Pasupula Tulasi¹, Gunturu Jaya Syamala²*, Y Ankamma Chowdary³, Inturi Venkata Rama Rao⁴ and Gunturu Uma Soundarya⁵

¹Master of Pharmacy, Department of Pharmaceutical Quality Assurance, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India
²Assistant Professor, Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India
³Dean, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India
⁴Professor, Department of Pharmaceutics, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India
⁵Research Scholar, Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India

*Corresponding Author: Gunturu Jaya Syamala, Assistant Professor, Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Dr. RVR NRI Institute of Technology, Deemed to be University, Pothavarappadu (V), Agiripalli (M), Vijayawada Rural, Eluru District, Andhra Pradesh, India.

Received: September 08, 2026; Published: October 13, 2026

Abstract

Introduction: Cefepime, a fourth-generation cephalosporin, is combined with enmetazobactam, a novel extended-spectrum β-lactamase (ESBL) inhibitor, to restore antibacterial activity against ESBL-producing Gram-negative organisms in complicated urinary tract infection. A simple, specific, stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of cefepime and enmetazobactam in combination.

Materials and Methods: Chromatographic separation was performed on a Waters Alliance e2695 system with a photodiode array (PDA) detector, using a Symmetry Shield RP-18 column (250 × 4.6 mm, 5 μm) and a mobile phase of methanol and potassium dihydrogen phosphate buffer (pH 2.5, adjusted with ortho-phosphoric acid) in the ratio 30:70 v/v, isocratically delivered at 1.0 mL/min. Detection was carried out at 281 nm, the isosbestic wavelength identified by PDA scanning. The method was validated for system suitability, specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), limit of quantitation (LOQ) and forced-degradation behaviour as per ICH guidelines.

Results: Cefepime and enmetazobactam eluted at 2.122 minutes and 3.858 minutes, respectively, with a resolution of 8.15, tailing factors of 1.08 and 0.95, and theoretical plate counts of 9825 and 7531. Linearity was established over 50 - 300 μg/mL for cefepime (R² = 0.99989) and 12.5 - 75 μg/mL for enmetazobactam (R² = 0.99969). Mean recoveries were 99.5% (cefepime) and 98.9% (enmetazobactam), with %RSD below 1% for system precision and below 2% for method and intermediate precision. LOD/LOQ were 0.6/2.0 μg/mL for cefepime and 0.15/0.5 μg/mL for enmetazobactam. The method remained robust to ± 10% variation in flow rate and ± 3% variation in the organic-phase ratio. Under ICH-recommended stress conditions, both analytes showed pronounced degradation under acidic, alkaline, oxidative and hydrolytic conditions (10 - 16%) and comparatively limited degradation under reductive, thermal and photolytic stress (< 4%), with peak-purity angle remaining below the purity threshold throughout.

Discussion: All validation parameters met ICH acceptance criteria, and the degradation profile confirms the expected susceptibility of the shared β-lactam pharmacophore to hydrolytic stress while demonstrating that the method can resolve the parent drugs from their degradation products.

Conclusion: The developed RP-HPLC-PDA method is simple, specific, accurate, precise, robust and stability-indicating, and is suitable for the routine simultaneous quality-control estimation of cefepime and enmetazobactam.

Keywords: Cefepime; Enmetazobactam; RP-HPLC; Method Development; Method Validation; Stability-Indicating; ICH Guidelines

References

1. Bassetti M and Peghin M. “How to manage KPC infections”. Therapeutic Advances in Infectious Disease 7 (2020): 2049936120912049.
2. Kaye KS., et al. “Effect of cefepime/enmetazobactam vs piperacillin/tazobactam on clinical cure and microbiological eradication in patients with complicated urinary tract infection or acute pyelonephritis: a randomized clinical trial”. Journal of the American Medical Association 13 (2022): 1304-1314.
3. US Food and Drug Administration. “FDA approves new antibacterial drug for complicated urinary tract infections as part of ongoing efforts to address antimicrobial resistance [cefepime and enmetazobactam, Exblifep]”. Silver Spring: FDA (2024).
4. Mameli M., et al. “Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of enmetazobactam and cefepime in human plasma”. Journal of Pharmaceutical and Biomedical Analysis 174 (2019): 655-662.
5. Jabeen S and Venkatappa S. “Stability indicating UPLC-MS/MS method for quantification and identification of cefepime and its degradants in API”. Journal of Applied Pharmaceutical Science 5 (2023): 199-207.
6. Shah CU and Satavara JR. “The review article on analytical method development and validation of antibiotic drugs cefepime and enmetazobactam for urinary tract infection”. International Journal of Research Publication and Reviews 3 (2025): 10234-10240.
7. International Council for Harmonisation. ICH Q2 (R1): Validation of analytical procedures: text and methodology. Geneva: ICH (2005).
8. Snyder LR., et al. “Practical HPLC method development”. 2^nd New York: John Wiley & Sons (1997).
9. Skoog DA., et al. “Fundamentals of analytical chemistry”. 9^th Belmont: Brooks/Cole, Cengage Learning (2014).

Citation

Citation: Gunturu Jaya Syamala., et al. “Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Ce- fepime and Enmetazobactam in Combination". Acta Scientific Pharmaceutical Sciences 10.6 (2026): 40-47.

Copyright

Copyright: © 2026 Gunturu Jaya Syamala., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.