Abstract
Previously, we have shown that dimethyl carbonate (DMC) is an effective green mobile-phase modifier for reversed-phase LC separation of polyaromatic hydrocarbons, particularly larger multi-ring compounds such as coronene. Ubidecarenone (CoQ10) and vitamin E (tocopheryl acetate) are highly hydrophobic, with long nonpolar chains (a 10-isoprene chain (C50) and a C16 saturated isoprenoid tail, respectively) attached to a substituted ring system. Reversed-phase LC methods for such compounds often require a completely nonaqueous mobile phase based on an alcohol with a more nonpolar solvent such as tetrahydrofuran or hexane. Our objective is to separate such compounds quickly with baseline resolution using a green mobile phase. Using 85% DMC:10% ethanol:5% water with a standard HPLC C18 column, the retention factor for tocopheryl acetate was a factor of 12 less as compared to a 95% methanol:5% water mobile phase. Separation with baseline resolution of COQH2, the reduced form of CoQ10, and CoQ10 was possible in <2 min using a 50% DMC:50% ethanol mobile phase with a standard C18 column as compared to 8 min using 50% acetonitrile:50% ethanol. Using a 1.8-micron C18 UHPLC column, the analysis time with baseline resolution was reduced to less than a minute with a 91% DMC:6% ethanol:3% water mobile phase. Analytical quality-by-design software (Minitab 17.1.0) was used to optimize the separation conditions for tocopheryl, tocopheryl acetate, CoQH2, and CoQ10, a mixture commonly found in supplement preparations. The optimum experimental conditions were a mobile phase of 76% DMC:16% ethanol:8% water running at 0.58 mL/min, and the column at 40 °C. Baseline resolution of all four compounds was possible in <1.75 min using the same UHPLC column. Analytical figures of merit showed LOQ values from 0.6 to 3 mg/L with linearity from 5 to 150 mg/L. Recovery of spiked samples was consistently close to 100%, and RSD values ranged from 0.2 to 2%. Analyzed supplements were generally close to the label claim with the exception of tocopheryl acetate in one sample. The greenness score of our analytical method, as assessed by MoGAPI, was 78, significantly higher than that for the USP method of 62. The proposed method's better attributes across redness (analytical performance), greenness (safety/environmental impact), and blueness (practicality) indicate it could be a superior alternative to the USP method.