Kiekens Filip, Van Daele Jeroen, Blancquaert Dieter, Van Der Straeten Dominique, Lambert Willy E, Stove Christophe P
Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium.
Laboratory of Functional Plant Biology, Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.
J Chromatogr A. 2015 Jun 12;1398:20-8. doi: 10.1016/j.chroma.2015.04.025. Epub 2015 Apr 20.
A stable isotope dilution LC-MS/MS method is the method of choice for the selective quantitative determination of several folate species in clinical samples. By implementing an integrated approach to determine both the plasma and red blood cell (RBC) folate status, the use of consumables and time remains limited. Starting from a single 300μl whole blood sample, the folate status in plasma and RBCs can be determined after separating plasma and RBCs and sequential washing of the latter with isotonic buffer, followed by reproducible lysis using an ammonium-based buffer. Acidification combines both liberation of protein bound folates and protein precipitation. Sample cleanup is performed using a 96-well reversed-phase solid-phase extraction procedure, similar for both plasma and RBC samples. Analyses are performed by UHPLC-MS/MS. Method validation was successfully performed based on EMA-guidelines and encompassed selectivity, carry-over, linearity, accuracy, precision, recovery, matrix effect and stability. Plasma and RBC folates could be quantified in the range of 1-150nmol/l and 5-1500nmol/l, respectively. This method allows for the determination of 6 folate monoglutamates in both plasma and RBCs. It can be used to determine short and long term folate status in both normal and severely deficient subjects in a single analytical sequence.
稳定同位素稀释液相色谱-串联质谱法是临床样本中几种叶酸物种选择性定量测定的首选方法。通过采用综合方法来确定血浆和红细胞（RBC）的叶酸状态，耗材的使用和时间仍然有限。从单个300μl全血样本开始，在分离血浆和红细胞并随后用等渗缓冲液对红细胞进行顺序洗涤后，使用铵基缓冲液进行可重复的裂解，即可确定血浆和红细胞中的叶酸状态。酸化结合了蛋白质结合叶酸的释放和蛋白质沉淀。使用96孔反相固相萃取程序进行样品净化，血浆和红细胞样品的操作相似。通过超高效液相色谱-串联质谱法进行分析。根据EMA指南成功进行了方法验证，包括选择性、残留、线性、准确性、精密度、回收率、基质效应和稳定性。血浆和红细胞叶酸的定量范围分别为1-150nmol/l和5-1500nmol/l。该方法可测定血浆和红细胞中的6种叶酸单谷氨酸。它可用于在单个分析序列中确定正常和严重缺乏受试者的短期和长期叶酸状态。
