Forced-degradation evaluation of erythromycin by HPLC and single quadrupole mass spectrometry

Importance of the Topic

Erythromycin is a widely used antibiotic whose low UV absorbance and complex impurity profile make stability and purity assessment challenging. Forced‐degradation studies help predict degradation pathways and support formulation development, regulatory submissions, and quality control.

Objectives and Study Overview

This work aimed to design a straightforward, stability‐indicating liquid chromatography–mass spectrometry (LC-MS) method for erythromycin. The method compares impurity profiles of a reference standard and acid‐stressed erythromycin, demonstrating that MS detection enhances impurity identification without extensive method optimization.

Methodology and Instrumentation

Sample preparation involved dissolving erythromycin standards and generating an acid‐degraded sample by exposure to 1 N HCl followed by quenching with NaHCO₃. Chromatographic separation used a Thermo Scientific Acclaim PolarAdvantage II column (3 µm, 3 × 150 mm) with a water/formic acid and methanol/water/formic acid gradient at 425 µL/min. Mass detection was performed on a Thermo Scientific ISQ EM single quadrupole in positive mode, with both full‐scan (350–1050 m/z) and selective ion monitoring (SIM) channels for known variants and impurities.

Instrumental Setup

• Vanquish Core Binary HPLC (pump, autosampler at 4 °C, column compartment at 40 °C)
• ISQ EM single quadrupole mass spectrometer with Autospray source (optimized for thermal lability at 250 °C ion transfer)
• Chromeleon 7.3 CDS for data acquisition and processing

Results and Discussion

Initial method development used the European Pharmacopeia system suitability standard. Tuning the ion source temperature minimized in-source fragmentation of erythromycin A (m/z 734.47) and enabled clear detection of related variants (e.g., m/z 718.47 for erythromycin B). Chromatographic conditions resolved erythromycin B from co-eluting impurities at m/z 716.4. In stressed samples, additional degradation products appeared (m/z 576–584 and 540 ranges), and five new low-molecular-weight impurities were observed. Relative impurity levels increased significantly under acidic stress, while the main API peak remained quantifiable by SIM despite minor co-elutions.

Benefits and Practical Applications

• Fit-for-purpose LC-MS method requiring minimal HPLC optimization
• Sensitive impurity profiling with < 3 µg injection, reducing sample consumption
• Mass‐selective detection overcomes co-elution and low UV absorbance challenges
• Rapid identification of forced‐degradation products without reference standards

Future Trends and Opportunities

High-resolution mass spectrometry could further elucidate structural details of unknown degradation products. Automated method development and advanced data‐processing algorithms will accelerate impurity identification. Extending this approach to other poorly UV-absorbing APIs and adopting greener solvents can enhance sustainability in pharmaceutical analysis.

Conclusion

The presented LC-MS approach offers a robust, low-effort solution for erythromycin stability testing. It delivers clear impurity profiles with minimal sample and method development, making it a valuable tool in early drug development and quality control.

Reference

1. European Pharmacopoeia monograph 0179: Erythromycin.
2. EDQM Information Leaflet: Erythromycin for system suitability CRS.
3. Chitneni SK et al., J. Chromatogr. A 1056 (2004) 111–120.