Determination of tizoxanide, the active metabolite of nitazoxanide, by micellar liquid chromatography using a monolithic column. Application to pharmacokinetic studies

Abstract

In this study, a micellar liquid chromatography (MLC) method is proposed for the determination of tizoxanide (TZ), the active metabolite of nitazoxanide (NX), using a micellar mobile phase consisting of 0.1 M sodium dodecyl sulphate, 8% n-propanol and 0.3% triethylamine in 0.02 M phosphoric acid buffered at pH 4. The method was successfully applied to the analysis of tizoxanide (TZ), the active metabolite of nitazoxanide (NX), in the presence of tinidazole (TIN) as an internal standard in the pure form, in real human urine and plasma without a previous extraction step. Analytical separation was performed in less than 10 min using a RP C18 monolithic column with UV detection at 240 nm. The validation study of the proposed method was successfully carried out in an assay range between 0.05 and 20 μg mL⁻¹ with the limit of detection (LOD) 0.016 μg mL⁻¹ and the limit of quantification (LOQ) 0.049 μg mL⁻¹. The method was fully validated in accordance with the ICH guidelines. The proposed method was successfully applied to quantitatively determine TZ in spiked human urine and plasma. It was also extended to the pharmacokinetic studies of TZ in real human urine and plasma samples.