Amisulpride is a clinically effective antipsychotic drug. It has been recommended for therapeutic drug monitoring in Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology. An integrated multicolumn two-dimensional liquid chromatography system was constructed. Two reversed-phase columns, Supersil ODS2 (150 mm×4.6 mm, 5 μm) and SinoChrom ODS-BP (150 mm×4.6 mm, 5 μm), with different hydrophobicities were employed in the first and second separation dimensions, respectively. A strong cation-exchange short column, Supersil SCX (10 mm×4.6 mm, 5 μm) was used to trap samples after the first dimensional separation. A two-position six-port valve was applied to change the flow path for transferring the samples. An auxiliary pump was used to change the mobile phase between the first dimensional column and the trapping column. An intact method for analyzing amisulpride in serum was developed using an integrated multicolumn two-dimensional liquid chromatography system. Serum samples were pretreated only by protein precipitation and centrifugation. In the protein precipitation step, a mixture of perchloric acid (6%, v/v) and methanol was used as the precipitation reagent, whose volume was three times that of the serum sample. The use of this reagent helped eliminate the obvious solvent effects resulting from the large injection volume (300 μL). Then, the samples were vortexed for 2 min and centrifuged for 5 min at a velocity of 10000 r/min. The supernatant was injected into the system directly. Acetonitrile/phosphate buffer (25 mmol/L, pH 3.0; 20∶80, v/v) and acetonitrile/phosphate buffer (25 mmol/L, pH 7.0; 25∶75, v/v) were used as mobile phases for the first and second dimensions, respectively, at a flow rate of 1 mL/min. The solvent strength and pH of the first dimensional eluent were adjusted at the two-dimensional chromatographic interface. Phosphate buffer (25 mmol/L, pH 3.0) was supplied at a rate of 1 mL/min by the auxiliary pump for adjustment. The adjustment process allowed amisulpride to remain cationic, thus leading to improved transfer and trapping efficiencies in strong cation-exchange columns, in the heart-cutting mode. The trapping time was determined to be between 4 and 5 min by a confirmation experiment. The use of a short trapping column and the appropriate mobile phase conditions allowed us to complete the analysis within 12 min. The established method was validated in detail by investigating the linearity, limit of detection (LOD), limit of quantification (LOQ), and recovery. A good linear relationship was observed between 10 and 200 ng/mL (r=0.9998). The LOD and LOQ were 7.28 ng/mL and 24.27 ng/mL, respectively. The high sensitivity of the validated method met the requirements of the therapeutic reference range of the Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology. The recovery of amisulpride spiked in a serum sample at 50 and 100 ng/mL were stabilized between 73.7% and 76.8%, which revealed the good stability of the established method. As opposed to the complicated traditional analytical methods, our method based on the automated integrated system is cost-effective and low-maintenance, thus being appropriate for routine therapeutic drug monitoring in clinical research. Moreover, our method is highly promising as the system cost is much lower than that of the popular LC-MS, while the capacity is sufficient for the determination of drugs in serum.
快速准确的治疗药物监测对于临床上确保患者用药有效性及安全性至关重要,同时也能够确定患者用药依从性,制定个性化给药方案。该文以两支疏水性略有差异的反相分离柱Supersil ODS2和SinoChrom ODS-BP,及强阳离子交换捕集柱Supersil SCX构建了基于集成化的多柱二维液相色谱系统。通过二维色谱接口,以pH 3.0的磷酸缓冲液调整第一维分离后的洗脱液组成,降低有机相含量并维持pH,改善了中心切割模式下样品转移和捕集的效率。利用该多柱二维液相色谱系统发展了血清中氨磺必利的二维液相色谱检测方法,血清样品经过高氯酸和甲醇混合液沉淀蛋白质并离心后直接300 μL大体积进样,以乙腈/磷酸缓冲液(25 mmol/L, pH 3.0)(20/80, v/v)作为第一维分离流动相,磷酸缓冲液(25 mmol/L, pH 3.0)作为捕集过程的稀释流动相,乙腈/磷酸缓冲液(25 mmol/L, pH 7.0)(25/75, v/v)作为第二维分离流动相,12 min内即可完成分析。方法在10~200 ng/mL的范围内线性相关性良好(r=0.9998)。样品在50 ng/mL和100 ng/mL两个加标浓度下的回收率稳定,在73.7%~76.8%之间。方法的检出限为7.28 ng/mL,定量限为24.27 ng/mL,能够满足《神经精神药理学治疗药物检测共识指南》中推荐的药物监控范围要求。由于该系统日常使用及维护成本较低,且能够实现自动化分析,故该方法适合在临床上用于治疗药物监测研究。
Keywords: amisulpride; multi-column two-dimensional liquid chromatography; serum; therapeutic drug monitoring.