Coupling Mass Detection with UV to Improve Method Sensitivity for Esters of Benzenesulfonic Acid in Analysis of Genotoxic Impurities

Importance of the Topic

An accurate and sensitive approach for monitoring genotoxic impurities (GTIs) is essential in pharmaceutical development and quality control. Alkyl esters of benzenesulfonic acid, formed during API salt formation or storage, can react with DNA and pose carcinogenic risks. Regulatory guidelines (ICH M7, FDA, EMEA) set stringent thresholds (TTC of 1.5 μg/day) for acceptable GTI levels over a lifetime exposure. A fast, robust UPLC method coupled with both UV and mass detection addresses the need for routine, high-throughput screening of these potentially harmful esters without time-consuming derivatization.

Objectives and Study Overview

This application note presents a streamlined UPLC method for simultaneous analysis of methyl, ethyl, and isopropyl benzenesulfonate esters. Goals include:

• Demonstrate method linearity, sensitivity, and specificity for UV and mass detection.
• Establish limits of detection (LOD) and quantification (LOQ) to meet regulatory ppm requirements relative to API dosage.
• Validate performance in the presence of a real-world sample: amlodipine besylate API.

Methodology and Instrumentation

Instrumentation and software:

• UPLC system: Waters ACQUITY UPLC H-Class with PDA detector (200–400 nm, 220 nm quantification) and QDa mass detector.
• Column: ACQUITY UPLC CSH C18, 1.7 μm, 2.1×50 mm, 40 °C, 0.6 mL/min flow.
• Mobile phases: A = ammonium acetate (optimized 1 mM for MS), B = methanol, gradient from 10% to 90% B.
• Mass settings: ESI+ for esters (SIR 190.0, 204.0, 218.0 Da), ESI– for benzenesulfonic acid (157.0 Da), capillary 1.4 kV, cone 6 V, probe 300 °C.
• Sample prep: Stock esters in methanol; serial dilutions in 20:80 methanol/5 mM ammonium acetate; amlodipine besylate API at 1–5 mg/mL.

Main Results and Discussion

Linearity and precision:

• UV detection: linear from 50–10,000 ng/mL (R² ≥ 0.9991).
• MS detection: linear from 1.5–500 ng/mL (R² ≥ 0.9984) using SIR mode.

Detection limits:

• UV LOQ: 50 ng/mL for methyl/ethyl, 100 ng/mL for isopropyl esters.
• MS LOQ: 15 ng/mL for methyl, 1.5 ng/mL for ethyl and isopropyl esters; corresponding to 7.5–1.5 ppm at 1 mg/mL API and 1.5–0.3 ppm at 5 mg/mL API.

Specificity and recovery in API matrix:

• No interfering peaks observed in unspiked amlodipine besylate API.
• Spiked recoveries at LOQ levels ranged from 90% to 110% (RSD ≤ 5.7%).

Benefits and Practical Applications of the Method

• Eliminates pre-column derivatization, reducing analysis time and complexity.
• Combines UV and MS detectors for orthogonal confirmation of peak identity.
• Single ion recording enhances sensitivity and selectivity for trace-level GTIs.
• Meets regulatory thresholds for routine QC of pharmaceutical APIs and products.

Future Trends and Potential Applications

Advancements may include:

• Expansion to other classes of genotoxic impurities and counter-ion esters.
• Integration with high-resolution MS for structural elucidation and impurity profiling.
• Automation and online monitoring in continuous manufacturing environments.
• Miniaturized UPLC-MS systems for at-line and remote testing.

Conclusion

The Waters ACQUITY UPLC H-Class system with PDA and QDa detectors provides a rapid, sensitive, and robust platform for quantifying methyl, ethyl, and isopropyl benzenesulfonate esters. This method achieves sub-ppm quantification limits, straightforward sample preparation, and reliable performance in API matrices, fulfilling ICH M7 requirements and enabling routine GTI monitoring in pharmaceutical QC laboratories.

Reference

1. Liu D.Q.; Sun M.; Kord A.S. Recent Advances in Trace Analysis of Pharmaceutical Genotoxic Impurities. J. Pharm. Biomed. Anal. 2010.
2. EMEA CHMP Guidelines on Limits of Genotoxic Impurities, CPMP/SWP/5199/02, 2006.
3. FDA CDER Guidance for Industry, Genotoxic and Carcinogenic Impurities in Drug Substances and Products, 2008.
4. ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals, 2014.
5. Taylor G.E.; Gosling M.; Pearce A. Low-Level Determination of p-Toluenesulfonate and Benzenesulfonate Esters in Drug Substance by HPLC/MS. J. Chromatogr. A 2006.