Development and Validation of Orthogonal RP‐HPLC Methods for the Determination of Organic Impurities in Penicillamine Drug Substance

ABSTRACT A stability‐indicating related substances method (M‐I) to determine penicillamine trisulfide and thiazolidine carboxylic acid and a non‐carryover impurities method (M‐II) to determine thiazolidine carboxamide, thiazolidine carbonitrile, and dihydrothiozole in penicillamine by using reverse phase high‐performance liquid chromatography (RP‐HPLC) have been developed. The stability‐indicating capability of M‐I was demonstrated by the successful separation of all process related impurities, and degradation products generated through forced degradation using the Unison UK‐C8 (250 mm × 4.6 mm, 3 µm) column in the gradient mode. Ascentis Express C18 (150 × 4.6 mm, 2.7 µm) column was used for the separation of non‐carryover impurities. A UV detector set at 210 nm was used to monitor all impurities. Validation of both methods adhered to ICH guidelines. Each method was made specific by separating the target analytes from each other in one method and the other impurities in another method and vice versa. Limit of detection and limit of quantitation values for impurities were found to be in the ranges of 0.001% to 0.005%, and 0.004% to 0.015%, respectively. The linear regression analysis of the calibration plots demonstrated a strong linear relationship between the response and concentration of impurities by achieving suitable correlation coefficient values (r 2 > 0.990). The recoveries of impurities from accuracy experiments were found in the range from 88.0 to 106.3%. These methods provide a thorough assessment of impurities associated with penicillamine.