Liquid chromatographic determination of CPZEN-45, a novel anti-tubercular drug, in biological samples

• Published in: Journal of pharmaceutical and biomedical analysis 2014-01, Vol.88, p.370-376
• Main Authors: Hanif, S.N.M., Hickey, A.J., Garcia-Contreras, L.
• Format: Article
• Language: English
• Subjects: Animals; Antitubercular Agents - analysis; Antitubercular Agents - pharmacokinetics; Azepines - analysis; Azepines - pharmacokinetics; Bronchoalveolar Lavage Fluid; Calibration; Chromatography, High Pressure Liquid; Chromatography, Liquid; CPZEN-45; Guinea pig; Guinea Pigs; HPLC; Infusions, Intravenous; Injections, Subcutaneous; Lung - drug effects; Male; Mycobacterium tuberculosis; Mycobacterium tuberculosis - drug effects; Reproducibility of Results; Software; Spleen - drug effects; Tissue Distribution; Tuberculosis; Validation
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2013.09.014

•We developed an efficient method (>96%) to extract CPZEN-45 from biological samples.•We validated an HPLC method to quantify CPZEN-45 in plasma, lung and spleen tissues.•The HPLC method was validated as per the FDA guidelines.•The limit of detection was 0.05μg/ml and the limit of quantitation was 0.29μg/ml.•This method can detect CPZEN-45 concentrations that are 5- to 21-fold smaller than MIC. CPZEN-45 is a new drug candidate being considered for the treatment of tuberculosis (TB). The aim of this study was to develop and validate a reverse-phase high-performance liquid chromatographic (HPLC) method suitable to determine CPZEN-45 concentrations in biological samples. CPZEN-45 was extracted from biological fluids and tissues (plasma, lung and spleen from guinea pig) by sequential extraction with acetonitrile and quantified by a Waters HPLC Alliance System coupled with a ZORBAX Bonus-RP column, guard column and UV detection at 263nm. The mobile phase was 20:80 acetonitrile:ultrapure-water with 0.05% TFA. The CPZEN-45 peak was eluted at 5.1min with no interference from the inherent peaks of plasma, bronchoalveolar lavage (BAL), lung or spleen tissues. Recovery of CPZEN-45 from biological samples was >96% of the spiked amount. The limit of detection was 0.05μg/ml and the limit of quantitation was 0.29μg/ml which was more than 5 and 21 times lower than the reported minimal inhibitory concentration of CPZEN-45 (MIC=1.56μg/ml for Mycobacterium tuberculosis and 6.25μg/ml for MDR-TB, respectively). Thus, HPLC method was deemed reliable, sensitive, reproducible and accurate for the determination of CPZEN-45 concentrations in plasma, BAL, lung and spleen tissues. Therefore, this method was used in subsequent studies in the guinea pig model to determine the disposition of CPZEN-45 after administration of solutions by the IV and SC routes.