A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product1

Details

• Alternative Title:
  Anal Bioanal Chem
• Personal Author:
  Fazili, Zia ; Whitehead, Ralph D. ; Paladugula, Neelima ; Pfeiffer, Christine M.
• Description:
  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.
• Subjects:
  Chromatography, Liquid Folic Acid High-Throughput Screening Assays Humans Leucovorin Limit Of Detection Oxidation-Reduction Solid Phase Extraction Tandem Mass Spectrometry Tetrahydrofolates
• Keywords:
  Anticoagulant Types Automated Solid Phase Extraction HmTHF MeFox Method Comparison Microbiologic Assay
• Source:
  Anal Bioanal Chem. 405(13):4549-4560
• Pubmed ID:
  23462981
• Pubmed Central ID:
  PMC5326548
• Document Type:
  Journal Article
• Funding:
  CC999999/ImCDC/Intramural CDC HHSUnited States/
• Volume:
  405
• Issue:
  13
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:942b2ecd9fcaa1e17c3563f8fd01d41628cd37032e12efaa5fb87a05a4906b5c
• Download URL:
  https://stacks.cdc.gov/view/cdc/44354/cdc_44354_DS1.pdf
• File Type:
  [PDF - 692.85 KB ]