Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE

The erythrocyte folate pool is reflective of an individual's long-term folate status; however, comprehensive quantitative determination of the various folate isoforms including polyglutamation (Glu(n)) status has posed an analytical problem. Factors complicating such analysis are the absence of authentic (isotope-labeled) standards and the large number of potential analytes. The present work presents high-throughput analytical methodology for the indirect comprehensive quantitation of the erythrocyte folate pool with commercially available standards.

METHODS

The erythrocyte folate pool was determined comprehensively by utilizing a cascade of three complementary ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) assays. In a first assay utilizing ion-pairing UPLC/MS/MS the relative (%) polyglutamation distribution (Glu(3-10)) of 5-methyltetrahydrofolate, tetrahydrofolate and 5-formyltetrahydrofolate is determined in a thermal extract obtained from packed erythrocytes, not requiring analytical standards. Quantitation of the erythrocyte folate pool was accomplished by performing two additional stable isotope dilution UPLC/MS/MS assays to determine whole blood and plasma folate content, utilizing commercially available [(13)C(5)]-labeled analogs of the Glu(1) analytes. Based on the values provided by each individual assay the comprehensive erythrocyte folate content could be calculated.

RESULTS

The various assays have been validated for intra- and inter-run precision, accuracy, linearity and are robust. The method was sensitive enough to measure the comprehensive erythrocyte folate distribution in a Down's syndrome patient with extremely low folate, bearing the C677T mutation in the gene encoding for methylenetetrahydrofolate reductase.

CONCLUSIONS

The erythrocyte folate pool can be comprehensively quantitated by running three complementary UPLC/MS/MS assays. The present assays are robust and allow for high-throughput analysis. The method can be utilized to support larger investigations that investigate the relationship between folate isoform and polyglutamation distribution and disease pathogenesis.