Fraction Collection for Isolating Impurities in Forced Degradation Studies

Importance of the topic

Forced degradation analysis provides critical insights into the chemical stability and degradation pathways of pharmaceutical compounds.
Accurate isolation and quantification of degradation products prevent misestimation of impurity levels and ensure regulatory compliance and product safety.

Study objectives and overview

The study aimed to isolate oxidative degradation products of loratadine (n-oxide and epoxide) using analytical-scale fraction collection and determine their relative response factors (RRFs) relative to the active pharmaceutical ingredient (API).
By obtaining pure fractions of impurities, the approach facilitates precise RRF calculation and improves mass balance assessment in forced degradation studies.

Methodology

The forced degradation protocol involved treating loratadine with 3% hydrogen peroxide at 70 °C for up to 90 minutes.
UPLC analysis was performed on a Waters ACQUITY UPLC H-Class system with PDA and QDa detectors.
Analytical separation used a BEH C18 column (2.1 × 50 mm, 1.7 µm) at 30 °C with a mobile phase of ammonium hydroxide, water, and acetonitrile at 254 nm detection.
Scale-up for fraction collection employed a 3.0 × 75 mm, 2.5 µm C18 column with increased injection volume and flow rate.

Instrumentation used

• Waters ACQUITY UPLC H-Class with PDA and QDa detectors
• Waters Fraction Manager-Analytical (WFMA)
• ACQUITY UPLC BEH C18 and XBridge C18 columns
• Electrospray ionization MS in positive mode (m/z 50–500)

Main results and discussion

Chromatographic data revealed a major impurity at 2.616 min (n-oxide, ~76% area) and a minor impurity at 3.828 min (epoxide, ~0.9% area).
Calibration for loratadine and impurities over 1–500 µg/mL was linear (R² > 0.996).
Determined RRFs: n-oxide ~1.1 and epoxide ~0.2; collected fractions yielded RRFs of ~1.2 and ~0.3, respectively.
Application of RRF correction improved mass balance from apparent values of 112–118% to corrected values of 109–111%.
Time-course degradation showed increasing conversion to n-oxide (49% at 90 min) with minimal epoxide formation.

Benefits and practical applications

• Analytical-scale fraction collection enables isolation of microgram quantities of degradation products for accurate quantification.
• Determined RRFs allow correction of response differences between API and impurities.
• Enhanced mass balance assessment supports regulatory documentation of stability studies.

Future trends and potential applications

• Extension of fraction collection techniques to other degradation pathways and pharmaceutical compounds.
• Integration with preparative-scale isolation and structural elucidation tools such as NMR and high-resolution MS.
• Automation of fraction collection workflows and data analysis through advanced software and AI integration.

Conclusion

Analytical-scale fraction collection coupled with UPLC-MS enables efficient isolation of oxidative degradation products and reliable determination of relative response factors.
Applying RRF corrections improves quantification accuracy and mass balance in forced degradation studies, strengthening pharmaceutical stability assessments.

References

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