Institute of Laboratory Medicine, LMU University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany.
Molecules. 2024 Aug 12;29(16):3829. doi: 10.3390/molecules29163829.
Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.
质谱（MS）是一种广泛应用的分析技术，包括医学诊断、法医毒物学、食品和水分析。定量化合物的金标准涉及使用稳定同位素标记的内标（SIL-IS）。然而，当这些标准不可用时、价格过高或极难合成时，会采用替代的外部定量技术。我们在此提出一种新颖、方便且廉价的定量方法——柱后注入定量（PCI）。作为概念验证，我们使用液相色谱-串联质谱（LC-MS/MS）展示了用于全血中环孢素免疫抑制剂的 PCI 定量。验证结果符合欧洲药品管理局（EMA）生物分析方法验证指南的标准，达到了 15%以下的变异系数和相对偏差的不精密度和不准确度。使用来自接受环孢素治疗的免疫抑制患者的匿名和剩余全血样本进行方法比较（PCI 定量与传统内标（IS）定量）。两种方法均表现出很强的一致性，Pearson 相关系数 r = 0.9532。这种新颖的 PCI 定量技术（使用目标分析物本身）扩展了 MS 中的定量选择，提供了可靠的结果，特别是当内标不可用时或价格过高时。通过本研究，我们旨在证明我们的创新 PCI 技术具有克服定量实际问题的巨大潜力，并提供如何将 PCI 纳入现有或新的 LC-MS 方法的指导。此外，与替代 PCI 技术相比，该研究还展示了一种更方便的方法来校正基质效应。
