液相層析飛行式質譜儀於功能性食品中多重藥物之篩檢分析

Screening Analysis of Multiple Drugs in Functional Foods by Liquid Chromatography Time-of-flight Mass Spectrometry

李欣樺(Hsin-Hua Li)

指導教授 ： 沈振峯

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2014年)

摘要

中草藥產品及保健食品中，常有為了提高產品功效而違法添加西藥成分。為了人們健康篩檢這些違法添加是必要的。對此，一般透過薄層層析(TLC)，紫外光分光光度計，氣相層析質譜儀(GC/MS)，高效液相層析儀（HPLC），液相層析串聯式質譜儀（LC/MS/MS）等方式進行篩檢。惟這些檢測方式都必須具備有多重標準品才能進行比對篩檢。本研究透過液相層析飛行式質譜儀(LC-TOF-MS)，在電灑法模式(ESI)下，針對萃取樣品進行全圖譜掃描(full scam)，並且在高、低離子源電壓條件下同時得到化合物精確質量及部分碎片離子資料。在無標準品情況下，嘗試以內建藥物清單資料庫進行多重藥物(72種)的快速篩檢分析，資料庫篩檢主要依據質量準確度。本實驗利用15種藥物製劑混合標準品作為篩檢測試樣品，在不同濃度下(1 μg/mL、2.5μg/mL、10μg/mL)進行篩檢分析，利用ChromaLynx XS數據庫軟體進行目標物篩檢比對並針對過程中所產生假陽性及假陰性之誤判情形進行探討。分析結果顯示干擾物之同位素峰、同分異構物、干擾物質量過於相近均可能造成假陽性的誤判，而質量誤差過大及濃度過低時易導致假陰性誤判情形。透過對主要數據庫篩檢參數適當調整-質量允差(25 mDa)、背景訊號界定值(20 counts)、及平滑化峰寬界定值(10 sec)，可以得到更準確的篩檢結果。在分析15種混合藥劑在不同濃度下1 μg/mL、2.5 μg/mL、10 μg/mL，初步篩檢正確率分別達75% (12 target/16 hits) ( 16 positive, 0 tentative, 57 negative)、75% (15 targets/20 hits) (20 positive, 0 tentative, 53 negative)及68% (15 targets/22 hits) (22 positive, 0 tentative, 51 negative)。透過進一步碎片離子比對後，在2.5 μg/mL及10 μg/mL情況下，可使正確率達100%，在1μg/mL下因15 targets僅檢出12 targets，即使經過比對後仍有3 targets未檢出(正確率為80%)。本實驗嘗試對市售7件中藥及3件健保食品樣品進行分析，初步可在3件樣品中篩檢出可能有caffeine、sildenafil及hydroxyacetildenafil的不當添加。

關鍵字

液相層析飛行式質譜儀 ； 目標物篩檢

並列摘要

In order to improve the efficacy the drug ingredients are often illegally added into the herbal and functional food products. It is necessary to screen the illegal adulterations to prevent the possible damage to human health. The screening of drug adulteration was usually made through the thin layer chromatography (TLC), UV spectrophotometry, gas chromatography mass spectrometry (GC/MS), high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS) screening. All the analytical methods are relied on numerous drug standards for comparing purposes. In this study, the liquid chromatography coupled with time-of-flight mass spectrometer (LC-TOF-MS) was applied for screening the multiple drug adulteration. In the absence of standards, the rapid screening analysis was perfomed based on a compound list database of multiple drugs (73 drugs, currently). After simple solvent extraction, the extracts were analyzed with the LC-ESI-TOF-MS operated under low and high source voltages simultaneously. Furthermore, the fragment ions spectra (obtained under high source voltage) of the positive hits were compared with available reference mass spectra from web mass spectra literary or literatures. The mixture of 15 pharmaceutical formulations was initially used as the test sample and it was diluted at three concentrations (1 μg / ml, 2.5 μg / ml, 10 μg / ml) during analyses. The target screening of the acquired LC-MS data was performed by using the ChromaLynx XS software, and ratios of correct identification, false positive and false negative hits were studied. It was found that the false positive hits were mainly due to the too closed exact masses of isotopic peak and structure isomers (same exact mass). Contrary, the reasons for the false negative results could be caused by unsatisfied error in exact mass measurement and too low concentration. To improve the rate of correct ID, the parameters of target screening method were optimized where the most critical ones are: the mass tolerance was set as 25 mDa, the background signal was set as 20 counts, and the smoothing the peak width defined value was 10 sec. By doing so, the rates of correct ID were 75% (12 target/16 hits) (16 positive, 0 tentative, 57 negative), 75% (15 targets/20 hits) (20 positive, 0 tentative, 53 negative) and 68% (15 targets/22 hits) (22 positive, 0 tentative, 51 negative) at 1 μg/mL, 2.5 μg/mL and 10 μg/mL, respectively. In addition, by comparing the detected fragment ions with the reference product ion spectra data, the rate of correct ID was improved to 100% at 2.5 μg/mL and 10 μg/mL. For sample prepared at 1μg /mL, 3 targets of the 15 items were not able to be clearly detected and rate of the correct ID was 80%. The developed method was applied for the screening of seven herbal products and three functional food samples. The adulterations of caffeine, sildenafil and hydroxyacetildenafil were tentatively identified in three screened samples

並列關鍵字

Liquid Chromatography Time-of-flight Mass Spectrometry ； Target Screening

參考文獻

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