Jump to ContentJump to Main Navigation
Show Summary Details
In This Section

Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

12 Issues per year

IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

SCImago Journal Rank (SJR) 2015: 0.873
Source Normalized Impact per Paper (SNIP) 2015: 0.982

See all formats and pricing
In This Section
Volume 44, Issue 4 (Apr 2006)


Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry

Desirée E.C. Smith
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Robert M. Kok
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Tom Teerlink
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Cornelis Jakobs
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Yvo M. Smulders
  • Department of Internal Medicine, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands and Institute for Cardiovascular Research, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
Published Online: 2011-09-21 | DOI: https://doi.org/10.1515/CCLM.2006.085


Background: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest.

Methods: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6min.

Results: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97–107%. We measured 8–72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327nmol/L and 23 to 363nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0–2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53–90% loss of the non-methylTHF fraction.

Conclusion: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.

Keywords: erythrocyte; folate; folate vitamer; liquid chromatography; mass spectrometry


  • 1.

    Shaw GM, Schaffer D, Velie EM, Morland K, Harris JA. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995; 6:219–26. [Crossref]

  • 2.

    Zittoun J. [Anemias due to disorder of folate, vitamin B12 and transcobalamin metabolism]. Rev Prat 1993; 43:1358–63.

  • 3.

    Voutilainen S, Rissanen TH, Virtanen J, Lakka TA, Salonen JT. Low dietary folate intake is associated with an excess incidence of acute coronary events: The Kuopio Ischemic Heart Disease Risk Factor Study. Circulation 2001; 103:2674–80. [Crossref]

  • 4.

    Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 2002; 346:476–83.

  • 5.

    Mason JB. Folate and colonic carcinogenesis: searching for a mechanistic understanding. J Nutr Biochem 1994; 5:170–5. [Crossref]

  • 6.

    Finkelstein JD, Martin JJ. Homocysteine. Int J Biochem Cell Biol 2000; 32:385–9. [Crossref]

  • 7.

    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111–3. [Crossref]

  • 8.

    Rozen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylenetetrahydrofolate reductase (MTHFR). Thromb Haemost 1997; 78:523–6.

  • 9.

    Bagley PJ, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci USA 1998; 95:13217–20. [Crossref]

  • 10.

    Levine S. Analytical inaccuracy for folic acid with a popular commercial vitamin B12/folate kit. Clin Chem 1993; 39:2209–10.

  • 11.

    Chanarin I. The megaloblastic anaemias. Oxford, UK: Blackwell Scientific, 1979:187–90.

  • 12.

    Belz S, Nau H. Determination of folate patterns in mouse plasma, erythrocytes, and embryos by HPLC coupled with a microbiological assay. Anal Biochem 1998; 265:157–66.

  • 13.

    Patring JD, Jastrebova JA, Hjortmo SB, Andlid TA, Jägerstad IM. Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection. J Agric Food Chem 2005; 53:2406–11. [Crossref]

  • 14.

    Bagley PJ, Selhub J. Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrical detection. Clin Chem 2000; 46:404–11.

  • 15.

    Eto I, Krumdieck CL. Determination of three different pools of reduced one-carbon-substituted folates. Anal Biochem 1980; 109:167–84. [Crossref]

  • 16.

    Fazili Z, Pfeiffer CM. 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 2004; 50:2378–81. [Crossref]

  • 17.

    Bertino JR, Coward JK, Cashmore A, Chello P, Panichajakul S, Horvath CG, et al. Polyglutamate forms of folate: natural occurrence and role as substrates in mammalian cells. Biochem Soc Trans 1976; 4:853–6. [Crossref]

  • 18.

    Matthews RG, Ghose C, Green JM, Matthews KD, Dunlap RB. Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes. Adv Enzyme Regul 1987; 26:157–71. [Crossref]

  • 19.

    Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr 1997; 65:1790–5.

  • 20.

    Rabinowitz JC. Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol 1963; 6:814–5.

  • 21.

    Blakley RL. The biochemistry of folic acid and related pteridines. In: Neuberger A, Tatum EL, editors. Frontiers of biology. London: North-Holland, 1969:92–4.

  • 22.

    Wright AJ, Finglas PM, Southon S. Erythrocyte folate analysis: saponin added during lysis of whole blood can increase apparent folate concentrations, depending on hemolysate pH. Clin Chem 2000; 46:1978–86.

  • 23.

    Pfeiffer CM, Gregory JF III. Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay: investigation with reversed-phase liquid chromatography. Clin Chem 1996; 42:1847–54.

  • 24.

    Kok RM, Smith DE, Dainty JR, Van Den Akker JT, Finglas PM, Smulders YM, et al. 5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism. Anal Biochem 2004; 326:129–38.

  • 25.

    Fazili Z, Pfeiffer CM, Zhang M, Jain R. Erythrocyte folate extraction and quantitative determination by LC/MS/MS: comparison of results with microbiologic assay. Clin Chem 2005; 51:2318–25. [Crossref]

  • 26.

    Owens JE, Holstege DM, Clifford AJ. Quantitation of total folate in whole blood using LC-MS/MS. J Agric Food Chem 2005; 53:7390–4. [Web of Science] [Crossref]

  • 27.

    Freisleben A, Schieberle P, Rychlik M. Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2003; 376:149–56.

  • 28.

    Vahteristo LT, Ollilainen V, Koivistoinen PE, Varo P. Improvements in the analysis of reduced folate monoglutamates and folic acid in food by high-performance liquid chromatography. J Agric Food Chem 1996; 44:477–82. [Crossref]

  • 29.

    Zhang G-F, Storozhenko S, Van Der Straeten D, Lambert WE. Investigation of the extraction behaviour of the main monoglutamate folates from spinach by liquid chromatography-electrospray ionisation tandem mass spectrometry. J Chromatogr A 2005; 1078:59–66.

  • 30.

    Gunter EW, Bowman BA, Caudill SP, Twite DB, Adams MJ, Sampson EJ. Results of an international round robin for serum and whole-blood folate. Clin Chem 1996; 42:1689–94.

  • 31.

    Suh JR, Herbig AK, Stover PJ. New perspectives on folate catabolism. Annu Rev Nutr 2001; 21:255–82. [Crossref]

About the article

Corresponding author: Dr. Yvo M. Smulders, Department of Internal Medicine, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Phone: +31-20-4444307, Fax: +31-20-4444313

Received: 2005-11-08

Accepted: 2006-01-16

Published Online: 2011-09-21

Published in Print: 2006-04-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2006.085. Export Citation

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

B. Van Guelpen
Scandinavian Journal of Clinical and Laboratory Investigation, 2007, Volume 67, Number 5, Page 459
Filip Kiekens, Jeroen Van Daele, Dieter Blancquaert, Dominique Van Der Straeten, Willy E. Lambert, and Christophe P. Stove
Journal of Chromatography A, 2015, Volume 1398, Page 20
Leon van Haandel and John F Stobaugh
Bioanalysis, 2013, Volume 5, Number 24, Page 3023
Nisha Padmanabhan, Dongxin Jia, Colleen Geary-Joo, Xuchu Wu, Anne C. Ferguson-Smith, Ernest Fung, Mark C. Bieda, Floyd F. Snyder, Roy A. Gravel, James C. Cross, and Erica D. Watson
Cell, 2013, Volume 155, Number 1, Page 81
Susanne H. Kirsch, Wolfgang Herrmann, Jürgen Geisel, and Rima Obeid
Analytical and Bioanalytical Chemistry, 2012, Volume 404, Number 3, Page 895
Minnamari Edelmann, Susanna Kariluoto, Laura Nyström, and Vieno Piironen
Food Chemistry, 2012, Volume 135, Number 3, Page 1938
Leon Haandel, Mara L. Becker, Todd. D. Williams, John F. Stobaugh, and J. Steven Leeder
Rapid Communications in Mass Spectrometry, 2012, Volume 26, Number 14, Page 1617
Jia Liu, Russell Pickford, Alan P. Meagher, and Robyn L. Ward
Analytical Biochemistry, 2011, Volume 411, Number 2, Page 210
Mara L. Becker, Leon van Haandel, Roger Gaedigk, Bradley Thomas, Mark F. Hoeltzel, Andrew Lasky, Hongying Dai, John Stobaugh, and James S. Leeder
Pharmacogenetics and Genomics, 2012, Volume 22, Number 4, Page 236
Joshua D. Williams and Myron K. Jacobson
Journal of Photochemistry and Photobiology B: Biology, 2010, Volume 99, Number 1, Page 49
Jill A. McKay, Yi K. Wong, Caroline L. Relton, Dianne Ford, and John C. Mathers
Molecular Nutrition & Food Research, 2011, Volume 55, Number 11, Page 1717
Veerle De Brouwer, Sergei Storozhenko, Jet C. Van De Steene, Sarah M.R. Wille, Christophe P. Stove, Dominique Van Der Straeten, and Willy E. Lambert
Journal of Chromatography A, 2008, Volume 1215, Number 1-2, Page 125
Susanne H. Kirsch, Jean-Pierre Knapp, Wolfgang Herrmann, and Rima Obeid
Journal of Chromatography B, 2010, Volume 878, Number 1, Page 68
Michèle Aké, Désiré Goin Bi Traye, Christophe Amin N’Cho, François Nicaise Bony, Gildas Komenan Gbassi, and Anglade Kla Malan
Revue Francophone des Laboratoires, 2008, Volume 2008, Number 407, Page 103
Siddharth Banka, Henk J. Blom, John Walter, Majid Aziz, Jill Urquhart, Christopher M. Clouthier, Gillian I. Rice, Arjan P.M. de Brouwer, Emma Hilton, Grace Vassallo, Andrew Will, Desirée E.C. Smith, Yvo M. Smulders, Ron A. Wevers, Robert Steinfeld, Simon Heales, Yanick J. Crow, Joelle N. Pelletier, Simon Jones, and William G. Newman
The American Journal of Human Genetics, 2011, Volume 88, Number 2, Page 216
Robert M. Kok, Desirée E.C. Smith, Rob Barto, Annemieke M.W. Spijkerman, Tom Teerlink, Henk J. Gellekink, Cornelis Jakobs, and Yvo M. Smulders
Clinical Chemical Laboratory Medicine, 2007, Volume 45, Number 7
Gillian Lindzon and Deborah L. O'Connor
Nutrition Research and Practice, 2007, Volume 1, Number 3, Page 163
Yuehua Huang, Stefanie Khartulyari, Megan E. Morales, Anna Stanislawska-Sachadyn, Joan M. Von Feldt, Alexander S. Whitehead, and Ian A. Blair
Rapid Communications in Mass Spectrometry, 2008, Volume 22, Number 16, Page 2403
Martin Kussmann, Michael Affolter, Kornél Nagy, Birgit Holst, and Laurent B. Fay
Mass Spectrometry Reviews, 2007, Volume 26, Number 6, Page 727
Anthony J. A. Wright, Jack R. Dainty, and Paul M. Finglas
British Journal of Nutrition, 2007, Volume 98, Number 04
Journal of Mass Spectrometry, 2007, Volume 42, Number 1, Page 127
Henk J. Blom, Gary M. Shaw, Martin den Heijer, and Richard H. Finnell
Nature Reviews Neuroscience, 2006, Volume 7, Number 9, Page 724

Comments (0)

Please log in or register to comment.
Log in