Validation of a method for determination of phospholipase A(2) and melittin in bee venom preparations by capillary electrophoresis

A method was validated for the determination of the 2 main components of be e venom, phospholipase A(2) and melittin, by capillary electrophesis (CE). Optimum resolution and selectivity were attained with a running electrolyte of 150 mM phosphoric acid, pH 1.8. The repeatability and day-to-day reprod ucibility of the migration times were better than 0.36 and 2.8%, respective ly. The repeatability and day-to-day reproducibility of the normalized peak areas were better than 1.3 and 2.6%, respectively. The response of the UV detector at 190 nm was linear over < 2 concentration decades, from 0.05 to 1.5 mg/mL, with correlation coefficients of 0.9994 for phospholipase A(2) a nd 0.9997 for melittin. The limits of detection and quantitation were 4.5 a nd 15 mu g/mL, respectively, for phospholipase A(2) and 1.6 and 6 mu g/mL, respectively, for melittin. The reproducibility of the measurements with 2 different CE instruments was satisfactory; the mean concentration and relat ive standard deviation (RSD) values for phospholipase A(2) and melittin wer e 14.4% (RSD, 1.3%) and 51.4% (RSD, 1.1%), respectively, with instrument I; the corresponding values with instrument II were 14.5% (RSD, 2.8%) and 52. 3% (RSD, 2.2%). The accuracy was estimated by comparison with a liquid chro matographic (LC) method. Differences between the CE and LC measurements wer e attributed to irreversible adsorption of the analytes on the LC column. T he recoveries of phospholipase A(2) and melittin with the CE method were 98 .8 and 101.7%, respectively.