Satoh Soichiro, Toyo'oka Toshimasa, Fukushima Takeshi, Inagaki Shinsuke
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):109-15. doi: 10.1016/j.jchromb.2007.04.003. Epub 2007 Apr 13.
The simultaneous determination of alpha-lipoic acid (LA) and DHLA (reduced form of LA) was carried out by HPLC with fluorescence detection. DHLA in the sample was first labeled with ABD-F at room temperature for 10 min and then the LA was labeled with SBD-F at 50 degrees C for 1 h after conversion to DHLA using the reducing agent, TCEP. The resulting fluorophores, ABD-DHLA and SBD-DHLA, were separated by reversed-phase chromatography and detected at 510 nm (excitation at 380 nm). Both fluorophors were completely separated without any interference of endogenous thiols and disulfides in the sample and sensitively detected by fluorimetry. The proposed method was applied to the assay of the LA supplement and the determination in human plasma after the oral administration of LA tablets. The concentration (%) of LA in the tablet was reasonable to the stated amount. Furthermore, the result of a time course study in the plasma after the administration of LA did not differ from a previous report. Thus, the present method seems to be applicable to the simultaneous determination of LA and DHLA in various biological specimens.
采用高效液相色谱荧光检测法同时测定α-硫辛酸（LA）和二氢硫辛酸（DHLA，LA的还原形式）。样品中的DHLA首先在室温下用ABD-F标记10分钟，然后使用还原剂三（2-羧乙基）膦（TCEP）将LA转化为DHLA后，在50℃下用SBD-F标记1小时。生成的荧光团ABD-DHLA和SBD-DHLA通过反相色谱法分离，并在510nm处检测（激发波长为380nm）。两种荧光团完全分离，不受样品中内源性硫醇和二硫化物的干扰，并通过荧光法进行灵敏检测。该方法应用于LA补充剂的含量测定以及口服LA片后人血浆中的含量测定。片剂中LA的含量（%）与规定量相符。此外，LA给药后血浆中的时间进程研究结果与先前报道无异。因此，本方法似乎适用于同时测定各种生物样品中的LA和DHLA。
