Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Abstract

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l −1 sodium borate buffer (pH 9.35) containing 20 mmol l −1sodium deoxycholate (SDC) and 10 mmol l −1 Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966–0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l −1, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.