SinS - Abstract

Abstract Information :

Detection of mutagenic nitrosamines in drug substance and product is an area of global concern. The polar nature and low molecular weight of some nitrosamines makes achieving chromatographic retention by LC challenging and presents possibilities for interference from other low molecular weight impurities. During development of an LC-MS/MS method for eight target nitrosamines in API, significant over-quantification of N-nitrosodimethylamine (NDMA) was observed. Spiking experiments confirmed this was due to isobaric interference from co-eluting DMF. At a spiking level of 100 ppm, 307% over-quantification of 1.0 ng/mL NDMA was recorded, highlighting the implications for quantification in pharmaceutical samples. Alternative MRM transitions were assessed, although accuracy improved, DMF co-elution remained problematic. Several stationary phase chemistries were found to provide enhanced retention and separation of NDMA and DMF. Additionally, fully porous particles, as opposed to solid core, were found to enhance hydrophobic retention of NDMA, sufficient to resolve it from DMF. Two alternative methods were developed and both demonstrated excellent linearity (R2 ≥ 0.99), accuracy (93.3 to 109.1%) and precision (%RSD 0.3 to 9.5%). Limits of detection ranged from 0.02 to 0.55 ppb with respect to drug substance (0.04 to 1.5 pg on column), significantly lower than limits specified by regulatory authorities. The improved chromatographic selectivity successfully improved NDMA quantification accuracy in the presence of DMF (accuracy = 104.0% and 102.7% at the 100 ppm spiking level). Highly selective MRM transitions for DMF were established to further safeguard against potential for NDMA over-quantification.