Validated UV-Visible Spectrophotometric Method for the Estimation of Aloe-emodin in Rhubarb Rhizomes

Abstract

Aim: To develop and validate a simple, cost-effective, accurate, and precise UV-Visible spectrophotometric method for the estimation of Aloe-emodin in rhizomes of Rhubarb Method: A UV-Visible spectrophotometric method for the estimation of Aloe-emodin was developed using a mixture of Methanol and water. The developed method was validated using ICH Q2 (R1) guidelines. Aloe-emodin solutions were scanned across the entire UV-visible range to identify the wavelength of maximum absorbance. The linearity of the method was established using the seven calibration standards over a concentration range of 0.1 μg/mL to 10 μg/mL. The accuracy in terms of percent difference and the precision in terms of percent relative standard deviation of the developed method was determined using three different quality control standard solutions (0.3, 5 & 9 μg/mL). Robustness of the proposed method was established by making slight changes in the co-solvent composition. The limit of detection (LOD) and the limit of quantification (LOQ) of the proposed method was calculated on the basis of slope and the standard deviation of the y-intercept of calibration curves. The Aloe-emodin content in the rhubarb rhizomes was estimated using the proposed method.  Results: The Aloe-emodin, when dissolved in a mixture of methanol and water (40:60 v/v) and scanned over 200 to 800 nm, showed maximum absorbance at 256 nm. The seven different calibration standards of Aloe-emodin, viz. 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL, and 10 μg/mL when analyzed at 265 nm, showed excellent linearity in the absorbance values with a correlation coefficient (r²) of 0.9999. The intra-day accuracy of the developed UV-Visible spectrophotometric method in terms of % Difference was found to be in the range of -6.6638 and 0.3083, whereas the inter-day accuracy was in the range of -0.9986 and 1.0768. The intra-day precision of the developed UV-Visible spectrophotometric method in terms of %RSD was found to be between 0.22 to1.85, whereas the inter-day values are in between 0.54 to 1.98. The variation (%RSD) during the robustness studies of the proposed UV-Visible spectrophotometric method was found to be below 2 %. The limit of detection (LOD) and limit of quantitation (LOQ) of the developed UV-Visible spectrophotometric method were found to be 0.037 µg/mL & 0.11 µg/mL, respectively, ensuring adequate sensitivity for routine quality control analysis. The developed method was successfully used for the estimation of Aloe-emodin in rhubarb rhizomes. Conclusion: A simple, cost-effective, precise, and accurate UV–visible spectrophotometric method was successfully developed and validated for the quantitative estimation of aloe-emodin. The analytical procedure was optimized using a co-solvent system containing a minimal and economical proportion of the organic phase, thereby enhancing method affordability while maintaining analytical reliability. Method validation parameters, including linearity, precision, accuracy, and reproducibility, demonstrated satisfactory performance within the established analytical range, confirming the robustness of the procedure. Owing to its operational simplicity and reduced solvent consumption, the validated UV–visible spectrophotometric method is suitable for routine quantitative estimation of aloe-emodin in rhubarb rhizomes and may serve as a practical tool for quality control in phytopharmaceutical analysis.