Fraction Collection for Isolating Impurities in Forced Degradation Studies

Importance of the Topic

Forced degradation studies are crucial for understanding the chemical stability and degradation pathways of pharmaceutical compounds. Isolating and quantifying impurities formed under stress conditions allow accurate assessment of drug safety and efficacy. Determining reliable relative response factors (RRFs) for impurities versus the active pharmaceutical ingredient (API) ensures proper quantification, supports regulatory compliance, and prevents mass balance errors during impurity profiling.

Study Objectives and Overview

This investigation aimed to develop an analytical-scale fraction collection approach to isolate two oxidative degradation products of loratadine (an N-oxide and an epoxide) from forced degradation samples. The isolated impurities were then used to generate calibration curves and determine their RRFs relative to the API. Finally, the impact of applying these RRFs on mass balance calculations during oxidative stress at 70 °C over 90 minutes was evaluated.

Methodology and Instrumentation

Forced degradation was performed by incubating loratadine with 3 % H2O2 at 70 °C for up to 90 minutes. Samples were analyzed using an ACQUITY UPLC H-Class system with PDA and QDa detectors. Chromatographic separation employed BEH C18 columns (2.1×50 mm, 1.7 µm for method development, and 3.0×75 mm, 2.5 µm for scale-up) at 30 °C, with a gradient of ammonium hydroxide buffer, water, and acetonitrile. Fraction collection was performed on Waters Fraction Manager-Analytical. Collected fractions were pooled over multiple injections, dried, lyophilized, and reconstituted in methanol for RRF determination.

Main Results and Discussion

The forced degradation profile revealed a major impurity at 2.616 min (76 % area, N-oxide) and a minor impurity at 3.828 min (0.88 % area, epoxide). Calibration curves for loratadine, N-oxide, and epoxide standards (1–500 µg/mL) showed high linearity (R2 > 0.996). Calculated RRFs were 1.0 for loratadine, 1.1 for N-oxide, and 0.2 for epoxide using reference standards, and similar values (1.2 and 0.3) from collected fractions.

• Stacked UV and MS TIC data confirmed impurity identities (base mass 399.2 Da).
• Scale-up collection yielded 188 µg of N-oxide and 12 µg of epoxide per 70 injections.
• Applying RRF corrections improved mass balance accuracy, reducing apparent overestimation to 109–111 % down to closer to 100 % (109–111 % corrected).

Benefits and Practical Applications

This approach demonstrates that analytical-scale fraction collection can efficiently isolate low-level degradation products for RRF determination without large sample requirements. Accurate RRFs enable precise impurity quantification, supporting quality control, regulatory submissions, and formulation stability studies.

Future Trends and Opportunities

Advanced fraction collection techniques integrated with high-resolution MS will further streamline impurity isolation. Automation of pooling and sample processing can enhance throughput. Extending this workflow to photolytic, hydrolytic, and thermal degradation pathways will broaden its applicability in drug development and quality assurance.

Conclusion

The study established a robust workflow for isolating and quantifying oxidative impurities of loratadine using analytical-scale fraction collection. Determined RRFs improved mass balance calculations and reinforced the method’s utility for forced degradation analysis. This strategy can be generalized to other APIs, facilitating more accurate impurity profiling.

Instrumentation Used

• ACQUITY UPLC H-Class with PDA and QDa detectors
• Waters Fraction Manager-Analytical (WFMA)
• ACQUITY UPLC BEH C18 columns (2.1×50 mm, 1.7 µm; 3.0×75 mm, 2.5 µm)
• Make-up pump with 0.1 % formic acid in methanol

References

• Chapter 621 Chromatography. United States Pharmacopeia and National Formulary, USP 37–NF 32 S1. United Book Press; 2014:6376–6385.