title: Determination of small halogenated carboxylic acid residues in drug substances by high performance liquid chromatography-diode array detection following derivatization with nitro-substituted phenylhydrazines.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Desheng
authors.initials: D
authors.lastname: Hou
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Department of Pharmacy, Nanyang Central Hospital, Nanyang 473000, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Fan
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Lingfei
authors.initials: L
authors.lastname: Han
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Ruan
authors.affiliation: Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Feng
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Electronic address: liuwenyuan8506@163.com.
authors.forename: Wenyuan
authors.initials: W
authors.lastname: Liu
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China; Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China. Electronic address: cpu_analyst@126.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Zheng
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D002264:Carboxylic Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D010659:Phenylhydrazines / Q000737:chemistry*; D011422:Propionates / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D002264:Carboxylic Acids; D006843:Hydrocarbons, Chlorinated; D004364:Pharmaceutical Preparations; D010659:Phenylhydrazines; D011422:Propionates; D014867:Water; C060574:2-nitrophenylhydrazine; C021974:2-chloropropionic acid; C007022:3-chloropropionic acid; C035775:2-bromopropionic acid; C032159:acetonitrile
keywords: Dehalogenation; Derivatization; Halogenated carboxylic acids; High performance liquid chromatography-diode array detection; Nitro-substituted phenylhydrazines; Residue determination