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

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 (13)C(5) 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.

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

“…Accordingly, measurement relies on polyglutamate removal, condensing polyglutamated forms into a single monoglutamate pool. This is typically accomplished with rat serum (18, 22), which has substantial glutamate carboxypeptidase activity (23). …”

Evaluating the performance of sample preparation methods for ultra‐performance liquid chromatography/mass spectrometry based serum metabonomics

“…Over 1000 features were detected from each of the serum samples. The higher chromatographic resolution and increased analytical sensitivity attained by using UPLC separation were extremely important for minimizing any co‐elution‐induced ionization suppression effects and maximizing the metabolic information obtainable …”

“…Since its introduction to metabolic profiling in 2005, ultra‐high‐performance liquid chromatography/mass spectrometry (UPLC/MS) has become widely used for metabolic profiling . Compared with other analytical methods, UPLC/MS offers the advantages of increased chromatographic resolution and analytical sensitivity at a higher sample throughput . UPLC/MS‐based metabonomic investigations have been performed to discover biomarkers of diseases and in many other applications .…”

“…[13][14][15][16][17] Compared with other analytical methods, UPLC/MS offers the advantages of increased chromatographic resolution and analytical sensitivity at a higher sample throughput. [18][19][20] UPLC/MS-based metabonomic investigations have been performed to discover biomarkers of diseases and in many other applications. 17,[21][22][23][24][25][26] Herein, UPLC coupled to hybrid quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) was applied to investigate the efficiency of different sample preparation methods for UPLC/MS-based metabonomic studies.…”

CHAPTER 11. The Chemistry of Folate

“…Recently, an automated 96-well plate isotope-dilution tandem mass spectrometry method and an ultra-performance liquid chromatography-tandem mass spectrometry method have been developed and validated for the quantification of synthetic folic acid in folate-fortified breads (Fazili and Pfeiffer 2004;Chandra-Hioe et al 2011).…”