High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H₃PO₄ with 0.2% triethylamine mobile phase was employed and separation was achieved with a C₁₈ analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9±0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 μg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 μg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 μg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 μg/ml. Results of the stability study indicated that when frozen at -80C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.