| Summary: | In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core–shell C<sub>18</sub> analytical column in an alkaline medium in order (i) to be compatible with the <i>o</i>-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λ<sub>ext</sub>/λ<sub>em</sub> = 340/460 nm) after post-column derivatization with <i>o</i>-phthalaldehyde and <i>N</i>-acetyl cysteine. The post-column derivatization parameters were optimized using the Box–Behnken experimental design, and the method was validated using the total error concept. The <i>β</i>-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L<sup>−1</sup>. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of −2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.
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