A method was developed for simultaneous determination of Clozapine, Quetiapine and Risperidone in human serum by fully automated online solid phase extraction (SPE)-high performance liquid chromatography. With Capcell MF Ph-1 column as SPE cartridge and Acclaim C-18 as analytical column, high selectivity could be achieved by this method according to the selective complementarity of the two columns. In the experiment, ACN-H2O was used as the SPE mobile phase with a flow rate of 1 mL/min and ACN-10 mmol/L NH4Ac was used as the analytical mobile phase with a flow rate of 1 mL/min. Serum samples were injected directly into the SPE column and the biological matrix was washed out with the loading solvent. By rotation of the switching valve, Clozapine, Quetiapine and Risperidone were eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase. The whole time of the online SPE purification and chromatographic separation of the analytes was 18 min. Calibration curve of Clozapine with good linearity (r = 0. 9996) was obtained in the range of 10-1800 mu g/L in human serum, and the recoveries at low, medium and high concentration levels were 118. 4%, 105. 0% and 105. 4%. Calibration curve of Quetiapine with good linearity (r = 0. 9997) was obtained in the range of 3. 6-640 mu g/L in human serum, and the recoveries at low, medium and high concentration levels were 112. 8%, 101. 1% and 101. 5%. Calibration curve of Risperidone with good linearity (r = 0. 9995) was obtained in the range of 0. 71-128 mu g/L in human serum, and the recoveries at low, medium and high concentration levels were 100.7%, 97. 2% and 98. 8%. In conclusion, the established automated online SPE-HPLC-UV method demonstrated good performance in terms of linearity, specificity, limits of quantification, and was successfully utilized to quantify Clozapine, Quetiapine and Risperidone in human serum.
