Semicarbazide Hydrochloride as Impurity in Drug Substances: a Validated LC-DAD-UV Method for Its Determination in Carbazochrome and Carbazochrome Sodium Sulfonate

Abstract

Carbazochrome (2-(1,2,3,6-tetrahydro-3-hydroxy-1-methyl-6-oxo-5H-indol-5-ylidene)-hydrazinecarboxamide, CBZ) and carbazochrome sodium sulfonate (5-[(aminocarbonyl)hydrazono]-2,3,5,6-tetrahydro-1-methyl-6-oxo-1H-indole-2-sulfonic acid sodium salt, CSS) are derivatives of adrenochrome currently employed mainly as hemostatic drugs. Since a semicarbazone group is present in both the structures of CBZ and CSS, semicarbazide hydrochloride (SEM) is used, as reagent, in their synthesis. Belonging to the hydrazine family of chemicals, SEM could possess some toxic potential. Accordingly, literature data showed that SEM causes osteolathyrism and induces toxic effects in the cardiovascular and skeletal systems. Moreover, SEM has a weak genotoxic activity in vitro while in vivo available data are not sufficient to state its genotoxicity. Based on these data, the determination of SEM as a synthesis-related impurity in CBZ and CSS drug substances is mandatory. Since there are no publications dealing with the determination of SEM in CBZ and CSS as well as in any other SEM-containing drugs, a new and unprecedented LC-DAD-UV method for its determination as impurity in CBZ and CSS drug substances was developed and validated. Due to the lack of chromophore, semicarbazide was converted into the corresponding UV-absorbing naphthalene-2-carbaldehyde semicarbazone (NCS) before the chromatographic analysis. The complete separation of NCS from the large concentration of the derivatization reagent and the parent drugs CBZ and CSS was accomplished through a liquid–liquid extraction (LLE) step followed by a chromatographic run of 10 min. The method was then successfully validated and its applicability demonstrated by the determination of SEM in real samples of CBZ and CSS.