Universal response method for the quantitative analysis of multi-components in josamycin and midecamycin using liquid...

Josamycin and midecamycin are consisted of three groups of components with different ultraviolet maximum absorption wavelengths (λ_max), which are 231 nm, 280 nm and 205 nm. The quantitative analysis of all these components is challengeable due to the absence of the respective reference substances. To address this problem, universal and reliable methods were developed using high performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) for the quantitative analysis of components in josamycin and midecamycin. The chromatographic conditions and CAD parameters setting were optimized. Subsequently, the components were identified using HPLC coupled with ion trap/time-of-flight mass spectrometry (IT/TOF MS). The developed methods were validated by assessing linearity, limit of quantitation (LOQ), accuracy, precision and robustness. Good separations were achieved for all components and the adjustment of the filter valve and power function value efficiently improved sensitivity. The developed methods were more comprehensive than current HPLC-UV method. The experimental results demonstrated good linearity with coefficients of determination (R²) greater than 0.999 in the range of 0.002-0.30 mg mL^-1. The limits of detection (LOD) were ranging from 1.8 to 2.0 μg·mL^-1. The intra-day and inter-day RSD values were less than 2.0 % (n = 6) and 5.6 % (n = 9) respectively. The recoveries were 95.0 %-124.0 % at the spiked concentration levels of 0.05 %, 0.50 %, 0.10 % and 2.5 % with relative standard deviations (RSDs, n = 3) lower than 2.0 %. Finally, the developed methods were successfully applied to the quantitative analysis of minor components and used main components (leucomycin A₃ and midecamycin A₁) as alternative reference substance of minor components. The overall results demonstrated that the HPLC-CAD was a good alternative for the quantitative analysis of multi-components in 16-membered macrolides.