AbstractThe chiral separation of simendan enantiomers using capillary electrophoresis was studied with β‐cyclodextrin (β‐CD) as chiral selector. The influences of the concentration and pH of borate buffer solution, β‐CD concentration and methanol content in the background electrolyte were investigated. These factors were compared with those in an HPLC with β‐CD as chiral mobile phase additive (CMPA‐HPLC). The quantification properties of the developed CE method were examined. A baseline separation of simendan enantiomers was achieved in the background electrolyte of 20 mmol/L borate buffer (pH 11.0) containing 12 mmol/L β‐CD‐methanol (50:50 in volume ratio). The CE method is comparable with CMPA‐HPLC in chiral resolution, although the optimal pH in CE (11.0) is much higher than that (6.0) in CMPA‐HPLC. This chiral CE method is applicable to the quantitative ananlysis and enantiomeric excess value determination of l‐simendan. Copyright © 2004 John Wiley & Sons, Ltd.