Clinical Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium.
Clinical Department of Laboratory Medicine, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium.
Clin Chem Lab Med. 2020 Apr 28;58(5):769-779. doi: 10.1515/cclm-2019-1237.
Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.
本研究旨在建立一种简单、快速、准确的超高效液相色谱串联质谱（UPLC-MS/MS）法，用于血清中视黄醇和α-生育酚的测定。目前已发表的 LC-MS/MS 方法要么需要复杂的提取程序（液液或固相），要么不符合血清精密度的理想要求（变异系数[CV]分别<6.8%和 6.9%）。方法：样品制备包括乙醇和乙腈的简单蛋白沉淀。采用稳定同位素标记的内标（IS）和自制校准曲线进行定量。分析采用 Acquity I-class Xevo TQ XS LC-MS/MS 进行。采用反相梯度洗脱，色谱运行时间为 6.0 min。与电喷雾电离（ESI）相比，UniSpray（US）作为一种离子化技术进行了比较。分析验证包括基质效应、与国家标准与技术研究所（NIST）标准和英国国家外部质量评估服务（UK NEQAS）样品相比的回收率和准确度。结果：视黄醇的日内和日间 CV 均<4.9%，α-生育酚的 CV 均<1.7%，均符合精密度的理想要求。与 NIST 标准相比，两种化合物的偏倚均<3.1%。该方法在整个测试范围内呈线性。与 ESI 相比，US 的定量下限（LLOQ）对视黄醇（0.022 与 0.043 mg/L）和α-生育酚（0.22 与 0.87 mg/L）均较低。视黄醇的基质效应不显著（<15%）。然而，对于α-生育酚，ESI 的基质效应平均为 54.0%，而 US 则没有。结论：本研究建立了一种快速、准确、灵敏的 UPLC-MS/MS 法，用于人血清中视黄醇和α-生育酚的测定，采用一步法样品预处理。US 离子化消除了α-生育酚的基质效应。
