Abstract
A method for the extraction and quantification of pretomanid in 40 μL of human plasma, by high performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection was developed and validated. Samples were prepared using liquid-liquid extraction and chromatographic separation was achieved on an Agilent Poroshell C18 column using an isocratic elution at a flow rate of 400 μL/min. Electrospray ionization with mass detection at unit resolution in the multiple reaction monitoring (MRM) mode on an AB Sciex API 3200 mass spectrometer was used. Over the validation period, accuracy, precision, selectivity, sensitivity, recovery and stability were assessed. The calibration range was 10 – 10 000 ng/mL. Inter- and intra-day precision, expressed as the coefficient of variation (%CV), was shown to be lower than 9% at all concentrations tested with accuracies between 95.2 and 110 %. The recovery was 72.4 % overall and reproducible at the low, medium and high end of the calibration range. The method was shown to be specific for pretomanid with no significant matrix effects observed. The validated method facilitated the analysis of pretomanid in plasma collected from adults with pulmonary TB as part of a clinical pharmacokinetic study.
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