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

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.

Figure

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

Keywords

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)

References

  1. 1.
    Yetley EA, Pfeiffer CM, Phinney KW, Fazili Z, Lacher DA, Bailey RL, Blackmore S, Bock J, Brody LC, Carmel R et al (2011) Biomarkers of folate status in the National Health and Nutrition Examination Survey (NHANES): a roundtable summary. Am J Clin Nutr 94:303S–312SCrossRefGoogle Scholar
  2. 2.
    Yang Q, Cogswell ME, Hamner HC, Carriquiri A, Bailey LB, Pfeiffer CM, Berry RJ (2010) Folic acid source, usual intake, and folate and vitamin B12 status in US adults: National Health and Nutrition Examination Survey (NHANES) 2003–2006. Am J Clin Nutr 91:64–72CrossRefGoogle Scholar
  3. 3.
    Fazili Z, Pfeiffer CM, Zhang M (2007) Comparison of serum folate species analyzed by LC-MS/MS with total folate measured by microbiologic assays and Bio-Rad radioassay. Clin Chem 53:781–784CrossRefGoogle Scholar
  4. 4.
    Yetley EA, Coates PM, Johnson CL (2011) Overview of a roundtable of NHANES monitoring of biomarkers of folate and vitamin B-12 status: measurement procedure issues. Am J Clin Nutr 94:297S–302SCrossRefGoogle Scholar
  5. 5.
    Pfeiffer CM, Zhang M, Lacher DA, Molloy AM, Tamura T, Yetley EA, Picciano M-F, Johnson CL (2011) Comparison of serum and red blood cell folate microbiologic assays for national population surveys. J Nutr 141:1402–1409CrossRefGoogle Scholar
  6. 6.
    Pfeiffer CM, Fazili Z, McCoy L, Zhang M, Gunter EW (2004) Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay. Clin Chem 50:423–432CrossRefGoogle Scholar
  7. 7.
    Fazili Z, Pfeiffer CM (2004) Measurement of folates in serum and conventionally prepared whole blood lysates: application of an automated 96-well plate isotope-dilution tandem mass spectrometry method. Clin Chem 50:2378–2381CrossRefGoogle Scholar
  8. 8.
    Fazili Z, Pfeiffer CM (2012) Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution–liquid chromatography–tandem mass spectrometry. J Nutr 143:108–113Google Scholar
  9. 9.
    Caudill SP, Schleicher RL, Pirkle JL (2008) Multi-rule quality control for the age-related eye disease study. Stat Med 27:4094–4106CrossRefGoogle Scholar
  10. 10.
    US Department of Health and Human Services (2001) Guidance for industry: bioanalytical method validation. Food and Drug Administration. http://www.fda.gov/downloads/Drugs/…/Guidances/ucm070107.pdf. Accessed 8 February 2013
  11. 11.
    National Institute of Standards and Technology (2008) Certificate of analysis, standard reference material 1955, homocysteine and folate in frozen human serum. https://www-s.nist.gov/srmors/certificates/1955.pdf?CFID=1979088&CFTOKEN=e0f2df69af7e3f9e-E52B4E5A-D3F5-ADF4-0BC8FFDC3BB1C0C6&jsessionid=f030b44ef80dbe9549eb287366473b7c6c3b. Accessed 9 October 2012
  12. 12.
    National Institute of Standards and Technology (2011) Certificate of analysis, standard reference material 1950, metabolites in human plasma. https://www-s.nist.gov/srmors/certificates/1950.pdf?CFID=1979097&CFTOKEN=aab280e30cbdde55-E52EE52D-FAB2-791A-ED2004079DDD64AD&jsessionid=f0308a1c78b0b3633f28572b4f1ba5775552. Accessed 9 October 2012
  13. 13.
    National Institute for Biological Standards and Control (2008) WHO international standard, vitamin B12 and serum folate, NIBSC code 03/178 (version 2.0, dated 04/04/2008). http://www.nibsc.ac.uk/documents/ifu/03-178.pdf. Accessed 9 October 2012
  14. 14.
    Taylor JK (1987) Quality assurance of chemical measurement. Lewis, Boca RatonGoogle Scholar
  15. 15.
    Kok RM, Smith DE, Dainty JR, van den Akker JT, Finglas PM, Smulders YM, Jakobs C, de Meer K (2004) 5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism. Anal Biochem 326:129–138CrossRefGoogle Scholar
  16. 16.
    Nelson BC, Satterfield MB, Sniegoski LT, Welch MJ (2005) Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry. Anal Chem 77:3586–3593CrossRefGoogle Scholar
  17. 17.
    Kirsch SH, Knapp J-P, Herrmann W, Obeid R (2010) Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry. J Chromatogr B 878:68–75CrossRefGoogle Scholar
  18. 18.
    Mönch S, Netzel M, Netzel G, Rychlik M (2010) Quantitation of folates and their catabolites in blood plasma, erythrocytes and urine by stable isotope dilution assays. Anal Biochem 398:150–160CrossRefGoogle Scholar
  19. 19.
    Büttner BE, Öhrvik VE, Witthöft CM, Rychlik M (2011) Quantification of isotope labeled and unlabeled folates in plasma, ileostomy and food samples. Anal Bioanal Chem 399:429–439CrossRefGoogle Scholar
  20. 20.
    Hannisdal R, Ueland PM, Svardal A (2009) Liquid chromatography–tandem mass spectrometry analysis of folate and folate catabolites in human serum. Clin Chem 55:1147–1154CrossRefGoogle Scholar
  21. 21.
    Kirsch SH, Herrmann W, Geisel J, Obeid R (2012) Assay of whole blood (6S)-5-CH3-H4folate using ultra performance liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 404:895–902CrossRefGoogle Scholar
  22. 22.
    Fraser CG, Hyltoft PP, Libeer JC, Ricos C (1997) Proposals for setting generally applicable quality goals based on biology. Ann Clin Biochem 34:8–12Google Scholar
  23. 23.
    Lacher DA, Hughes JP, Carroll MD (2010) Biological variation of laboratory analytes based on the 1999–2002 National Health and Nutrition Examination Survey. National Health Statistics report no. 21. National Center for Health Statistics, Hyattsville, MDGoogle Scholar
  24. 24.
    Hannisdal R, Ueland PM, Eussen SJ, Svardal A, Hustad S (2009) Analytical recovery of folate degradation products formed in human serum and plasma at room temperature. J Nutr 139:1415–1418CrossRefGoogle Scholar

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

Personalised recommendations