Ultra high performance liquid chromatography coupled with high resolution quantitation mass spectrometry method development and validation for determining genotoxic 2,5-dichlorobenzoyl chloride in MLN9708 drug substance

• A conservative surrogate strategy was utilized to quantify a genotoxic impurity in an investigational drug, which was acknowledged by FDA.
• HRQMS was used since conventional MS/MS methodology gave poor selectivity due to non-specific fragmentation upon CAD.
• The novel UPLC/HRQMS method was validated based on International Conference on Harmonization (ICH) guideline.

A novel reversed-phase ultra high performance liquid chromatography coupled with high resolution quantitation mass spectrometry (UHPLC/HRQMS) method was developed to quantify 2,5-dichlorobenzoyl chloride (DCBC), a genotoxic impurity, in MLN9708 drug substance. A surrogate strategy was utilized whereby DCBC was intentionally hydrolyzed to 2,5-dichlorobenzoic acid (DCBA) to provide a stable and reliable detection target. The hydrolysis approach was conservative since the measured signal represented the sum of DCBC and DCBA in MLN9708 drug substance, and such approach was acknowledged and accepted by food and drug administration (FDA). HRQMS was used as the detection method since conventional MS/MS methodology gave poor sensitivity and selectivity due to non-specific fragmentation of carbon dioxide loss upon collision activation dissociation. Profile algorithm mass spectrometry data were acquired with mass resolving power (MRP) of 60,000. Quantitation was based on the extracted ion chromatography (EIC) peak area signal, which was extracted at m/z 188.9515 with a mass extraction window (MEW) of 5 ppm. The UHPLC/HRQMS method was validated based on International Conference on Harmonization (ICH) guidelines, which included selectivity, limit of detection (LOD), limit of quantitation (LOQ), repeatability, linearity, accuracy, and stability.

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