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Abstract
Optimization of important conditions for the reversed‐phase high‐performance liquid chromatographic method was done for the separation of the active ingredients in Marcaine® adrenaline injections (bupivacaine hydrochloride 2.5 mg and adrenaline 5.0 µg). Simultaneous influence of several conditions, such as the mobile phase composition, pH of the mobile phase, and temperature, on important chromatographic criteria for the separation, was investigated. The separation factor values defined the optimal conditions, which were confirmed by analysing the appropriate mathematical models. The 3‐D graphs, constructed with sixty‐four experimental points, were investigated, and the results showed that the optimal separation was achieved with the mobile phase of methanol–water (65:35 v/v), by adjusting pH to 3.5 and the temperature range from 20°C to 30°C. The optimized method was validated and the obtained results were statistically evaluated.