Piperine, the major bioactive compound, was found in Piper nigrum. This present investigation aimed to find the factors that can affect high-performance liquid chromatography (HPLC) conditions for the evaluation of piperine content from Thai herbal medicines. Box–Behnken design for the response surface methodology was selected to assess the interaction between three factors (% acetonitrile in the mobile phase, flow rate, and detection wavelength) and five outputs (retention time, peak area, theoretical plate, tailing factor, and capacity factor). The optimal HPLC system was found to be a mobile phase containing 35% acetonitrile with a flow rate of 1.2 ml/minute at wavelength 254 nm. The evolved HPLC condition was subjected to system suitability and robustness testing. The results indicated a slight change to the acetonitrile ratio affected the retention time, peak area, tailing factor, and capacity factor. This HPLC method was reliable and was applied to determine the piperine constituent in seven Thai herbal recipes, ranging from 0.256 ± 0.064 to 22.284 ± 0.802 mg/g extract. Overall, the experimental design was very helpful in studying the factors that affect the HPLC conditions and robustness of this method, which was discovered to be uncomplicated to carry out and acceptable for the exploration of piperine in Thai herbal medicines.
Key words: Thai traditional formula, Piper nigrum, piperine, Box-Behnken design, response surface methodology, HPLC, system suitability testing
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