FRACTION COLLECTION FOR ISOLATING IMPURITIES IN FORCED DEGRADATION STUDIES

Significance of the Topic

Forced degradation studies are essential for understanding drug stability and ensuring accurate impurity profiling. Determining relative response factors (RRFs) for degradation products is crucial to avoid over- or underestimating impurity levels, which can impact mass balance and regulatory compliance.

Objectives and Study Overview

This study aimed to isolate oxidative degradation impurities of loratadine via small-scale fraction collection. By pooling multiple injections, the collected fractions were used to establish calibration curves and calculate RRFs for the N-oxide and epoxide impurities relative to the active pharmaceutical ingredient (API).

Methodology

Oxidative degradation was induced by exposing loratadine to 3% hydrogen peroxide at 70 °C for up to 90 minutes. Samples were analyzed at defined intervals (30, 60, 90 minutes). Forced degradation runs were performed on a 2.1×50 mm UPLC column with PDA and QDa detection, followed by scale-up fraction collection on a 3.0×75 mm column to accumulate sufficient impurity mass. Collected fractions were dried, lyophilized, and reconstituted in methanol for quantification.

Instrumentation

• ACQUITY UPLC H-Class with PDA and QDa detectors
• Waters Fraction Manager-Analytical (WFMA)
• Columns: ACQUITY UPLC BEH C18 (1.7 µm, 2.1×50 mm) and XBridge BEH C18 (2.5 µm, 3.0×75 mm)
• QDa MS settings: ESI positive mode, 50–500 m/z range, capillary voltage 1.5 kV, cone voltage 15 V
• Software for fraction scheduling and simulation

Main Results and Discussion

Chromatographic analysis revealed a dominant N-oxide impurity eluting at 2.616 minutes (76% area) and a minor epoxide at 3.828 minutes (0.88% area). Calibration curves for loratadine and impurities exhibited excellent linearity (R2 ≥0.996). Calculated RRFs were 1.0 for loratadine, 1.1 for N-oxide, and 0.2 for epoxide. Fraction collection yielded approximately 188 µg of N-oxide and 12 µg of epoxide. Applying RRF corrections improved mass balance accuracy, adjusting apparent mass balances of 112–118% down to 109–111%.

Benefits and Practical Applications

• Enables microgram-scale isolation of degradation impurities
• Facilitates direct determination of RRFs without reliance on external standards
• Improves impurity quantification accuracy in forced degradation studies
• Supports mass balance calculations for regulatory submissions

Future Trends and Opportunities

Advancements may include integration of high-resolution mass spectrometry for structural confirmation, automation of fraction collection workflows, miniaturized fraction collectors for low-volume applications, and predictive modeling of degradation pathways to guide targeted impurity isolation.

Conclusion

Fraction collection on an analytical scale allows efficient isolation of multiple degradation products in a single run. Pooled injections can deliver microgram-level impurity masses suitable for calibration and RRF determination. Incorporation of RRF factors refines mass balance calculations and enhances the accuracy of forced degradation analysis.

References

• United States Pharmacopeia and National Formulary. Chapter <621> Chromatography. USP 37–NF 32 S1. Baltimore, MD: United Book Press, Inc.; 2014:6376–85.