Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 03, Hradec Králové, Czech Republic.
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 03, Hradec Králové, Czech Republic.
Talanta. 2024 Dec 1;280:126658. doi: 10.1016/j.talanta.2024.126658. Epub 2024 Aug 2.
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/x), with 1/x 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.
本研究评估了在超高效超临界流体色谱与质谱联用中，通过不同的样品前处理方法和考察不同的校正模型来确定和减少基质效应的方法。本研究采用了五种样品前处理方法，包括蛋白质沉淀、液-液萃取、支持液萃取以及基于“结合和洗脱”和“干扰物去除”模式的固相萃取，重点关注 8 种维生素 E 形式（包括α-、β-、γ-和δ-生育酚和生育三烯酚）从血浆中的基质效应和回收率。基质效应评估包括使用和比较外部和内部校正，使用三种模型，即无转换和无加权（1/x）、1/x 加权和对数转换的最小二乘。对数转换的校正模型提供了最低的%误差和最佳的拟合。此外，校准模型的类型不仅显著影响数据的拟合，而且在基于校准曲线斜率比较评估基质效应时也会影响基质效应。事实上，基于所使用的校准模型，α-生育酚的基质效应计算值从校准斜率的+92%到-72%不等，而δ-生育三烯酚的基质效应计算值则从-77%到+19%不等。因此，通过 Matuszewski 的萃取后添加方法在六个浓度水平上计算基质效应非常重要。事实上，即使使用稳定同位素标记的内标（SIL-IS）进行补偿，所有优化的样品前处理方法都表现出很强的浓度依赖性。即使在测试的校准范围内只涵盖一个数量级，也观察到了各个浓度水平和化合物之间的显著差异。在具有更宽校准范围的方法中，错误地使用校准斜率比较而不是萃取后添加方法可能会导致基质效应的假阴性结果。在所选的维生素 E 示例中，当以干扰物去除模式使用时，固相萃取受基质效应的影响最小，但支持液萃取的回收率最高。我们表明，校准模型、使用 SIL-IS 和分析物浓度水平在基质效应中起着至关重要的作用。此外，具有相似物理化学性质和接近保留时间的化合物的基质效应可能会有很大差异。因此，在所有生物分析应用中，通常在一个分析运行中同时测定不同的分析物，除了样品前处理、SIL-IS 和色谱外，还需要仔细选择数据处理。
