Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Eight Related Substances in Milrinone API

A simple, quick, and economical reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of related substance in milrinone API was developed and validated. The method has shown adequate separation of milrinone and eight kinds of related substance. Chromatographic separation was achieved on an Agilent ZORBAX Rx-C8 (250 × 4.6 mm, 5 µm) column at wavelength of 220 nm, using a mobile phase acetonitrile: phosphate buffer (85:15) in an isocratic elution mode at a flow rate of 1.0 mL min⁻¹. The method was validated in terms of system suitability, specificity, linearity, range, accuracy, intermediate precision, and robustness. A linear relationship between peak area and concentration of milrinone and its related substances was observed in a level of 10–300% (determination coefficient, r² ≥ 0.998), respectively. The method showed acceptable levels of precision (%RSD ≤ 2), accuracy (> 96% recovery), robustness (< 10% content difference), and stability (> 96% recovery) over varied environment and laboratory conditions. The validation results suggested that the developed method is sensitive enough and repeatable and could be used for qualitative and quantitative assessment of eight kinds of related substance in milrinone. As a significant part of drug development process, stress testing is also performed to identify the degradation products and validate the stability-indicating power of our analytical methods, in which milrinone is subjected to acidic, base, oxidation, thermal and photolytic stress environment. To sum up, the proposed method is suitable for purpose in quality-control laboratories for quantitative analysis of the drugs individually, as it is simple and rapid with good precision and accuracy.