Use of a Triple Detection (UV-ELSD-MS) System for Mass Balance in the Forced Degradation of Pharmaceuticals

Importance of the Topic

The accurate assessment of drug stability under stress conditions is essential in pharmaceutical development to ensure product safety and efficacy. Mass balance—accounting for the parent drug and all degradation products—is a regulatory requirement for forced degradation studies. Employing multiple, orthogonal detection methods enhances confidence in impurity quantitation and structural identification.

Objectives and Study Overview

This study evaluated a triple‐detection system combining photodiode array (PDA), evaporative light scattering (ELSD) and mass detection (QDa) for mass balance in forced degradation of glimepiride. Key aims were to determine relative response factors (RRF) for related impurities, confirm degradation pathways by mass spectrometry, and demonstrate accurate mass balance using Empower 3 FR2 software.

Methodology and Instrumentation

• System: ACQUITY UPLC H-Class with Column Manager and Triple Detection (PDA, ELSD, QDa) including an isocratic solvent manager and splitter.
• Column: BEH C18, 1.7 µm, 2.1 × 50 mm at 30 °C.
• Mobile phases: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile; isocratic 60% A:40% B; flow rate 0.8 mL/min; injection 2 µL.
• Detectors: PDA (210–400 nm, 228 nm monitoring), ELSD (25 psi, 55 °C nebulizer), QDa mass detection for impurity identification.
• Sample preparation: Standards of glimepiride and related compounds B/C in 55:45 methanol:water; forced degradation via 0.1 M HCl at 40 °C for up to 7 days.

Main Results and Discussion

• PDA provided a linear detector response for API and impurities, whereas ELSD exhibited a non-linear signal requiring logarithmic transformation for calibration.
• Calculated RRF values using the ratio of UV slope to log(ELSD) slope: compound B fell outside the 0.8–1.2 acceptance range and was treated according to USP <621> guidance.
• Mass detection confirmed the presence and relative abundance of related compounds, with compound C showing higher peak intensity than B in QDa.
• Mass balance determinations incorporating corrected impurity areas in Empower 3 FR2 yielded recoveries within 2% deviation, meeting regulatory criteria.

Benefits and Practical Applications

• Triple detection enhances reliability of stability assessments by compensating for detector‐specific biases.
• Log‐transformed ELSD data allow quantitation of non‐UV‐active impurities without additional chromophores.
• Integrated software workflows streamline RRF application and mass balance reporting, reducing manual calculations.

Future Trends and Opportunities

• Incorporation of alternative universal detectors (e.g., charged aerosol detection) for broader compound coverage.
• Utilization of high‐resolution MS to provide definitive structural elucidation of degradation products.
• Automation and machine‐learning algorithms for dynamic RRF prediction and real‐time mass balance monitoring.

Conclusion

The combined PDA–ELSD–QDa platform demonstrated robust performance in forced degradation studies by enabling accurate quantitation of API and impurities and reliable mass balance calculations. Adoption of this approach can improve data integrity in stability testing and support regulatory compliance.

Reference

1. United States Pharmacopeia. Chapter <621> Chromatography. USP 37–NF 32; 2014. p. 6376–85.
2. Mark AN, Andreas K, Patrick JJ. Role of Mass Balance in Pharmaceutical Stress Testing. In: Pharmaceutical Stress Testing. CRC Press; 2011. p. 233–53.