HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

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 18 analytical column in an alkaline medium in order (i) to be compatible with the o -phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λ ext /λ em = 340/460 nm) after post-column derivatization with o -phthalaldehyde and N -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 β -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 -1 . 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.