透過極致效能合相層析和實驗設計建立具選擇性之尿液檢體中新興影響精神物質分析方法

Abstract

新興影響精神物質(New psychoactive substances, NPS)的出現，對於個人健康及公共衛生造成重大的威脅。目前濫用藥物檢驗實驗室主要利用液相層析串聯質譜儀來分析新興影響精神物質，但液相層析串聯質譜儀分離分析物的過程耗時，無法快速地分析大量檢體。且有越來越多新興影響精神物質的同分異構物出現在非法藥物市場，它們的結構相似、滯留時間接近以及幾乎相同的碎片離子，使得液相層析串聯質譜在區分新興影響精神物質及它們的同分異構物造成挑戰。極致效能合相層析(UltraPerformance Convergence Chromatography™, UPCC)的發展，對新興影響精神物質的分析提供了一個全新的選擇。合相層析相較於液相層析有許多優勢，擁有類似氣體的低黏滯性及高擴散性，能夠更快速且有效率的分離分析物，協助快速分析大量檢體。並且合相層析能夠藉由調整壓力和溫度來調節移動相溶劑化能力來獲得樣品分離的高解析度和選擇性，展現出能夠分離同分異構物的優勢，相當適合用於分析新興影響精神物質和其大量的同分異構物。除此之外，合相層析使用的移動相為二氧化碳，便宜、安全、無毒、不易燃及容易回收，且能夠降低有毒、具揮發性的有機溶劑的使用，大幅降低分析成本及環境傷害。 本研究以極致效能合相層析(UltraPerformance Convergence Chromatography™, UPCC)串聯質譜儀開發同時檢測尿液檢體中185項新興影響精神物質與常見傳統濫用藥物的分析方法，利用實驗設計 (design of experiment)方法協助層析分離條件系統性地最佳化，最終合相層析條件為: 固定相使用BEH管柱，移動相共溶劑MeOH/IPA (85/15, v/v)，管柱溫度為55℃，自動化背壓調節器設定為2040 psi，流速為1.7 ml/min，甲酸銨添加劑濃度為25 mM，在此條件下同分異構物JWH-019和JWH-122間的解析度能夠達到1.18。此合相層析方法經過方法確校，包含線性、精確度、準確度，確效結果顯示大部分化合物判定係數大於0.99，準確度介於80-120%，95%的化合物精確度之相對標準差值小於15%。完成確效後與現行實驗室常規液相層析串聯質譜法進行比較，結果顯示對於過去液相層析不易分離之同分異構物，合相層析能夠提供良好的分離效果。本研究亦將合相層析串聯質譜法運用於分析真實濫用藥物尿液檢體，結果顯示和實驗室常規液相層析串聯質譜法分析結果相符。我們預期極致效能合相層析未來能夠提供全新的快速、可信賴、具有選擇性且能和液相層析互補的濫用藥物分析平台。

The emergence of new psychoactive substances (NPS) poses significant threats to individual health and public health. Currently, analytical laboratories primarily utilize liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analyzing NPS. However, LC-MS/MS methods for separating analytes are time-consuming and cannot rapidly analyze a large number of samples. Moreover, the increasing presence of NPS isomers in the illicit drug market, which have similar structures, close retention times, and nearly identical fragment ions, poses challenges for LC-MS/MS in differentiating NPS and their isomers. The development of UltraPerformance Convergence Chromatography™ (UPCC) offers a new option for the analysis of NPS. Convergence chromatography, compared to liquid chromatography, has many advantages, including gas-like low viscosity and high diffusion capabilities, enabling faster and more efficient separation of analytes and facilitating the rapid analysis of a large number of samples. Additionally, convergence chromatography can achieve high resolution and selectivity in sample separation by adjusting the pressure and temperature to modulate the solvating power of the mobile phase, proving advantageous for separating isomers and particularly suitable for analyzing NPS and their numerous isomers. Moreover, the mobile phase used in convergence chromatography is carbon dioxide, which is inexpensive, safe, non-toxic, non-flammable, and easy to recover, significantly reducing the use of toxic, volatile organic solvents, thereby lowering analysis costs and environmental impact. This study develops a method using UPCC-MS/MS for simultaneous detection of 185 new psychoactive substances and common traditional illicit drugs in urine samples. The design of experiments (DOE) approach assists in systematically optimizing chromatographic conditions. The final conditions for convergence chromatography include using a BEH column, a mobile phase co-solvent of MeOH/IPA (85/15, v/v), a column temperature of 55°C, an automated back-pressure regulator (ABPR) set at 2040 psi, a flow rate of 1.7 ml/min, and an ammonium formate additive concentration of 25 mM, achieving a resolution of 1.18 between the isomers JWH-019 and JWH-122. Method validation, including linearity, precision, and accuracy, shows that most compounds have a coefficient of determination greater than 0.99, accuracy between 80-120%, and the relative standard deviation of precision for 95% of the compounds is less than 15%. Upon completion of validation, a comparison with the current routine liquid chromatography-tandem mass spectrometry indicates that convergence chromatography provides good separation for isomers previously difficult to separate by liquid chromatography. This study also applies convergence chromatography-tandem mass spectrometry to the analysis of real drug abuse urine samples, showing results consistent with the liquid chromatography-tandem mass spectrometry method. We anticipate that UltraPerformance Convergence Chromatography will offer a new, rapid, reliable, selective, and complementary analytical platform to the conventional liquid chromatography for drug abuse analysis in the future.