Development and Validation of a High-Performance Liquid Chromatography (HPLC) Method for the Quantitative Determination of Caffeine in Rat Plasma for In Vivo Bioanalysis

Abstract

Caffeine is one of the most widely consumed methylxanthine compounds and is rapidly absorbed into the bloodstream following oral administration. Quantitative analysis of caffeine in biological matrices requires a selective and validated analytical method because blood plasma contains numerous endogenous compounds that may interfere with the analysis. This study aimed to develop, validate, and apply a high-performance liquid chromatography (HPLC) method for the quantitative determination of caffeine in rat plasma following in vivo oral administration. This/’  experimental laboratory study was conducted by optimizing the mobile phase composition using distilled water and methanol at ratios of 50:50, 60:40, and 70:30 (v/v), with ultraviolet (UV) detection. The optimized chromatographic conditions were evaluated through a system suitability test and validated in accordance with the FDA Bioanalytical Method Validation Guidance, including linearity, precision, accuracy, selectivity, limit of detection (LoD), and limit of quantification (LoQ). The validated method was subsequently applied to determine plasma caffeine concentrations at 1, 2, and 3 hours after oral administration in rats. The optimized mobile phase, consisting of distilled water and methanol (50:50, v/v), produced satisfactory chromatographic separation and fulfilled all system suitability criteria. The method demonstrated excellent linearity, with correlation coefficients (r) of 0.9973 based on peak area and 0.9986 based on peak height. The precision test yielded a %RSD of 1.454%, while the accuracy test showed recovery values of 105.67%, 100.19%, and 104.96%. The average chromatographic resolution was 2.17, whereas the LoD and LoQ were 0.38 mg/L and 1.15 mg/L, respectively. The mean plasma caffeine concentrations were 2.54 mg/L at 1 hour, 1.79 mg/L at 2 hours, and 1.43 mg/L at 3 hours after oral administration. The developed HPLC method fulfilled the validation criteria and proved to be reliable for the quantitative determination of caffeine in rat plasma. Therefore, this method is suitable for in vivo pharmacokinetic and bioanalytical studies involving caffeine.