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

Anal Bioanal Chem. 2013 May;405(13):4549-60. doi: 10.1007/s00216-013-6854-9. Epub 2013 Mar 6.


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.

MeSH terms

  • Chromatography, Liquid
  • Folic Acid / blood*
  • High-Throughput Screening Assays*
  • Humans
  • Leucovorin / blood*
  • Limit of Detection
  • Oxidation-Reduction
  • Solid Phase Extraction
  • Tandem Mass Spectrometry
  • Tetrahydrofolates / blood*


  • Tetrahydrofolates
  • 5,10-methenyltetrahydrofolate
  • Folic Acid
  • Leucovorin
  • 5-methyltetrahydrofolate