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

Significance of the topic

p-Toluenesulfonate esters are common counterions in pharmaceutical salt formation but can generate genotoxic impurities when reacting with alcohol solvents. Regulatory agencies (FDA, EMA) require these impurities to remain below a 1.5 µg/day threshold for long-term treatments. Sensitive and reliable quantitation is therefore critical to ensure patient safety and meet quality control standards in drug manufacturing.

Aims and overview of the study

This study describes the development and validation of an HPLC-UV method for simultaneous quantitation of four p-toluenesulfonate impurities (methyl, ethyl, propyl, isopropyl) in the active pharmaceutical ingredient aprepitant. Key performance characteristics—linearity, detection limits, quantification limits, and recovery—were evaluated.

Methodology and Used Instrumentation

• Chromatographic system: Thermo Scientific Vanquish Flex Quaternary UHPLC with DAD FG semi-micro flow cell.
• Column: Acclaim Polar Advantage II (150 × 2.1 mm, 2.2 µm) at 35 °C.
• Mobile phase: 15 mM ammonium acetate (A) and methanol (B) using a gradient from 60% to 73% B over 6 minutes, flow rate 0.3 mL/min.
• Standards: Individual 1 mg/mL stocks in acetonitrile, mixed to 10 µg/mL, then diluted to calibration levels (0.01–2.5 µg/mL); LOD standard at 0.005 µg/mL.
• Sample preparation: Aprepitant dissolved at 1 mg/mL in 50:50 water/acetonitrile and filtered; spiked with impurity standards for recovery studies.
• Data system: Thermo Scientific Chromeleon 7 CDS for acquisition and processing.

Results and Discussion

• Linearity: Excellent correlation (R² > 0.9998) across 0.01–2.5 µg/mL for all four analytes.
• Detection limits: LOD values ranged from 3.3 to 4.1 ng/mL; LOQ from 9.4 to 10.8 ng/mL.
• Recovery: Spike recoveries at 0.01, 1, and 2.5 µg/mL ranged from 90% to 99%, with isopropyl-TSF at 73% recovery at the LOQ level but still within acceptable limits.
• Dynamic range: Detector response supported accurate quantitation over more than two orders of magnitude and relative peak-area measurements down to 0.02% of the API signal.

Benefits and practical applications

This method enables routine quality control and in-process monitoring of genotoxic p-toluenesulfonate impurities in aprepitant and other APIs. Its high sensitivity and broad dynamic range fulfill regulatory requirements, ensuring drug safety and compliance in pharmaceutical development and manufacturing.

Future trends and potential applications

Emerging opportunities include integrating automated sample preparation, coupling HPLC-UV screening with mass spectrometry for confirmation, expanding the approach to other alkyl sulfonates, and applying Quality by Design and advanced data analytics to further optimize impurity control strategies.

Conclusion

The presented HPLC-UV method delivers rapid, precise, and highly sensitive quantitation of four genotoxic p-toluenesulfonate esters in an aprepitant API matrix. With low nanogram-per-milliliter detection limits, strong linearity, and robust recoveries, it meets stringent regulatory standards for genotoxic impurity assessment.

References

1. Zacharis C.K., Vastardi E. Application of analytical quality by design principles for the determination of alkyl p-toluenesulfonate impurities in Aprepitant by HPLC: validation using total-error concept. J. Pharm. Biomed. Anal. 2018, 150, 152–161.
2. U.S. FDA. Guidance for Industry: Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approach. December 2008.
3. European Medicines Agency. Guidelines on the limits of genotoxic impurities. EMEA/CHMP/QWP/251344/2006, 2006.