利用毛細管液相層析搭配紫外線偵測器和基質輔助雷射脫附游離質譜法檢測硫辛酸

Trace analysis of lipoic acid by capillary liquid chromatography with ultraviolet detection and matrix-assisted laser desorption ionization mass specetrometry

蔡佳汝(Chia-Ju Tsai)

指導教授 ： 馮嘉嫻

高雄醫學大學/藥學院/香粧品學系碩士班/碩士(2011年)

摘要

硫辛酸(lipoic acid，LA)普遍存在原核及真核生物內，是個理想的抗氧化劑，其在生物體能量產生過程中可做為粒腺體內多種酵素的輔助因子。本研究利用毛細管液相層析結合紫外線偵測器(CapLC-UV)和基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)，建立一套快速、簡單且高偵測感度的硫辛酸定量分析方法。此方法利用柱前衍生法提高硫辛酸偵測靈敏度，衍生前先使用還原試劑NaBH4將硫辛酸還原成二氫硫辛酸(dihydrolipoic acid，DHLA)，之後加入衍生試劑4-bromomethyl-6,7-dimethoxycoumarin (Br-DMC)進行親核性取代反應，即可得到最終衍生產物。在本研究中針對影響衍生反應和分散液液微萃取法(DLLME)萃取尿液中硫辛酸之不同參數進行探討，並使其最佳化。硫辛酸在經過衍生步驟後可提升離子化能力，衍生物在以α-cyano-4-hydroxycinnamic acid (CHCA)為基質之分析環境下，在標準品和尿液檢品中，可於m/z 683測得衍生物。硫辛酸分離條件採梯度沖提法並使用C18的毛細管層析管柱，由ACN和0.1%甲酸水溶液組成之移動相，且於345 nm偵測。在標準品和尿液檢品硫辛酸的線性範圍分別為0.1-40 μM和0.1-20 μM，具有良好的線性其相關係數為0.999。本研究方法之最低偵測極限：利用CapLC-UV分析在標準品和尿液檢品中分別是0.01 μM和0.03 μM，而利用MALDI-TOF MS分析在標準品和尿液檢品中分別是0.008 μM和0.02 μM。我們成功地建立硫辛酸之檢測分析方法，並可應用至市售健康食品、化粧品及尿液檢體。

關鍵字

硫辛酸 ； 衍生反應 ； 毛細管液相層析 ； 基質輔助雷射脫附游離飛行時間質譜儀

並列摘要

Lipoic acid (LA, or 6,8-thioctic acid) is an essential cofactor for mitochondrial enzymes and ideal antioxidant present in prokaryotic and eukaryotic cells. The rapid, simple and sensitive capillary liquid chromatography coupled with ultraviolet detector (CapLC-UV) and matrix-assisted laser desorption ionization time-of-flight mass specetrometry (MALDI-TOF MS) methods for the determination of lipoic acid were established. This method employed a pre-column derivatization with 4-bromomethyl-6,7-dimethoxycoumarin (Br-DMC). LA was labeled with Br-DMC after it was changed into dihydrolipoic acid (DHLA) by using the reducing agent, NaBH4. Some important parameters affected the derivatization and extraction of LA from urine by dispersive liquid-liquid microextraction (DLLME) were investigated and optimized. Ionization ability of LA derivative was increased and LA derivative was detected at m/z 683 in standard solution and urine with matrix, α-cyano-4-hydroxycinnamic acid (CHCA), by MALDI-TOF MS. The gradient separation was carried out by using reversed-phase C18 column with the mobile phase consisting of acetonitrile-0.1% formaic acid solution and monitored at 345 nm. The linear response was ranged from 0.1 to 40 μM and 0.1 to 20 μM in standard solution and urine respectively, with a correlation coefficient of 0.999. The detection limits was 0.01 μM and 0.03 μM by CapLC-UV, and the detection limits was 0.008 μM and 0.02 μM by MALDI-TOF MS in standard solution and urine respectively. We successfully applied these methods to detect LA in dietary supplements, cosmetic products and urine.

並列關鍵字

lipoic acid ； derivatization ； capillary liquid chromatography ； matrix-assisted laser desorption ionization time-of-flight mass specetrometry

參考文獻

參考文獻

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