title: A simple and sensitive method to analyze genotoxic impurity hydrazine in pharmaceutical materials.
abstract: Hydrazine (N2H4) is a known genotoxic impurity that typically needs to be controlled down to low ppm level in pharmaceutical development. Hydrazine, however, is a challenging molecule to analyze using conventional analytical techniques due to its physical and chemical properties (e.g. lack of chromophore, absence of any carbon atom, low molecular weight, high polarity and volatility). Additionally, analysis in pharmaceutical samples commonly encounters significant interference from matrix components that greatly overshadow the response of hydrazine. This work describes a simple, accurate and sensitive reversed-phase liquid chromatography-UV derivatization method for determination of trace amount hydrazine in pharmaceutical materials featuring three prominent strategies to address the problems associated with hydrazine analysis. First, the derivatization reaction attaches chromophores to hydrazine, which greatly increases its sensitivity by UV-vis detection. Secondly, the derivatization reaction generates a lambda max that is well-shifted away from the absorption wavelengths of pharmaceutical matrix interferences. Thirdly, from a separation standpoint, the derivatization further removes matrix interference effects through chromatography by achieving higher resolution of the derivative product from the active pharmaceutical ingredient (API) and its related impurities for accurate quantitation for trace level of genotoxic impurities (GTIs). 2-Hydroxy-1-Naphthalaldehyde (HNA) was chosen as the derivatizing reagent, and the resulting hydrazone product has a maximum UV absorbance at wavelength of 406/424nm which is in the visible range. Since most drug substance and impurities have UV absorbance ranging from 190 to 380nm, interference from the matrix was minimized and the appropriate selectivity was obtained, the detection limit is 0.25ppm (0.25μg/g API). This method was validated and applied as a generic method to determine hydrazine for pharmaceutical process control and drug material release.
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Jenny
authors.initials: J
authors.lastname: Wang
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States.
authors.forename: Samuel
authors.initials: S
authors.lastname: Yang
authors.affiliation: Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States. Electronic address: zhang.kelly@gene.com.
authors.forename: Kelly
authors.initials: K
authors.lastname: Zhang
mesh_terms: D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004340:Drug Contamination; D006834:Hydrazines / Q000032:analysis*; D057230:Limit of Detection; D009153:Mutagens / Q000032:analysis*; D004364:Pharmaceutical Preparations / Q000032:analysis; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D006834:Hydrazines; D009153:Mutagens; D004364:Pharmaceutical Preparations; C029424:hydrazine
keywords: 2-Hydroxy-1-Naphthaldehyde; Derivatization; Genotoxic impurity (GTI); HPLC; Hydrazine; Matrix interference; Trace analysis