Development and Validation of a Unique Stability‐Indicating Reversed‐Phase HPLC Method for the Determination of <scp>l</scp> ‐Penicillamine (R‐Enantiomer) Content in Penicillamine API: Derivatization With Marfey's Reagent Toward Improved Sensitivity

ABSTRACT Penicillamine is a thiol drug mostly used to treat cystinuria, rheumatoid arthritis, and Wilson's disease. To evaluate l ‐penicillamine (R‐enantiomer) content in penicillamine drug substance, a gradient reversed‐phase high‐performance liquid chromatography method that is simple, sensitive, and stability‐indicating has been developed, optimized, and validated. Marfey's reagent is employed as a derivatization agent. Enantiomeric separation was achieved on a Poroshell HPH C18 stationary phase. Mobile phase consists of an aqueous 0.5% orthophosphoric acid buffer as mobile phase A and acetonitrile as mobile phase B. Gradient elution was performed at a flow rate of 0.8 mL/min, the column oven temperature was set at 25°C, and detection was at 340 nm. The resolution between l ‐penicillamine (R‐enantiomer) derivative and penicillamine derivative is found to be more than 10.0. The detector response is found to be linear over the concentration range of 0.03–0.30 µg/mL. LOD and LOQ levels of l ‐penicillamine (R‐enantiomer) derivative are found to be 0.01 and 0.03 µg/mL, respectively. The recovery of l ‐penicillamine (R‐enantiomer) is 90.7%. Specificity, precision, linearity, accuracy, and robustness parameters are all verified for the proposed method.