Analytical and Bioanalytical Chemistry

, Volume 405, Issue 13, pp 4549–4560

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

  • Zia Fazili
  • Ralph D. WhiteheadJr
  • Neelima Paladugula
  • Christine M. Pfeiffer
Original Paper


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


Automated solid phase extraction MeFox hmTHF Method comparison Microbiologic assay Anticoagulant types 

Supplementary material

216_2013_6854_MOESM1_ESM.pdf (326 kb)
ESM 1(PDF 325 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Zia Fazili
    • 1
  • Ralph D. WhiteheadJr
    • 1
  • Neelima Paladugula
    • 1
  • Christine M. Pfeiffer
    • 1
  1. 1.Division of Laboratory Sciences, National Center for Environmental HealthCenters for Disease Control and PreventionAtlantaUSA

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