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.
Owens
Janel E
JE
Department of Nutrition, University of California, Davis, California 95616, USA.
Holstege
Dirk M
DM
Clifford
Andrew J
AJ
D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D005492:Folic Acid / Q000097:blood*; D006801:Humans; D008163:Luminescent Measurements; D013058:Mass Spectrometry / Q000379:methods*
D005492:Folic Acid