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

Importance of the topic

The accurate detection and quantification of genotoxic impurities in active pharmaceutical ingredients (APIs) is critical to ensuring patient safety and regulatory compliance. Genotoxic impurities, such as alkyl p-toluenesulfonates, can arise during synthesis and pose carcinogenic risks at trace levels. Regulatory guidelines from the ICH, FDA and EMA set stringent threshold of toxicological concern (TTC) values, necessitating analytical methods capable of confirming identity and quantifying these impurities at low ng/mL levels.

Goals and overview of the study

This study aimed to develop a single-run analytical workflow for aprepitant, an antiemetic API, to (1) confirm the identity of related process and product impurities, including the genotoxic methyl-p-toluenesulfonate (methyl-TSF), using single quadrupole mass detection and (2) accurately quantify methyl-TSF by sensitive UV detection. The method was validated against ICH M7 TTC limits for short-term treatment.

Methodology and instrumentation

Samples of aprepitant were prepared in water/acetonitrile (50/50) at 1 mg/mL. A methyl-TSF stock (1 mg/mL) and calibration standards (0.01–2.5 µg/mL) were prepared in acetonitrile/water (50/50). UHPLC separation was performed on a Thermo Scientific Acclaim Polar Advantage II column (150 × 2.1 mm, 2.2 µm) with an ammonium acetate/methanol gradient at 0.3 mL/min and 35 °C, injecting 10 µL.

Mass screening used six SIM channels targeting [M+H]+ or [M+NH4]+ ions of known impurities. Optimization of MS source parameters was automated via the Autospray feature in Chromeleon software, adjusting gas flows, vaporizer and transfer tube temperatures to maximize sensitivity. UV detection at 225 nm provided quantitation.

Instrumentation used

• Thermo Scientific Vanquish Flex Quaternary UHPLC system
• Thermo Scientific ISQ EM single quadrupole mass spectrometer with HESI source
• Chromeleon 7.2.9 chromatography data system

Main results and discussion

Impurity screening of aprepitant revealed five detectable peaks. SIM scans confirmed four known impurities including methyl-TSF; one predicted impurity yielded only trace MS signal and was not confirmed by UV. Full-scan spectra of the methyl-TSF peak matched the standard reference, with no co-elution.

UV calibration of methyl-TSF was linear (R2 = 0.9999) over 0.01–2.5 µg/mL. The LOD was 3.3 ng/mL and LOQ 9.4 ng/mL. Spiking experiments at LOQ, TTC-based limit (0.96 µg/mL) and upper range (2.5 µg/mL) into a methyl-TSF free aprepitant sample yielded recoveries of 93–99%, demonstrating method accuracy.

Quantitative analysis of a commercial aprepitant batch indicated a methyl-TSF level of 0.010 µg/mL (RSD 5.1%), well below the 0.96 µg/mL TTC-derived limit.

Benefits and practical applications

• Combined UHPLC-UV and single quadrupole MS allows simultaneous impurity identification and quantitation in a single run.
• Autospray intelligent source optimization simplifies MS parameter tuning for non-expert users.
• The UV method provides sensitivity an order of magnitude below the TTC limit, meeting regulatory requirements.
• High recovery and linearity support robust QA/QC implementation during API development.

Future trends and possibilities

Advances in LC-MS instrumentation, such as higher resolution mass analyzers and enhanced ion source designs, will further improve sensitivity and selectivity for trace genotoxic impurities. Automated method development tools may reduce setup time and user variability. Expanding this workflow to other API classes and impurity profiles can streamline impurity control across pharmaceutical pipelines.

Conclusion

A UHPLC-UV method coupled with single quadrupole MS was successfully developed for the confirmation and quantification of genotoxic methyl-TSF in aprepitant. The approach meets stringent regulatory TTC thresholds, offers ease of use through Autospray optimization and achieves low ng/mL sensitivity and accurate recovery, supporting API purity assessment in early development and QC laboratories.

Reference

• EMEA/CHMP/QWP/251344/2006. Guidelines on the limits of genotoxic impurities.
• FDA CDER 2008 Guidance for Industry. Genotoxic and Carcinogenic Impurities in Drug Substances and Products.
• ICH M7 (R1). Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals.
• Zacharis C.K., Vastardi E. (2018). Application of AQbD principles for determination of alkyl p-toluenesulfonates in aprepitant by HPLC. J. Pharm. Biomed. Anal. 150:152–161.
• Elati C.R. et al. (2007). Convergent approach to the synthesis of aprepitant. Tetrahedron Lett. 48:8001–8004.