Comparison of HPLC with pre-column derivatization and HPLC-FLD for the determination of 4 alkaloids in the Zhi-Zi-Hou-Pu decoction

OBJECT To develop HPLC with pre-column derivatization and HPLC-FLD method for determination of 4 alkaloids in zhi-zi-hou-pu decoction,and compare with each other.METHODS①HPLC with pre-column derivatization method:the separation was carried on a Lichrospher C_(18) column utilizing an isocratic elution with methanol and water in the ratio of 57:43(v/v).The flow rate was 1.0mL·min_(-1).the UV detection wavelength was 257nm.②HPLC-FLD method:The analysis was performed on a Lichrospher C_(18) column utilizing a mobile phase of water containing 10mM sodium hexanesulfonate adjusted to pH 2.5 with phosphoric acid and methanol in the ratio of 85:15(v/v).The flow rate was 1.0mL·min~(-1).The fluorescence detector:the excitation wavelength was 281nm;the emission wavelength was 302nm.RESULTS①HPLC with pre-column derivatization method:The calibration curve was linear over the range of 0.05μg·mL~(-1) for four alkaloids with r≥0.9994.The average recoveries ranged from 96.2%to 98.8%with RSDs≤3.4%.②HPLC-FLD method:The calibration curve was linear over the range of 0.025～1μg·mL~(-1) for four alkaloids with r≥0.9998.The average recoveries ranged from 100.0%to 101.4%with RSDs≤3.0%.CONCLUSION The two established method are accurate,specific and sensitive,and can be applied to analyze 4 alkaloids Zhi-Zi-Hou-Pu decoction.Compared to HPLC with pre-column derivatization method,HPLC-FLD method is more simple and sensitive.