請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47525
標題: | 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31種濫用藥物 Determination of 31 Illicit Drugs in Oral Fluid Using Ultra-high Performance Liquid Chromatography/Tandem Mass Spectrometry with Isotope-Dilution Techniques |
作者: | Han-Hsuan Tsai 蔡函烜 |
指導教授: | 陳家揚 |
關鍵字: | 極致液相層析/質譜/質譜儀,同位素稀釋技術,電灑游離,基質效應,大氣壓光游離,濫用藥物, UHPLC/MS/MS,isotope-dilution techniques,electrospray ionization,matrix effect,atmospheric pressure photoionization,drug of abuse, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 相對於尿液或血液,採集唾液檢體以篩檢濫用藥物相對快速簡便且不具侵入性,不需專業技能即可操作,採樣時亦可在現場監控,因此也減少檢體被摻假、掉包的情形。唾液中的藥物為由血液分佈而來,亦較能反應採樣當時體內血液之藥物濃度。上述優點使唾液作為濫用藥物篩檢的實用性逐漸受到重視。
液相層析/質譜/質譜儀不需化學衍生即可游離極性且低揮發性的化合物,使其在藥物檢測上受到注目。極致液相層析(ultra-high performance liquid chromatography,UHPLC)技術的出現,更大幅縮短了液相層析時間且提升了分析靈敏度。本研究開發並驗證以電灑游離(ESI)及大氣壓光游離(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精確定量唾液中七種鴉片類與代謝物、十二種安非他命類、五種氟硝西泮/愷他命和其代謝物、五種古柯與代謝物及兩種5-羥色胺類迷幻藥等共31種化合物之檢驗方法。 本研究以兩倍體積去離子蒸餾水(Milli-Q water)稀釋120 μL唾液之後,添加穩定同位素標定內對照標準品,並以14,800 rpm(16,162 ×g)高速離心20分鐘,沉澱蛋白質等大分子物質,離心後即上機分析,大幅減少一般進行固相萃取耗費的人力、時間與耗材。本研究的前處理方式之基質於ESI+對大多數分析物的離子抑制效應約在-28%至78%之間,於APPI+則約在40%至100%之間,而前處理回收率約為83%至108%。 本研究之方法於ESI+之最低可檢濃度範圍約為0.003–0.39 ng/mL;方法最低可定量濃度範圍為0.011–1.29 ng/mL。實驗亦利用ESI+測試三種不同添加濃度之定量準確度與精密性,其測試濃度範圍從0.6 ng/mL至540 ng/mL;定量誤差範圍大多<15%,相對標準偏差(RSD)則大多低於14.7%。儀器穩定性方面,利用ESI+同日檢測的RSD約在0.6–13.9%之間;異日檢測的RSD則約在0.6–14.0%之間。 本研究另外開發以micro-elution plates(Oasis MCX)進行極低沖提體積的固相萃取方法。經過micro-elution plates處理後分析物之基質效應在ESI上皆小於10%,大多數分析物在APPI上之基質效應也低於20%;micro-elution plates處理之回收率則約為59%到86%。所有分析物在ESI上之最低可檢濃度皆低於0.91 ng/mL(最低可定量濃度限皆小於 3.02 ng/mL),在APPI上分析物之最低可檢濃度則介於0.009–1.29 ng/mL之間(最低可定量濃度介於0.032 ng/mL至4.31 ng/mL)。本方法利用ESI之定量準確度與精密度方面,大多數分析物之誤差小於25%,相對標準偏差則多小於16%。 本研究採用Ascentis Express RP-amide管柱進行部分實驗,其內部填充的靜相為利用熔融核(fused-core)技術,其實心顆粒之外層包覆有孔殼層(porous-shell)的吸附劑。本研究發現在使用該管柱分析時可達與一般粒徑小於2 μm(sub-2 μm)之UHPLC管柱相同的層析時間(9分鐘)與優異的分離效能,且在相同層析條件下管柱背壓不到sub-2 μm UHPLC管柱的一半(僅4,000 psi),因此相當適合在傳統HPLC系統下操作,即能達到UHPLC之效能。 本研究亦測試了某廠商販售宣稱與一般唾液具有相同性質之人工唾液,觀察其與真實唾液是否具有相同的基質效應或回收率,評估其作為替代真實唾液進行研究的合適性。與真實空白唾液樣本相比,人工唾液之黏滯性較低,加上其對分析物無論在ESI或是APPI游離源造成之基質效應皆比真實唾液明顯許多,因此可能並不適合作為真實空白唾液之替代品進行研究。 本研究將唾液樣本前處理方式簡化至稀釋、離心而後過濾,縮短了以往極為耗時的前處理工作。另一方法為使用micro-elution plates可同時萃取96個唾液樣本,相較於傳統固相萃取管(SPE cartridges)亦節省了萃取後揮發與回溶的時間,大幅提升樣本前處理速度的同時亦可有效降低基質效應。同位素稀釋技術的運用則可大幅降低在前處理過程中分析變異以及分析物訊號受基質不同程度影響造成的定量不確定性。本研究利用極致液相層析只需9分鐘即可完成31項分析物的定性與定量(包含管柱再平衡時間),不但大幅縮短儀器分析所需時間,同時也因層析波峰變窄而改善訊雜比,大幅提升檢測的靈敏度(多數分析物於UHPLC管柱之靈敏度較於使用HPLC管柱時高二至五倍),因此非常適合需於短時間內分析數量龐大且藥物濃度低的唾液檢體,以監測藥物濫用的趨勢或用於檢測目標族群之使用情形。 Collection of oral fluid is easy and non-invasive, and can be done under surveillance to reduce the chances of adulteration or exchange of the specimens. Concentrations of drugs in the oral fluid can usually be related to those in plasma, which represent the body burden at sampling. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is suitable for analyzing polar, non-volatile compounds and no needs to derivatize drugs comparing with gas chromatography-mass spectrometry (GC-MS). Besides, ultra-high performance liquid chromatography (UHPLC) provides better separation and much higher sample throughput than traditional HPLC. This study developed and validated a method using isotope-dilution UHPLC-MS/MS with selective reaction monitoring to analyze seven opiates and metabolites, twelve amphetamines-type stimulants (ATS), flunitrazepam/ketamine and their three metabolites, five cocaine and metabolites and two serotonin-based hallucinogens in the oral fluid. The sensitivity and matrix effects on positive electrospray ionization (ESI+) and atmospheric pressure photoionization (APPI+) were compared. One hundred and twenty micro-liter of oral fluid was diluted with two times of Milli-Q water then the mixture was spiked with isotope-labeled internal standards. The sample was centrifuged for 20 min at 14,800 rpm (16,162 ×g), and the supernatant was filtered before instrumental analysis. The ion suppression of most analytes at ESI+ and APPI+ ranged from -28% to 78% and 40% to 100%, respectively, and the recoveries of sample preparation most ranged from 83% to 108%. Limits of detection (LODs) and limits of quantification (LOQs) of the analytes at ESI+ in oral fluid were about 0.003–0.39 ng/mL and 0.011–1.29 ng/mL, respectively. The accuracy and the precision of the method were tested on ESI+ at three spiked levels, which the tested concentration ranged from 0.6 ng/mL to 540 ng/mL, and the bias (accuracy) and the relative standard deviation (precision, RSDs) were lower than 15% and 14.7%, respectively. The intra- and inter-day RSDs of analysis tested at ESI+ were found to be about 0.6% to 13.9% and 0.6% to 14.0%, respectively. This study also evaluated the performance of micro-elution plates of Oasis MCX on sample preparation, an adsorbent for solid-phase extraction (SPE) with ultra-low elution volumes. The ion suppression of oral fluid using the SPE was most below 10% at ESI+ and was smaller than 20% at APPI+ for most analytes. The recovery of this procedure ranged from 59% to 86% for most analytes. The LODs of the analytes using the SPE were all below 0.91 ng/mL at ESI+ (LOQs were smaller than 3.02 ng/mL) and ranged from 0.009 ng/mL to 1.29 ng/mL for most analytes at APPI+ (LOQs ranged from 0.032 ng/mL to 4.31 ng/mL). The bias percent and the RSDs were below 25% and 16% for most analytes at ESI+, respectively. This study investigated the separation of the 31 illicit drugs on the Ascentis Express RP-amide column. The RP-amide column is packed with fused-core particles coating with porous-shell stationary phase, which provides fast mass transfer and good separation efficiency that is comparable to a sub-2 μm UHPLC column. In addition, the backpressure of the RP-amide column was 4,000 psi (only half of that of a sub-2 μm column) at the same chromatographic conditions; therefore, it could be applied on conventional HPLC systems. This study investigated the difference of the matrix effects and the recoveries between OraFlx Negative (an artificial oral fluid) and oral fluid was investigated in this study to evaluate if OraFlx Negative is a suitable alternative to oral fluid for method development. The result showed that the viscosity of OraFlx Negative was much lower than that of oral fluid; besides, analytes spiked in OraFlx Negative were more susceptible to ion suppression than that in oral fluid either at ESI or APPI; therefore, OraFlx Negative may not be a suitable substitute to oral fluid for method development. This method simplified the sample pretreatment to dilution and centrifugation; besides, SPE by micro-elution plates was also time- and labor-saving compared with traditional SPE cartridges (by eliminating the steps of post-extraction evaporation and reconstitution) and could significantly reduce the matrix effects. The isotope-dilution technique decreased the quantitative inaccuracy resulting from the analytical variations during sample preparation and from matrix effects. The chromatographic time was shortened to only 9 min per run using UHPLC (including column re-equilibration), and the sensitivity was improved for two to five times for most analytes comparing with that using a HPLC column because of the narrower peak widths of analytes, which significantly improved the signal-to-noise ratio. Therefore, this method could handle a large number of oral fluid samples containing trace amount of illicit drugs. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47525 |
全文授權: | 有償授權 |
顯示於系所單位: | 環境衛生研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf 目前未授權公開取用 | 11.29 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。