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 406/424 nm which is in the visible range. Since most drug substance and impurities have UV absorbance ranging from 190 to 380 nm, interference from the matrix was minimized and the appropriate selectivity was obtained, the detection limit is 0.25 ppm (0.25 μg/g API). This method was validated and applied as a generic method to determine hydrazine for pharmaceutical process control and drug material release.
Hydrazine; Genotoxic impurity (GTI); Matrix interference; HPLC; Derivatization; 2-Hydroxy-1-Naphthaldehyde; Trace analysis
Citation: Jenny Wang, Samuel Yang, Kelly Zhang A Simple And Sensitive Method To Analyze Genotoxic Impurity Hydrazine In Pharmaceutical Materials doi:10.1016/j.jpba.2016.04.038
Received: 23 February 2016, Revised: 22 April 2016, Accepted: 24 April 2016, Available online: 27 April 2016
Copyright: © 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
We have developed a method for the sensitive and accurate quantitation of hydrazine in pharmaceutical materials using a simple derivatization reaction and RPLC-UV. Selection of the derivatization agent, 2-Hydroxy-1-Naphthaldehyde, was a key step toward this analytical approach, which generates a derivatized product that meets the specific requirements of our analytical strategies. The derivatization effectively shifts the resultant hydrazone product away to higher wavelengths in the UV spectrum where API matrix components do not interfere with the analysis. Secondly, the derivatization reaction generates a product that can be separated on a reversed-phase LC method with high resolution from the rest of the API matrix. A specific LC-UV method using an Eclipse XDB-C18 column was tailored to achieve the desired chromatography with the HNA-hydrazone product and was demonstrated for suitable specificity, linearity/range, accuracy and precision. The LOQ of the method was determined to be 1 ppm (w/w) based on the average signal-to-noise ratio of 6 replicate injections of a 2 mg/mL API and was adequate for sensitive quantification of hydrazine in pharmaceutical materials.