Klencsár Balázs, Balcaen Lieve, Cuyckens Filip, Lynen Frédéric, Vanhaecke Frank
Ghent University, Department of Analytical Chemistry, Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
Janssen R&D, Pharmacokinetics, Dynamics & Metabolism, Turnhoutseweg 30, 2340 Beerse, Belgium.
Anal Chim Acta. 2017 Jun 29;974:43-53. doi: 10.1016/j.aca.2017.04.030. Epub 2017 Apr 26.
A novel quantification approach, compensating for the effect of gradient elution on the instrumental response during reversed phase high-performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry analysis, has been developed and validated. As a proof of concept, diclofenac and its related compounds, including its major metabolite i.e. 4'-hydroxy-diclofenac, have been quantitatively determined in human plasma matrix. An inductively coupled plasma - tandem mass spectrometer has been applied for the interference-free determination of Cl as ClH using H as a reaction gas in the collision/reaction cell. The effect of the eluent composition on the instrumental response for Cl has been thoroughly investigated for the most common organic solvents in reversed phase high-performance liquid chromatography, i.e. methanol and acetonitrile. A proper mathematical function describing the effect of the eluent composition on the sensitivity for Cl, monitored as ClH, permitted adequate correction for the otherwise detrimental effect of gradient elution for both solvents. Validation using synthetically degraded diclofenac samples spiked with its major metabolite, 4'-hydroxy-diclofenac, demonstrated appropriate accuracy (recovery for 4'-hydroxy-diclofenac between 95 and 105%) and >90% and >80% recovery for Cl using acetonitrile and methanol, respectively. When applied to spiked human plasma samples (the most important matrix in drug metabolism studies), a satisfactory accuracy (recovery of 92-98%) and precision (<4% RSD) were established for both 4'-hydroxy-diclofenac and diclofenac. The limit of quantification for Cl (as diclofenac) using the novel method was 0.05 mg L. This value can be significantly improved (to 0.002 mg L) via on-line sample pre-concentration using a trapping chromatographic column and a time-programmable 10 ports/2 positions micro valve.
一种新型定量方法已被开发并验证，该方法可补偿反相高效液相色谱 - 电感耦合等离子体 - 串联质谱分析过程中梯度洗脱对仪器响应的影响。作为概念验证，已在人血浆基质中对双氯芬酸及其相关化合物（包括其主要代谢物即4'-羟基双氯芬酸）进行了定量测定。电感耦合等离子体 - 串联质谱仪已用于在碰撞/反应池中以H作为反应气体对Cl进行无干扰测定，以ClH形式检测。对于反相高效液相色谱中最常见的有机溶剂即甲醇和乙腈，已全面研究了洗脱液组成对Cl仪器响应的影响。一个适当的数学函数描述了洗脱液组成对以ClH形式监测的Cl灵敏度的影响，从而能够对两种溶剂梯度洗脱的不利影响进行充分校正。使用掺入其主要代谢物4'-羟基双氯芬酸的合成降解双氯芬酸样品进行验证，结果表明具有适当的准确度（4'-羟基双氯芬酸的回收率在95%至105%之间），使用乙腈和甲醇时Cl的回收率分别>90%和>80%。当应用于加标的人血浆样品（药物代谢研究中最重要的基质）时，4'-羟基双氯芬酸和双氯芬酸均具有令人满意的准确度（回收率为92 - 98%）和精密度（<4%相对标准偏差）。使用该新方法测定Cl（以双氯芬酸计）的定量限为0.05 mg/L。通过使用捕集色谱柱和时间可编程的10端口/2位置微型阀进行在线样品预浓缩，该值可显著提高（至0.002 mg/L）。
