Fluorometric determination of pantothenic acid in human urine by isocratic reversed-phase ion-pair high-performance liquid chromatography with post-column derivatization

We describe here a method for the determination of pantothenic acid, vitamin B5, in human urine by isocratic reversed-phase ion-pair HPLC with post-column derivatization. Pantothenic acid in urine was separated using a Tosoh ODS-80Ts (4.6 i.d. × 250 mm) column with phosphate-sodium hydroxide buffer (pH 7.0) containing dodecyltrimethylammonium chloride. Following the isolation of pantothenic acid it was decomposed to pantoic acid and β-alanine by alkali treatment. The product β-alanine was post-derivatized to the fluorescent 1-alkylthio-2-alkylisoindole with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid. In the proposed method, a urine sample can be directly injected into a HPLC system without any pre-clean up treatment. The limit of detection was 3 pmol (ca. 650 pg) per 20 μL of urine at a signal-to-noise ratio of 5:1 and the limit of quantification was 5 pmol (ca. 1000 pg) per 20 μL of urine, which was sufficiently sensitive for the determination of pantothenic acid in human urine. The total time required for the analysis was ca. 25 min. The proposed method can be used to assess the pantothenic acid content of human urine as an alternative to the standard microbioassay for pantothenic acid.