2-nitrophenylhydrazine and acetonitrile

A method for the determination of small halogenated carboxylic acid (HCA) residues in drug substances is urgently needed because of the potential of HCAs for genotoxicity and carcinogenicity in humans. We have now developed a simple method, involving derivatization followed by high performance liquid chromatography-diode array detection (HPLC-DAD), for the determination of six likely residual HCAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 2-bromopropionic acid and 3-chloropropionic acid) in drug substances. Different nitro-substituted phenylhydrazines (NPHs) derivatization reagents were systematically compared and evaluated. 2-Nitrophenylhydrazine hydrochloride (2-NPH·HCl) was selected as the most suitable choice since its derivatives absorb strongly at 392 nm, a region of the spectrum where most drug substances and impurities absorb very weakly. During the derivatization process, the commonly used catalyst, pyridine, caused rapid dechlorination or chlorine substitution of α-halogenated derivatives. To avoid these unwanted side reactions, a reliable derivatization method that did not use pyridine was developed. Reaction with 2-NPH·HCl using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as coupling agent in acetonitrile-water (70:30) at room temperature for 2h gave complete reaction and avoided degradation products. The derivatives were analyzed, without any pretreatment, using gradient HPLC with detection in the near visible region. Organic acids commonly found in drug substances and other impurities did not interfere with the analysis. Good linearity (r>0.999) and low limits of quantitation (0.05-0.12 μg mL(-1)) were obtained. The mean recoveries were in the range of 80-115% with RSD <5.81% except for 3-CPA in ibuprofen which was 78.5%. The intra- and inter-day precisions were expressed as RSD <1.98% and <4.39%, respectively. Finally, the proposed method was successfully used for the residue determination of the six HCAs in eight drug substance samples.

Topics: Acetonitriles; Carboxylic Acids; Chromatography, High Pressure Liquid; Hydrocarbons, Chlorinated; Pharmaceutical Preparations; Phenylhydrazines; Propionates; Water

In general, spectrophotometric methods (inductively coupled plasma or atomic absorption spectrophotometry) are used for the assay of magnesium stearate (Mg-St). In this study, a new rapid, selective assay method was developed for Mg-St in pharmaceutical formulations. The method was based on isocratic reverse-phase liquid chromatography using a mobile phase of acetonitrile-water (80:20, v/v) after precolumn derivatization with 2-nitrophenyl hydrazine for sensitive UV detection. Margaric acid was used as an internal standard and the substances were detectable at 230 nm or 400 nm. Using a short (2 cm) HPLC column reduced the analytical time to 5 min. Validation of the newly developed method was performed in accordance with the International Conference on Harmonization guidelines. The linearity range for Mg-St was 0.00-0.04 mg/ml (as the concentration of injected sample solution) and their correlation factor was 0.9998. The determination and detection limits for Mg-St were 6 microg and 2 microg, respectively. The proposed method was successfully applied to the determination of trace amounts of Mg-St in commercially available tablets with a high recovery percentage, good accuracy, and precision.

Topics: Acetonitriles; Chromatography, High Pressure Liquid; Hydrazines; Pharmaceutical Preparations; Phenylhydrazines; Sensitivity and Specificity; Stearic Acids; Water