| Objectives:Ketoconazole, a broad-spectrum antifungal agent, has significant antibacterial activity against superficial and deep fungal and strong inhibition on CYP450. Berberine (Ber), an isoquinoline-type alkaloid, has significant antibacterial effect. It was reported that there was synergistic antibacterial effect with combination therapy of berberine and ketoconazole in vitro. But neither LC-MS method nor the in vivo study about combination of ketoconazole and berberine in rats has not been reported according to our knowledge. The aim of this study is to develop an LC-MS method for simultaneous determination of berberine and ketoconazole in rat plasma, to provide a reliable detection method for pharmacokinetics study and explore the relationship between synergistic antibacterial effect and pharmacokinetics. This study can also provide accurate data for clinical combination therapy, developing anti-pathogen compound and preventing the emergence of drug resistant strains.Methods:According to FDA bioanalytical method validation guidelines, a sensitive LC-MS method was developed for quantitation of berberine and ketoconazole in rat plasma, which was validated for its selectivity, linearity, LLOQ, accuracy, precision, recovery, stability and matrix effect. The validated method was applied to study the pharmacokinetics of berberine and ketoconazole in rats. Sprague-Dawley rats were divided into alone administration group and coadministration group with oral administration at dose of berberine 60mg/kg and ketoconazole 10mg/kg, and berberine (1mg/kg) i.v. group. Rat plasma sample was obtained from the oculi chorioideae vein before and after administration, plasma sample was extracted by liquid-liquid extraction (LLE) method and analyzed by LC-MS method. The pharmacokinetic parameters of study were calculated using DAS special Software.Results:Linear range of this method was over the concentration range of 0.05-600 ng/mL for berberine and 0.4-6000 ng/mL for ketoconazole with coefficients of correlation above 0.995. The lower limits of quantification (LLOQ) were 0.05 ng/mL for berberine and 0.4 ng/mL for ketoconazole, respectively. The inter-and intra-day precision (CV) values were within 12.2% and accuracy (RE) ranged from-7% to 9%. The extraction recovery was on average 61.5% for berberine and 99.8% for ketoconazole. The result indicated that this method was consistent with FDA bioanalytical method validation guidelines.Comparing co-administration group with alone administration group, values for ketoconazole AUC0-8 (1.6 fold), Cmax (2.0 fold) were higher and CL/F was 57.2% lower. Similarly, values for berberine AUC0-8 (1.9 fold), Cmax (1.5 fold) were higher and CL/F was 53.4% lower. In addition, female rats AUC0-8 (15-20 fold) and Cmax (4-10 fold) were higher than those of male rats, while CL/F (20-25fold) was lower than that of male rats. In i.v. group of berberine, the main pharmacokinetics parameters were detected to be AUC0-8 218±14μg/L*h, Cmax 127±30 ng/mL and CL/F 4.6±0.3 L/h/kg, respectively.Conclusions:A sensitive LC-MS method has been developed for the first time and validated for the simultaneous quantification of berberine and ketoconazole in rat plasma, which was applied successfully in rat pharmacokinetic study. The method is conformed with good reproducibility, high sensitivity and wide linear range, which is conducive to future research. This method can also be improved for quantitation of other alkaline drugs. At present, this is the first time that the coadministration pharmacokinetic behavior of berberine and ketoconazole in rats has been reported. There was potential drug interaction between berberine and ketoconazole in rats, but no significant increase of plasma concentration and AUC was observed in present study, which implied that synergistic antibacterial effect may be mainly related with pharmacodynamics. There was significant gender indifference of ketoconazole pharmacokinetics in rats, and faster metabolism was found in male rats. No gender indifference of berberine pharmacokinetics was observed in rats.
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