Galba Jaroslav, Michalicova Alena, Parrak Vojtech, Novak Michal, Kovac Andrej
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; Department of Pharmaceutical analysis and nuclear pharmacy, Faculty of Pharmacy of Comenius University, Odbojarov 10, 832 32 Bratislava, Slovak Republic.
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 84510 Bratislava, Slovak Republic; AXON Neuroscience SE, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovak Republic.
J Pharm Biomed Anal. 2016 Jan 5;117:85-90. doi: 10.1016/j.jpba.2015.08.026. Epub 2015 Aug 22.
We developed and validated a simple and sensitive ultra-high performance liquid chromatography (UHPLC) method for the analysis of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and kynurenine (Kyn) in rat plasma. Analytes were separated on Acquity UPLC HSS T3 column (2.1 mm×50 mm, 1.8 μm particle size) using a 4 min ammonium acetate (pH 5) gradient and detected by fluorescence and positive ESI mass spectrometry. Sample preparation involved dilution of plasma, deproteinization by trichloroacetic acid and centrifugation. The procedure was validated in compliance with the FDA guideline. The limits of quantification (LOQ) were 0.3 μM for Kyn and from 1.5 to 3 μM for Phe, Tyr, Trp. The method showed excellent linearity with regression coefficients higher than 0.99. The accuracy was within the range of 86-108%. The inter-day precision (n=5 days), expressed as % RSD, was in the range 1-13%. The benefit of using UHPLC is a short analysis period and thus, a very good sample throughput. Using this method, we analyzed plasma samples and detected significant changes of Kyn and Phe in transgenic rat model for tauopathies.
我们开发并验证了一种简单且灵敏的超高效液相色谱（UHPLC）方法，用于分析大鼠血浆中的苯丙氨酸（Phe）、酪氨酸（Tyr）、色氨酸（Trp）和犬尿氨酸（Kyn）。分析物在Acquity UPLC HSS T3柱（2.1 mm×50 mm，粒径1.8 μm）上分离，采用4分钟的醋酸铵（pH 5）梯度洗脱，并通过荧光和正离子电喷雾质谱检测。样品制备包括血浆稀释、用三氯乙酸脱蛋白和离心。该方法按照美国食品药品监督管理局（FDA）指南进行了验证。犬尿氨酸的定量限（LOQ）为0.3 μM，苯丙氨酸、酪氨酸、色氨酸的定量限为1.5至3 μM。该方法显示出良好的线性，回归系数高于0.99。准确度在86 - 108%范围内。日间精密度（n = 5天），以相对标准偏差（% RSD）表示，在1 - 13%范围内。使用UHPLC的好处是分析周期短，因此样品通量非常高。使用该方法，我们分析了血浆样本，并在tau蛋白病转基因大鼠模型中检测到犬尿氨酸和苯丙氨酸的显著变化。
