Using of HPLC coupled with coulometric detector for the determination of biotin in pharmaceuticals.

The method for the determination of biotin by high performance liquid chromatography (HPLC) coupled with coulometric detector is presented here. Chromatographic and detection conditions were tested. A LiChrospher 60RP-select B column (250 mm x 4 mm; 5 microm) and the mobile phase containing 0.24 mol/L aqueous solution of acetic acid and acetonitrile in the ratio 85:15 (v/v) were found as the most suitable. The flow rate was 1 mL/min and the injected volume of the sample was 20 microL. The hydrodynamic voltammogram of biotin was measured and according to obtained data the detection parameters were set--channel I 600 mV, channel II 900 mV, sensitivity 1 microA. The developed method has been validated. The calibration curve is linear in the range 15-3600 ng/mL, correlation coefficient is 0.9998, limits of detection and quantification are 5 and 15 ng/mL, respectively. Recovery of the spiked samples was 98.67% with R.S.D. 0.255% on average. The developed method has been successfully applied for determination of biotin in pharmaceutical preparations.