Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.
Labor Berlin-Charité Vivantes GmbH, Department of Laboratory Medicine and Toxicology, Sylter Str. 2, 13353 Berlin, Germany.
Clin Chem Lab Med. 2020 Apr 28;58(5):709-718. doi: 10.1515/cclm-2019-1001.
Background Accurate assessment of kidney function is needed for a variety of clinical indications and for research. The measurement of the serum clearance of iohexol has emerged as a feasible method to reach this objective. We report the analytical validation and clinical application of a new high-performance liquid chromatography (HPLC) - tandem mass spectrometry (MS/MS) assay to quantify iohexol in human serum. Specificity was enhanced due to the use of method specific acceptance limits for relative ion (RI) intensities. Methods The internal standard ioversol was added to 50 μL serum prior to protein precipitation with methanol. Linear gradient elution was performed on a Waters Oasis® HLB column. Three transitions for both iohexol and ioversol were monitored allowing calculation of RIs. Measurements acquired during method validation were used as a training set to establish stricter acceptance criteria for RIs which were then tested retrospectively on clinical routine measurements (86 measurements) and on mathematically simulated interferences. Results The method was linear between 5.0 μg/mL (lower limit of quantification [LLOQ]) and 100.3 μg/mL iohexol. Intraday and interday imprecision were ≤2.6% and ≤3.2%, respectively. Bias was -1.6% to 1.5%. All validation criteria were met, including selectivity, recovery, extraction efficiency and matrix effects. Retrospectively acceptance limits for RIs could be narrowed to ±4 relative standard deviations of the corresponding RIs in the training set. The new limits resulted in an enhanced sensitivity for the simulated interferences. Conclusions Criteria for validation were met and the assay is now used in our clinical routine diagnostics and in research.
为了满足各种临床指征和研究的需要，需要准确评估肾功能。目前，已将血清碘海醇清除率的测量作为实现这一目标的可行方法。我们报告了一种新的高效液相色谱（HPLC）-串联质谱（MS/MS）分析方法，用于定量人血清中碘海醇，该方法具有分析验证和临床应用。由于使用了特定方法的相对离子（RI）强度接受标准，因此提高了特异性。
在 50 μL 血清中加入内标碘海醇，然后用甲醇沉淀蛋白质。在 Waters Oasis®HLB 柱上进行线性梯度洗脱。监测碘海醇和碘呋醇的三个跃迁，允许计算 RI。在方法验证期间获得的测量值用作训练集，以建立更严格的 RI 接受标准，然后对临床常规测量（86 次测量）和数学模拟干扰进行回顾性测试。
该方法在 5.0 μg/mL（定量下限 [LLOQ]）和 100.3 μg/mL 碘海醇之间呈线性。日内和日间精密度分别≤2.6%和≤3.2%。偏差为-1.6%至 1.5%。所有验证标准均符合要求，包括选择性、回收率、提取效率和基质效应。可以将 RI 的接受限制在训练集中相应 RI 的 4 个相对标准偏差内，从而回顾性地缩小 RI 的接受限制。新的限制提高了对模拟干扰的灵敏度。
满足了验证标准，该方法现已用于我们的临床常规诊断和研究中。
