Identity confirmation and accurate quantitation of a genotoxic impurity in an active pharmaceutical ingredient by UHPLC-UV coupled to a single quadrupole mass detector

Significance of the Topic

Accurate detection and quantitation of genotoxic impurities in active pharmaceutical ingredients is critical to ensure patient safety and comply with regulatory limits defined by ICH M7, FDA, and EMA guidelines. Methyl-p-toluenesulfonate is a common genotoxic contaminant arising from p-toluenesulfonic acid used during synthesis. Establishing a sensitive and reliable analytical workflow during early drug development stages helps control impurity levels below toxicological threshold of concern values.

Objectives and Overview of the Study

This study aimed to develop a single-run method that confirms the identity of related impurities in the antiemetic aprepitant and quantifies its genotoxic impurity methyl-p-toluenesulfonate. The approach couples ultrahigh-performance liquid chromatography with UV detection and a single quadrupole mass spectrometer for simultaneous quantitation and identity confirmation.

Methodology and Instrumentation

The method employs a Thermo Scientific Vanquish UHPLC system with an Acclaim Polar Advantage II column (150 × 2.1 mm, 2.2 µm) operated at 35 °C. Mobile phases consist of 15 mM ammonium acetate in water and methanol under a gradient at 0.3 mL/min. UV detection at 225 nm provides quantitation, while a Thermo Scientific ISQ EM single quadrupole mass detector operating in positive HESI mode records full scans (m/z 100–650) and six targeted SIM scans. An Autospray intelligent source optimization feature simplifies gas and temperature settings based on flow rate. Chromeleon 7 CDS software integrates instrument control, data acquisition, and processing.

Main Results and Discussion

Screening of two commercial aprepitant samples confirmed four related impurities including methyl-p-toluenesulfonate by matching retention times and mass spectra. The method achieved excellent linearity for methyl-p-toluenesulfonate from 0.01 µg/mL to 2.5 µg/mL (R2 = 0.9999). Limits of detection and quantification were 3.3 ng/mL and 9.4 ng/mL, respectively. Recovery rates spiked into a blank aprepitant matrix ranged from 93 % to 99 %. Quantitative analysis of the spiked sample yielded 10 ng/mL methyl-p-toluenesulfonate with RSD 5.1 %, well below the 960 ng/mL threshold based on a 125 mg daily dose.

Benefits and Practical Applications

• Single-run workflow delivers both quantitation and identity confirmation of genotoxic impurities
• UV detection provides sensitivity at low nanogram per milliliter levels
• Mass detection via SIM scans enables reliable peak assignment without separate standards
• Autospray settings offer user-friendly optimization of ion source parameters

Future Trends and Opportunities

Advances in intelligent source tuning and data processing may further automate impurity profiling and expand multi-analyte screening. Integration with higher-resolution mass spectrometers and adoption of green solvent systems could enhance sensitivity, selectivity, and environmental sustainability. Ongoing regulatory emphasis on genotoxic impurity control will drive demand for robust, high-throughput workflows.

Conclusion

The combined UHPLC-UV and single quadrupole mass detection method provides a sensitive, reliable, and user-friendly platform for confirmatory analysis and quantitation of genotoxic impurities in pharmaceutical ingredients during early development.

Reference

European Medicines Agency. Guidelines on the Limits of Genotoxic Impurities. 2006. EMEA/CHMP/QWP/251344/2006
FDA CDER. Guidance for Industry Genotoxic and Carcinogenic Impurities in Drug Substances and Products. December 2008.
ICH M7 (R1). Assessment and Control of DNA Reactive Impurities in Pharmaceuticals. March 2017.