Accurate and reliable quantitation of p-toluenesulfonates genotoxic impurities in an active pharmaceutical ingredient by HPLC-UV

Importance of the topic

The presence of p-toluenesulfonate impurities in active pharmaceutical ingredients poses a significant genotoxic risk. These sulfonate esters can form during salt‐formation steps when p-toluenesulfonic acid reacts with common alcohol solvents, generating potentially carcinogenic by‐products that demand precise quantitation.

Regulatory agencies such as the U.S. FDA and EMA enforce a threshold of toxicological concern of 1.5 µg/day for genotoxic impurities in long‐term treatments, driving the need for robust analytical methods capable of detecting trace levels reliably.

Objectives and study overview

This work aims to develop and validate a high‐performance liquid chromatography method with UV detection (HPLC‐UV) for simultaneous quantitation of four p-toluenesulfonate esters—methyl, ethyl, propyl, and isopropyl p-toluenesulfonates—in an aprepitant drug substance.

Key goals include assessment of linearity, determination of limits of detection and quantification, and evaluation of recovery performance across relevant concentration ranges.

Methodology

• Preparation of individual 1 mg/mL stock solutions of each sulfonate ester in acetonitrile, combined to form calibration mixtures ranging from 0.01 to 2.5 µg/mL.
• Sample solutions of aprepitant at 1 mg/mL in water/acetonitrile (50/50 v/v), spiked at 0.01, 1, and 2.5 µg/mL to assess recoveries.
• Chromatographic separation on a Polar Advantage II column (150 × 2.1 mm, 2.2 µm) using a gradient of 15 mM ammonium acetate and methanol at 0.3 mL/min, column temperature 35 °C, UV detection at 225 nm.

Instrumentation

• Thermo Scientific™ Vanquish™ Flex Quaternary UHPLC system with quaternary pump, autosampler (4 °C), column compartment (35 °C), and diode array detector FG (semi-micro flow cell).
• Chromeleon™ 7 Chromatography Data System for acquisition and processing.
• Acclaim™ Polar Advantage II column (150 × 2.1 mm, 2.2 µm).

Results and discussion

• Linearity: All four sulfonate esters showed excellent linear response (R² > 0.9998) over 0.01–2.5 µg/mL.
• Sensitivity: Limits of detection ranged from 3.3 to 4.1 ng/mL; limits of quantification from 9.4 to 10.8 ng/mL.
• Recovery: Spike recoveries were 90–99% at 0.01–2.5 µg/mL levels, with isopropyl-TSF slightly lower (73%) at the lowest level but acceptable.
• Dynamic range: The UV detector provided a wide dynamic range enabling quantitation of impurities down to 0.02% relative to the API peak.

Benefits and practical applications

• Sensitive monitoring of genotoxic impurities at regulatory threshold levels.
• Broad linear range supports both trace‐level detection and routine impurity profiling.
• High recovery and reproducibility ensure reliable quality control in pharmaceutical manufacturing.

Future trends and applications

• Integration of analytical quality by design (AQbD) principles to further optimize robustness.
• Extension to other genotoxic impurities and drug substances requiring trace‐level control.
• Potential coupling with mass spectrometry for orthogonal confirmation and enhanced specificity.

Conclusion

The presented HPLC-UV method delivers high sensitivity, excellent linearity, and reliable recovery for four p-toluenesulfonate genotoxic impurities in aprepitant, meeting stringent regulatory requirements and demonstrating suitability for routine QC applications.

References

1. Zacharis C.K.; Vastardi E. Application of analytical quality by design principles for the determination of alkyl p-toluenesulfonates impurities in Aprepitant by HPLC: Validation using total‐error concept. Journal of Pharmaceutical and Biomedical Analysis. 2018;150:152–161.
2. U.S. Department of Health and Human Services, FDA/CDER. Guidance for Industry: Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approach. December 2008.
3. European Medicines Agency, CHMP. Guidelines on the limits of genotoxic impurities. EMEA/CHMP/QWP/251344/2006.