A Novel UPLC Method Development for Quantifying Active and Inactive Ingredients in a Commercial Energy Drink Product Using Chemometric Optimization Methodology

The content or matrix of commercial energy drinks, which are increasingly consumed, has been changed over time to enhance the products’ competitiveness and effectiveness. This necessitates the development of new analytical methods for quality control and routine analysis. In this regard, a novel ultra-performance liquid chromatography with photodiode array detection (UPLC-PDA) method was developed using a chemometric design and optimization methodology. The method is designed to effectively separate and quantify caffeine (CAF), the active ingredient, and potassium sorbate (PS), the inactive ingredient, in a commercial energy drink with short runtime and high resolution. A 3³-full factorial experimental design was employed, considering key factors such as column temperature (X₁), phosphate buffer percentage (X₂), and mobile phase flow rate (X₂). The chromatograms of the 27 samples containing CAF and PS in the design matrix were recorded using a Waters Acquity® BEH C₁₈ column (100 mm × 2.1 mm i.d., 1.7 µm) with the detection at 284 nm. The ICRF values were computed from the recorded chromatograms. A quadratic second-order model was established between the design matrix (independent variables) and ICRF (dependent variable). From the model, optimal experimental conditions were identified. The optimal chromatographic conditions were found to be a column temperature of 58.9 °C, a flow rate of 0.24 mL/min, and a phosphate buffer system percentage of 59.3% (v/v) + methanol of 40.7% (v/v) with 0.4 mL triethylamine (TEA)/L, achieving the best separation and shortest analysis time. Calibration curves for CAF and PS were established within the concentration ranges of 2.0–26.0 µg/mL and 4.0–28.0 µg/mL, respectively. Excellent linearity was observed, with high determination coefficients (r² = 0.9996 for CAF and r² = 0.9994 for PS). The limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.18 µg/mL and 0.59 µg/mL for CAF and 0.20 µg/mL and 0.65 µg/mL for PS, respectively. The elution times were 1.29 min for CAF and 2.81 min for PS, with a total analysis runtime of 4.0 min. The results revealed that this method could serve as a new, cost-effective alternative for the quality control and routine analysis of samples containing CAF and PS. The method was validated through analysis of independent test samples, standard addition samples, and intra-day and inter-day samples, for example yielding satisfactory accuracy (percent mean recovery; 100.7 for CAF and 100.5 for PS) and precision (RSD %; 1.48 for CAF and 2.02 for PS) in applying the newly developed UPLC procedure. The ICH Q2(R1) guideline was followed for method validation to ensure compliance with international standards. This newly developed UPLC method was successfully implemented to analyze the active and inactive ingredients in a commercial energy drink product with a short runtime and low reagent consumption. The results revealed that this method could serve as a new, cost-effective alternative for the quality control and routine analysis of samples containing CAF and PS.