Determination of Total Folate Content and Polyglutamate Folate Distribution in Winged Beans Using UPLC-MS/MS
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Abstract:
An ultra-high performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method was established to determine the total folate content and polyglutamate folate distribution in winged beans. Coarsely chopped winged beans were boiled to inactivate endogenous polyglutamate hydrolase and degrade folate-binding proteins so that the bound folate was converted to free form. After filtration, the extracted folate in various forms was converted to 5-methyl tetrahydrofolate polyglutamyl (5MTHFGlun), salt was removed using a C18 SPE cartridge, and UPLC was performed to separate the different forms of 5MTHFGlun. The content of 5MTHFGlun in different forms were determined using positive electrospray ionization (ESI+) under multiple reaction monitoring (MRM) mode. Quantitative analysis was conducted using PteGlu(1–6) as the internal standard and 5MTHFGlu(1–6) as external standards. The method showed high sensitivity, with a limit of detection of 40~1133 fmol and a limit of quantification of 120~3400 fmol. The precision of determination ranged from 0.5% to 6.8% and the average recovery was 43%. The results showed that the predominant form of polyglutamate folate in winged bean was pentaglutamyl folate (65%), followed by 5MTHF, accounting for 29.6%; other forms accounted for only 5.6%. The total folate content in fresh winged beans was 1.1 μmol/100 g, which was significantly higher than that in spinach and broccoli. This method is fast, sensitive, and accurate, and is suitable for determining total folate and polyglutamyl folate distribution in different vegetables.
