Nakov Natalija, Petkovska Rumenka, Ugrinova Liljana, Kavrakovski Zoran, Dimitrovska Aneta, Svinarov Dobrin
Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jun 15;992:67-75. doi: 10.1016/j.jchromb.2015.04.029. Epub 2015 Apr 25.
Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (Ibu) in human plasma without a need of derivatization or other complexities such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1% (v/v) acetic acid in mixture of methanol and water (90:10%, v/v) as a mobile phase. Two types of extraction procedure for Ibu and Ketoprofen (internal standard, IS) were optimized using Full factorial 3(2) design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50μL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1-50mg/L with a lower limit of detection of 0.02mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400mg rac-Ibu.
开发并验证了一种高效液相色谱-串联质谱法（HPLC-MS/MS），用于直接测定人血浆中的R-和S-布洛芬（Ibu），无需衍生化或其他复杂操作，如柱后注入溶剂或试剂。在方法开发过程中，使用实验设计研究了关键步骤，以实现可靠且耐用的分析方法。在Lux纤维素手性柱上，以甲醇和水（90:10%，v/v）的混合物中含0.1%（v/v）乙酸作为流动相，实现了R-Ibu和S-Ibu的LC-MS/MS分离。使用全因子3(2)设计（液液萃取，LLE）和D-最优实验设计（固相萃取，SPE）优化了Ibu和酮洛芬（内标，IS）的两种萃取程序。使用50μL血浆时，LLE和SPE的回收率分别达到了出色的80%（平均值）和95%（平均值）。评估了两种萃取程序的基质效应，包括高脂血症和溶血血浆。对基质效应的广泛研究表明，LLE产生的提取物比SPE更纯净。R-Ibu和S-Ibu体外相互转化的研究结果表明，在pH、温度和整个分析过程的影响下不会发生相互转化。符合欧洲药品管理局（EMA）生物分析方法验证指南的验证数据表明，所提出的方法在0.1-50mg/L范围内提供准确且可重复的结果，检测下限为0.02mg/L。通过测定口服400mg消旋Ibu后人血浆中的R-Ibu和S-Ibu，证明了该方法的适用性。
