Determination of hydroxylamine genotoxic impurity by derivatization in penicillamine drug substance by GCHS-MS

Abstract

The aim of the present research is to develop and validate highly sensitive and selective method for the quantitate determination of hydroxylamine by derivatization with acetone in presence of pyridine as a base in penicillamine drug substance by using hyphenated technique. Hydroxylamine was determined by GCHS-MS method using Gs BP-624, 30 mts × 0.32 mm × 1.8 µm column. Temperature program was set by maintaining 60 °C for 3 min, and then increased by 40 °C per minute to 200 °C and hold for 11.5 min. Methanol and acetonitrile was used as diluents. Injector temperature was 250 °C. Split ratio is 1:5. Head space oven, sample line and transfer line temperatures are 100 °C, 150 °C and 170 °C respectively. Validation of the method was performed as per standard guideline “International Conference on Harmonization” (ICH) guideline Q2(R1). This developed GCHS-MS method is able to quantitate 0.1 ppm of hydroxylamine. Under these GCHS-MS conditions hydroxylamine was quantified by selective reaction monitoring m/z of 73 as its acetone oxime derivative at ppm level. The developed GCHS-MS method was validated and resulting to show the developed method is specific, sensitive, linear, accurate and precise. Hence the GCHS-MS method is well suited for the quantification of hydroxylamine genotoxic impurity as its acetone oxime derivative in penicillamine drug substance.