Validated RP-HPLC method for simultaneous determination of doxorubicin and 5F: application in nanoliposome formulation development and pharmacokinetic studies

Objective The study aims to develop a reversed-phase high-performance liquidchromatography (RP-HPLC) method for the simultaneous determination of two antitumor drugs, doxorubicin (DOX) and ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), in KM mice plasma following intravenous administration. Methods Nanoliposomes co-loaded with DOX and 5F were prepared using thin-film hydration method and administered intravenously to KM mice. Liquid-liquid extraction with methanol as the extraction solvent was employed to isolate DOX and 5F from plasma samples prior to Chromatographic analysis. Chromatographic separation of DOX and 5F was performed on an Agilent 1,260 series HPLC system fitted with Hypersil ODS C 18 column. The mobile phase consisted of methanol/1% glacial acetic acid (40:60, v/v) at a flow rate of 1.0 mL min −1 . Detection wavelength was set at 242 nm and the column temperature was maintained at 30 °C ± 2 °C. Naproxen was selected as standard (IS). To assess matrix effect and specificity, blank liposome formulation spiked with DOX and 5F was disrupted with methanol and analyzed by HPLC; meanwhile, matrix effect in plasma was evaluated using six individual blank plasma samples with post-extraction spiking at three QC concentration levels (0.125, 0.25 and 0.5 μg mL −1 ) for both analytes. The method was validated strictly in accordance with ICH guidelines. Results The linear ranges of DOX and 5F were 0.125–2.0 μg mL −1 and 0.0625–0.6 μg mL −1 , respectively. Both analytes achieved favorable linearity with correlation coefficients greater than 0.998. Recoveries from quality control samples were 84.2%–85.5% for DOX and 86.2%–87.1% for 5F. The intra-day and inter-day precisions were less than 10% for all QC samples. The limits of quantitation (LOQ) were 60 ng mL −1 for DOX and 20 ng mL −1 for 5F. Matrix effect of DOX and 5F in blank liposome formulation and plasma exerted no significant influence on the separation and detection of DOX and 5F under the established RP-HPLC conditions. Conclusion The developed HPLC method exhibited satisfactory precision, accuracy and acceptable limits of quantitation. The validated assay was proven suitable for the pharmacokinetic studies of DOX and 5F in mice.