A Simple And Sensitive Method To Analyze Genotoxic Impurity Hydrazine In Pharmaceutical Materials
Authors: Jenny Wang, Samuel Yang, Kelly Zhang
Abstract:
Hydrazine (N2H4) is a genotoxic impurity that must be tightly controlled at low ppm levels during pharmaceutical development. However, due to its unique physical and chemical properties (e.g., lack of chromophore, absence of carbon atom, low molecular weight, high polarity, and volatility), hydrazine poses challenges for analysis using conventional techniques. Moreover, the analysis of hydrazine in pharmaceutical samples often encounters significant interference from matrix components, overwhelming the hydrazine response. In this study, we present a straightforward, precise, and sensitive method that combines reversed-phase liquid chromatography with UV derivatization to accurately determine trace amounts of hydrazine in pharmaceutical materials. This method employs three key strategies to address the challenges associated with hydrazine analysis. Firstly, the derivatization reaction introduces chromophores to hydrazine, greatly enhancing its sensitivity through UV-visible detection. Secondly, the derivatization reaction generates a lambda max that is deliberately shifted away from the absorption wavelengths of matrix interferences commonly found in pharmaceuticals. Thirdly, from a separation standpoint, the derivatization enhances chromatography resolution between the derivative product and the active pharmaceutical ingredient (API) and its related impurities, effectively mitigating matrix interference effects and enabling accurate quantitation of trace levels of genotoxic impurities (GTIs). The derivatizing reagent used in this method is 2-Hydroxy-1-Naphthalaldehyde (HNA), which yields a hydrazone product with maximum UV absorbance at a visible wavelength of 406/424 nm. This choice of wavelength minimizes interference from the matrix, as most drug substances and impurities have UV absorbance within the range of 190 to 380 nm. As a result, the method achieves excellent selectivity with a detection limit of 0.25 ppm (0.25 μg/g API). Validation studies confirm the reliability of this generic method for determining hydrazine levels in pharmaceutical process control and drug material release.
Keywords
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/).
Conclusion
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.
