Characterization of products formed by forced degradation of Etodoalc using LC/MS/MS

Importance of the Topic

Forced degradation studies play a critical role in pharmaceutical development by accelerating the breakdown of drug products under stress conditions. They help in elucidating degradation pathways, assessing potential toxicity of byproducts, informing shelf-life predictions, and guiding formulation and manufacturing optimizations.

Objectives and Study Overview

This work aimed to characterize degradation products of the nonsteroidal anti-inflammatory drug Etodolac subjected to acid, base, oxidative, thermal, and photolytic stress. By applying accelerated conditions, the study sought to identify key impurities using rapid LC/MS/MS analysis and to map degradation pathways in support of stability assessment and regulatory guidelines.

Methodology and Instrumentation

Stress Conditions:

• Acid hydrolysis: 5 M HCl at 60 °C for 8 hours
• Base hydrolysis: 5 M NaOH at 80 °C for 8 hours
• Oxidation: 30 % H₂O₂ at 80 °C for 8 hours
• Thermal: solid Etodolac at 80 °C for 48 hours
• UV exposure: 1.2 million lux-hours on a 500 ppm solution

Sample Preparation:
After stress treatment, solutions were neutralized where required, diluted to 500 ppm, and subjected to preparative HPLC fractionation prior to MS analysis.

Instrumentation:

• Preparative HPLC with fraction collector for isolation of impurities
• Nexera UHPLC coupled to Shimadzu LCMS-8040 triple quadrupole MS
• Analytical column: Shim-pack XR ODS (100 × 2 mm, 3 µm)
• Mobile phase: water (pH 3 with formic acid) and acetonitrile (50:50 v/v)
• Flow rate 0.4 mL/min, column 40 °C, UV detection at 225 nm
• ESI source with nebulizing gas 2 L/min, drying gas 15 L/min

Main Results and Discussion

Extent of degradation:

• 100 % under acid hydrolysis
• 68 % under oxidative stress
• 6 % under UV exposure
• 5 % under basic hydrolysis
• 1 % under thermal stress

Key impurities identified by precursor m/z and MS/MS fragmentation:

• m/z 190 and 244 in acid-degraded samples, sharing a common fragment at m/z 172
• m/z 304 under oxidative and UV conditions, exhibiting product ions at m/z 188 and 130

Fragmentation data enabled proposal of probable structures for each degradation product and elucidation of degradation pathways, highlighting the conversion of the pyranocarboxylic core under acid and oxidative conditions.

Benefits and Practical Applications

This rapid LC/MS/MS approach offers:

• Detailed structural information on trace impurities without extensive isolation effort
• Efficient screening of multiple stress conditions in a single analytical run
• Data to support formulation optimization, stability protocol design, and regulatory submissions

Future Trends and Opportunities

Advances likely to enhance forced degradation studies include:

• Integration of high-resolution MS for more precise mass assignments
• Automation of sample preparation and data processing
• Application of UHPLC-HRMS coupling for faster separations and deeper impurity profiling
• Use of predictive software and machine learning to anticipate degradation pathways

Conclusion

A fast and reliable LC/MS/MS method was developed on the Shimadzu LCMS-8040 platform, enabling comprehensive identification of Etodolac degradation products across acid, base, oxidative, thermal, and photolytic stresses. The study provides critical insight for stability testing, formulation development, and regulatory planning.

References

• Aneesh TP and Rajasekaran A International Journal of Biological & Pharmaceutical Research 3(5) 2012 699-702
• George Ngwa Drug Delivery Technology 10(5) 2010
• Strickmann DB et al Journal of Pharmaceutical and Biomedical Analysis 25 2001 977-984
• ICH Q1A(R2) Stability Testing of New Drug Substances and Products Nov 2003