Impurity Profiling and a Stability Indicating Advanced Liquid Chromatographic Method Development and Validation for the Estimation of Related Impurities of Anagrelide

Abstract

A novel, streamlined and highly selective isocratic liquid chromatographic method has been developed for the quantification of impurities present in anagrelide. Exceptional resolution between anagrelide and its impurities was accomplished utilizing a Waters Nova Pack C18 column (250 mm × 4.6 mm, 4 µm), employing a phosphate buffer with a pH of 4.4 as mobile phase A and a blend of acetonitrile and methanol with buffer in a 30:40:30 v/v/v ratio as mobile phase B. The column oven was maintained at 35 ºC and the mobile phase flowed at a constant rate of 1.0 mL/min, while detection was set at 254 nm. Rigorous validation was conducted to assess accuracy, precision, specificity, linearity and sensitivity. Validation studies have unequivocally established the HPLC method’s precision, specificity, rapidity, reliability and reproducibility. Linearity was ascertained for both anagrelide and its impurities, exceeding an R2 value of 0.95. The limits of detection (LOD) and limit of quantitation (LOQ) were determined to be more than sufficient for precise estimation. This method was validated in accordance with the stringent ICH guidelines. The relative standard deviation (RSD) for intra- day and inter-day precision remained consistently below 5%. Percentage recovery demonstrated excellent agreement, affirming the simplicity, specificity, precision and enhanced accuracy of method for determining related substances in pharmaceutical substances and various anagrelide-containing dosage forms.