Analytical and Bioanalytical Chemistry

, Volume 401, Issue 3, pp 1035–1042 | Cite as

Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads

Original Paper
First Online:
Received:
Revised:
Accepted:

Abstract

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl-l-glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of 13C5 labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018–14 μg FA/g of fresh bread (r 2 = 0.997) and 9.3–900 ng 5-MTHF/g of fresh bread (r 2 = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79–110 μg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200–300 μg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors.

Figure

Chromatogram of labelled folic acid using UPLC-MS/MS

Keywords

Folic acid 5-MTHF UPLC MS/MS HPLC-MS Fortification Bread Quantitation 

Abbreviations

5-MTHF

5-Methyltetrahydrofolate

CID

Collision-induced dissociation

CV

Coefficient of variation

Da

Dalton

ESI

Electrospray ionization

FA

Folic acid

HPLC

High-performance liquid chromatography

IS

Internal standard

LC-MS

Liquid chromatography-mass spectrometry

LOD

Limit of detection

LOQ

Limit of quantitation

MS/MS

Tandem mass spectrometry

m/z

Mass-to-charge ratio

SPE

Solid-phase extraction

SIM

Single-ion monitoring

SRM

Selected reaction monitoring

UPLC

Ultra-performance liquid chromatography

This is a preview of subscription content, log in to check access.

Supplementary material

216_2011_5156_MOESM1_ESM.pdf (13 kb)
ESM 1 (PDF 13.0 kb)

References

  1. 1.
    MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1:1027–1031Google Scholar
  2. 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):1832–1835CrossRefGoogle Scholar
  3. 3.
    Mathews TJ, Honein MA, Erickson JD (2002) Spina bifida and anencephaly prevalence—United States, 1991–2001. Morb Mortal Wkly Rep 51(13):9–11Google Scholar
  4. 4.
    De Wals P, Tairou F, Van Allen MI, Uh SH, Lowry RB, Sibbald B, Evans JA, Van Den Hof MC, Zimmer P, Crowley M, Fernandez B, Lee NS, Niyonsenga T (2007) Reduction in neural-tube defects after folic acid fortification in Canada. N Engl J Med 357(2):135–142CrossRefGoogle Scholar
  5. 5.
    Oakley GP Jr (2009) The scientific basis for eliminating folic acid-preventable spina bifida: a modern miracle from epidemiology. Ann Epidemiol 19(4):226–230. doi: 10.1016/j.annepidem.2009.01.016 CrossRefGoogle Scholar
  6. 6.
    Stokes P, Webb K (1999) Analysis of some folate monoglutamates by high-performance liquid chromatography-mass spectrometry. I. J Chromatogr A 864(1):59–67. doi: 10.1016/S0021-9673(99)00992-9 CrossRefGoogle Scholar
  7. 7.
    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):4760–4768CrossRefGoogle Scholar
  8. 8.
    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):149–156Google Scholar
  9. 9.
    Freisleben A, Schieberle P, Rychlik M (2003) Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry. Anal Biochem 315(2):247–255CrossRefGoogle Scholar
  10. 10.
    Pawlosky RJ, Flanagan VP, Pfeiffer CM (2001) Determination of 5-methyltetrahydrofolic acid in human serum by stable-isotope dilution high-performance liquid chromatography-mass spectrometry. Anal Biochem 298(2):299–305CrossRefGoogle Scholar
  11. 11.
    Pawlosky RJ, Flanagan VP (2001) A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods. J Agric Food Chem 49(3):1282–1286CrossRefGoogle Scholar
  12. 12.
    Pawlosky RJ, Flanagan VP, Doherty RF (2003) A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods. J Agric Food Chem 51(13):3726–3730CrossRefGoogle Scholar
  13. 13.
    Pawlosky RJ, Hertrampf E, Flanagan VP, Thomas PM (2003) Mass spectral determinations of the folic acid content of fortified breads from Chile. J Food Compos Anal 16(3):281–286CrossRefGoogle Scholar
  14. 14.
    Leporati A, Catellani D, Suman M, Andreoli R, Manini P, Niessen WMA (2005) Application of a liquid chromatography tandem mass spectrometry method to the analysis of water-soluble vitamins in Italian pasta. Anal Chim Acta 531(1):87–95CrossRefGoogle Scholar
  15. 15.
    Patring JDM, Jastrebova JA (2007) Application of liquid chromatography-electrospray ionisation mass spectrometry for determination of dietary folates: Effects of buffer nature and mobile phase composition on sensitivity and selectivity. J Chromatogr A 1143(1–2):72–82CrossRefGoogle Scholar
  16. 16.
    Kok RM, Smith DEC, Dainty JR, Van Den Akker JT, Finglas PM, Smulders YM et al (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(2):129–138CrossRefGoogle Scholar
  17. 17.
    Rychlik M (2004) Revised folate content of foods determined by stable isotope dilution assays. J Food Compos Anal 17(3–4):475–483CrossRefGoogle Scholar
  18. 18.
    Patring J, Wandel M, Jägerstad M, Frølich W (2009) Folate content of Norwegian and Swedish flours and bread analysed by use of liquid chromatography-mass spectrometry. J Food Compos Anal 22(7–8):649–656CrossRefGoogle Scholar
  19. 19.
    Pfeiffer CM, Rogers LM, Gregory Iii 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):407–413CrossRefGoogle Scholar
  20. 20.
    Zhang GF, Maudens KE, Storozhenko S, Mortier KA, Van Der Straeten D, Lambert WE (2003) Determination of total folate in plant material by chemical conversion into para-aminobenzoic acid followed by high performance liquid chromatography combined with on-line postcolumn derivatization and fluorescence detection. J Agric Food Chem 51(27):7872–7878CrossRefGoogle Scholar
  21. 21.
    Nielsen AC (2008) Special report on top brandsGoogle Scholar
  22. 22.
    Yazynina E, Johansson M, Jägerstad M, Jastrebova J (2008) Low folate content in gluten-free cereal products and their main ingredients. Food Chem 111(1):236–242CrossRefGoogle Scholar
  23. 23.
    Hibbert G (2006) Data analysis for chemistry. Oxford University Press, New YorkGoogle Scholar
  24. 24.
    Osseyi ES, Wehling RL, Albrecht JA (2001) HPLC determination of stability and distribution of added folic acid and some endogenous folates during breadmaking. Cereal Chem 78(4):375–378CrossRefGoogle Scholar
  25. 25.
    Gujska E, Majewska K (2005) Effect of baking process on added folic acid and endogenous folates stability in wheat and rye breads. Plant Foods Hum Nutr 60(2):37–42CrossRefGoogle Scholar
  26. 26.
    Öhrvik V, Öhrvik H, Tallkvist J, Witthöft C (2010) Folates in bread: retention during bread-making and in vitro bioaccessibility. Eur J Nutr 49(6):365–372CrossRefGoogle Scholar
  27. 27.
    Anderson WA, Slaughter D, Laffey C, Lardner C (2010) Reduction of folic acid during baking and implications for mandatory fortification of bread. Int J Food Sci Technol 45(6):1104–1110CrossRefGoogle Scholar
  28. 28.
    Rader JI, Weaver CM, Angyal G (2000) Total folate in enriched cereal-grain products in the United States following fortification. Food Chem 70(3):275–289CrossRefGoogle Scholar
  29. 29.
    FSANZ (2006) Draft assessment report proposal P295 consideration of mandatory fortification with folic acid. FSANZ, CanberraGoogle Scholar
  30. 30.
    Alaburda J, de Almeida AP, Shundo L, Ruvieri V, Sabino M (2008) Determination of folic acid in fortified wheat flours. J Food Compos Anal 21(4):336–342CrossRefGoogle Scholar
  31. 31.
    Whittaker P, Tufaro PR, Rader JI (2001) Iron and folate in fortified cereals. J Am Coll Nutr 20(3):247–254Google Scholar
  32. 32.
    Kea D (2008) Nutritional value of bread. J Cer Sci 48:243–257CrossRefGoogle Scholar
  33. 33.
    Shakur YA, Rogenstein C, Hartman-Craven B, Tarasuk V, O’Connor DL (2009) How much folate is in canadian fortified products 10 years after mandated fortification? Can J Public Health 100(4):281–284Google Scholar
  34. 34.
    Lucock M (2000) Folic acid: nutritional biochemistry, molecular biology, and role in disease processes. Mol Genet Metab 71(1–2):121–138CrossRefGoogle Scholar
  35. 35.
    Finglas PM, Witthöft CM, Vahteristo L, Wright AJA, Southon S, Mellon FA et al (2002) Use of an oral/intravenous dual-label stable-isotope protocol to determine folic acid bioavailability from fortified cereal grain foods in women. J Nutr 132(5):936–939Google Scholar
  36. 36.
    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(2):423–432CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Food Science and Technology, School of Chemical EngineeringUniversity of New South WalesSydneyAustralia
  2. 2.Mark Wainwright Analytical Centre, Bioanalytical Mass Spectrometry FacilityUniversity of New South WalesSydneyAustralia
  3. 3.School of ChemistryUniversity of New South WalesSydneyAustralia

Personalised recommendations