Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Anal Chim Acta. 2018 Jan 25;999:69-75. doi: 10.1016/j.aca.2017.10.016. Epub 2017 Nov 8.
Coated Blade Spray (CBS) is a technology that efficiently integrates sample preparation and direct coupling to mass spectrometry (MS) on a single device. In this article, we present CBS-tandem mass spectrometry (CBS-MS/MS) as a novel tool for the rapid and simultaneous determination of four commonly used immunosuppressive drugs (ISDs) in whole blood: tacrolimus (TAC) and cyclosporine-A (CycA), which are calcineurin inhibitors; and sirolimus (SIR) and everolimus (EVR), which are both mTOR (mechanistic target of rapamycin) inhibitors. Given that CBS extracts via free concentration, analytes that are largely bound to plasma proteins or red blood cells provide considerably lower extraction recovery rates. Therefore, we defy the solventless philosophy of SPME-based techniques, like CBS, by performing the analyte-enrichment step via direct immersion in a solvent-modified matrix. The assay was linear within the evaluated range of concentrations (between 1 and 100 ng/mL for EVR/SIR/TAC and 10-1000 ng/mL for CycA), and the limits of quantification were determined to be 10 ng/mL for CycA and 1 ng/mL for EVR/SIR/TAC. Good accuracy (87-119%) and linearity (r ≥ 0.99) were attained over the evaluated range for all ISDs. Interassay imprecision (CV) determined from incurred sample reanalysis was ≤10% for all ISDs. Our method was validated using Liquichek™ whole blood immunosuppressant quality control (QC) standards purchased from Bio-Rad. Concentrations determined by CBS-MS/MS were inside the range specified by Bio-Rad and within 15% of the expected mean value for all ISDs at all QC levels. Furthermore, the effect of different hematocrit levels (20, 45, and 70%) in the entire calibration range was carefully studied. No statistical differences (RSD ≤ 7%) in the calibration curve slopes of ISDs in blood were observed. CBS offers a simpler workflow than that of traditional methods; it eliminates the need for chromatographic separation and provides a clean extract that allows for long-term MS instrumental operation with minimal maintenance. Additionally, because CBS integrates all analytical steps into one device, it eliminates the risk of instrumental carry-over and can be used as a low-cost disposable device for sample preparation and analysis. Fully-automated sample preparation simplifies the method and allows for total analysis times as short as 3 min with turn-around times of less than 90 min.
涂层刀片喷雾（CBS）是一种将样品制备和直接与质谱（MS）耦合到单个设备中的高效技术。在本文中，我们将 CBS-串联质谱（CBS-MS/MS）作为一种新工具，用于快速同时测定全血中的四种常用免疫抑制剂（ISD）：他克莫司（TAC）和环孢素-A（CycA），它们是钙调神经磷酸酶抑制剂；以及西罗莫司（SIR）和依维莫司（EVR），它们都是 mTOR（雷帕霉素靶蛋白）抑制剂。由于 CBS 通过自由浓度提取，因此与血浆蛋白或红细胞大量结合的分析物提供的提取回收率要低得多。因此，我们通过直接浸入溶剂改性基质中来执行分析物富集步骤，从而违背了基于 SPME 的技术（如 CBS）的无溶剂理念。该测定法在评估浓度范围内（EVR/SIR/TAC 为 1 至 100ng/mL，CycA 为 10-1000ng/mL）呈线性，定量限确定为 10ng/mL CycA 和 1ng/mL EVR/SIR/TAC。对于所有 ISD，在评估范围内均获得了良好的准确性（87-119%）和线性（r≥0.99）。使用从 Bio-Rad 购买的 Liquichek™全血免疫抑制剂质量控制（QC）标准品对方法进行验证。通过 CBS-MS/MS 确定的浓度在 Bio-Rad 规定的范围内，并且在所有 QC 水平下，所有 ISD 的浓度均在预期平均值的 15%以内。此外，还仔细研究了整个校准范围内不同红细胞压积水平（20%、45%和 70%）的影响。在血液中，ISD 的校准曲线斜率没有统计学差异（RSD≤7%）。CBS 提供了比传统方法更简单的工作流程；它消除了色谱分离的需要，并提供了清洁的提取物，允许长时间进行 MS 仪器操作，而无需进行大量维护。此外，由于 CBS 将所有分析步骤集成到一个设备中，因此消除了仪器交叉污染的风险，并且可以作为用于样品制备和分析的低成本一次性设备使用。全自动样品制备简化了方法，总分析时间最短可达 3 分钟，周转时间不到 90 分钟。
