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

Significance of the Topic

The assessment of drug stability under forced degradation conditions is a key step in pharmaceutical development and quality control. Traditional liquid chromatography with ultraviolet detection may fail to resolve all impurities or detect low UV absorbing compounds. The combination of high performance liquid chromatography and single quadrupole mass spectrometry offers a simple and sensitive approach to characterize degradation profiles, assign peaks based on mass to charge ratio and minimize sample consumption.

Objectives and Study Overview

This study aimed to develop a stability indicating LCMS method for erythromycin, a macrolide antibiotic with low UV absorbance and multiple biosynthetic variants. A comparison of impurity profiles between an erythromycin reference standard and samples stressed by acidic degradation was performed. The goals included minimization of method development effort, confirmation of impurity identity without chemical standards and achievement of adequate sensitivity with microgram sample injections.

Methodology and Instrumentation

Sample preparation involved dissolution of erythromycin standards and stressed mixtures in water acetonitrile formic acid solutions. Forced degradation was induced by treatment with hydrochloric acid followed by neutralization with sodium bicarbonate.

The chromatographic system consisted of a Vanquish Core Binary HPLC equipped with an Acclaim PolarAdvantage II column 3 micron 3 x 150 mm. Mobile phases were water with 0.1 percent formic acid and methanol water 9 to 1 with 0.1 percent formic acid. A gradient from 50 to 100 percent organic over 20 minutes at 425 microliter per minute flow rate was applied. Column compartment was held at 40 degrees Celsius and autosampler at 4 degrees.

Mass spectrometry detection was performed on an ISQ EM single quadrupole instrument operating in positive ion mode. Full scan from m z 350 to 1050 was combined with selected ion monitoring for known erythromycin variants and degradation products. Source parameters were adjusted to minimize thermal fragmentation by reducing the ion transfer tube temperature.

Chromeleon chromatography data system software was used for data acquisition component identification and quantitative evaluation via extracted ion chromatograms.

Results and Discussion

Initial method development with system suitability standard revealed coeluting thermal fragments of erythromycin A. Lowering the ion transfer tube temperature eliminated in source degradation peaks and improved quantitation. Chromatographic conditions were optimized to resolve erythromycin B from impurities sharing the same nominal mass m z 716.4 by selecting a polar embedded stationary phase and appropriate organic modifier.

The final method achieved baseline separation of the main components of the suitability mixture with injections of less than three micrograms. Analysis of the stressed erythromycin sample showed increased number and relative abundance of lower molecular weight impurities and the appearance of novel degradation species not present in the reference material. Relative impurity levels were assessed by normalizing extracted ion peak areas to erythromycin A.

Method Benefits and Practical Applications

• Fit for purpose and generic chromatographic conditions require minimal optimization
• Sensitive detection of low UV absorbing antibiotics with microgram level injections
• Mass selective peak assignment removes the need for reference standards of impurities
• Ability to tolerate co elution of species with distinct mass to charge ratios
• Reduction of sample consumption prevents column overload and saves early development material

Future Trends and Potential Applications

Advances in mass spectrometry such as quadrupole orbitrap and time of flight systems may further enhance impurity characterization. Integration of automated method scouting and multi attribute monitoring workflows could streamline forced degradation studies. The approach demonstrated here for erythromycin can be extended to other pharmaceutical compounds with poor UV response or complex degradation pathways. Hyphenation with tandem mass spectrometry and high resolution detection will enable definitive impurity identification without chemical standards.

Conclusion

A simple stability indicating LCMS method for erythromycin was developed rapidly using a generic HPLC gradient and single quadrupole mass spectrometer. The method provided sensitive and selective quantitation of the antibiotic and its degradation products with microgram injections. Mass detection overcame the limitations of UV absorbance and enabled assignment of coeluting species based on m z values. The workflow reduces development time and sample demand while delivering comprehensive impurity profiles.

Reference

1. European Directorate for the Quality of Medicines and HealthCare European Pharmacopoeia monograph 0179 erythromycin
2. European Pharmacopoeia information leaflet reference standard erythromycin for system suitability CRS batch 1
3. Chitneni SK Identification of impurities in erythromycin by liquid chromatography mass spectrometric detection Journal of Chromatography A 2004 1056 111 120