A Validated HPTLC Method for Simultaneous Analysis of Eugenol and Piperine in a Siddha Formulation

A high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous analysis of eugenol and piperine in the Siddha formulation Amukkara choornam. TLC was performed on aluminum foil-backed silica gel 60F254 plates with toluene-ethyl acetate 9:3 as mobile phase. Calibration plots for both eugenol and piperine were linear in the range 1–12 µg. The polynomial regression data for the calibration plots were indicative of good linear relationships with r2 = 0.9979 and 0.9980 for eugenol and piperine respectively. The RF values of eugenol and piperine were 0.73 ± 0.03 and 0.28 ± 0.01, respectively. The method was validated for sensitivity, accuracy, precision, and robustness. Minimum detectable amounts were 0.121 ng per band for eugenol and 0.0102 µg per band for piperine. The eugenol and piperine content of the formulation were 0.207 µg g−1 and 1.992 mg g−1, respectively. Recovery of eugenol and piperine was greater than 97%. The method enabled simple, sensitive, precise, accurate, and specific analysis of eugenol and piperine in the formulation.