ISOCRATIC DETERMINATION OF DACARBAZINE AND RELATED IMPURITIES 2-AZAHYPOXANTHINE AND 5-AMINO-IMIDAZOLE-4-CARBOXAMIDE BY HPLC ON AN AVIDIN PROTEIN COLUMN

Abstract

The separation of dacarbazine and its two related impurities, 5-amino-imidazole-4 carboxamide (AIC) and 2-azahypoxanthine (AHX) was investigated by capillary electrophoresis and high performance liquid chromatography. Baseline separation of dacarbazine and its related impurities was achieved by capillary electrophoresis (CE) on a fused silica capillary (70 cm × 50 μm, i.d) with an electrolyte buffer of methanol: phosphate buffer (0.025 M, pH 4) - 2:98 v/v at an applied voltage of 15 kV and detection at 220 nm. The CE method could not be used for the quantitation of impurities because at high concentration, the dacarbazine peak overlapped the nearest impurity peak. The initial HPLC method developed on an octadecylsilane (ODS) column successfully separated the impurities from the dacarbazine, but required a longer time of analysis (>50 min). Another HPLC method developed on an avidin protein column separated the drug and its impurities with a run time of less than 27 min and was chosen for method development and validation. Dacarbazine and its two impurities were separated isocratically on avidin column using a mobile phase of 2-propanol: phosphate buffer (pH 7, 0.02 M) 4:96 v/v at a flow rate of 0.6 mL/min and detection at 230 nm. Calibration curves were prepared for each impurity from 100–1000 ng/mL. The detector response was linear (r2 >0.997) for each individual impurity down to 100 ng/mL which represented 0.5% w/w of the dacarbazine concentration. Accuracy and precision of analysis for each impurity were less than 3 % at 100 ng/mL. The limits of quantitation and detection (s/n >3) for both impurities were 100 ng/mL and 50 ng/mL, respectively. Linearity was also demonstrated for dacarbazine in the 15–25 μg/mL range with a correlation coefficient greater than 0.998.