Novel HPLC Methods for the Assessment of Some Cephalosporins

Abstract

Herein, a new, simple, sensitive, and reliable three bio-analytical methods were introduced for the determination of two cephalosporins in the human plasma– namely: cefprozil and cephalexin. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) and with ultraviolet (LC-UV) detection methods were developed and validated for the determination of cis- and trans-cefprozil diastereoisomers. Moreover, LC-UV method was developed and validated for the determination of cephalexin in the human plasma. For cefprozil determination, the human plasma samples were subjected to protein precipitation after the addition of cephalexin as internal standard. Chromatographic separation of the two diastereoisomers from the endogenous interfering components of the human plasma was achieved using Xbridge C18 column (5µm, 4.6×150mm) and maintained at 25° C. The LC-MS/MS method utilized the multiple reaction monitoring (MRM) transitions 390.1 to 208.1 m/z for cefprozil and 348.1 to 158.2 m/z for cephalexin detection, while the wavelength 292 nm was used for the UV detection. Both methods provided good linearity for the determination of cis- and trans-cefprozil diastereoisomers within the ranges of 0.05-10.00 and 0.02-1.00 μg/ml respectively. The methods were validated and applied successively to study the bio-equivalence of the two cefprozil diastereoisomers in pharmaceutical products. The maximum plasma levels (Cmax) detected for cefprozil for the brand and generic products were 10.0 and 9.9 μg/ml, respectively, as determined using the LC-MS/MS method compared to 10.5 and 10.6 μg/ml, respectively, as determined using the LC-UV method. The pharmaceutical products were found to be bio-equivalent after analysis using both methods. The pharmacokinetics data of the reference product were statistically compared over the two methods and resulted in insignificant P-values. This comparison confirm both methods reproducibility, reliability and ability to quantify cefprozil diastereoisomers in the human plasma.