Capillary Electrophoresis and High-Performance Liquid Chromatography Determination of Polyglutamyl 5-Methyltetrahydrofolate Forms in Citrus Products

The 5-methyltetrahydrofolate (5mTHF) polyglutamates in citrus products were analyzed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Folate species were purified from citrus products and concentrated from 2- to 100-fold using combined folate-affinity chromatography and C18 extraction. Seven polyglutamyl 5mTHFs were found in most not-from-concentrate (NFC) orange juices (OJ) in total amounts of ∼1 nmol/mL, with varying distributions of individual polyglutamates. Folate amounts and distributions were also measured in orange fractions, single-strength OJ from concentrate, NFC grapefruit juice, and citrus peel molasses. Models containing ascorbic acid had folate thermal degradation rates one-seventh that of models without ascorbic acid. Pasteurization studies demonstrated that folate loss was <2% for commercial OJ pasteurization conditions (i.e., 93 °C for 5 s, 88 °C for 15 s, and 82 °C for 30 s). Both methods were precise, reproducible, and potentially faster than traditional analytical procedures requiring enzymatic deconjugation and microbial assays. Keywords: Capillary electrophoresis; citrus byproducts; 5-methyltetrahydrofolate; high-performance liquid chromatography; orange juice