Kunicki Paweł K, Grymm Maciej T, Pawiński Tomasz, Szulczyk Daniel, Waligóra Marcin, Kopeć Grzegorz
Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Warsaw, Poland.
Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Warsaw, Poland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 1;1252:124443. doi: 10.1016/j.jchromb.2024.124443. Epub 2024 Dec 29.
A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics - Shimadzu) with a manual injector (50 μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150 × 4.6 mm, 5 μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CHCN:HO:0.5 M KHPO:85 % HPO (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8 mL/min. Ethyl acetate (4 mL) was used for 10-min liquid-liquid extraction from 0.4 mL alkalized plasma sample. Detection was performed at λ = 245 nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4 min (RIO), 5.4 min (BOS), 8.9 min (MAC) and 7.8 min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5-1000 ng/mL for RIO, 10-2000 ng/mL for BOS and 20-2000 ng/mL for MAC, with r of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15 % (≤20 % for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3 h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.
肺动脉高压（PAH）治疗中相当大比例的无效治疗可能与治疗剂量不足或不依从有关。本研究的目的是开发一种简单的分析方法，适用于测定PAH患者血浆中选定药物：利奥西呱（RIO）、波生坦（BOS）和马昔腾坦（MAC）的含量。采用配有手动进样器（50 μL定量环）的等度高效液相色谱 - 紫外系统（Spectra Physics - 岛津）。色谱分析在室温下使用Suplecosil LC - CN柱（150×4.6 mm，5 μm）进行，该柱由Supelguard保护柱保护。分离使用流动相：CHCN:HO:0.5 M KHPO:85% HPO（172:324.2:3.7:0.1，v/v），流速为1.8 mL/min。用4 mL乙酸乙酯从0.4 mL碱化血浆样品中进行10分钟的液 - 液萃取。检测在λ = 245 nm处进行，该波长是在信号强度和基质干扰之间折中的选择。分析物的保留时间分别为：利奥西呱4.4分钟（RIO）、波生坦5.4分钟（BOS）、马昔腾坦8.9分钟（MAC），加洛帕米（内标，GAL）为7.8分钟。该方法在以下范围内呈线性并进行了校准：利奥西呱为5 - 1000 ng/mL，波生坦为10 - 2000 ng/mL，马昔腾坦为20 - 2000 ng/mL，利奥西呱的r为0.9991，波生坦为0.9983，马昔腾坦为0.9949。在给定范围内，该方法以所需的精密度和准确度确保了可靠的结果：≤15%（LLOQ时≤20%）。没有明显的残留效应。该方法已成功用于PAH治疗患者依从性的初步研究，能够监测利奥西呱、波生坦和马昔腾坦。在给药前（C0）和给药后3小时（C3）采集的样品中评估药物浓度。对于利奥西呱、波生坦和马昔腾坦，所开发的方法适用于C0和C3样品，如果使用可进行稳态药物测定。所提出的方法可推荐给配备基本高效液相色谱仪器的实验室，作为用于治疗药物监测（TDM）和依从性研究的有吸引力的分析工具。
