Mixed Micellar Electrokinetic Chromatographic Analysis of Colistin, Polypeptide Antibiotic, Using Laser-Induced Fluorescence Detection

Abstract

The main goal of this work was to quantify the detection of colistin at low levels in urine samples through the practical application of mixed surfactant micellar electrokinetic chromatography–laser-induced fluorescence (MEKC-LIF) analysis method using its advantage of sensitivity and to examine direct injection of biological samples. Colistin (po- lymyxin E) has neither strong UV chromophore nor fluorophore. So, its assay for metabolism, pharmacokinetics studies for bioavailability and bioequivalence are difficult because of poor detectability. Therefore an enhanced UV or fluores-cence detection by chemical derivatization is required. MEKC-LIF method was proposed for colistin with a 488/520 nm argon-ion laser using a pre-CE derivatization with fluorescein isothiocyanate (FITC). Borate buffer was used as background buffer (BGB). The different parameters affecting the proposed derivatization reaction including concentration of the derivatizing reagent, reaction time and temperature were studied and optimized. The derivative was stable for up to 3 days. Different micelles (TX-100 and SDS) were examined as BGB additives separately but negative-charged mixed micelles (SDS/TX-100) were shown to be the best additive to BGB for the analysis of colistin particularly in human urine as they enhance both selectivity and sensitivity of the proposed method. BGB was used with pH 9.5, 10 kV, 8 s inj time, capillary length 75 cm × 75 μm ID (66 cm effective length), detection was LIF Ex 488 nm; Em 520 nm. The method was applied to colistin analysis in human urine and the recovery was > 98% (n = 5). LOD and LOQ in urine after pre-column derivatization using FITC were 100 and 250 ng/ml, respectively. Urine samples were analysed by direct injection without sample pre-treatment. The mechanism of enhancement of fluorescence of the derivative by surfactant was proposed.