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Microemulsion eluents have been found to have excellent potential uses in high-performance liquid chromatography (HPLC). Here, a novel, environmentally benign and simple method using concentration/flow-rate double-gradient elution using a microemulsion eluent was used to separate water- and fat-soluble vitamins simultaneously and rapidly. Preliminary screening experiments were performed to determine the optimum column type, surfactant concentration, co-surfactant to surfactant ratio, oil, mobile phase pH and microemulsion concentration. The resolution and analysis time were simultaneously optimized using concentration/flow-rate double-gradient elution. The optimized method simultaneously separated water- and fat-soluble vitamins using a Venusil ASB C8 column and a combination of isocratic and linear gradient elution modes using a microemulsion mobile phase (solvent A) consisting of 3.5% (w/w) sodium dodecyl sulfate, 10.5% (w/w) n -butanol, 0.8% (w/w) n -octanol and 85.2% (w/w) water and water (solvent B) at pH 2.50. The optimum detection wavelength was 283 nm. The method was validated and used to analyze a solid pharmaceutical sample. [ABSTRACT FROM AUTHOR]