. Le, W. R. Cullen, and K. J. Reimer, Talanta., 41, 495 (1994).
49. C. J. Hwang, and S. J. Jiang, Anal. Chim. Acta., 289, 205 (1994).
50. J. Wang, M. J. Tomlinson, and J. A. Caruso, J. Anal. At. Spectrom., 10, 601 (1995).
51. P. Bavazzano, A. Perico, K. Rosendahl, and P. Apostoli, J. Anal. At. Spectrom., 11, 521 (1996).
52. R. Cornelis, and J. D. Kimpe, J. Anal. At. Spectrom., 9, 945 (1994).
53. L. Hagel and J. C. Janson, J. Chromatogr. Libr., 51A, A267 (1992).
54. L. R. Snyder and J. J. Kirkland, "Introduction to modern Liquid Chromatography" (1979).
55. D. L. Allara and W. J. Howe, "Size Exclusion Chromatography" (1980).
56. M. Potschka and P. L. Dubin, "Strategies in Size Exclusion Chromatography", Anaheim, California (1995).
57. Wu. Chi-san, "Handbook of Size Exclusion Chromatography", Wayne, New Jersey (1995).
58. Training course, Liquid Chromatography School, Waters.
59. P. T. Kissinger, Anal. Chem., 49, 883 (1977).
60. L. Stryer, Biochemistry., W. H. Freeman and Company, New York (1995).
61. B. A. Bidlingmeyer, J. Chromatographic Science., 18, 525 (1980).
62. H. P. J. Bennett, J. Chromatogr., 226, 501 (1983).
63. K. Hunter, At. Spectrosc., 15, 17 (1994).
64. S. H. Tam and G. Horlick, J. Anal. At. Spectrom., 2, 745 (1987).
65. G. J. Batterham, N. C. Munksgaard and D. L. Parry, J. Anal. At. Spectrom., 12, 1277 (1997).
66. P. Schramel, Fresenius Z. Anal. Chem., 320, 233 (1985).
67. M. G. Minnich and R. S. Houk, J. Anal. At. Spectrom., 13, 167 (1998).
68. R. I. Botto and J. J. Zhu, J. Anal. At. Spectrom., 9, 905 (1994).
69. J. Yang, T. S. Conver, J. A. Koropchak and D. A. Leihhty, Spectrochimica Acta Part B., 51, 1491 (1996).
70. 行政院國家科學委員會精密儀器發展中心出版, 科儀新知, 第20卷, 第一期, 21.
71. I. B. Brenner, A. Zander, M. Plantz and J. Zhu, J. Anal. At. Spectrom., 12, 273 (1997).
72. D. R. Wiederin and R. S. Houk, Appl. Spectrosc., 45, 1408 (1991).
73. R. A. Copeland, "Method for Protein Analysis", Chapman and Hall (1994).
74.曹鳳琪，國立清華大學原子科學研究所碩士論文 (1995).
75. B. Wallaeys, R. Cornelis, E. Belpaire and N. Lameire, "in Trace Element Analytical Chemistry in Medicine and Biology 4", ed. P. Schramel, Walter de Gruyter, Berlin, 451 (1987).
76. H. Schluter and W. Zidek, J. Chromatogr., 639, 17 (1993).
77.柯富祥，國立清華大學原子科學研究所博士論文 (1996).
78. D. Kimpe, J. Cornelis, R. Mees and V. Holder, "in Trace Element in Man and Animals", Proceeding of the Eighth International Symposium, TEMA 8, ed. Anke, M., 845 (1993).
79. R. L. Warren, J. H. Steve, Fresenius J. Anal. Chem., 355, 202 (1996).
80.夏台陵，國立清華大學原子科學研究所碩士論文 (2000).
81.黃傳捷，國立清華大學原子科學研究所博士論文 (1997).