Simultaneous quantitation of trace level hydrazine and acetohydrazide in pharmaceuticals by benzaldehyde derivatization with sample ‘matrix matching’ followed by liquid chromatography–mass spectrometry

• ‘Matrix matching’ strategy for the derivatization of hydrazine and acetohydrazide.
• Simultaneous quantitation of trace level hydrazine and acetohydrazide.
• Successful application of the robust method in various API matrices.

Hydrazine and acetohydrazide are potential genotoxins and therefore need to be controlled in APIs and drug products to ppm levels for patient safety in cases where there is a reasonable probability of either of them being present. They are structurally related and could both be formed in the same chemical process under certain circumstances. However, no previous studies have reported simultaneous trace level quantification of these two compounds. Herein, a chemical derivatization scheme using benzaldehyde followed by LC–MS analysis is presented to address that need. During method development, unexpectedly high recoveries were encountered and presented a major challenge. A systematic investigation was undertaken to understand the benzaldehyde derivatization reaction and determine the underlying causes of the unacceptable recovery. It was found that this was due to the presence of the counter ion of the API in the sample matrix. Employing a ‘matrix matching’ sample preparation strategy, which involved acidifying the derivatization reaction medium with benzoic acid, gave similar reaction rates for the chemical derivatization in the presence and absence of the API salt and accordingly more consistent recoveries. Resultantly, a robust method for simultaneous quantification of hydrazine and acetohydrazide (1–100 ppm) was successfully developed and validated.