Highly sensitive and selective high-performance liquid chromatography method for bioequivalence study of cefpodoxime proxetil in rabbit plasma via fluorescence labeling of its active metabolite

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2013-09, Vol.934, p.34-40
• Main Authors: Ahmed, Sameh, Abdel-Wadood, Hanaa M., Mohamed, Niveen A.
• Format: Article
• Language: English
• Subjects: Animals; Benzofurazan; Bioequivalence study; Buffers; cefotaxime; cefpodoxime; Cefpodoxime acid; Cefpodoxime Proxetil; Ceftizoxime - analogs & derivatives; Ceftizoxime - blood; Ceftizoxime - metabolism; Ceftizoxime - pharmacokinetics; Chromatography; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; cross-over studies; detection limit; Fluorescence; Fluorescent Dyes - chemistry; Formulations; guidelines; high performance liquid chromatography; Marking; Metabolites; Oxazoles - chemistry; Pharmacokinetic; pharmacokinetics; phosphates; Prodrug; Rabbits; Sensitivity and Specificity; Sulfonamides - chemistry; Therapeutic Equivalency; urinary tract diseases; wavelengths
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.06.036

•Highly sensitive and selective method for the determination of CFA in rabbit plasma.•This is the first HPLC-FL method concerning the quantitation of CFA in plasma.•The developed method was 50 times more sensitive than reported HPLC methods.•Plasma sample clean-up involves a simple protein precipitation procedure with no reconstitution step.•The developed HPLC-FL method was applied successfully for bioequivalence study. Cefpodoxime proxetil (CFP), a broad-spectrum third-generation cephalosporin, has been used most widely in the treatment of respiratory and urinary tract infections. For bioequivalence study of CFP in rabbit plasma, it was necessary to develop a highly sensitive and selective high-performance liquid chromatographic (HPLC) method with fluorescence (FL) detection. The pre-column labeling of cefpodoxime acid (CFA) (active metabolite) with an efficient benzofurazan type fluorogenic reagent, 4-N,N-dimethyl aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was carried out in the present study in 100mM borate buffer (pH=8.5) at 50°C for 15min. The obtained fluorescent products were separated on C18 column with an isocratic elution of the mobile phase, which consists of 10mM phosphate buffer (pH=3.5)/CH3CN (70:30, v/v). The fluorescent product (DBD-CFA) was detected fluorimetrically at 556nm with an excitation wavelength of 430nm. Cefotaxime sodium was used as internal standard. The method was validated according to the requirements of US-FDA guidelines. The correlation coefficient of 0.999 was obtained in the concentration ranges of 10–1000ngmL−1. The limits of detection and quantification (S/N=3) were 3 and 10ngmL−1, respectively. Plasma CFA levels were successfully determined in rabbit with satisfactory precision and accuracy. The proposed HPLC-FL method was successfully applied to study bioequivalence in rabbits for two formulations of different brands contained CFP (prodrug) in a randomized, two-way, single-dose, crossover study and all pharmacokinetic parameters for the two formulations were assessed.