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A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product

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Abstract

Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine eight-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers [5-methyltetrahydrofolate (5-methylTHF), folic acid (FA), plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF] and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 μL instead of 275 μL) by using 96-well SPE plates with 50 mg instead of 100 mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, two replicates/day) for three serum quality control pools was 2.8–3.6 % for 5-methylTHF (19.5–51.1 nmol/L), 6.6–8.7 % for FA (0.72–11.4 nmol/L), and ≤11.4 % for the minor folate forms (<1–5 nmol/L). The mean (±SE) recoveries of folates spiked into serum (3 days, four levels, two replicates/level) were: 5-methylTHF, 99.4 ± 3.6 %; FA, 100 ± 1.8 %; minor folates, 91.7–108 %. SPE extraction efficiencies were ≥85 %, except for THF (78 %). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method [n = 150, r = 0.99 for 5-methylTHF, FA, and total folate (tFOL, sum of folate forms)] and produced slightly higher tFOL (5.6 %) and 5-methylTHF (7.3 %) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h), resulting in improved SPE efficiency and recovery compared to the eight-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ∼2 h, and all relevant folate forms can be accurately measured using a small serum volume.

High-throughput LC-MS/MS method for population monitoring of serum folate forms

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Acknowledgment

No specific sources of financial support. The findings and conclusions in this report are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry.

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Correspondence to Christine M. Pfeiffer.

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Published in the topical collection Functional Foods and Dietary Supplements with guest editors Melissa M. Phillips and Catherine A. Rimmer.

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Fazili, Z., Whitehead, R.D., Paladugula, N. et al. A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product. Anal Bioanal Chem 405, 4549–4560 (2013). https://doi.org/10.1007/s00216-013-6854-9

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