High-Throughput Method for the Quantitation of Total Folate in Whole Blood Using LC-MS/MS

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.

Keywords: Folate; erythrocyte; mass spectrometry; para-aminobenzoic acid