Determination of Biologically Active Chloro-Nitro-Substituted Benzofuroxans by High-Performance Liquid Chromatography

Chloro-nitro-derivatives of benzofuroxan are compounds exhibiting pronounced biological (antibacterial, fungicidal, and NO-donor) and other types of activities along with low toxicity. For further pharmaceutical development and dosage form design based on them, effective quality control methods that allow the selective and sensitive detection of active ingredients are needed. Here, for 4,6-dinitro- 5,7-dichlorobenzofuroxan (4,6-DN-5,7-DCBF), 5-nitro-4,6-dichlorobenzofuroxan (5-N-4,6-DCBF), and process-related impurities of their synthesis, a method for the simultaneous determination of analytes by high-performance liquid chromatography with diode matrix detection was introduced. The chromatographic behavior of the analytes was studied using different reversed phases. The separation of the components was successful with amide and nitrile phases. Optimal conditions for the chromatographic separation were established using the Discovery RP Amide C16 (4.6×250 mm) columns and the isocratic elution with a mixture of 0.05 % H 3 PO 4 – acetonitrile – methanol (51:34:15, vol. %) at the detection wavelengths of 245 and 207 nm. The linearity of the calibration graphs was achieved in the range of 0.5–200 μg/mL with the detection limits up to 0.12 μg/mL. The developed method was applied for the purification of chloro-nitro-derivatives of benzofuroxan in the mixtures and dosage forms based on them.