Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal; CIBIT/ICNAS - Coimbra Institute for Biomedical Imaging and Translational Research, University of Coimbra, Coimbra, Portugal.
Pharmaceutical Services, Coimbra Hospital and University Centre, Coimbra, Portugal.
J Pharm Biomed Anal. 2020 Mar 20;181:113109. doi: 10.1016/j.jpba.2020.113109. Epub 2020 Jan 14.
Direct oral anticoagulants (DOACs) have been commonly used for the treatment of venous thromboembolism and for the prevention of stroke in patients with atrial fibrillation. Despite not being initially recommended, monitoring DOACs plasma concentrations is now recognized as essential in emergency situations and in special populations. Moreover, the inter-individual variability found in real studies as well as the high reported non-adherence are corroborating the importance of determining the individual relationship between administered doses, plasma concentrations and pharmacological effects. Therefore, accurate but user-friendly bioanalytical techniques are required to monitor DOACs plasma concentrations in routine clinical practice and phase IV clinical trials. Herein, a fast and simple high performance liquid chromatography (HPLC) method coupled to diode array detection (DAD) was developed, validated and applied to quantify the four currently marketed DOACs (apixaban, edoxaban, dabigatran and rivaroxaban). Sample preparation was performed by solid phase extraction followed by evaporation and concentration of the analytes. Chromatographic separation was accomplished within 6 min on a reversed-phase column (octadecyl-silica packing material; 55 mm × 4 mm, 3 μm particle size), applying a mobile phase composed of an aqueous solution of formic acid (0.1 %, v/v) and acetonitrile, pumped with a gradient elution at 30 °C. The proposed method was linear (r ≥ 0.993) within the concentration ranges of 0.017-5.28 μg mL, 0.066-5.28 μg mL, 0.033-5.28 μg mL and 0.017-5.28 μg mL for apixaban, dabigatran, edoxaban and rivaroxaban, respectively, all of them including the expected range of therapeutic concentrations. Overall, intra- and inter-day trueness of quality control samples, including at the lower limit of quantification (LLOQ), varied between -12.98 to 5.79 %, while imprecision was lower than 16.43 %, supporting that the method is accurate and precise in accordance to international guidelines. Recovery and stability were also assessed and allowed the method to be applied in clinical practice, during therapeutic drug monitoring.
直接口服抗凝剂（DOACs）已广泛用于治疗静脉血栓栓塞症和预防心房颤动患者的中风。尽管最初不建议进行监测，但在紧急情况下和特殊人群中，监测 DOACs 血浆浓度现在被认为是必不可少的。此外，实际研究中发现的个体间变异性以及高报告的不依从性证实了确定给予剂量、血浆浓度和药理作用之间的个体关系的重要性。因此，需要准确但用户友好的生物分析技术来监测常规临床实践和 IV 期临床试验中的 DOACs 血浆浓度。在此，开发、验证并应用一种快速简便的高效液相色谱（HPLC）法结合二极管阵列检测（DAD）来定量四种目前市售的 DOACs（阿哌沙班、依度沙班、达比加群和利伐沙班）。样品制备通过固相萃取进行，然后蒸发和浓缩分析物。在反相柱（十八烷基硅烷填充材料；55mm×4mm，3μm粒径）上，在 30°C 下以梯度洗脱的方式，于 6 分钟内完成色谱分离，流动相由含 0.1%（v/v）甲酸的水溶液和乙腈组成。所提出的方法在阿哌沙班、达比加群、依度沙班和利伐沙班的浓度范围分别为 0.017-5.28μg mL、0.066-5.28μg mL、0.033-5.28μg mL 和 0.017-5.28μg mL 内呈线性（r≥0.993），均包括预期的治疗浓度范围。总的来说，包括定量下限（LLOQ）在内的质控样品的日内和日间准确度变化在-12.98%至 5.79%之间，而精密度低于 16.43%，表明该方法符合国际指南的准确性和精密度要求。还评估了回收率和稳定性，允许该方法在临床实践中用于治疗药物监测。
