Abstract
A simple and sensitive reversed phase HPLC method has been developed for the simultaneous quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried powder formulation for inhalation, without any use of an internal standard. Good resolution of the analytes was achieved on a Luna C18 (2), 150×4.6mm, 5μm, 100Å column using gradient elution with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of 1.0mL/min in a total run time of 25min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were detected at wavelengths (retention times) of 269nm (3.80min), 296nm (7.94min) and 330nm (17.46min), respectively. The assay was linear for all analytes in the concentration range 2.5-100μg/mL (correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and 1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the three drugs were 99.6-106.8% with intra- and inter-day precisions (as relative standard deviation) of <7%. The method was successfully applied to an evaluation of content uniformity and freedom from interference by l-leucine of a spray-dried combination powder for inhalation.