Determination of Cefepime and Related Compounds Using HPLC with UV Detection

September 1, 2008
Brian De Borba

Dionex Corporation

,
Jeff Rohrer

,
Deanna Hurum

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2008, Volume 0, Issue 0

Cephalosporins contain a four-member β-lactam ring that is inherently strained and prone to hydrolysis and photolysis, limiting stability and leading to degradation products that may be toxic (1). In addition, synthetic byproducts are generated and persist during production of these antibiotics including cefepime. Analysis of cefepime purity is particularly challenging due to the presence of such isomeric synthetic impurities. The Acclaim® 120 C18, 3 μm can be used to meet and exceed the criteria set by the USP for determining related substances and assaying the purity of cefepime.

Deanna Hurum, Brian De Borba, and Jeff Rohrer, Dionex Corporation

Cephalosporins contain a four-member β-lactam ring that is inherently strained and prone to hydrolysis and photolysis, limiting stability and leading to degradation products that may be toxic (1). In addition, synthetic byproducts are generated and persist during production of these antibiotics including cefepime. Analysis of cefepime purity is particularly challenging due to the presence of such isomeric synthetic impurities. The Acclaim® 120 C18, 3 μm can be used to meet and exceed the criteria set by the USP for determining related substances and assaying the purity of cefepime.

Experimental

A Dionex UltiMate® 3000 system with a DGP-3600M pump, an FLM-3100 flow manager (a TCC-3200 column compartment could be used) a WPS-3000T autosampler, and a VWD-3400 detector was used in this work. An Acclaim 120 C18, 3 μm Analytical (2.1 × 150 mm) was used for all separations with a flow rate of 0.20 mL/min. Analytes were detected by UV absorption at 254 nm. A 1 μL injection volume was used throughout. Chromeleon® Chromatography Management Software was used for system control and data processing.

Figure 1

Results and Discussion

Due to the smaller column dimensions and the 3 μm particle size, the USP gradient method can be considerably shortened and still meet the USP requirements. Figure 1 illustrates the separation possible with a gradient method that removes 20 min from the run time for each injection. This shortened gradient meets the USP criteria for peak asymmetry, resolution between cefepime and cefepime related substances A and B, theoretical plates, and capacity factor. The precision of this shortened gradient was tested with retention time RSDs of 0.06% or less and peak area RSDs of 0.82% or less (Table I). Resolution is significantly improved, including the clear separation of an additional peak with a retention time of 9.39 min. This peak is not described in the current USP monograph, but it is recognized as impurity F in EP method 2126.

Table I: Precision of replicate injections of 1.4 mg/mL cefepime system suitability standard, n=10

Conclusion

In this study, the Acclaim 120 C18 column, an HPLC L1 column with good steric selectivity, was successfully used for the determination of cefepime and cefepime related substances. The methods reduced the time needed for analysis by 40% and improved the resolution as compared to the current USP method. Additionally, the low flow rates used in this method save time and resources spent on mobile phase preparation and reduce waste production.

Reference

(1) Rabouan-Guyon, S.M.; Guet, A.F.; Courtois, P.Y.; Barthes, D.M.C. "Stability study of Cefepime in Different Infusion Solutions." Int. J. Pharm., 1997 154, 185–190.

Acclaim, Chromeleon, and UltiMate are registered trademarks of Dionex Corporation.

Dionex Corporation

1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088-3603

tel. (408) 737-0700, fax (408) 730-9403

www.dionex.com