RP-HPLC Method for Simultaneous Quantification of Withanolides in <i>Withania somnifera</i>

Background: Withanolides are the primary bioactive components of Withania somnifera (L.) Dunal. Among them, withanolide A, and 12-deoxy-withastramonolide are the herb’s most physiologically active ingredients. The steroidal lactones are isomers, which makes them difficult to separate. Aim: In the current research, a simple, precise, accurate, and decent RP-HPLC method has been developed, optimised and validated for simultaneous quantification and separation. Methods: Resolution was conducted on Gemini, Phenominex C18 (250mm X 4.6mm, 5 μ) column at 40oC utilising binary mobile phases of acetonitrile and ammonium acetate (10 mM) in gradient mode with 1.0 mL/min flow rate and observed at 230 nm. The total run time was twenty-five minutes. The procedure complied with ICH guidelines, covering the limit of detection, specificity, linearity, quantification, precision, accuracy, and robustness. Results: The standard curves of both relevant analytes displayed a linear pattern with regression values > 0.999 in the 1–10 μg/mL range. For both herbal markers, 1 μg/mL was the lowest limit of quantification. The method’s accuracy was 100.38-100.93 % and 99.13-100.75 % for with withanolide A, and 12-deoxy-withastramonolide respectively. The precision of within and between days was found in the tolerable limit of >2% for both herbal markers. Conclusion: The suggested method worked well for simultaneously analyzing the extracted samples and identifying withanolides.