Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
J Chromatogr A. 2013 Oct 11;1311:115-20. doi: 10.1016/j.chroma.2013.08.076. Epub 2013 Aug 28.
A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh performance liquid chromatography tandem mass spectrometry, IP-RP-UHPLC-MS/MS, was developed for quantitative determination of intracellular coenzyme A (CoA), acetyl CoA, succinyl CoA, phenylacetyl CoA, flavin mononucleotide, (FMN), flavin adenine dinucleotide, (FAD), NAD, NADH, NADP, NADPH. Dibutylammonium acetate (DBAA) was used as volatile ion pair reagent in the mobile phase. Addition of DBAA to the sample solutions resulted in an enhanced sensitivity for the phosphorylated coenzymes. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), was added to keep CoA in the reduced form. Isotope dilution mass spectrometry (IDMS) was applied for quantitative measurements for which culture derived global U-(13)C-labeled cell extract was used as internal standard. The analytical method was validated by determining the limit of detection, the limit of quantification, repeatability and intermediate precision. The method was successfully applied for quantification of coenzymes in the cell extracts of Saccharomyces cerevisiae.
建立了一种基于离子对反相超高效液相色谱-串联质谱（IP-RP-UHPLC-MS/MS）的快速、灵敏、特异的分析方法，用于定量测定细胞内辅酶 A（CoA）、乙酰辅酶 A、琥珀酰辅酶 A、苯乙酰辅酶 A、黄素单核苷酸（FMN）、黄素腺嘌呤二核苷酸（FAD）、NAD、NADH、NADP、NADPH。在流动相中使用二丁基铵乙酸盐（DBAA）作为挥发性离子对试剂。向样品溶液中添加 DBAA 可增强磷酸化辅酶的灵敏度。添加三（2-羧乙基）膦盐酸盐（TCEP·HCl）可使 CoA 保持还原形式。采用同位素稀释质谱法（IDMS）进行定量测量，使用培养衍生的全球 U-（13）C 标记细胞提取物作为内标。通过测定检测限、定量限、重复性和中间精密度对分析方法进行了验证。该方法成功应用于酿酒酵母细胞提取物中辅酶的定量分析。
