Enderle Yeliz, Witt Lukas, Wilkens Heinrike, Grünig Ekkehard, Haefeli Walter E, Burhenne Jürgen
Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University, Germany.
Department of Respiratory Medicine, Saarland University Hospital, Homburg, Saar, Germany.
J Pharm Biomed Anal. 2017 Sep 5;143:291-298. doi: 10.1016/j.jpba.2017.05.052. Epub 2017 Jun 10.
Combination treatment with endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors (PDE5I) improved efficacy of pulmonary arterial hypertension (PAH) therapy. However, drug-drug interactions, variable exposure, non-adherence can influence plasma levels. For these reasons, drug quantification may be advantageous particularly in patients with poor treatment responses. We developed, validated, and applied an assay for the simultaneous quantification of ambrisentan, bosentan, macitentan, sildenafil, and tadalafil as well as their main (and partly active) metabolites in human plasma. This method is based on LC-MS/MS separation for a rapid and sensitive quantification with stable isotopically labelled analogues as internal standards for each drug and metabolite. Sample preparation was carried out using a solid phase extraction protocol based on Oasis HLB material. The separation was achieved on a Kinetex C18 column and multiple reaction monitoring in negative ionization mode was used for sensitive detection. The calibrations were linear for all analytes with correlation coefficients >0.99 within the concentration range observed under a therapeutic PAH dosing scheme with lower limits of quantification between 0.34ng/mL (OH-ambrisentan) and 10ng/mL (despropyl-macitentan). Intra- and inter-day precision at LLOQ and QC levels ranged between 2.03% and 19.8%, and 0.65% and 14.0%, respectively. The sample turnover time was 12min. The applicability of this versatile LC/MS/MS assay was verified by the successful analysis of clinical routine samples of patients on PAH medication. This new method allows for the first time to assess trough drug and metabolite levels of the currently approved PDE5I and ERAs in PAH patients, thus enabling for measurement of samples in clinical routine.
内皮素受体拮抗剂（ERA）与磷酸二酯酶5抑制剂（PDE5I）联合治疗可提高肺动脉高压（PAH）的治疗效果。然而，药物相互作用、暴露量变化、不依从性会影响血浆水平。因此，药物定量可能具有优势，特别是对于治疗反应不佳的患者。我们开发、验证并应用了一种检测方法，可同时定量人血浆中的安立生坦、波生坦、马昔腾坦、西地那非和他达拉非及其主要（部分具有活性）代谢产物。该方法基于液相色谱-串联质谱（LC-MS/MS）分离，以稳定同位素标记类似物作为每种药物和代谢产物的内标，实现快速、灵敏的定量。采用基于Oasis HLB材料的固相萃取方案进行样品制备。在Kinetex C18柱上实现分离，并采用负离子模式下的多反应监测进行灵敏检测。在PAH治疗给药方案下观察到的浓度范围内，所有分析物的校准曲线均呈线性，相关系数>0.99，定量下限在0.34 ng/mL（OH-安立生坦）至10 ng/mL（去丙基马昔腾坦）之间。LLOQ和质控水平的日内和日间精密度分别在2.03%至19.8%和0.65%至14.0%之间。样品周转时间为12分钟。通过对PAH用药患者的临床常规样本进行成功分析，验证了这种通用LC/MS/MS检测方法的适用性。这种新方法首次能够评估PAH患者中目前已获批的PDE5I和ERA的谷值药物和代谢产物水平，从而能够在临床常规中对样本进行检测。
