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 (lambda(ext)/lambda(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 beta-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 center dot 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.

Automated summary

This study demonstrates the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples. The method was optimized using experimental design and validated using the total error concept. The proposed method showed satisfactory results and was successfully applied to the analysis of the drug in human urine samples.