The report presents robust and high throughput methods, based on liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD), for the simultaneous determination of major metabolites of ibuprofen (IBU), namely 2-hydroxyibuprofen and carboxyibuprofen (method A) as well as creatinine (Crn) (method B) in human urine. The assays primarily involve straightforward sample purification. For both methods, the chromatographic separation of the analytes is achieved within 8 min at room temperature on Poroshell 120 SB-C18 (75 × 4.6 mm, 2.7 µm) column using gradient elution. The eluents consisted of 0.1% formic acid in water and acetonitrile (method A) or water and methanol (method B) delivered at a flow rate of 1 or 0.5 mL/min, respectively. In relation to metabolites of IBU, the assay linearity was observed within 0.06-0.5 g/L in urine, while the Crn assay linearity was demonstrated within 0.5-30 mmol/L in urine. The limit of quantification for IBU metabolites was determined to be 0.06 g/L, and 0.5 mmol/L for Crn. These methods were successfully applied to urine samples delivered by ten apparently healthy donors showing that the HPLC-ELSD assays are suitable for human urine screening.
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