Folate vitamers

Freisleben, A., Sehieberle, P, Ryehlik, M. (2003 May). Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass speetrometry. Anal. Bioanal. Chem., 376 2), 149-156. 

Figure 10.1. Folate vitamers, the folate antagonist methotrexate, molybdopterln, and tetrahydrobiopterin. Relative molecular masses (Mr) tetrahydrofolic acid, 445.4 methotrexate, 454.5 and tetrahydrobiopterin, 290.3.

Matherly LH, Goldman DI. Membrane transport of folates. Vitam Horm 2003 66 403-56. 

The diversity of folate vitamers is attributed to different oxidation states of the pteridine ring, the number of glutamic acid residues, and the type of one-carbon unit attached at positions 5 and/or 10 relative to the nitrogen atom [5,6], as illustrated in Figure 6.2. 

FIGURE 6.2 Various folate vitamers differ in the one-carbon unit that can be substituted at N-5, N-10, or N-5 and N-10 positions. (From Gerald F. Combs Jr, The Vitamins Folate, 4th ed.. Academic Press, San Diego, 2012. With permission.)... 

The main folate vitamers occurring in fruits and vegetables are different from those found in animal and cereal products [16]. There is also evidence that the vitamers have different bioavailability [17] and stability [18]. A clear need existed for an analytical method that would be capable of independently quantifying each specific folate vitamer. Liquid chromatography (LC) seemed an ideal and logical technology for this purpose. 

In addition, sensitivity has been identified as another weakness of HPLC [25]. In spinach, 5-formyltetrahydrofolate was not detected using a fluorescence detector [26] and in other foods its level was close to the limit of detection, leading to inaccurate quantification [19]. It was not until the late 1990s that mass spectrometry found application as a highly spedlic detector for individual folate vitamers that can... 

Jastrebova et al. (2011) compared the use of HPLC and UPLC, both coupled with ultra-violet and fluorescence detection, to determine folate in foods. In addition to reduced run times, the limit of detection was lower dne to improved signal-to-noise ratio for folate vitamers [40]. 

The m/z values monitored in SRM mode for various folate vitamers are shown in Table 6.1. For quantitative purposes, commercially available Cj-labeled internal standards (Merck-Eprova, Schaffhausen, Switzerland) are added at the start of the sample extraction. 

De Brouwer et al. (2010) identified 5 folate vitamers in their study 5-methyltet-rahydrofolate (the predominant vitamer), 5,10-methenyltetrahydrofolate and tetrahy-drofolate (the most labile vitamers), folic acid, and 10-formyl folic acid. This study reported that folate levels in wild-type rice were approximately 20 pg/lOO g lower than in bio-fortified rice [41], While separation of folate vitamers was obtained in 8 min run time, a previous LC-MS/MS method required 20 min [49]. Also, UPLC improved the sensitivity by reducing the limit of quantitation. Another study documented that endogenous 5-methyltetrahydrofolate in bread ranged between 1.3 and 3.4 pg/100 g [14]. Measurements of folic acid and 5-methyltetrahydrofolate in flour and infant milk formula have been successfully made using this method, described in the latter study. 

The other factor that needs to be taken into consideration during sample processing is the stability of folate vitamers. Stability of the vitamers differs with respect to the susceptibility to oxidative degradation, thermal, pH, and ultra-violet light [51]. Interconversion of folate vitamers occurs during sample preparation and analysis, for example, 5,10-methylenetetrahydrofolate dissociates from tetrahydrofolate and formaldehyde in the presence of mercaptoethanol (antioxidant) at low pH [49]. Furthermore, it has been argued that 5,10-methenyltetrahydrofolate is difficult to analyze on a reversed-phase (RP-18) HPLC column with a low pH mobile phase [13]. This is because interconversion of 10-formyltetrahydrofoIate to 5,10-methenyltetrahydrofolate occurs at low pH [18]. 

Ruggeri, S., Vahteristo, L. T., Aguzzi, A., Finglas, P., and Camovale, E. 1999. Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography. J. Chromatogr. A. 855 237-245. 

Figure 25.1 Structures of folic acid and naturally occurring folate vitamers. The formulae and the table show all possible variations of folates that have been detected in foods.

The most widely used method to quantitate folates is a microbiological assay (MBA), which provides a total figure for all folate vitamers. During this bioassay, the growth of Lactobacillus casei ssp. rhamnosus, for which folate is essential, is measured in a specifically deficient nutrient solution. The medium is then supplemented with a sample or a folate standard, and the resulting growth is measured at intervals by turbidity, which conveniently can be measured in an ultraviolet-visible (UV-vis) spectrophotometer. 

The microbiological assay is not able to differentiate between single folate vitamers. 

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