Determination of phosphoamino acids derivatized with 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein by micellar electrokinetic chromatography

A sensitive analytical protocol for determining phosphoamino acids using capillary electrophoresis coupled with laser-induced fluorescence detection has been developed. The technique involved the derivatization of the phosphoamino acids with fluorescent reagent 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the analyses of the derivatives by micellar electrokinetic chromatography with laser induced fluorescence detection (MEKC-LIF). Different variables that affect derivatization (DTAF concentration, pH, temperature and time) and separation (kind of surfactant, pH and concentration of buffer) were studied. The baseline separation of three phosphoamino acids could be obtained in less than 11 min with good reproducibility. There was a linear relationship between the peak area of the analyte and its concentration, with correlation coefficients in the range of 0.9979–0.9997. The concentration detection limits (signal to noise = 3) with respect to each single phosphoamino acid were in the range of 0.5–1 nM. The developed method was successfully applied for the determination of phosphoamino acids in the hydrolyzed phosphorylated protein samples.