System for simultaneous RP-HPLC analysis of aloin and curcumin

Abstract

A chromatographic system for the simultaneous RP-HPLC quantification of aloin and curcumin, consisting of a C18 column, a PDA detector, a programmable solvent feed module and an optimized mobile phase of acetonitrile, methanol and water.

Description

Field of invention

[0001] The invention relates to a chromatographic analysis system for the development and validation of a simultaneous RP-HPLC method for the quantitative determination of aloin and curcumin. Background of the invention

[0002] The analytical quantification of plant bioactive compounds such as aloin and curcumin is essential for ensuring the quality, safety, and efficacy of pharmaceutical and nutraceutical preparations. Although both compounds offer significant therapeutic benefits, their simultaneous determination presents a technical challenge due to differences in polarity, UV absorption, and chromatographic behavior. Conventional chromatographic methods often lack sufficient peak separation, precision, or sensitivity, resulting in poor reproducibility and limited applicability for regulatory compliance. While reversed-phase high-performance liquid chromatography (RP-HPLC) offers high resolution and sensitivity, its effectiveness depends on optimized instrument configuration, mobile phase selection, and method validation.There remains a need for a reliable system for the simultaneous analysis of aloin and curcumin under standardized operating conditions and with validated performance parameters. The invention fulfills this need through an integrated chromatographic system that enables method development, optimization, and complete validation in accordance with ICH and FDA guidelines for aloin-curcumin mixtures. Summary of the invention:

[0003] The invention provides an analytical system for the simultaneous RP-HPLC quantification of aloin and curcumin. The system comprises a chromatographic apparatus with a C18 stationary phase, a PDA detector, and programmable units for flow control and wavelength monitoring. By systematically varying the composition of the mobile phase (acetonitrile, methanol, and water), the system ensures optimal peak separation, sharpness, and symmetry. The system enables the preparation of standard solutions from aloin-curcumin mixtures and offers controlled injection, detection, and chromatographic evaluation for precise quantitative analysis.

[0004] The system also includes modules for complete method validation, including specificity, linearity, accuracy, precision, robustness, and stability tests. It can be operated under various flow rates, wavelengths, and temperature conditions to evaluate the stability and robustness of the method. The system allows the determination of the limit of detection (LOD) and limit of quantification (LOQ) based on the slope and standard deviation of the calibration curves. By providing consistent and reproducible chromatographic results, the invention offers a validated RP-HPLC system suitable for routine quality control and research applications with aloin-curcumin formulations. Detailed description

[0005] The system consists of a high-performance liquid chromatograph (HPLC) with a programmable solvent pump, manual injector, PDA detector, and a C18 analytical column (approx. 4.6 mm × 7.5 mm, particle size approx. 3.5 µm). The chromatographic platform includes a temperature-controlled column oven and software for data acquisition, peak purity analysis, and calibration management. Before measurement begins, the system is calibrated and purged to ensure a stable baseline and precise wavelength positioning.

[0006] The mobile phase is selected through iterative experiments with acetonitrile, methanol, and water mixtures. The instrument enables precise solvent dosing for determining peak shape, resolution, peak symmetry, theoretical plate number, and purity index. Optimized solvent ratios ensure clear separation of aloin and curcumin without interference. The instrument allows flow rate adjustments between 0.4 and 1.2 ml / min for robustness testing.

[0007] The system supports the preparation of standard solutions through controlled dilution steps. A stock solution is prepared by dissolving an aloin-curcumin mixture in a solvent, achieving a concentration of 1000 µg / ml. Further dilution to 100 µg / ml and subsequent dilution series allow the preparation of calibration standards in the linear range of 500–4000 ng / ml. These standard preparations are injected into the system to generate calibration curves.

[0008] During chromatographic operation, the system maintains a flow rate of 1 ml / min, with detection wavelengths of 254 nm for aloin and 425 nm for curcumin. The instrument provides measurable retention times of approximately 2.3 minutes for aloin and 4.5 minutes for curcumin. Peak parameters such as area, height, tailing factor, and theoretical plate number are continuously monitored.

[0009] The system suitability module assesses whether the device meets predefined performance criteria. Multiple injections are analyzed to determine the relative standard deviation (%RSD), resolution, tailing factor, and retention time precision. The system also enables specificity testing through injections of blanks, standards, and samples to ensure the absence of interfering peaks.

[0010] In the validation phase of the system, linearity is investigated using calibration curves with high correlation coefficients for both analytes. Accuracy is confirmed by recoveries at 80%, 100%, and 120%. Precision is validated through studies of variability within a single day, between different days, and over a 100-day period, with relative standard deviations (%RSD) within acceptable limits in each case.

[0011] Robustness is evaluated by varying the flow rate, temperature, and wavelength, demonstrating that minor changes do not compromise chromatographic integrity. Robustness is further ensured by assessing method performance under different users and conditions. The limit of detection (LOD) and limit of quantification (LOQ) are calculated using system-generated statistical parameters based on the slope and standard deviation of the calibration curve.

[0012] Overall, the system functions as a complete analytical environment for the development, optimization, and validation of a reversed-phase HPLC method for the simultaneous determination of aloin and curcumin. Its structured modules ensure compliance with regulatory requirements and reproducible results suitable for industrial and research laboratories.

Claims

[1] A chromatographic system for the simultaneous RP-HPLC quantification of aloin and curcumin, consisting of a C18 column, a PDA detector, a programmable solvent feed module and an optimized mobile phase of acetonitrile, methanol and water. [2] System according to claim 1, configured for the production and analysis of calibration standards in the concentration range of 500-4000 ng / ml for determining the linearity of the method. [3] System according to claim 1, designed to support validation parameters according to ICH and FDA guidelines, including specificity, accuracy, precision, robustness and stability. [4] System according to claim 1, which can be operated under varying flux rates, wavelengths and temperatures for robustness testing and the determination of the limit of detection (LOD) and limit of quantification (LOQ).