Analysis of High Potency Mutagenic Impurities: Identification and the Role of Response Factor Variation on Quantitative Accuracy
25 Jun 2019

Impurities in an API can significantly affect stability, efficacy, and patient safety. Recently, hundreds of lots of Angiotensin II Receptor drugs have been recalled due to the presence of high potency mutagenic impurities (nitrosamines) found in these products as a result of a manufacturing process change. Guidance for the control of mutagenic impurities in pharmaceuticals has been provided by ICH in M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk.

Analysis of mutagenic impurities is challenging due to the very high toxicity of these compounds which then requires very low detection limits for quantitative methods (parts per billion). This also places a much higher burden on the analytical screening methods required to identify unexpected or unknown mutagenic impurities in pharmaceutical products as screening at low levels requires high instrument sensitivity and low background noise. In addition, limitations on the commercial availability of impurity standards can require alternative quantitation strategies including relative quantitation versus a surrogate standard. This has the potential to adversely affect quantitative accuracy.

In this webinar, we will explore the regulations covering mutagenic impurities. We will also review relevant analytical methodologies for characterization of mutagenic impurities through the lens of a case study showing the synthesis of amino-drug intermediates prepared via the reduction of aromatic nitro groups to the corresponding amines. Reduction of nitro groups to amines has been reported to result in the formation of nitroso and hydroxyamine impurities. The resulting amino-drug intermediates were characterized using Quadrupole Time of Flight Liquid Chromatography Mass Spectrometry (QTOF-LCMS) to identify potential mutagenic impurities. This process was facilitated using differential analysis software to identify low level impurities. Preparative fraction collection was used for isolation of individual impurities allowing for further structure confirmation by NMR and FTIR and toxicological review of the resulting structures was used to asses potential safety concerns. A comparison of the response factors for structural analogs containing nitroso, hydroxyamine, amino and nitro groups was performed as a means to determine the accuracy of relative quantitation as this approach is commonly used due to the lack of commercially available standards of these impurities. Quantitative methods for the nitrosamine impurities were developed and validated using triple quadrupole liquid chromatography mass spectrometry (QqQ-LCMS).

Key Learning Objectives:

  • Understanding regulatory expectations for impurities in drug substance and product with an emphasis on mutagenic impurities
  • Impurities specification requirements based on ICH Q3 and M7 guidelines
  • Approaches for identification, isolation and structure confirmation of low-level impurities

Agilent Technologies