全自动固相萃取与液质联用方法测定人体血清中可乐定血药浓度

目的: 为可乐定临床研究建立一种特异、灵敏、高效的固相萃取(SPE)联合LC-MS/MS定量分析方法。 方法: 使用200 μL血清,加入内标可待因和氨水溶液混匀,使用96 孔SPE自动萃取装置进行样品制备。HPLC色谱柱为Symmetry Shield RP18(2.1 mm×50 mm,3.5 μm);流动相为甲醇-10 mmol·L^-1乙酸铵(pH 5.3)(65∶ 35)。采用串联四级杆质谱在ESI正离子电离模式下,应用多反应监测(MRM)进行测定:可乐定m/z 230.1→213.1,内标m/z 300.3→215.2。分析时间2.0 min。 结果: 在0.10~100.0 ng·mL^-1的范围内本方法线性良好(r>0.99),最低定量限为0.10 ng·mL^-1,日内与日间精密度(RSD)≤11.5%,准确度(RE)≤±10.4%,平均萃取回收率为66.4%(n=15)。稳定性实验结果表明,可乐定在不同环境中均能够保持稳定。 结论: 本文建立了用于血清中可乐定浓度测定的LC-MS/MS方法。该方法高效、灵敏、特异,通过了全面的方法学考察,各项指标均符合要求,已经成功用于可乐定的临床研究。

Objective: To develop a specific,sensitive and efficient SPE-LC-MS/MS method for the determination of clonidine in human serum. Method: After being mixed with IS (codeine) and ammonia water,200 μL of serum was extracted using 96-well plate automatic SPE device.The HPLC separation was performed on a Symmetry Shield RP18 (2.1 mm×50 mm,3.5 μm) using methanol-ammonium acetate (pH 5.3) ( 65∶ 35) as mobile phase,and the isocratic flow rate was 0.2 mL·min^-1.Detection was performed on a tandem quadrapole mass spectrometer using positive electro-spray ionization.Multiple reactions monitoring (MRM) scan was applied to detect mass transitions of 230.1→213.1 for clonidine and 300.3→215.2 for the IS,respectively.Each analytical run was only 2.0 min. Results: The calibration curves were linear over the concentration range of 0.10-100.0 ng·mL^-1 (r>0.99) and the lower limit of quantification (LLOQ) was 0.10 ng·mL^-1.The intra-and inter-day precisions(RSD) were ≤ 11.5% and accuracy (RE) was ≤±10.4%.The average extraction recovery was 66.4% (n=15).The analyte proved to be stable under a variety of storage condition. Conclusion: A sensitive and specific LC-MS/MS method for the determination of clonidine in serum was developed.The method was fully validated with respect to specificity,accuracy,precision and stability.The results indicate that the present quantitative method is suitable to the analysis of clonidine in human serum.