Owens Janel E, Holstege Dirk M, Clifford Andrew J
Department of Nutrition, University of California, Davis, California 95616, USA.
J Agric Food Chem. 2007 May 2;55(9):3292-7. doi: 10.1021/jf063648p. Epub 2007 Mar 31.
A high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method for red blood cell (RBC) folate analysis was developed from a previously described manual (M) LC-MS/MS method. The HT LC-MS/MS method used 96-well plates in which RBC folates were hydrolyzed with concentrated HCl in the presence of the [13C6]pABA internal standard (IS). The pH of the hydrolysate was adjusted to 5.0 before cleanup using 96-well plate OASIS HLB SPE cartridges. The analyte and IS were eluted with ethyl acetate/hexane (95:5, v/v) and methylated with methanol and trimethylsilyldiazomethane. The methylated analyte and IS were quantified with LC-MS/MS as previously described. The HT LC-MS/MS method was validated by determining the recovery of six different folate vitamers, which were quantitatively recovered (84-105% with CV<9.0%). RBC folate concentrations in whole blood samples correlated between HT and M LC-MS/MS methods (r=0.922, p<0.0001 for n=43 samples) and between the HT LC-MS/MS method and a chemiluminescence assay (r=0.664, p<0.001 for n=325 samples). Comparison of the results between HT LC-MS/MS and chemiluminescence methods with Bland-Altman difference plots and by ROC curve analysis indicates that the chemiluminescence assay underreports RBC folate concentrations. The HT LC-MS/MS method allows for high-throughput sample preparation for the analysis of RBC folate.
一种用于红细胞（RBC）叶酸分析的高通量液相色谱串联质谱（HT LC-MS/MS）方法是在先前描述的手动（M）LC-MS/MS方法基础上开发的。HT LC-MS/MS方法使用96孔板，在[13C6]对氨基苯甲酸内标（IS）存在的情况下，用浓盐酸水解RBC叶酸。在使用96孔板OASIS HLB固相萃取柱进行净化之前，将水解产物的pH值调节至5.0。分析物和内标用乙酸乙酯/己烷（95:5，v/v）洗脱，并用甲醇和三甲基硅基重氮甲烷进行甲基化。甲基化的分析物和内标如前所述用LC-MS/MS进行定量。通过测定六种不同叶酸维生素类似物的回收率对HT LC-MS/MS方法进行了验证，这些类似物被定量回收（回收率为84-105%，变异系数<9.0%）。全血样本中的RBC叶酸浓度在HT和M LC-MS/MS方法之间具有相关性（对于n = 43个样本，r = 0.922，p < 0.0001），并且在HT LC-MS/MS方法和化学发光测定之间也具有相关性（对于n = 325个样本，r = 0.664，p < 0.001）。通过Bland-Altman差异图和ROC曲线分析比较HT LC-MS/MS和化学发光方法的结果表明，化学发光测定低估了RBC叶酸浓度。HT LC-MS/MS方法允许对RBC叶酸分析进行高通量样品制备。
