臺灣博碩士論文加值系統

生物胺是一種低分子含氮化合物，主要由胺基酸經脫羧作用後形成。生物胺廣泛分佈在各種蔬果與食品中，對生物的溫度調節、蛋白質合成、腸道免疫等生理功能非常重要。然而，若人體攝入過量的生物胺，就可能會對人體造成過敏或中毒反應，症狀包括有噁心、呼吸窘迫、頭痛、心悸、紅疹、低血壓或高血壓等。在目前的生物胺調查相關文獻中，使用非衍生化的前處理方法搭配LC-MS/MS檢測蔬菜樣品的文獻相對較少，因此本研究以非衍生化方法搭配LC-MS/MS，針對蔬菜樣品開發同時檢測九種生物胺之分析方法，並將此方法應用於國產蔬菜中生物胺的檢測上。本研究取2 g的均質樣品，加入5% 甲酸之水溶液10 mL震盪後，再超音波震盪後離心，取上清液用濾膜過濾至離心管中，取過濾後的濾液和等量的純水混合後以LC-MS/MS 分析。在添加標準品之純水樣品中，每種生物胺線性範圍皆能達到R2值大於0.99，兩種蔬菜樣品(蘿美心、高麗菜)的精密度的變異係數皆小於20%，萃取效率皆在70 - 120%的範圍。此外，以本研究之方法分析18種不同的蔬菜後發現，18件不同的蔬菜樣品皆含有腐胺與亞精胺，在小番茄、牛番茄與菠菜中均有檢驗到酪胺與組織胺，此結果與過往文獻敘述與分析結果相似1,2，不過本研究的台灣菠菜分析出的生物胺濃度比其他研究中不同產地(義大利與巴西)的菠菜多出至少一倍。本研究建立的方法可以應用在蔬菜樣品上的生物胺濃度檢測，且前處理時間較短，步驟較簡單，適合分析大量的蔬菜樣品生物胺濃度檢測，期望未來以此方法分析各種蔬菜樣品生物胺濃度，評估計算出各種蔬菜的營養指數或風險指數。

Biogenic amines is a group of low-molecular-weight nitrogen-containing compounds, which is mainly formed by decarboxylation of amino acids. Biogenic amines are widely distributed in various fruits, vegetables and foods, and are very important for biological functions such as temperature regulation, protein synthesis, and intestinal immunity. However, if the human body consumes excessive amounts of biogenic amines, it may cause allergic or toxic reactions to the human body. Symptoms include nausea, respiratory distress, headache, palpitations, rash, hypotension or hypertension, etc. In the current literature related to the investigation of biogenic amines, there are relatively few literatures on the use of non-derivatization preparation combined with LC-MS/MS to detect biogenic amines in vegetable samples. The samples were developed to detect nine kinds of biogenic amines at the same time, and this method was applied to the detection of biogenic amines in domestic vegetables. In this method, 2 g of homogeneous samples were taken, 10 mL of 5% formic acid aqueous solution was added to shake, and then ultrasonically shaken and centrifuged. Water was mixed and analyzed by LC-MS/MS. In the pure water sample added with the standard, the linear range of each biogenic amine can reach values greater than 0.99. For 2 vegetables spiked at 3 levels, the CV% were less than 20%. and the extraction efficiency was in the range of 70 - 120%. In addition, after analyzing 18 different vegetables with the method of this study, it was found that all 18 different vegetable samples contained putrescine and spermidine, and tyramine and histamine were detected in cherry tomatoes, beef tomatoes and spinach. This result is similar to the previous literature description and analysis results1,2, but the concentration of biogenic amines analyzed in Taiwan spinach in this study is at least double that of spinach from different origins (Italy and Brazil) in other studies. The method established in this study can be applied to the detection of biogenic amines on vegetable samples, and the pretreatment time is short and the steps are relatively simple. It is suitable for the analysis of a large number of vegetable samples for the detection of biogenic amine concentration. It is expected that this method will be used to analyze various vegetable samples in the future. concentration, evaluate and calculate the nutritional index or risk index of various vegetables.

中文摘要 ......i
英文摘要 ......ii
誌謝......iv
目錄......v
表目錄......viii
圖目錄......ix
第一章 緒論......1
1.1 前言......1
1.2 蔬果中的生物胺......2
1.3 生物胺的樣品前處理方法......2
1.3.1 直接稀釋和蛋白質沉澱......3
1.3.2 固液萃取(SLE)......3
1.3.3 超音波輔助萃取(UAE)......4
1.3.4 固相萃取(SPE)......4
1.4 生物胺的分析方法......5
1.4.1 液相層析結合紫外光分析(LC-UV)......5
1.4.2 液相層析結合螢光分析(LC-FL)......5
1.4.3 液相層析串聯質譜(LC-MS/MS)......6
1.4.4 離子配對層析法(IPC)......7
1.4.5 生物胺的分析方法總結......7
1.5 研究動機......8
第二章 材料與方法......9
2.1 藥品及實驗材料......9
2.1.1 標準品......9
2.1.2 試劑及材料......9
2.2 儀器設備及器材......9
2.3 標準品溶液配置......10
2.4 樣品前處理方法......10
2.5 液相層析串聯質譜方法......11
2.5.1 生物胺液相層析條件......11
2.5.2 液相層析質譜參數設定......11
2.6 生物胺方法性能測試......12
2.6.1 線性與定量極限測試......12
2.6.2 精密度與準確度測試......12
2.6.3 基質效應測試......12
2.6.4 萃取效率測試......13
2.7 實際蔬菜樣品分析......13
第三章 結果與討論......14
3.1 標準品之測試......14
3.1.1 生物胺標準品質譜分析......14
3.1.2 LC-MS/MS生物胺標準品......14
3.2 樣品萃取方法及條件測試......16
3.2.1 萃取液選擇測試......16
3.2.2 超音波震盪測試......16
3.2.3 前處理萃取後淨化測試......17
3.2.4 實際蔬菜樣品的初步分析......17
3.2.5 分析添加樣品的層析管柱與動相溶液比例測試......18
3.3 方法性能測試......19
3.3.1線性範圍與定量極限......19
3.3.2精密度與準確度......19
3.3.3萃取效率......20
3.4 蔬菜樣品分析......21
第四章 結論......22
參考文獻......51
英文論文......56

1Moret, S., Smela, D., Populin, T. & Conte, L. S. A survey on free biogenic amine content of fresh and preserved vegetables. Food chemistry 89, 355-361 (2005).
2Dala-Paula, B. M., Starling, M. d. F. V. & Gloria, M. B. A. Vegetables consumed in Brazilian cuisine as sources of bioactive amines. Food Bioscience 40, doi:10.1016/j.fbio.2020.100856 (2021).
3Michalski, R., Pecyna-Utylska, P. & Kernert, J. Determination of ammonium and biogenic amines by ion chromatography. A review. Journal of Chromatography A 1651, 462319 (2021).
4Shalaby, A. R. Significance of biogenic amines to food safety and human health. Food research international 29, 675-690 (1996).
5Banks, C. N. & Adams, M. E. Biogenic amines in the nervous system of the cockroach, Periplaneta americana following envenomation by the jewel wasp, Ampulex compressa. Toxicon 59, 320-328 (2012).
6Bouchereau, A., Guénot, P. & Larher, F. Analysis of amines in plant materials. Journal of Chromatography B: Biomedical Sciences and Applications 747, 49-67 (2000).
7Bulushi, I. A., Poole, S., Deeth, H. C. & Dykes, G. A. Biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation—a review. Critical reviews in food science and nutrition 49, 369-377 (2009).
8Del Rio, B. et al. The biogenic amines putrescine and cadaverine show in vitro cytotoxicity at concentrations that can be found in foods. Scientific reports 9, 1-7 (2019).
9Anal, A. K. et al. Food safety risks in traditional fermented food from South-East Asia. Food Control 109, 106922 (2020).
10Saaid, M., Saad, B., Ab Rahman, I., Ali, A. S. M. & Saleh, M. I. Extraction of biogenic amines using sorbent materials containing immobilized crown ethers. Talanta 80, 1183-1190 (2010).
11Wei, F. et al. Irradiated Chinese Rugao ham: Changes in volatile N-nitrosamine, biogenic amine and residual nitrite during ripening and post-ripening. Meat Science 81, 451-455 (2009).
12Regulation, E. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union 338, 1-29 (2005).
13Anlı, R. E. & Bayram, M. Biogenic amines in wines. Food Reviews International 25, 86-102 (2008).
14Zhang, X. et al. Determination of 6 biogenic amines in food using high-performance liquid chromatography-tandem mass spectrometry without derivatization. J Chromatogr A 1653, 462415, doi:10.1016/j.chroma.2021.462415 (2021).
15Zhang, X. et al. Determination of 8 biogenic amines in aquatic products and their derived products by high-performance liquid chromatography-tandem mass spectrometry without derivatization. Food Chem 361, 130044, doi:10.1016/j.foodchem.2021.130044 (2021).
16Jastrzȩbska, A., Piasta, A. & Szłyk, E. Application of ion chromatography for the determination of biogenic amines in food samples. Journal of analytical chemistry 70, 1131-1138 (2015).
17Papageorgiou, M. et al. Literature update of analytical methods for biogenic amines determination in food and beverages. TrAC Trends in Analytical Chemistry 98, 128-142 (2018).
18Palermo, C., Muscarella, M., Nardiello, D., Iammarino, M. & Centonze, D. A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages. Analytical and bioanalytical chemistry 405, 1015-1023 (2013).
19Manetta, A. C. et al. Evaluation of biogenic amines in wine: determination by an improved HPLC-PDA method. Food Control 62, 351-356 (2016).
20La Torre, G. L., Saitta, M., Potortì, A. G., Di Bella, G. & Dugo, G. High performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry for sensitive determination of bioactive amines in donkey milk. Journal of Chromatography A 1217, 5215-5224 (2010).
21Nguyen, A., Aerts, T., Van Dam, D. & De Deyn, P. Biogenic amines and their metabolites in mouse brain tissue: Development, optimization and validation of an analytical HPLC method. Journal of Chromatography B 878, 3003-3014 (2010).
22Mayer, H., Fiechter, G. & Fischer, E. A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese. Journal of Chromatography a 1217, 3251-3257 (2010).
23Shukla, S., Park, H.-K., Kim, J.-K. & Kim, M. Determination of biogenic amines in Korean traditional fermented soybean paste (Doenjang). Food and Chemical Toxicology 48, 1191-1195 (2010).
24Andic, S., Genccelep, H. & Kose, S. Determination of biogenic amines in herby cheese. International Journal of Food Properties 13, 1300-1314 (2010).
25Lázaro, C. A. et al. Validation of an HPLC methodology for the identification and quantification of biogenic amines in chicken meat. Food Analytical Methods 6, 1024-1032 (2013).
26Gong, X., Qi, N., Wang, X., Lin, L. & Li, J. Ultra-performance convergence chromatography (UPC2) method for the analysis of biogenic amines in fermented foods. Food chemistry 162, 172-175 (2014).
27Pawar, R. S., Grundel, E., Fardin-Kia, A. R. & Rader, J. I. Determination of selected biogenic amines in Acacia rigidula plant materials and dietary supplements using LC–MS/MS methods. Journal of Pharmaceutical and Biomedical Analysis 88, 457-466 (2014).
28Tahmouzi, S., Khaksar, R. & Ghasemlou, M. Development and validation of an HPLC-FLD method for rapid determination of histamine in skipjack tuna fish (Katsuwonus pelamis). Food Chemistry 126, 756-761 (2011).
29Zhang, X. et al. Determination of 8 biogenic amines in aquatic products and their derived products by high-performance liquid chromatography-tandem mass spectrometry without derivatization. Food Chemistry 361, 130044 (2021).
30Sagratini, G. et al. Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography–tandem mass spectrometry. Food Chemistry 132, 537-543 (2012).
31Zhang, Y.-j. et al. A review of pretreatment and analytical methods of biogenic amines in food and biological samples since 2010. Journal of Chromatography A 1605, 360361 (2019).
32Zhu, L. et al. A novel ultra-performance liquid chromatography hyphenated with quadrupole time of flight mass spectrometry method for rapid estimation of total toxic retronecine-type of pyrrolizidine alkaloids in herbs without requiring corresponding standards. Food Chemistry 194, 1320-1328 (2016).
33Jia, S., Kang, Y. P., Park, J. H., Lee, J. & Kwon, S. W. Determination of biogenic amines in Bokbunja (Rubus coreanus Miq.) wines using a novel ultra-performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry. Food chemistry 132, 1185-1190 (2012).
34Önal, A., Tekkeli, S. E. K. & Önal, C. A review of the liquid chromatographic methods for the determination of biogenic amines in foods. Food chemistry 138, 509-515 (2013).
35Soufleros, E., Bouloumpasi, E., Zotou, A. & Loukou, Z. Determination of biogenic amines in Greek wines by HPLC and ultraviolet detection after dansylation and examination of factors affecting their presence and concentration. Food Chemistry 101, 704-716 (2007).
36沈振峰 & 何國榮. 液相層析質譜術在藥物分析之應用. 藥物食品分析 3, 243-258 (1995).
37Kushnir, M. M., Rockwood, A. L. & Bergquist, J. Liquid chromatography–tandem mass spectrometry applications in endocrinology. Mass spectrometry reviews 29, 480-502 (2010).
38Sagratini, G. et al. Simultaneous determination of eight underivatised biogenic amines in fish by solid phase extraction and liquid chromatography-tandem mass spectrometry. Food Chem 132, 537-543, doi:10.1016/j.foodchem.2011.10.054 (2012).
39Knox, J. H. & Laird, G. R. Soap chromatography—a new high-performance liquid chromatographic technique for separation of ionizable materials: dyestuff intermediates. Journal of Chromatography A 122, 17-34 (1976).
40Eksborg, S., Lagerström, P.-O., Modin, R. & Schill, G. Ion-pair chromatography of organic compounds. Journal of Chromatography A 83, 99-110 (1973).
41Ochi, N. Simultaneous determination of eight underivatized biogenic amines in salted mackerel fillet by ion-pair solid-phase extraction and volatile ion-pair reversed-phase liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1601, 115-120 (2019).
42Nishigaki, A. et al. Simultaneous determination of cationic and anionic surfactants in environmental water samples by ion-pair liquid chromatography/mass spectrometry. Current Chromatography 7, 57-64 (2020).
43Kaczmarkiewicz, A., Nuckowski, Ł., Studzińska, S. & Buszewski, B. Analysis of antisense oligonucleotides and their metabolites with the use of ion pair reversed-phase liquid chromatography coupled with mass spectrometry. Critical Reviews in Analytical Chemistry 49, 256-270 (2019).
44Karaca, S. A. & Ugur, D. Y. A stability indicating ion-pair lc method for the determination of asenapine in pharmaceuticals. Journal of the Chilean Chemical Society 62, 3325-3329 (2017).
45Zhang, Y.-n., Yu, H., Ma, Y.-j. & Cui, G. Imidazolium ionic liquids as mobile phase additives in reversed phase liquid chromatography for the determination of iodide and iodate. Analytical and bioanalytical chemistry 410, 7347-7355 (2018).
46Wang, G. & Tomasella, F. P. Ion-pairing HPLC methods to determine EDTA and DTPA in small molecule and biological pharmaceutical formulations. Journal of Pharmaceutical Analysis 6, 150-156 (2016).
47Ito, S., Del Bino, S., Hirobe, T. & Wakamatsu, K. Improved HPLC conditions to determine eumelanin and pheomelanin contents in biological samples using an ion pair reagent. International journal of molecular sciences 21, 5134 (2020).
48Campbell, T. D., Febrian, R., Kleinschmidt, H. E., Smith, K. A. & Bracher, P. J. Quantitative analysis of glycine oligomerization by ion-pair chromatography. ACS omega 4, 12745-12752 (2019).
49Srijaranai, S., Burakham, R., Deming, R. L. & Khammeng, T. Simplex optimization of ion-pair reversed-phase high performance liquid chromatographic analysis of some heavy metals. Talanta 56, 655-661 (2002).
50Park, U.-J., Choi, K.-H., Lee, J.-S., Cho, E.-H. & Yu, K.-H. Reversed-phase ion-pair liquid chromatography for the purification of 177Lu. Journal of Radioanalytical and Nuclear Chemistry 310, 339-346 (2016).
51Dala-Paula, B. M., Maria de Fátima, V. S. & Gloria, M. B. A. Vegetables consumed in Brazilian cuisine as sources of bioactive amines. Food Bioscience 40, 100856 (2021).
52Angulo, M. F., Flores, M., Aranda, M. & Henriquez-Aedo, K. Fast and selective method for biogenic amines determination in wines and beers by ultra high-performance liquid chromatography. Food chemistry 309, 125689 (2020).
53 Pearson, G. Perchloric acid. Advances in Inorganic Chemistry and Radiochemistry 8, 177-224 (1966).