European Food Research and Technology

, Volume 236, Issue 1, pp 17–28

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

Original Paper

DOI: 10.1007/s00217-012-1849-x

Cite this article as:
Ringling, C. & Rychlik, M. Eur Food Res Technol (2013) 236: 17. doi:10.1007/s00217-012-1849-x


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.


10-Formyldihydrofolate LC–MS/MS Food folate Stable isotope dilution assay Postcolumn infusion 

Supplementary material

217_2012_1849_MOESM1_ESM.pdf (59 kb)
Supplementary material 1 (PDF 59 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  1. 1.Division BIOANALYTIK Weihenstephan, Research Center for Nutrition and Food Sciences (Z I E L)Technische Universität MünchenFreisingGermany
  2. 2.Chair of Analytical Food ChemistryTechnische Universität MünchenFreisingGermany

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