利用LC-ESI-MS 與蛋白質沉澱法檢測人類血漿中之第二代抗精神疾病藥物

Analysis of secondary generation antipsychotics in human plasma by LC-ESI-MS with protein precipitating pretreatment.

蕭如君

指導教授 ： 謝有容

國立交通大學/理學院/理學院應用科技學程/碩士(2011年)

https://doi.org/10.6842/NCTU.2011.00778

摘要

Olanzapine、quetiapine 與aripiprazole 為新一代非典型抗精神疾病藥
物。已有許多文獻使用液-液萃取與固相萃取的方式萃取人類全血、血漿與
血清中的這些藥物，並以液相層析串聯質譜做檢測。這些方法通常需要繁
雜、昂貴且耗時的前處理步驟。在這個研究中，我們希望能以快速並簡單
的蛋白質沉澱方式來萃取人類血漿中的這些藥物並以液相層析串聯質譜定
量。
首先，我們探討 LC-ESI-MS 的參數，包含乾燥氣體流速、霧化壓力
以及毛細管的壓力與溫度。在上述最佳化的條件下使用20 mM 乙酸銨加
入0.15% 三乙胺，以乙酸調整至pH 4.0 與乙腈做為移動相以梯度沖提。
實驗中以mirtazapine 做為內標準品，olanzapine、quetiapine、aripiprazole
與mirtazapine 均以正離子模式偵測。偵測質荷比分別為313、384、448 與
266。Olanzapine 與quetiapine 範圍在2.5 至 100 ng/ml 與 100 至 800
ng/ml之間；aripiprazole 範圍在50 至350 ng/ml 之間有良好的線性關係，
相關係數為在0.9926 至 0.9965 之間。三分析物olanzapine、quetiapine 與
aripiprazole 之最低偵測極限分別為0.94、0.95 與1.35 ng/ml。
ii
在真實樣品的部分，我們先以62.5% 的甲醇萃取人類血漿中的
olanzapine、quetiapine 與aripiprazole，並以14000 rpm 離心 7 分鐘，取
出上清液，加入內標準品，以此作為萃取條件，有良好的回收率與準確性。
此方法相較於傳統樣品前處理方式有更簡便的操作步驟同時兼顧良好之準
確性與再現性。

關鍵字

液相層析串聯質譜 ； 蛋白質沉澱 ； 血漿

並列摘要

Olanzapine, quetiapine and aripiprazole were new generation atypical
antipsychotics. Several methods have been developed to determinate these drugs
in human blood, plasma, or serum with different analyzing methods. For LC/MS,
liquid-liquid extraction or solid-phase extraction pretreatments were usually
used before analysis. These pretreatment methods were complex, expensive, and
time consuming. In this study, a rapid and effective protein precipitating
procedure was used for extraction of antipsychotics in human plasma for LC/MS
analysis.
First, the parameters of MS including flow rate of dry gas, pressure of
nebulize gas, capillary voltage and temperature were optimized for each analytes
for best sensitivity. Mirtazapine was added as internal standard which has
similar structure construction with analyte olanzapine, quetiapine, and
aripiprazole. A three analytes and internal standard were observed under positive
mode with m/z 313, 384, 448 and 266 corresponding to related [M + H]+
adducts. By using acetonitrile and H2O containing 20 mM ammonium acetate
and 0.15% triethylamine as mobile phase, 3 analytes and spiked internal
standard were separated within 9 minutes. Organic solvents with different
polarity were compared for both extracting and interferences removing (most
iv
were serum albumins) efficiency. Methanol was chosen as extraction organic
solvent for best recovery and better resolution of HPLC separation. Then,
different extraction organic solvent volume was optimized for lowest sample
dilution, which affects the signal and sensitivity in MS. Under optimized
conditions, all analytes have good linear range from 2.5 to 100 ng/ml, 100 to
800 ng/ml, 50 to 350 ng/ml for olanzapine, quetiapine and aripiprazole
separately, which were suitable for actual clinical dosed concentrations. The
coefficients of determination (r2) were between 0.9926 to 0.9965 and the
estimated detection limits were 0.94, 0.95 and 1.35 ng/ml for olanzapine,
quetiapine and aripiprazole. The analytes performing good recoveries in
proposed method in real human serum sample. The method presented in this
study was related simple but existed good extraction efficiency for all three
analytes and removed most of interferences contained in human serum samples,
which providing a better way of sample pretreatment for LC/MS analysis.

並列關鍵字

LC-ESI-MS ； protein precipitate ； plasma

參考文獻

second ed.; Global View Publishing: 2002.

鄭州, 2005.

olanzapine in human blood by liquid chromatography-tandem mass

spectrometry" J. Chromatogr. B 2002, 767, 163-168.

"High performance liquid chromatographic methods for the determination