Quantitation of Whole-Blood Total Folate within Defined MTHFR C677T Genotype Groups by Isotope Dilution–Liquid Chromatography–Tandem Mass Spectrometry Differs from Microbiologic Assay1–3

Abstract

Standardization of folate measurement is needed for accurate assessment of folate status. We compared the measurement of whole-blood folate by isotope dilution–liquid chromatography–tandem MS (ID-LC-MS/MS) with the historical gold standard microbiological assay (MA) using 3 common calibrators within the frame of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Seventy-three whole-blood samples with an even distribution of MTHFR C677T genotypes (24 CC, 24 CT, 24 TT) were prepared, and total folate was determined by ID-LC-MS/MS and MA using the following calibrators: 5-methyltetrahydrofolate (5-methylTHF) (Merck), folic acid (FA) (Merck), and FA (Sigma). To compare the methods, 5-formyltetrahydrofolate (5-formylTHF) was excluded in the ID-LC-MS/MS summation of total folate, because it is likely that the majority of 5-formylTHF detected is a pyrazino-s-triazine oxidation product of 5-methylTHF. MA whole-blood folate measured by using the FA calibrators was consistently higher than with the 5-methylTHF calibrator. Differences between dilutions and analysis of spiked whole-blood samples showed a nonlinear response, with overrecovery of 5-methylTHF by ~23% toward the higher end of the MA calibration range. Significant proportional biases between ID-LC-MS/MS and MA were found in all comparisons except when the MA was calibrated with 5-methylTHF and a higher sample dilution of 1:1600 (regression slope: 1.05; P= 0.31; intercept-21, P = 0.16). Calibration bias and matrix effects in the MA underscore the need for a formally accepted whole-blood folate reference method. ID-LC-MS/MS procedures have the potential to offer a high degree of accuracy; however, further work is needed to determine the origin of the pyrazino-s-triazine derivative.