在这项研究中,使用不同的样品制备方法和对不同校准模型的研究,对具有质谱检测的超高效超临界流体色谱中基体效应测定和减少的方法进行了评估。五种样品制备方法,包括蛋白质沉淀,液-液萃取,支持的液体萃取,以及基于“结合和洗脱”和“干扰物去除”模式的固相萃取,优化了8种维生素E的基质效应和回收率,包括α-,β-,γ-,和δ-生育酚和生育三烯酚,从等离子体矩阵效应评估包括使用三种模型对外部和内部校准的使用和比较,即,没有变换和没有加权的最小二乘(1/x0),1/x2加权,并进行对数变换。具有对数变换的校准模型提供了最低的%误差和最佳拟合。此外,在根据校准曲线斜率的比较进行评估时,校准模型的类型不仅会显著影响数据的拟合,还会影响矩阵效应。的确,基于使用的校准模型,从校准斜率计算的基体效应对于α-生育酚为92%至-72%,对于δ-生育三烯酚为-77%至19%。因此,通过Matuszewski的后提取方法在六个浓度水平下计算基质效应至关重要。的确,对于所有优化的样品制备方法,观察到强烈的浓度依赖性,即使使用稳定的同位素标记的内标(SIL-IS)进行补偿。观察到单个浓度水平和化合物之间的显着差异,即使测试校准范围只覆盖一个数量级。在具有更宽校准范围的方法中,不适当使用校准斜率比较而不是提取后添加方法可能会导致基质效应的假阴性结果。在维生素E的选定例子中,当用于干扰物去除模式时,固相萃取受基体效应的影响最小,但支持的液体提取导致最高的回收率。我们证明了校准模型,使用SIL-IS,分析物浓度水平在基体效应中起着至关重要的作用。此外,对于具有相似的理化性质和接近的保留时间的化合物,基体效应可能存在显着差异。因此,在所有生物分析应用中,通常在一次分析运行中确定不同的分析物,除了样品制备方法外,还需要仔细选择数据处理,SIL-IS,和色谱。
The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both \"bind and elute\" and \"interferent removal\" modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to - 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski\'s post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.