An effective normal phase HPLC method was developed and validated for stereoselective determination of propiconazole enantiomers in water, soil and grape. The performances of five different chiral stationary phases were evaluated for the separation of propiconazole enantiomers. And the effects of organic alcohols in the mobile-phase and column temperature on the enantioseparation were also investigated. Baseline separation of propiconazole enantiomers was successfully obtained on an OD-H column with n-hexane–ethanol (95 : 5, v/v) as the mobile phase at a flow rate of 0.6 mL min−1 with UV detection at 220 nm and temperature at 25 °C. Based on this enantioseparation method, enantioselective analysis methods for this fungicide in water, soil and grape matrices were developed and validated. Parameters including linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ) were investigated. The mean recoveries from three matrices were satisfactory, ranging from 89.6% to 99.8%, with relative standard deviations in the range of 1.6–9.5% at three fortification levels. LOD for the four stereoisomers in water, soil and grape were less than 0.02 mg kg−1, whereas LOQ did not exceed 0.05 mg kg−1. The methodology was also successfully applied for the enantioselective analysis of real samples, confirming the application of this method.
