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Analysis of seven folates in food by LC–MS/MS to improve accuracy of total folate data

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Abstract

An LC–MS/MS method for analyzing seven folates in food was developed and validated. 5-Methyltetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolic acid, tetrahydrofolate and folic acid were quantified using a stable isotope dilution assay (SIDA) with deuterated analogues as internal standards. Additionally, 10-formyldihydrofolate and 5,10-methenyltetrahydrofolate were quantified using deuterated internal standards different in structure. Due to interconversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate and 10-formyldihydrofolate to 10-formylfolic acid during sample preparation, a SIDA was not considered because of a resulting double calculation of the amounts interconverting. [2H4]-5-methyltetrahydrofolate was used as internal standard for 5,10-methenyltetrahydrofolate, due to a similar retention time, and [2H4]-10-formylfolic acid as well as [2H4]-5-methyltetrahydrofolate was used for 10-formyldihydrofolate, because no internal standards co-elute. To confirm that no matrix effects affect the quantitation of 5,10-methenyltetrahydrofolate and 10-formyldihydrofolate, postcolumn infusion experiments were performed. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection and limit of quantitation. The latter parameters were partly obtained by application of a dual-isotope label design including [13C5]-labeled folates. The amounts of 5,10-methenyltetrahydrofolate in the purified extracts of different food samples ranged between 0.3 and 1.3 % and for 10-HCO-H2folate between 0.05 and 8 % of the total folate amount. Correction for incomplete recovery of the latter folate during cleanup indicates even higher contents. Therefore, especially 10-formyldihydrofolate should not be neglected to obtain accurate results for folates.

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References

  1. Arnesen E, Refsum H, Bonaa KH, Ueland PM, Forde OH, Nordrehaug JE (1995) Serum total homocysteine and coronary heart-disease. Int J Epidemiol 24(4):704–709

    Article  CAS  Google Scholar 

  2. Czeizel AE, Dudas I (1992) Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 327(26):1832–1835

    Article  CAS  Google Scholar 

  3. Robinson K (2000) Homocysteine, B vitamins, and risk of cardiovascular disease. Heart 83(2):127–130

    Article  CAS  Google Scholar 

  4. Beck WS (1991) Diagnosis of megaloblastic-anemia. Annu Rev Med 42:311–322

    Article  CAS  Google Scholar 

  5. Food and Nutrition Board—Institute of Medicine (1998) Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academy Press, Washington, DC

    Google Scholar 

  6. Gregory JF, Bhandari SD, Bailey LB, Toth JP, Baumgartner TG, Cerda JJ (1992) Relative bioavailability of deuterium-labeled monoglutamyl tetrahydrofolates and folic acid in human subjects. Am J Clin Nutr 55(6):1147–1153

    CAS  Google Scholar 

  7. Rychlik M (2009) Die Vitamine der Folatgruppe, Eine Übersicht. Deut Lebensm-Rundschau 105(5):292–296

    CAS  Google Scholar 

  8. Annesley TM (2003) Ion suppression in mass spectrometry. Clin Chem 49(7):1041–1044

    Article  CAS  Google Scholar 

  9. Freisleben A, Schieberle P, Rychlik M (2003) 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 376(2):149–156

    CAS  Google Scholar 

  10. Gutzeit D, Moench S, Jerz G, Winterhalter P, Rychlik M (2008) Folate content in sea buckthorn berries and related products (Hippophae rhamnoides L. ssp rhamnoides): LC-MS/MS determination of folate vitamer stability influenced by processing and storage assessed by stable isotope dilution assay. Anal Bioanal Chem 391(1):211–219

    Article  CAS  Google Scholar 

  11. Buettner BE, Ohrvik VE, Witthoft CM, Rychlik M (2011) Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples. Anal Bioanal Chem 399(1):429–439

    Article  CAS  Google Scholar 

  12. Vishnumohan S, Arcot J, Pickford R (2010) Naturally-occurring folates in foods: Method development and analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Food Chem 125(2):736–742

    Article  Google Scholar 

  13. Zhang GF, Storozhenko S, van der Straeten D, Lambert WE (2005) Investigation of the extraction behavior of the main monoglutamate folates from spinach by liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr A 1078(1–2):59–66

    CAS  Google Scholar 

  14. Garratt LC, Ortori CA, Tucker GA, Sablitzky F, Bennett MJ, Barrett DA (2005) Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom 19(17):2390–2398

    Article  CAS  Google Scholar 

  15. Phillips KM, Ruggio DM, Ashraf-Khorassani M, Haytowitz DB (2006) Difference in folate content of green and red sweet peppers (Capsicum annuum) determined by liquid chromatography-mass spectrometry. J Agric Food Chem 54(26):9998–10002

    Article  CAS  Google Scholar 

  16. de Brouwer V, Storozhenko S, van de Steene JC, Wille SMR, Stove CP, van der Straeten D, Lambert WE (2008) Optimisation and validation of a liquid chromatography-tandem mass spectrometry method for folates in rice. J Chromatogr A 1215(1–2):125–132

    Google Scholar 

  17. Wang C, Riedl KM, Somerville J, Balasubramaniam VM, Schwartz SJ (2011) Influence of high-pressure processing on the profile of polyglutamyl 5-methyltetrahydrofolate in selected vegetables. J Agric Food Chem 59(16):8709–8717

    Article  CAS  Google Scholar 

  18. Munyaka AW, Oey I, Verlinde P, van Loey A, Hendrickx M (2009) Acidification, crushing and thermal treatments can influence the profile and stability of folate poly-gamma-glutamates in broccoli (Brassica oleracea L. var. italica). Food Chem 117(3):568–575

    Article  CAS  Google Scholar 

  19. de Brouwer V, Zhang G, Storozhenko S, van der Straeten D, Lambert WE (2007) pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates. Phytochem Anal 18(6):496–508

    Article  Google Scholar 

  20. Wilson SD, Horne DW (1984) High-performance liquid-chromatographic determination of the distribution of naturally-occurring folic acid derivatives in rat liver. Anal Biochem 142(2):529–535

    Article  CAS  Google Scholar 

  21. O’Broin JD, Temperley IJ, Brown JP, Scott JM (1975) Nutritional stability of various naturally occurring monoglutamate derivatives of folic acid. Am J Clin Nutr 28(5):438–444

    Google Scholar 

  22. Pfeiffer CM, Rogers LM, Gregory JF (1997) Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography. J Agric Food Chem 45(2):407–413

    Article  CAS  Google Scholar 

  23. Ruggeri S, Vahteristo LT, Aguzzi A, Finglas P, Carnovale E (1999) Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography. J Chromatogr A 855(1):237–245

    Article  CAS  Google Scholar 

  24. Konings EJ, Roomans HH, Dorant E, Goldbohm RA, Saris WH, van den Brandt PA (2001) Folate intake of the Dutch population according to newly established liquid chromatography data for foods. Am J Clin Nutr 73(4):765–776

    CAS  Google Scholar 

  25. Kariluoto MS, Vahteristo LT, Piironen VI (2001) Applicability of microbiological assay and affinity chromatography purification followed by high-performance liquid chromatography (HPLC) in studying folate contents in rye. J Sci Food Agric 81(9):938–942

    Article  CAS  Google Scholar 

  26. Baggott JE, Tamura T (2001) Metabolism of 10-formyldihydrofolate in humans. Biomed Pharmacother 55(8):454–457

    Article  CAS  Google Scholar 

  27. Freisleben A, Schieberle P, Rychlik M (2002) Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays. J Agric Food Chem 50(17):4760–4768

    Article  CAS  Google Scholar 

  28. Tyagi A, Penzkofer A, Batschauer A, Wolf E (2009) Thermal degradation of (6R, S)-5,10-methenyltetrahydrofolate in aqueous solution at pH 8. Chem Phys 358(1–2):132–136

    Article  CAS  Google Scholar 

  29. Vogelgesang J, Haedrich J (1998) Limits of detection, identification and determination: a statistical approach for practitioners. Accred Qual Assur 3(6):242–255

    Article  CAS  Google Scholar 

  30. Baggott JE, Johanning GL, Branham KE, Prince CW, Morgan SL, Eto I, Vaughn WH (1995) Cofactor role for 10-formyldihydrofolic acid. Biochem J 308(Part 3):1031–1036

    CAS  Google Scholar 

  31. Baggott JE, Johanning GL (1999) 10-Formyl-dihydrofolic acid is bioactive in human leukemia cells. J Nutr 129(7):1315–1318

    CAS  Google Scholar 

  32. Baram J, Chabner BA, Drake JC, Fitzhugh AL, Sholar PW, Allegra CJ (1988) Identification and biochemical-properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells. J Biol Chem 263(15):7105–7111

    CAS  Google Scholar 

  33. Holcapek M, Jandera P (1999) Interpretation of electrospray and atmospheric pressure chemical ionization mass spectra of 10-formyl-7,8-dihydrofolic acid and 5-formyl-5,6,7,8-tetrahydropteroic acid. Rapid Commun Mass Spectrom 13(14):1423–1426

    Article  CAS  Google Scholar 

  34. Pedersen JC (1988) Comparison of γ-glutamyl hydrolase (conjugase; EC 3.4.22.12) and amylase treatment procedures in the microbiological assay for food folates. Br J Nutr 59(2):261–271

    Article  CAS  Google Scholar 

  35. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75(13):3019–3030

    Article  CAS  Google Scholar 

  36. Rychlik M, Netzel M, Pfannebecker I, Frank T, Bitsch I (2003) Application of stable isotope dilution assays based on liquid chromatography-tandem mass spectrometry for the assessment of folate bioavailability. J Chromatogr B 792(2):167–176

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant from the Deutsche Forschungsgemeinschaft (RY, 19/7-2). Moreover, the authors gratefully acknowledge the support by the Faculty Graduate Center Weihenstephan of TUM Graduate School at Technische Universität München, Germany.

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Authors and Affiliations

  1. Division BIOANALYTIK Weihenstephan, Research Center for Nutrition and Food Sciences (Z I E L), Technische Universität München, Alte Akademie 10, 85350, Freising, Germany

    Christiane Ringling & Michael Rychlik

  2. Chair of Analytical Food Chemistry, Technische Universität München, Alte Akademie 10, 85350, Freising, Germany

    Michael Rychlik

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  1. Christiane Ringling
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  2. Michael Rychlik
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Correspondence to Michael Rychlik.

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Ringling, C., Rychlik, M. Analysis of seven folates in food by LC–MS/MS to improve accuracy of total folate data. Eur Food Res Technol 236, 17–28 (2013). https://doi.org/10.1007/s00217-012-1849-x

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  • DOI: https://doi.org/10.1007/s00217-012-1849-x

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