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

Abstract

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。

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.