Quantitation of whole-blood total folate within defined MTHFR C677T genotype groups by isotope dilution-liquid chromatography-tandem mass spectrometry differs from microbiologic assay

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. © 2012 American Society for Nutrition.