Development and Validation of a Quantitative Method for Plasma Folates Jack Maggiore, PhD , Zhen Wu, Karen Urek, Erlo Roth, MD, Andrea Gruszecki, ND, David Quig, PhD Doctor’s Data, Inc. ◊ Saint Charles, Illinois Abstract Materials and Methods Results Sample Collection Folic Acid Dose vs Detection Sample Stability Background: An increased interest in folate analysis has arisen from an enhanced ability to detect single nucleotide polymorphisms (SNPs) in the genes that transcribe the enzymes of folate metabolism. Defects in folate metabolism affect the remethylation of homocysteine to methionine. The C677T and T677T methyltetrahydrofolate reductase (MTHFR) SNPs occur at a frequency of 32.4% and 7.4%, respectively, in the general US population (1). When consuming folate-rich foods or supplements, the majority of individuals may experience excessive plasma levels of unmodified folic acid (UMFA), which has been associated with cellular hyperplasia and neoplasia(2). Folinic acid and 5-methyltetrahydrofolate (5-MTHF) are synthetic folate supplements recommended by clinicians for those with specific MTHFR C677T heterozygous (C/T) and homozygous (T/T) genotypes to provide a source of folates while minimizing undesired levels of UMFA in circulation. Immunoassays that determine total folates in serum or red blood cells cannot differentiate between UMFA and the other folates. To aid in the monitoring of the folate status, we developed a method for measuring UMFA and the folate metabolites folinic acid, 5-MTHF, and tetrahydrofolate (THF) using liquid chromatography with tandem mass-spectrometry (LC-MS/MS). Methods: Plasma samples prepared in 100 mM ascorbic acid were mixed with internal standards and extracted by polymeric reversed phase solid phase extraction cartridges. Certified standards traceable to the National Institute of Standards and Technology (NIST) were acquired to derive a 5-point calibration curve for each analyte. Calibrators, NIST standard reference materials (SRM 1955), and plasma extracts were analyzed on an Agilent 6490 LC-MS/MS with chromatographic separation achieved on a C18 analytical column, permitting identification and quantitation of the folate compounds of interest. Analytical performance characteristics evaluated include precision, linearity, recovery, and stability. Results: The intra-assay and total imprecision coefficients of variation (CVW and CVT) (n=42) in plasma samples were determined for UMFA: 4.0% and 7.0% at 6.5 nmol/L, and 3.7% and 7.7% at 9.6 nmol/L; folinic acid: 4.5% and 10.9% at 45.2 nmol/L, and 4.1% and 11.6% at 51.7 nmol/L; 5-MTHF: 1.0% and 4.9% at 46.7 nmol/L, and 1.1% and 5.6% and at 57.2 nmol/L; and THF: 5.3% and 14.6% at 2.7 nmol/L, and 3.9% and 12.9% at 5.0 nmol/L. Linearity range (n=11) and percent recovery was confirmed in spiked plasma samples for UMFA: 1.0-425.0 nmol/L, 98.3%-107.6%; folinic acid: 0.15- 402.4 nmol/L, 95.7%-108.6%; 5-MTHF: 1.2-632.8 nmol/L, 99.7%-104.1%; and THF: 0.25-405.2 nmol/L, 98.6%-109.0%. Stability of all analytes in acidified plasma samples was demonstrated for 8-days stored at -20oC, and 4-days stored at 4oC. Conclusion: This LC-MS/MS method developed for assessing plasma folate status has been validated to be analytically precise, accurate, and sensitive for the measurement of UMFA, folinic acid, 5-MTHF, and THF. Sample suitability has been established for the expedited temperature-controlled transportation of acidified plasma aliquots from remote locations to a central laboratory for analysis. Studies are underway to use this method to assess reference intervals for the folate analytes in those with previously determined MTHFR C677T genotypes. References: 1) J Med Genet. 2003;40:601-605. 2) J Nutr. 2015; 145(3):520-531. Venous whole blood is collected into collection tubes with EDTA and centrifuged to yield plasma. To enhance stability, plasma samples are immediately acidified using 10 mg/mL ascorbic acid. Acidified plasma is maintained below 4oC during transit and at -80oC upon receipt in the laboratory until analysis. The samples are extracted by polymeric reversed phase SPE cartridge to remove protein and water-soluble impurities. Extracted samples are mixed with internal standards and eluted with formic acid. The supernatants are then analyzed using an Agilent 6490 LC-MS/MS with Jet Stream Electrospray Ionization. The internal standards employed in this method are folic acid-13C5, 15N, and 5-MTHF-13C5, which are components of their respective extraction solutions, added to every calibrator, calibration verifier, quality control sample, and patient sample prior to SPE. Chromatographic separation is achieved on a C18 analytical column which facilitates resolution and identification of the compounds of interest. Multiple reaction monitoring (MRM) transition ions are detected by MS/MS for each analyte of interest while a precursor ion is further broken down into a fragment ion for greatly improved selectivity. 5-point calibration curves are generated for each measurand with each analytical run. Gravimetrically prepared calibrators from sourced and certified components are run in duplicate for each calibration point to span the linear range of the method. Bi-level calibration verifiers are prepared from a secondary certified source, and are run immediately following the calibrators. Human plasma-based quality control pools at two distinct levels are analyzed with each run. A procedural blank is run initially with each analytical run, and at four additional scheduled times within the load list to verify the integration baseline. Sample Preparation Sample Analysis Conclusions The folates play an essential role as cofactors in the remethylation cycle of homocysteine. This method allows for the simultaneous detection and quantitation of the primary folate components in blood to permit the assessment of folate status in response to dietary and oral supplement therapy. All folate compounds demonstrate 4-day stability at 4oC and 8-day stability at -20oC simulated transit conditions when plasma is acidified with 10 mg/mL ascorbic acid. Linearity testing demonstrates both sensitivity and a robust analytical range. Analytical precision permits ability to assess subtle changes in response to therapeutic interventions. Studies are underway to use this method to assess reference intervals and optimal dosing and collection times for the folate analytes in those with previously determined MTHFR C677T genotypes. Precision (n = 42) Low High Mean (nmol/L) Total %CV UMFA 6.5 7.0% 9.6 7.7% Folinic Acid 45.2 10.9% 51.7 11.6% 5-MTHF 46.7 4.9% 57.2 5.6% THF 2.7 14.6% 5.0 12.9% Sample Chromatograms Calibration Standard Linearity and Recovery (n = 11) Linearity Range (nmol/L) Recovery Range UMFA 1.0 – 425.0 98.3 – 107.6% Folinic Acid 0.15 – 402.4 95.7 – 108.6% 5-MTHF 1.2 – 632.8 99.7 – 104.1% THF 0.25 – 405.2 98.6 – 109.0% Participant Sample