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. 2010 Nov 1;878(29):2949-58.
doi: 10.1016/j.jchromb.2010.08.043. Epub 2010 Sep 9.

A liquid chromatography-tandem mass spectrometric method for quantitative determination of native 5-methyltetrahydrofolate and its polyglutamyl derivatives in raw vegetables

Chao Wang  1 , Ken M Riedl, Steven J Schwartz
Affiliations

A liquid chromatography-tandem mass spectrometric method for quantitative determination of native 5-methyltetrahydrofolate and its polyglutamyl derivatives in raw vegetables

Chao Wang et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

Folate deficiency is a prevalent phenomenon worldwide especially in underprivileged countries. Polyglutamyl 5-methyltetrahydrofolate (5MTHF) species are the naturally occurring principle folate in store-bought vegetables. Here we report a simple and complete extraction method for the determination of native polyglutamyl 5-methyltetrahydrofolate in vegetables using high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS). Coarsely chopped samples (18 different vegetables) were steamed to inactivate glutamylase enzymes and liberate folate from binding proteins and extracted in a reducing buffer with (13)C(5) 5MTHF stable isotope added as internal standard. The polyglutamyl 5-methyltetrahydrofolate species were separated in 9 min on a C(18) column using a reversed phase system. HPLC eluate was interfaced with a triple quadrupole mass spectrometer operated in electrospray positive mode. The respective pseudomolecular cation of each polyglutamyl 5-methyltetrahydrofolate species was selected for fragmentation to a common daughter ion for detection. We quantitated polyglutamyl 5-methyltetrahydrofolate in store-bought vegetables from families Brassicaceae, Asteraceae and Amaranthaceae (including mustard greens, romaine lettuce and Swiss chard) of which most have not been quantitated previously. Most vegetables from Asteraceae and those from Amaranthaceae contained similar amounts of monoglutamyl 5MTHF and polyglutamyl 5MTHF while Brassicaceae were dominated by polyglutamyls and endive species (Asteraceae) contained mainly monoglutamyl 5MTHF. The precision of the method for the various polyglutamyl 5-methyltetrahydrofolate forms was 1-9% RSD, recovery 84-91%, limit of detection 64-658 fmol and limit of quantitation 193-1994 fmol. Herein we describe a rapid, sensitive and selective HPLC-MS/MS technique to quantitate polyglutamyl 5-methyltetrahydrofolate species. This method may be suitable for analyzing the polyglutamyl 5-methyltetrahydrofolate profile of inherent folates in a wide range of leafy green vegetables.

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Figures

Fig. 1
Fig. 1
Chemical structure of polyglutamyl 5-methyltetrahydrofolate.
Fig. 2
Fig. 2
HPLC–MS/MS chromatogram of polyglutamyl 5-methyltetrahydrofolate (5MTHF-Glun) in broccoli rabe extract. Steamed broccoli rabe (Brassica rapa) was freeze-ground in liquid nitrogen and extracted in boiling reducing buffer for 10 min. *5MTHF-Glu8 for which authentic standard was unavailable was quantified by parent > daughter MS/MS transition and elution order consistent with species.
Fig. 3
Fig. 3
Effect of steaming time on the polyglutamyl 5-methyltetrahydrofolate (5MTHF-Glun) species of cauliflower (Brassica oleracea). Cauliflower was steamed prior to grinding in liquid nitrogen and then extracted in boiling reducing buffer for 10 min. The values were determined according to individual calibration curves for 5MTHF-(Glu)1–7 while the MS/MS response of 5MTHF-(Glu)8 was estimated by extrapolation of the trend in response for 5MTHF-(Glu)5–7. Data represented the mean of two replicates. (A) Absolute amount of polyglutamyl 5MTHF species and total 5MTHF as a function of steaming time. (B) Relative profile (% relative to total) of polyglutamyl 5MTHF species after boiling (no steaming). (C) Profile of polyglutamyl 5MTHF species after steaming for 10 min followed by boiling extraction.
Fig. 4
Fig. 4
Polyglutamyl 5-methyltetrahydrofolate (5MTHF-Glun) profiles in vegetables from various plant families. Levels determined by HPLC–MS/MS using authentic standards for 5MTHF-(Glu)1–7 while the MS/MS response of 5MTHF-(Glu)8 was estimated by extrapolation of the trend in response for 5MTHF-(Glu)5–7. Values as x-axis represent polyglutamyl chain length. (A) Average profile (% relative to total) of nine vegetables from family Brassicaceae. (B) Average of two members of family Amaranthaceae: Spinacia oleracea (spinach) and Beta vulgaris (Swiss chard). (C) Average of two members of family Asteraceae Taraxacum officinale Wigg (dandelion greens) and Lactuca sativa L. (romaine lettuce). (D) Average of five members of family Asteraceae: Cichorium endivia (curly endive, frisée and escarole), Cichorium intybus (radicchio and Belgian endive) [39].
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