1*Dept of Pharmaceutical Chemistry, SMBT College of Pharmacy Dhamangaon, Affiliated to Savitribai Phule Pune University, Nashik, 422403, India. Email: ssshaharwale.yipb@gmail.com
1Dept of Pharmaceutical Chemistry, SMBT College of Pharmacy Dhamangaon, Affiliated to Savitribai Phule Pune University, Nashik, 422403, India
2Dept of Pharmacognosy, SMBT College of Pharmacy Dhamangaon, Affiliated to Savitribai Phule Pune University, Nashik, 422403, India
Objective: The main objective of the study was to develop and validate a robust Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method for the simultaneous estimation of Diosgenin and Piperine in bulk as well as in human plasma samples, with Nintedanibethanesulfonate as an internal standard (IS).
Method: Chromatographic separation was performed on Phenomenex C18 column (250 mm X 4.6 mm, 5µ). Methanol and 0.05% Ortho-phosphoric acid in water in the ratio of 50:50 % V/V was used as the mobile phase at a flow rate of 1 mL/min. Diosgenin and Piperine have different absorption maxima, so the detection was carried out at 210 nm and 343 nm, respectively. In plasma analysis, the method for the extraction was optimized by a protein precipitation extraction method using methanol because it is cost-effective with high drug recovery.
Results: Consequently, this method has demonstrated excellent linearity with concentrations ranging from 0.40-16.00 µg/mL and 0.15-6.00 µg/mL for Diosgenin and Piperine, respectively. Therefore, the retention times were approximately 3.65, 5.62, and 6.52 minutes for the IS, Diosgenin, and Piperine, respectively. On the other hand, the percentage recovery from the extracts was significantly high, as confirmed by the results, which were >92% for both compounds under investigation, specifically Diosgenin, 92.87% and Piperine, 93.09%. Moreover, the precision, as well as accuracy, of all the levels such as LLOQ, LQC, MQC, HQC, were all within acceptable limits.
Conclusion: A simple, sensitive, and accurate RP-HPLC method for the analysis of Diosgenin and Piperine has been successfully developed. The efficiency of the single internal standard using dual-wavelength detection is impressive, thus making the method suitable for its routine bio-analytical application/pharmacokinetic study for Diosgenin and Piperine.
Keywords: Chromatography, Diosgenin, Piperine, linearity, extraction.
How to cite this article: Shaharwale SS, Shinde NV, Bhosale S, Ushir Y. Validated RP-HPLC Method for Simultaneous Bioanalytical Quantification of Diosgenin and Piperine in Human Plasma Using Internal Standard Calibration. Int J Drug Deliv Technol. 2026;16(2): 107-120; DOI: 10.25258/ijddt.16.2.14
Source of support: Nil.
Conflict of interest: None