RP-HPLC Method for Simultaneous Determination of Zingerone, Berberine, and Piperine in Ayush Kadha: Greenness and Whiteness Evaluation

Abstract

RP-HPLC method for simultaneous determination of Zingerone, Berberine, and Piperine present in Ayush Kadha was developed and validated. The separation was achieved on the C18 column (250 mm × 4.6 mm, 5 µm) using isocratic elution with 0.1% trifluoroacetic acid:methanol:acetonitrile (35:60:5, v/v/v) at flow rate of 1.0 mL min⁻¹. The detection wavelength was set at 269 nm. For Zingerone, Berberine, and Piperine, the retention times were found to be 4.417 min, 6.550 min, and 15.983 min, respectively. The limit of detection (LOD)/limit of quantification (LOQ) were found to be 0.0007/0.002 µg mL⁻¹, 0.001/0.003 µg mL⁻¹, and 0.19/0.59 µg mL⁻¹, for Zingerone, Berberine, and Piperine, respectively. The method was linear in the range of 1.5–6 µg mL⁻¹ for all the markers. Mean % recoveries for Zingerone, Berberine, and Piperine were found to be within the acceptance limit of 90–110%, and the % RSD for precision was found to be less than 2.0%. The proposed method can be used as a quality control tool for formulations containing a combination of Zingiber officinale, Tinospora cordifolia, and Piper nigrum. Greenness of the method was assessed using different tools, namely AGREE, AGREEprep, and RGBfast, and whiteness was assessed by the BAGI tool. This HPLC method demonstrates novelty by offering a greener, more efficient approach with superior sensitivity and validated robustness for the simultaneous determination of Zingerone, Berberine, and Piperine in Ayush Kadha.