Determination of folate monoglutamates in biofortified rice with UPLC-MS/MS

Folates are essential nutrients for human beings. To reduce folate deficiencies and consequences thereof, such as spina bifida, in developing areas like Northern China, biofortification of rice by means of genetic modification is one of the approaches. We successfully overexpressed two plant genes involved in the folate pathway in rice (1) and evaluated these interventions by analysing 6 monoglutamate folates present in rice matrix, by a validated ultra high performance chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Rice was homogenized and heated, followed by a a tri-enzyme treatment (a-amylase, protease and deconjugase). Subsequently, the extract was centrifuged, ultrafiltrated, and injected on an Acquity UPLC (R) HSS T3 column (150mm x 2.1mm, 1.8-mu m particle size), coupled to tandem mass spectrometric detection. The method was validated based on linearity, sensitivity, precision, accuracy, recovery, stability and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 mu g/100g and 0.12 and 0.91 mu g/100g, respectively. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 mu g/100g rice. 5-Methyl-THF was the dominant natural folate form (+/- 90% of total folate content for biofortified rice). This shows the successful implementation of this UPLC method for the rapid, quantitative determination of folates