Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection.

Abstract

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.