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標題: 利用HPLC-ELSD對三酸甘油酯種類分析方法之建立
The analysis and method development of the triglyceride species by using HPLC-ELSD
作者: 辜鈺惠
Ku, Yu-Hui
關鍵字: edible oil;食用油;triacylglycerol;evaporation light scattering detector (ELSD);三酸甘油酯;光揮發散射偵測器
出版社: 食品暨應用生物科技學系所
引用: 王三郎:應用微生物學(二版)。高立圖書有限公司,80-84(1999)。 李敏雄等:食品分析。藝軒圖書出版社,533-578(2005)。 何宥心:利用Mucor rouxii生產γ-次亞麻油酸之生長動力學研究與發酵饋料策略的應用。國立成功大學化學工程研究所碩士論文(2007)。 林瑩禎、李河水、華傑:機能性油脂在加工食品的應用研究。食品所(2002)。 周于嵐:脂質攝取對脂質代謝之影響。食品工業,39:22-29(2007)。 陳淑華:營養學(二版)。華香園出版社,103-106(1996)。 黃明利、黃文哲:應用微生物(三版)。精華出版社,65-71(1992)。 彭瑄第:微生物之多元不飽和脂肪酸。食品工業,39:46-52(2007)。 張為憲、李敏雄、呂政義…等:食品化學(初版)。華香園出版社,72-77 (1995)。 賴滋漢、金安兒:食品加工學加工篇(再版)。富林出版社,385-419 (2003)。 Aitzetmüller, K. (1975). The liquid chromatography of lipids : A critical review. Journal of Chromatography A, 113, 231-266. Andrikopoulos, N. K. (2002). Triglyceride species compositions of common edible vegetable oils and methods used for their identification and quantification. Food Review International, 18, 71-102. Anonymous.(2003). Waters 2420 ELS detector theory of operation. In: Waters 2420 Evaporative light scattering detector operator’ guide, edn. Pp. 1–15. The Waters Corporation, USA. Anonymous. 黃豆加工流程圖。大統益股份有限公司。Available from: 5 July 2009. AOAC. (2000). AOAC official method 996.06, fat (total, saturated, and unsaturated) in foods. In: Official methods of analysis of AOAC international (17th), edn. Pp. 20–24. Maryland: AOAC International. Barron, L. J. R. and Santa-María, G. (1989). HPLC analysis of complex mixtures of triglycerides using gradient elutions and an ultraviolet detector. Chromatographia, 28, 183-188. Blight, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917. Buchgraber, M., Ulberth, F., Emons, H. and Anklam, E. (2004). Triacylglycerol profiling by using chromatographic techniques. European Journal of Lipid Science and Technology, 106, 621-648. Carelli, A. A. and Cert, A. (1993). Comparative study of the determination of triacylglycerol in vegetable oils using chromatographic techniques. Journal of Chromatography A, 630, 213-222. Christie, W. W. (1992). Detectors for high-performance liquid chromatography of lipids with special reference to evaporative light-scattering detection. In: Advances in lipid methodology-one, edn (edited by W.W. Christie). Pp. 239–271. The Oily Press, Ayr, UK. Christie, W. W. (1987). High-performance liquid chromatography: theoretical considerations and equipment. In:HPLC and lipids, edn (edited by W.W. Christie). Pp. 8–41. Pergamon Press. Cunha, S. C. and Oliveira, M. B. P. P. (2006). Discrimination of vegetable oils by triacylglycerols evaluation of profile using HPLC/ELSD. Food Chemistry, 95, 518-524. Davies, R. J. (1992). Scale up of yeast technology. In: Industrial application of single cell oils, edn (edited by D. J. Kyle, and C. Ratledge). Pp. 196-218. American Oil Chemists’ Society, Champaign, IL. Easterling, E. R., French, W. T., Hernandez, R. and Licha, M. (2009). The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis. Bioresource Technology, 100, 356-361. El-Hamdy, A. H. and Perkins, E. G. (1981a). High performance reversed-phase chromatography of natural triglyceride mixtures. Journal of the American Oil Chemists'' Society, 58, 49-53. El-Hamdy, A. H. and Perkins, E. G. (1981b). High-performance reversed-phase chromatography of natural triglyceride mixtures: critical pair separation. Journal of the American Oil Chemists'' Society, 58, 867-872. Gaita, R. (2006). A revered phase HPLC method using evaporative light scattering detection (ELSD) for monitoring the reaction and quality of biodiesel fuels, Grace Davison discovery science. Available from: Accessed 30 May 2009. Ganger, A., Karande, A. A. and Rajasenkharan, R. (2001). Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis. Biochemical Journal, 360, 471-479. Genge, B. R., Wu, L. N. Y. and Wuthier, R. (2003). Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection. Analytical Biochemistry, 322, 104-115. Goiffon, J. P., Reminiac, C. and Furon, D. (1981). Application de la chromatographie haute performance à l''analyse des triglycérides des corps gras. II Grandeurs de rétention des triglycérides. Revue Francaise des Corps Gras, 28, 199–206. Haley, J. E. and Jack, R. C. (1974). Stereospecific analysis of triacylglycerols and major phosphoglycerides from Lipomyces lipoferus. Lipids, 9, 679-681. Harrison. (1927). Rhodotorula spp :Taxonomic Classification. Available from: Accessed 30 June 2009. Hierro, M. T., Tomás, M. C., Fernández-Martín, F. and Santa-María, G. (1992). Determination of the triglyceride composition of avocado oil by high-performance liquid chromatography using a light-scattering detector. Journal of Chromatography B, 607, 329-338 Holčapek, M., Lísa, M., Jandera, P. and Kabátová, N. (2005). Quantitation of triacylglycerols in plant oils using HPLC with APCI-MS, evaporative light-scattering, and UV detection. Journal of Separation Science, 28, 1315-1333. Jump, D. B. and Clarke, S. D. (1999). Regulation of gene expression by dietary fat. Annual Review of Nutrition, 19, 63-90. Lee, K. T., Jones, K. C. and Foglia, T. A. (2002). Separation of structured lipids by high-performance liquid chromatography. Chromatographia, 55, 197-201. Letter, W. S. (1993). A qualitative method for triglyceride analysis by HPLC using an evaporative light scattering detector. Journal of Liquid Chromatography & Related Technologies, 16, 225-239. Li, Q., Du, W. and Liu, D. (2008). Perspectives of microbial oils for biodiesel production. Microbiol Biotechnol, 80, 749-756. McNabb, T. J. (2000). The analysis of polar lipid classes by high performance liquid chromatography/evaporative light scattering detection. Kingston, RI 02881, USA – The State University of Rhode Island. Megoulas, N. C. and Koupparis, M. A. (2005a). Twenty years of evaporative light scattering detection. Critical reviews in analytical chemistry, 35, 301-316. Megoulas, N. C. and Koupparis, M. A. (2005b). Development and validation of a novel HPLC/ELSD method for the direct determination of tobramycin in pharmaceuticals, plasma, and urine. Analytical and Bioanalytical Chemistry, 382, 290-296. Moreau, R. A. (2005). The evaporative light-scattering detector as a tool for the analysis of lipids by HPLC. In: HPLC of Acyl Lipids, edn (edited by J-T (Ken) Lin, and T. A. Mckeon). Pp. 93-115. HNB. Nikolova-Damyanova, B. (1997). Reversed phase high performance liquid chromatography: general principles and application to the analysis of fatty acids and triacylglycerols. In: Advance in lipid methodology-four, edn (edited by W. W. Christie). Pp. 193-251. The Oily Press. Pauls, R. E. (1983). A time normalization study of the separation of olive oil triglycerides. Journal of the American Oil Chemists'' Society, 60, 819-822. Pei, P. T., Henly, R. and Ramachandran, S. (1975). New application of high pressure reversed-phase liquid chromatography in lipids. Lipids, 10, 152-156. Plattner, R. D., Spencer, G. F., Kleinman, R. (1977). Triglyceride separation by RP-HPLC. Journal of the American Oil Chemists'' Society, 54, 511-515. Plattner, R. D.(1981a). High performance liquid chromatography of triglycerides: Controlling selectivity with reverse phase columns. Journal of the American Oil Chemists'' Society, 58, 638-642 Plattner, R. D. (1981b). High performance liquid chromatography of triglycerides. Methods in Enzymology, 72, 21-34. Phillips, F. C., Erdahl, W. L., Nadenicek, L. J., Nutter, J. A., Schmit, J. A. and Privett, O. S.(1984a). Analysis of triglyceride species by high-performance liquid chromatography via a flame lonization detector. Lipids, 19, 142-150. Phillips, F. C., Erdahl, W. L., Schmit, J. A. and Privett, O. S.(1984b). Quantitative analysis of triglyceride species of vegetable oils by high-performance liquid chromatography via a flame lonization detector. Lipids, 19, 880-887. Podlaha, O., Töregård, B. (1982). A system for identification of triglycerides in RP-HPLC chromatograms based on equivalent carbon numbers. Journal of High Resolution Chromatography. 5, 553-558. Privett, O. S. and Erdahl, W. L. (1978). An improved flame ionization detector for high performance liquid chromatography. Analytical Biochemistry, 84, 449-461. Ratledge, C. (2004). Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86, 807-815. Ratledge, C. (2005). Single cell oils for the 21st century. In: Single cell oil, edn (edited by Z. Cohen, and C. Ratledge). Pp. 1-20. American Oil Chemists’ Society, Champaign, IL. Ratledge, C. and Cohen, Z. (2008). Microbial and algal oils: Do they have a future for biodiesel or as commodity oils? Lipid Technology, 20, 155-160. Ratlesge, C. (1989). Biotechnology of oils and fats. In: Microbial lipids Volume 2, edn (edited by C. Ratledge and S. G. Wilkinson). Pp. 567-668. Academic Press Inc. Rombaut, R., Clercq, N. D., Foubert, I., and Dewettinck, K. (2009). Triacylglycerol analysis of fats and oils by evaporative light scattering detecton. Journal of the American Oil Chemists'' Society, 86, 19-25. Schunk, T. C. and Burke, M. F. (1993). Bonded phase conformation and salvation effects on the stationary phase structure in reversed-phase liquid chromatography. Journal of Chromatography A, 656, 289-316. Scott, R. P. W. (1993). Control of retention by molecular interactions in reversed-phase chromatography. Journal of Chromatography A, 656, 51-68. Shimiziu, S., Kawashima, H., Shinmen, Y., Akimoto, K. and Yamada, H. (1988). Production of eicosapentaenoic acid by Mortierella fungi. Journal of the American Oil Chemists'' Society, 65, 1455-1459. Smit, H., Ykema, A., Verbree, E. C., Verwoert, I. I. G.. S., and Kater, M. M. (1992). Production of cocoa butter equivalents by yeast mutants. In: Industrial application of single cell oils, edn (edited by D. J. Kyle, and C. Ratledge). Pp. 185-195. American Oil Chemists’ Society, Champaign, IL. Stolyhwo, A., Colin, H., Martin, M. and Guiochon, G. (1984). Study of the qualitative and quantitative properties of the light-scattering detector. Journal of Chromatography, 288, 253-275. Thijssen, M. A. and Mensink, R. P. (2005). Fatty acids and atherosclerotic risk. Handbook of experimental pharmacology, 170, 165-194. Tsimidou, M. and Macrae, R. (1984). Influence of injection solvent on the reversed-phase chromatography of triglycerides. Journal of Chromatography A, 285, 178-181. Ward, O. P. and Singh, A. (2005). Omega-3/6 fatty acids: Alternative sources of production. Process Biochemistry, 40, 3627-3652. Yongmanitchai, W. and Ward, O. P. (1991). Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Applied and Environmental Microbiology, 57, 419-425. Young, C. S. and Dolan, J. W. (2003). Success with evaporative light-scattering detection. Available from: Accessed 30 August 2008.
飲食攝取之油脂在人體生理上有許多功能,其脂肪酸多寡與組成之改變,會影響細胞膜及細胞核上的訊息傳遞路徑,進而去控制特定轉錄因子的活性表現。因此,飲食脂肪與許多慢性疾病及免疫反應失常有關。飲食中脂質約95%為三酸甘油酯(Triacylglycerol, TG),其它為磷脂質、膽固醇等。而三酸甘油酯之結構相當複雜,會因三酸甘油酯中三個脂肪酸的雙鍵位置cis/trans結構和R/S光學異構物而有不同結構。
本實驗利用HPLC-ELSD來分析食用大豆油之三酸甘油酯中三個脂肪酸種類,利用許多參數,包括:ECN、CN、DB、Log α及Log β,探討分析三酸甘油酯種類方法之可靠性,另外以批式發酵培養產油酵母菌Rhodotorula glutinis,探討發酵前活化菌體與發酵後菌體之三酸甘油酯中三個脂肪酸種類變化。
實驗結果顯示,分離不同食用油利用AlltimaTM HP C18 Hi-Load分析管柱有較好之解析度。在漂移管溫度40 ℃及霧化氣體流速之壓力20 psi 為ELSD偵測器最適條件。ELSD對油脂偵測極限可以達到1.25 μg,遠低於RI對油脂之偵測,修飾Goiffen et al.(1981)的方法,依照其參數應用在HPLC-ELSD對油脂分析,實驗結果顯示由於 ELSD不會有溶劑波峰出現,在理論推演時也不需加入內標,利用ECN、Log α、DB等參數,即可判定其三酸甘油酯種類。本實驗經由不同時間收集波峰收集液,再經由氣相層析分析確認比對之三酸甘油酯種類,其結果Intra-day小於3.50%,Inter-day小於2.26%,都可以使用HPLC-ELSD做油脂分析。
依上述方法測定R. glutinis活化菌體與發酵菌體之三酸甘油酯中三個脂肪酸的種類,發現發酵前與發酵後之三酸甘油酯種類不同,可能因微生物在限氮培養環境下,進而累積對生理有利之三酸甘油酯種類,其詳細原因還未了解,需進一步深入探討。

The dietary intake of fats in the body features many physiological functions. The amount of fatty acid and compositional changes will affect the physical characteristic of cell membranes and signal transduction path of the nucleus, and control the activity performance of specific transcription factors. The amounts of intake of dietary fats are related to a number of chronic diseases and abnormal immune responses. About 95% of the lipids in an average meal are constituted of triacylglycerol (TG), and the remainders are phospholipids, cholesterol and others. The structure of triacylglycerol is rather complex because the cis/trans double bond structure of the three fatty acids in TG molecules and the R/S optical isomerism come in different structures.
This experiment used HPLC-ELSD to analyze the three types of fatty acid contained in the triacylglycerol of edible soybean oil. We utilized several parameters including ECN, CN, DB, Log α and Log β to explore the reliability of the analyzing methods of triacylglycerol types, and on the other hand, also to cultivate oleaginous yeast (Rhodotorula glutinis) using batch fermentation method to explore the bacterial activation, before and after fermentation, on the changes of three fatty acid types in triacylglycerol.
The experimental results indicated that using the AlltimaTM HP C18 Hi-Load analysis column to separate different edible oils yielded a higher resolution. The optimal conditions for using ELSD are that of a drift tube temperature of 40 ℃ and nebulizer nitrogen pressure of 20 psi. The ELSD detection limit of fats reached 1.25 μg, which is well below the RI detection of fats and of which has a modified Goiffen et al.(1981) method. As the experimental results indicate, there is no occurrence of an ELSD solvent peak, so it is not necessary to add internal standard into the course of theoretical deduction, but only necessary to employ parameters such as ECN, Log α, and DB to identify its triacylglycerol types. During the experiment we gathered the peak collecting fluids at different time intervals using gas chromatography analysis to confirm the matching triacylglycerol types. Results show that the intra-day reading was lower than 3.5%, and that inter-day reading was lower than 2.26%, confirming that they are able to use HPLC-ELSD to analyze fats.
When the above-mentioned method was used to test the three fatty acid types of triacylglycerol during the process of R. glutinis bacterial activation and bacterial fermentation, we discovered that the types of triacylglycerol are different before and after fermentation. This might be due to further accumulation of triacylglycerol types towards physiological benefits when existing in the environment of nitrogen deficit cultivation of microorganisms. The detailed reasons for this have not yet been understood and still await in-depth exploration.
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