以極致液相層析/串聯式質譜儀及同位素稀釋技術分析唾液中之濫用藥物

Determination of Abused Drugs in Oral Fluid Using Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry with Isotope-Dilution Techniques

王怡婷(I-Ting Wang)

指導教授 ： 陳家揚

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.03054

摘要

使用唾液作為篩檢樣本，具有收集不具侵入性、較無侵犯隱私的優點；由於非醫護人員也能進行樣本收集，採樣時能就近進行監控，以免樣本被置換的情形。再者，唾液中之藥物及由血漿分布而來，較能反應採樣當時體內之濃度，因此使用唾液樣本在濫用藥物檢測上漸受重視。
本研究開發以電灑游離（ESI）、大氣壓化學游離法（APCI）以及光化學游離法（APPI）作為質譜儀的游離源，搭配極致液相層析與同位素稀釋技術定量唾液中四種鴉片類與代謝物、五種安非他命類、三種氟硝西泮和代謝物、五種古柯和代謝物等共17種化合物之檢驗方法。每個化合物之質譜儀參數皆分別最適化並偵測最佳之母離子（precursor ion）和兩個訊號最強的子離子（product ion），以達到四個鑑定點（identification point）的專一性要求，本研究亦發現新型的親水性作用層析管柱（hydrophilic interaction chromatography, HILIC）對17種代測藥物的滯留效果比HSS T3管柱之滯留效果差.
在樣品前處理方面，本研究將唾液樣品以兩倍體積去離子蒸餾水 (distilled and deionizationed water, DDW) 稀釋後，添加穩定同位素標定內標準品 (internal standard)，並以14,800 rpm ( 16,162 ×g )高速離心20分鐘，取上清液分析，此步驟大幅減縮一般使用固相萃取所消耗的人力、時間與耗材。本研究發現，唾液基質對大多數代測藥物的離子抑制 (ion suppression) 約在28%至78%之間，前處理回收率約為81–108%。另一方面，使用管柱後分流的方式未能有效降低唾液樣本基質效應，而使用大氣壓化學游離法以及光化學游離法亦對基質效應並無改善。
17種待測藥物分別在電灑游離、大氣壓化學游離、光化學游離情況下之定量極限分別為0.11−0.87 ng/mL, 0.02–0.74 ng/mL和0.02−0.43 ng/mL，雖然APCI 及APPI 並無法改善基質效應所造成的離子訊號抑制，但是本研究之17種待測藥物在此兩種游離源下較為靈敏，因此定量極限較ESI為低。
本研究成功利用同位素標定內標準品定量唾液中微量的待測藥物，相對標準偏差及誤差大多小於百分之15；同日與異日儀器穩定性評估，誤差與相對標準偏差亦小於15%。
本研究完成開發以液相層析/質譜/質譜儀分析唾液中重要濫用藥物及代謝物之方法，探討唾液基質對於不同游離方式之基質效應，並大幅簡化唾液樣本前處理方式。利用極致液相層析每個樣本上機時間只需7.5分鐘，大幅縮短檢測所需時間並改善靈敏度，適合於短時間內分析大量唾液檢體，提高藥物濫用防制之執行力。

關鍵字

液相層析/質譜/質譜儀 ； 同位素稀釋技術 ； 電灑游離 ； 大氣壓化學游離 ； 光化學游離 ； 基質效應 ； 濫用藥物

並列摘要

Collection of oral fluid for drug testing is non-invasive, easy, and can be done under surveillance or other difficult conditions in clinics and workplace. This study analyzed four opiates and metabolite, five amphetamines, flunitrazepam and its two metabolites, and five cocaine and metabolites in the oral fluid with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) at selected-reaction monitoring (SRM) with isotope-dilution techniques. The most intense product ions from the most abundant precursor ions were used for quantification, and the next abundant product ions were used for confirmation. A BEH-HILIC column could not retain the 17 analytes well than that in HSS-T3 column.
This study simplified the sample preparation of oral fluid. A 100-µL sample of oral fluid was diluted with two times of deionized and distilled water (DDW) then was spiked with isotope-labelled internal standards. The sample was centrifuged for 20 min at 14,800 rpm (16,162 ×g), and the supernatant was collected for analysis. The recovery of sample preparation ranged from 81% to108%. For positive electrospray ionization (ESI+), the ion suppression of most analytes ranged from 28% to 78%; a post-column flow split (1:5) did not reduce the matrix effect. For positive atmospheric pressure chemical ionization (APCI+) and atmospheric pressure photoionization (APPI+), the ion suppression of most analytes ranged from 45% to 89% and from 74% to 96%, respectively.
Limits of quantification on the 17 drugs in oral fluid on ESI, APCI and APPI were ranged from 0.11−0.87 ng/mL, 0.02−0.74 ng/mL and 0.02−0.43 ng/mL, respectively. The methods were more sensitive on APCI and APPI than on ESI.
Methods were validated using spiked oral fluid at three levels on the three different ionization probes. The error percentage (accuracy) and relative standard deviations (precision) of intra-day and inter-day quantifications were most smaller than 15%.
This study extensively investigated the matrix effects of oral fluid on three ionization probes. The sample preparation was much simplified and the chromatographic time was only 7.5 min per run, with a sensitivity reached ppt levels.

並列關鍵字

UPLC/MS/MS ； isotope-dilution techniques ； electrospray ionization ； atmospheric pressure chemical ionization ； atmospheric pressure photoionization ； matrix effect ； illicit drugs

參考文獻

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