臺灣博碩士論文加值系統

摘要

近年來酵母食品中的硒化合物備受注目，臨床研究發現每日攝取200 μg有機硒酵母可以降低癌症的發生率與死亡率。開發一套靈敏、簡單的分析技術測定健康食品中selenoamino acids的含量是必要的。
本研究針對selenoamino acids發展毛細管電泳 / 氦鎘雷射激發螢光偵測法。利用2,3-naphthalenedicarboxaldehyde ( NDA ) 與非螢光性的selenocystine、Se-methylcysteine、D-methionine、selenomethionine進行管柱前衍生，以管壁塗佈polydimethylacrylamide ( PDMA ) 之毛細管，成功分離四種NDA- selenoamino acids衍生物。各分析物遷移時間的相對標準偏差( RSD )為2.3 ~ 2.6 %，訊號面積的RSD值為3.6 ~ 8.6 % ( n = 7 )。Selenocystine與Se-methylcysteine的檢量線線性範圍分別為50 ~ 500 μM與0.15 ~ 10 μM，D-methionine與D, L- selenomethionine鏡像異構物的線性範圍分別為0.1 ~ 10 μM與1 ~ 100 μM。4種selenoamino acids偵測極限範圍為0.03 ~ 25 μM。結合酵素萃取方法進行酵母標準參考樣品SELM-1中selenomethionine的分析，實驗結果顯示L-selenomethionine偵測含量僅為已知含量的50.5 %。分析值與標示值差異顯著的主要原因是酵素萃取不完全，因此在酵母樣品的前處理方面仍需進一步改良。

目錄

摘要……………………………………………………………………….I
目錄………………………………………………………………………II
表目錄…………………………………………………………………...V
圖目錄…………………………………………………………………..VI

內容
壹、緒論
1.1 毛細管電泳的原理………………………………………………….1
1.2 毛細管電泳-雷射激發螢光偵測……………………………………3
1.2.1 螢光偵測方法…………………………………………………4
1.3 硒…………………………………………………………………….6
1.3.1 硒的生理作用………………………………………………....7
1.3.2 無機硒與有機硒……………………………………………....8
1.3.3 硒的攝取……………………………………………………..10
1.3.4 有機硒的分析方法…………………………………………..10
1.4 研究目的…………………………………………………………...28
貳、實驗部份
2.1 儀器設備…………………………………………………………...29
2.2 化學藥品…………………………………………………………...34
2.3 分析物結構與pKa值……………………………………………..36
2.4 實驗方法
2.4.1 電泳緩衝溶液的配製………………………………………..37
2.4.2 樣品儲存溶液的配製………………………………………..37
2.4.3 有機硒化合物標準品溶液衍生步驟………………………..37
2.4.4 硒酵母標準參考樣品 ( SELM-1 ) 萃取步驟………………38
2.4.5 硒酵母標準參考樣品 ( SELM-1 ) 萃取液衍生反應………38
2.4.6 毛細管內壁塗佈程序………………………………………..38
2.4.7 PDMA塗佈毛細管處理與樣品注射方式…………………...39
參、結果與討論
3.1 PDMA塗佈毛細管…………………………………………………41
3.2 α-環糊精對selenoamino acids分離之影響………………………..43
3.3 最佳實驗條件探討
3.3.1 衍生反應……………………………………………………..55
3.3.2 衍生pH值的影響……………………………………………56
3.3.3 衍生反應時間的影響………………………………………..60
3.3.4 衍生反應溫度………………………………………………..60
3.3.5 電泳緩衝溶液濃度之影響………………………………......63
3.3.6 電泳緩衝溶液pH值之影響………………………………....63
3.3.7 管柱塗佈的再現性…………………………………………..66
3.3.8 進樣時間之影響…………………………………………......70
3.3.9 最佳分析條件………………………………………………..70
3.3.10 檢量線與偵測極限之探討………………………………....76
3.3.11 分析再現性……………………………………………..…..84
3.4 真實樣品分析……………………………………………………...86
肆、結論……………………………………………………………….90
伍、參考文獻…………………………………………………………92

表目錄

表( 1 ) 氣相層析法分析selenoamino acids之報導…………………..12
表( 2 ) 高效能液相層析法分析selenoamino acids之報導…………...15
表( 3 ) 毛細管電泳分析selenoamino acids之報導…………………...22
表( 4 ) NDA-selenoamino acids之移動時間的ANOVA分析…………68
表( 5 ) NDA-selenoamino acids之訊號面積的ANOVA分析…………69
表( 6 ) NDA-selenoamino acids之檢量線……………………………...78
表( 7 ) 水溶液樣品中NDA-selenoamino acids的分析再現性……….85
表( 8 ) SELM-1標準參考樣品中L-SeMet的分析結果……………….89

圖目錄

圖( 1 ) 毛細管電泳系統裝置圖………………………………………...2
圖( 2 ) 雷射激發螢光偵測器內部元件構造………………………….32
圖( 3 ) 毛細管電泳/雷射激發螢光偵測器裝置圖……………………33
圖( 4 ) 一般毛細管分離NDA-selenoamino acids之電泳圖…………42
圖( 5 ) 毛細管壁塗佈PDMA的步驟…………………………………44
圖( 6 ) PDMA塗佈毛細管分離NDA-selenoamino acids的電泳圖.....45
圖( 7 ) 環糊精分子結構圖……………………………………………..47
圖( 8 ) 環糊精分子結構側視圖………………………………………..48
圖( 9 ) 環糊精與分析物於水溶液中的作用模式……………………..49
圖( 10 ) 緩衝溶液中添加不同濃度α- CD對NDA-selenoamino acids分離的影響……………………………………………………51
圖( 11 ) 緩衝溶液中α-CD濃度對selenoamino acids移動時間的影
響………………………………………………………………52
圖( 12 ) 緩衝溶液中α-CD濃度對D-與L-SeMet解析度的影響…...54
圖( 13 ) NDA與一級胺化合物的衍生機制…………………………...57
圖( 14 ) NDA與SeMet的衍生反應……………………………………58
圖( 15 ) 衍生反應pH對selenoamino acids訊號面積的影響………..59
圖( 16 ) 衍生反應時間對selenoamino acids訊號面積的影響……….61
圖( 17 ) 衍生反應溫度對selenoamino acids訊號面積的影響………62
圖( 18 ) 電泳緩衝溶液濃度對selenoamino acids移動時間的影響…64
圖( 19 ) 電泳緩衝溶液濃度對selenoamino acids訊號面積的影響…65
圖( 20 ) 電泳緩衝溶液pH值對NDA-selenoamino acids移動時間的影響………………………………………………………………67
圖( 21 ) 進樣時間對NDA-SeCys訊號面積及理論板數的影響…….71
圖( 22 ) 進樣時間對NDA-Se-mcys訊號面積及理論板數的影響…..72
圖( 23 ) 進樣時間對NDA-D-Met訊號面積及理論板數的影響…….73
圖( 24 ) 進樣時間對NDA-D-SeMet訊號面積及理論板數的影響….74
圖( 25 ) 進樣時間對NDA-L-SeMet訊號面積及理論板數的影響.….75
圖( 26 ) NDA-selenoamino acids衍生產物電泳圖………………….…77
圖( 27 ) NDA-SeCys的檢量線…………………………………………79
圖( 28 ) NDA-Se-mcys的檢量線………………………………………80
圖( 29 ) NDA-D-Met檢量線…………………………………………...81
圖( 30 ) NDA-D-SeMet的檢量線……………………………………...82
圖( 31 ) NDA-L-SeMet的檢量線………………………………………83
圖( 32 ) SELM-1標準參考樣品之電泳圖……………………………..87

伍、參考文獻

[1] Hayes, M. A., Ewing, A. G., Anal. Chem., 1992, 64, 512-516.
[2] Swaile, D. F., Sepaniak, M. J., Anal. Chem., 1991, 63, 179-184.
[3] Hogan, B. L., Yeung, E. S., Anal. Chem., 1992, 64, 2841-2845.
[4] Dolnlk, V., Dolnlkova, J., J. Chromatogr. A, 1995, 716, 269-277.
[5] Schneede, J., Ueland, P. M., Anal. Chem., 1995, 67, 812-819.
[6] Kenney, B. F., J. Chromatogr., 1991, 546, 423-430.
[7] Lalljie, S. P. D., Vindevogel, J., Sandra, P., J. Chromatogr. A, 1993, 652, 563-569.
[8] Karovicova, J., Polonsky, J., Drdak, M., Simko, P., Vollek, V., J. Chromatogr., 1993, 638, 241-246.
[9] Lagoutte, D., Lombard, G., Nisseron, S., Papet, M. P., Saint-Jalm, Y., J. Chromatogr. A, 1994, 684, 251-257.
[10] Thompson, C. O., Trenerry, V. C., Kemmery, B., J. Chromatogr. A, 1995, 704, 203-210.
[11] Bachmann, K., Boden, J., Haumann, I., J. Chromatogr., 1992, 626, 259-265.
[12] Chao, Y. C., Whang, C. W., J. Chromatogr. A, 1994, 663, 229-237.
[13] Zlotorzynska, E. D., Dlouhy, J. F., J. Chromatogr. A, 1994, 685, 145-153.
[14] Lurie, I. S., Klein, R. F. X., Dal Cason, T. A., LeBelle, M. J., Brenneisen, R., Weinberger, R. E., Anal. Chem., 1994, 66, 4019-4026.
[15] Baba, Y., J. Chromatogr. B., 1996, 687, 271-302.
[16] Shihabi, Z. K., J. Chromatogr. A., 1999, 853, 349-354.
[17] Schiewe, J., Mrestani, Y., Neubert, R., J. Chromatogr. A., 1995, 717, 255-259.
[18] Lin, C. E., Chang, C. C., Lin, W. C., Lin, E. C., J. Chromatogr. A., 1996, 753, 133-138.
[19] Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D., Anal. Chem. 1987, 59, 1230-1232.
[20] Smith, R. D., Loo, J. A., Edmonds, C. G., Barinaga, C. J., Udseth, H. R., J. Chromatogr. 1990, 516, 157-165.
[21] Hallen, R. W., Shumate, C. B., Siems, W. F., Tsuda, T., Hill, H. H., J. Chromatogr. 1989, 480, 233-245.
[22] Deterding, L. J., Parker, C. E., Perkins, J. R., Moseley, M. A., Jorgenson, J. W., Tomer, K. B., J. Chromatogr., 1991, 554, 329-338.
[23] Moseley, M. A., Deterding, L. J., Jorgenson, J. W., Tomer, K. B., Anal. Chem., 1991, 63, 109-114.
[24] Cai, J., Henion, J., J. Chromatogr. A., 1995, 703, 667-692.
[25] Jin, W., Zhang, J., J. Chromatogr. A., 2000, 868, 101-107.
[26] Jorgenson, J. W., Lukacs, K. D., Anal. Chem., 1981, 53, 1298-1302.
[27] Gassmann, E., Kuo, J. E., Zare, R. N., Science. 1985, 230, 813-814.
[28] Chen, D. Y., Adelhelm, K., Cheng, X. L., Dovichi, N. J., Analyst.
1994, 119, 349-352.
[29] Couderc, D. F., Causse, E., Bayle, C., Electrophoresis. 1998,
19, 2777-2790.
[30] Bardelmeijer, H. A., Waterval, J. C. M., Lingeman, H., Hof, R. V., Bult, A., Underberg, W. J. M., Electrophoresis. 1997, 18, 2214-2227.
[31] Schwartz, K., Foltz, C. M., J. Am. Chem. Soc., 1957, 79, 3292-3293.
[32] Combs, G. F., Gray, W. P., Pharmacology. Therapeutics, 1998, 79, 179-192.
[33] Arthur, J. R., Can. J. Physiol. Pharmacol., 1991, 69, 1648-1652.
[34] Fleet, J. C., Nutr. Rev., 1997, 55, 277-279.
[35] Combs, G. F., Clark, L. C., Turnbull, B. W., Biomed. Environ. Sci., 1997, 10, 227-234.
[36] Schrauzer, G. N., J. Nutr., 2000, 130, 1653-1656.
[37] Maxon, A. L., Dubois, K. P., Potter, R. L., J. Pharmacol. Exptl. Therap., 1941, 72, 184-196.
[38] Meve, J., Vertongen, F., Molle, L., Clinics in endocrinology and metabolism, 1985, 14, 629-656.
[39] Arruda, M. A. Z., Gallego, M., J. Anal. At. Spectrum, 1994, 9, 657-662.
[40] Ferrer, E., Monleon, J., Sci. Total Environ, 1999, 227, 139-143.
[41] Bronzetti, G., Croce, C. D., J. Environ. Pathol. Toxicol. Oncol., 1993, 12, 59-71.
[42] Cunha, S. D., Filho, F. M. A., Sci. Total Environ, 2003, 301, 51-54.
[43] Calle-guntinas, M. B., Brunori, C., Scerbo, R., Chiavarini, S., Quevauviller, P., J. Anal. Atom. Spectrometry, 1997, 12, 1041-1046.
[44] Mendez, S. P., Bayon, M. M., Gonzalez, E. B., Sanz-Medel, A., J. Anal. Atom. Spectrometry, 1999, 14, 1333-1337.
[45] Pelaez, M. V., Bayon, M. M., Alonso, I. G., Sanz-Medel, A., J. Anal. Atom. Spectrometry, 2000, 15, 1217-1222.
[46] Devos, C., Sandra, K., Sandra, P., J. Pharam. Biomed. Anal., 2002, 27, 507-514.
[47] Vonderheide, A. P., Montes-Bayon, M., Caruso, J. A., Analyst, 2002, 127, 49-53.
[48] Iscioglu, B., Henden, E., Anal. Chim. Acta., 2004, 505, 101-106.
[49] Yang, L., Mester, Z., Sturgeon, R. E., Anal. Chem., 2004, 76, 5149-5156.
[50] Yang, L., Sturgeon, R. E., McSheehy, S., Mester, Z., J. Chromatogr. A, 2004, 1055, 177-184.
[51] Gilon, N., Potin-Gautier, M., Astruc, M., J. Chromatogr. A, 1996, 750, 327-334.
[52] Bird, S. M., Ge, H., Uden, P. C., Tyson, J. F., Block, E., Denoyer, E., J. Chromatogr. A, 1997, 789, 349-359.
[53] Bird, S. M., Uden, P. C., Tyson, J. F., Block, E., Denoyer, E., J. Anal. Atom. Spectrometry, 1997, 12, 785-788.
[54] Hammel, C., Kyriakopoulos, A., Rosick, U., Behne, D., Analyst, November 1997, 122, 1359-1363.
[55] Mendez, S. P., Gonzales, E. B., Fernandez, S., Maria, L., Medel, A. S., J. Anal. Atomic Spectrom., 1998, 13, 893-898.
[56] Casiot, C., Szpunar, J., Lobinski, R., Potin-Gautier, M., J. Anal. Atom. Spectrometry, 1999, 14, 645-650.
[57] Zheng, J., Ohata, M., Furuta, N., Kosmus, W., J. Chromatogr. A, 2000, 874, 55-64.
[58] Mendez, S. P., Gonzales, E. B., Medel, A. S., J. Anal. At. Spectrom., 2000, 15, 1109-1114.
[59] Sutton, K. L., Ponce de Leon, C. A., Ackley, K. L., Sutton, R. M. C., Stalcup, A. M., Caruso, J. A., Analyst, 2000, 125, 281-286.
[60] Marchante-Gayon, J. M., Thomas, C., Feldmann, I., Jakubowski, N., J. Anal. At. Spectrom., 2000, 15, 1093-1102.
[61] Montes-Bayon, M., B’Hymer, C., Ponce de Leon, C., Caruso, J. A., J. Anal. Atomic Spectrom., 2001, 16, 945-950.
[62] Vonderheide, A. P., Wrobel, K., Kannamkumarath, S. S., B’Hymer, C., Montes-Bayon, M., Caruso, J. A., J. Agric. Food Chem., 2002, 50, 5722-5728.
[63] Wrobel, K., Kannamkumarath, S. S., Wrobel, K., Caruso, J. A., Anal. Bioanal. Chem., 2003, 375, 133-138.
[64] Dumont, E., Cremer, K. D., Hulle, M. V., Chery, C. C., Vanhaecke, F., Cornelis, R., J. Anal. At. Spectrom., 2004, 19, 167-171.
[65] Pilar, V., Ignacio, L. G., Beatriz, M. M., Natalia, C., Manuel, H. C., Anal. Chim. Acta., 2005, 535, 49-56.
[66] Albert, M., Demesmay, C., Rocca, J. L., Fresenius J. Anal. Chem., 1995, 351, 426-432.
[67] Gilon, N., Potin-Gautier, M., J. Chromatogr. A, 1996, 732, 369-376.
[68] Michalke, B., Schramel, P., Electrophoresis, 1998, 19, 270-275.
[69] Michalke, B., Schramel, P., J. Chromatogr. A, 1998, 807, 71-80.
[70] Casiot, C., Alonso, M. C. B., Boisson, J., Donard, O. F. X., Potin-Gautier, M., Analyst, 1998, 123, 2887-2893.
[71] Michalke, B., Schramel, O., Kettrup, A., J. Anal. Chem., 1999, 363, 456-459.
[72] Sutton, K. L., Sutton, R. M. C., Stalcup, A. M., Caruso, J. A., Analyst, 2000, 125, 231-234.
[73] Zheng, J., Greschonig, H., Liu, F., Kosmus, W., Trace Elements and Electrolytes, 2000, 17, 40-47.
[74] Mounicou, S., McSheehy, S., Szpunar, J., Potin-Gautier, M., Lobinski, R., J. Anal. At. Spectrom., 2002, 17, 15-20.
[75] Casiot, C., Donard, O. F. X., Potin-Gautier, M., Spectrochimica Acta Part B, 2002, 57, 173-187.
[76] Chang, S. Y., Chiang, H. T., Electrophoresis, 2002, 23, 2913-2917.
[77] Day, J. A., Kannamkumarath, S. S., Yanes, E. G., Montes-Bayon, M., Caruso, J. A., J. Anal. Atomic Spectrom., 2002, 17, 27-31.
[78] Sun, B., Macka, M., Haddad, P. R., J. Chromatogr. A, 2004, 1039, 201-208.
[79] Bendahl, L., Gammelgaard, B., J. Anal. At. Spectrom., 2004, 19, 143-148.
[80] Kannamkumarath, S. S., Wrobel, K., Wuilloud, R. G., Talanta, 2005, 66, 153-159.
[81] Wang, J, Mannino, S., Camera, C., Chatrathi, M. P., Scampicchio,
M., Zima, J., J. Chromatogr. A., 2005, 1091, 177-182.
[82] Infante, H. G., Rayman, M., Wahlen, R., Entwisle, J., Norris, P., Hearn, R., Catterick, T., J. Anal. At. Spectrom., 2004, 19, 1529-1538.
[83] Polatajko, A., Sliwka Kaszynska, M., Dernovics, M., Ruzik, R.,
Encinar, J. R., Szpunar, J., J. Anal. At. Spectrom., 2004, 19, 114-120.
[84] Hong, W., Ohman, M., Blomberg, L. G., J. Chromatogr. A., 2001, 924, 59-70.
[85] Villiers, A., Compt. Rend. Acad. Sci. Paris., 1891, 112, 536-543.
[86] 陳鑫昌，「利用毛細管電泳結合線上濃縮方法分離奈磺酸鹽之
機制探討」，國立中央大學，碩士論文，民國92年。
[87] Quang, C., Khaledi, M. G., J. Chromatogr., 1995, 692, 253-265.
[88] Matchett, M. W., Branch, S. K., Jeffferies, T. M., J. Chromatogr., 1995, 705, 351-361.
[89] Mendez, S. P., Gonzalez, E. B., Medel, A. S., Anal. Chimica Acta., 2000, 416, 1-7.
[90] Roach, M. C., Harmony, M. D., Anal. Chem., 1987, 59, 411-415.
[91] Bostick, J. M., Strojek, J. W., Metcalf, T., Kuwana, T., Applied Spectroscopy, 1992, 46, 1532-1539.
[92] McSheehy, S., Pohl, P., Szpunar, J., Potin-Gautier, M., Lobinski, R., J. Anal. At. Spectrom., 2001, 16, 68-73.
[93] Encinar, J. R., Ruzik, R., Buchmann, W., Tortajada, J., Lobinski, R., Szpunar. J., Analyst, 2003, 128, 220-224.
[94] B’Hymer, C., Caruso, J., J. Anal. At. Spectrom., 2000, 15,
1531-1539.
[95] Wolf, W. R., Zainal, H., Yager, B., Fresenius J. Anal. Chem., 2001,370, 286-290.
[96] Dumont, E., De Cremer, K., Van Hulle, M., Chery, C. C.,
Van-haecke, F., Cornelis, R., J. Anal. At. Spectrom., 2004, 19,
167-171.
[97] Uden, P. C., Boakye, H. T., Kahakachchi, C., Hafezi, R., Nolibos, P., Block, E., Johnson, S., Tyson, J. F., J. Anal. At. Spectrom., 2004, 19, 65-73.
[98] Mester, Z., Willie, S., Yang, L., Sturgeon, R. Caruso, J. A., Fernandez, M. L., Fodor, P., Goldschmidt, R. J., Goenaga-Infante, H., Lobinski, R., Maxwell, P., McSheehy, S., Polatajko, A., Sadi, B. B. M., Sanz-Medel, A., Scriver, C., Szpunar, J., Wahlen, R., Wolf, W., Anal. Bioanal. Chem., 2006, 385, 168-180.