Simultaneous Quantitative Analysis of Clarithromycin and Ranitidine, Probe Inhibitors of P-Glycoprotein and OCT1, to Evaluate Potential Pharmacokinetic Influence of Potential Transporter Substrates

Abstract

Clartihromycin and ranitidine are accepted probe inhibitors in drug/drug interaction studies (DDI) with substrates of the multidrug transporters P-glycoprotein and OCT1. We assayed both drugs in human plasma, urine, and feces using LC–MS/MS with positive mass transition mode (AB Sciex API 2000 with turbo-ion spray) with fexofenadine as an internal standard. After protein denaturation with acetonitrile/water (50:50, v/v), the samples were centrifuged and the supernatants (10 µL) were injected into the chromatographic system (column: Supelco Ascentis^® C18, 3 µm, 2.1 × 100 mm). The chromatography was performed with isocratic elution plied using ammonium formate buffer [(5 mM; pH 3.0)/acetonitrile, 40:60, v/v] as mobile phase at a flow rate of 200 μL min⁻¹. The chromatograms were evaluated online with the internal standard method using peak-area-ratios for linear regression analysis weighted by 1/x (x = concentration) for the validation ranges in plasma between 0.005 and 2.0 µg mL⁻¹, and for urine and feces between 0.005 and 10.0 µg mL⁻¹. The method was shown to possess sufficient specificity, accuracy, precision and stability without matrix effects, thereby fulfilling current bioanalytical guidelines. The assay was suitable to simultaneous quantitative analysis of clarithromycin and ranitidine in plasma, urine, and feces of a DDI with trospium chloride to exclude major influence of trospium on the pharmacokinetics of the probe inhibitors. The assay is superior to other methods as it enables for the first time, quantification of the drugs in feces.