臺灣博碩士論文加值系統

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&;#33096;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.

摘 要.................................................................................................................. i
Abstract ............................................................................................................. iii
誌謝................................................................................................................. v
縮寫表................................................................................................................ vi
目錄.............................................................................................................. viii
圖目錄................................................................................................................ xi
表目錄.............................................................................................................. xiii
一、 緒論........................................................................................................... 1
二、 背景與理論介紹....................................................................................... 2
2.1 液相層析儀............................................................................................... 2
2.1.1 液相層析儀器裝置............................................................................. 2
2.1.1.1 流動相儲存槽與溶劑處理系統 .............................................. 3
2.1.1.2 幫浦系統 ................................................................................ 4
2.1.1.3 樣品注射系統 ........................................................................ 5
2.1.1.4 液相層析之分析管柱 ............................................................. 6
2.1.1.5 保護管柱 ................................................................................ 6
2.1.1.6 偵測器 ................................................................................... 7
2.2 液相層析串聯質譜儀............................................................................. 11
2.3 游離源..................................................................................................... 12
2.3.1 電灑游離法....................................................................................... 14
2.3.2 大氣壓化學游離法........................................................................... 15
2.3.3 大氣壓光學游離法........................................................................... 16
2.4 質量分析器............................................................................................. 17
2.4.1 四極柱質量分析器........................................................................... 17
2.4.2 離子阱............................................................................................... 18
ix
2.4.3 飛行時間分析器............................................................................... 20
2.5 液相層析串聯質譜儀之介面................................................................. 21
2.5.1 液相直接導入介面........................................................................... 22
2.5.2 傳送帶介面....................................................................................... 22
2.5.3 加熱濃縮線....................................................................................... 22
2.5.4 熱灑法............................................................................................... 23
2.5.5 粒子束介面....................................................................................... 23
2.5.6 電灑法界面....................................................................................... 24
2.5.7 離子灑法界面................................................................................... 26
2.6 精神分裂症............................................................................................. 27
2.7 第二代抗精神疾病藥物......................................................................... 27
2.8 分析物簡介與文獻回顧......................................................................... 29
2.8.1 Olanzapine ....................................................................................... 29
2.8.2 Quetiapine ........................................................................................ 30
2.8.3 Aripiprazole ..................................................................................... 31
三、 實驗......................................................................................................... 34
3.1 儀器裝置................................................................................................. 34
3.2 藥品、詴劑與樣品................................................................................. 34
3.3 實驗方法................................................................................................. 34
3.4 液相層析儀條件..................................................................................... 34
3.5 質譜參數................................................................................................. 35
3.6 標準品溶液之配製................................................................................. 36
3.7 萃取方法................................................................................................. 38
四、 結果與討論............................................................................................. 39
4.1 游離源最佳化......................................................................................... 39
x
4.1.1 乾燥氣體流量................................................................................... 40
4.1.2 霧化壓力之測詴............................................................................... 41
4.1.3 毛細管電壓....................................................................................... 42
4.1.4 毛細管溫度....................................................................................... 43
4.2 萃取條件測詴......................................................................................... 45
4.2.1 不同溶劑測詴................................................................................... 47
4.2.2 不同溶劑比例................................................................................... 49
4.2.3 不同離心時間測詴........................................................................... 51
4.2.4 定量校正曲線與再現性結果.......................................................... 52
4.3 同日與異日間之回收率與再現性......................................................... 55
4.4 不同實驗室差異..................................................................................... 58
五、 結論......................................................................................................... 60
六、 參考文獻................................................................................................. 62

(1) Douglas, A. S.; Holler, F. J.; Crouch, S. R., Principles of Instrumental
Analysis. 5 ed.; Harcourt Colleage Publisher: Belmont, 1997.
(2) Douglas, A. S.; Holler, F. J.; Crouch, S. R., Fundamentals of Analysis
Chemistry. eighth ed.; Thomson Publisher,: Arlington, 2004.
(3) Willoughby, R.; Sheehan, E.; Mitrovich, S., A Global View of LC/MS.
second ed.; Global View Publishing: 2002.
(4) 呂路線; 石玉中; 邱亞峰, 精神分裂癥的診斷與護理. 中原農民出版社:
鄭州, 2005.
(5) 瞿發林, 精神疾病現代藥物治療手冊. 1 ed.; 第二軍醫大學出版社:
2005.
(6) Berna, A.; Ackermann, B.; Ruterbories, K.; Glass, S., "Determination of
olanzapine in human blood by liquid chromatography-tandem mass
spectrometry" J. Chromatogr. B 2002, 767, 163-168.
(7) Shimokawa, Y.; Akiyama, H.; Kashiyama, E.; Koga, T.; Miyamoto, G.,
"High performance liquid chromatographic methods for the determination
of aripiprazole with ultraviolet detection in rat plasma and brain:
Application to the pharmacokinetic study" J. Chromatogr. B 2005, 821,
8-14.
(8) Kollroser, M.; Schober, C., "Direct-injection high performance liquid
chromatography ion trap mass spectrometry for the quantitative
determination of olanzapine, clozapine and N-desmethylclozapine in
human plasma" Rapid Commun. Mass Spectrom. 2002, 16, 1266-1272.
(9) Raggi, M. A.; Casamenti, G.; Mandrioli, R.; Izzo, G.; Kenndler, E.,
"Quantitation of olanzapine in tablets by HPLC, CZE, derivative
spectrometry and linear voltammetry" J. Pharm. Biomed. Anal. 2000, 23,
973-981.
(10) Davis, P. C.; Wong, J.; Gefvert, O., "Analysis and pharmacokinetics of
quetiapine and two metabolites in human plasma using reversed-phase
HPLC with ultraviolet and electrochemical detection" J. Pharm. Biomed.
Anal. 1999, 20, 271-282.
(11) Zhou, Z. L.; Li, X.; Li, K. Y.; Me, Z. H.; Cheng, Z. E.; Peng, W. X.; Wang,
F.; Zhu, R. H.; Li, H. D., "Simultaneous determination of clozapine,
olanzapine, risperidone and quetiapine in plasma by high-performance
liquid chromatography-electrospray ionization mass spectrometry" Journal
of Chromatography B,Analytical Technologies in the Biomedical and Life
Sciences 2004, 802, 257-262.
(12) Bellomarino, S. A.; Brown, A. J.; Conlan, X. A.; Barnett, N. W.,
"Preliminary evaluation of monolithic column high-performance liquid
63
chromatography with tris(2,2 '-bipyridyl)ruthenium(II) chemiluminescence
detection for the determination of quetiapine in human body fluids" Talanta
2009, 77, 1873-1876.
(13) Saar, E.; Gerostamoulos, D.; Drummer, O. H.; Beyer, J., "Identification and
quantification of 30 antipsychotics in blood using LC-MS/MS" J. Mass
Spectrom. 2010, 45, 915-925.
(14) Basavaiah, K.; Rajendraprasad, N.; Vinay, K. B., "Microtitrimetric
deterimination of a drug content of pharamaceuticals containing
olanzapine in-aqueous medium" CI&CEQ 2009, 15, 77-81.
(15) Hilaert, S.; Snoeck, L.; Van den Bossche, W., "Optimization and validation
of a capillary zone electrophoretic method for the simultaneous analysis of
four atypical antipsychotics" J. Chromatogr. 2004, 1033, 357-362.
(16) Musenga, A.; Saracino, M. A.; Spinelli, D.; Rizzato, E.; Boncompagni, G.;
Kenndler, E.; Raggi, M. A., "Analysis of the recent antipsychotic
aripiprazole in human plasma by capillary electrophoresis and
high-performance liquid chromatography with diode array detection" Anal.
Chim. Acta 2008, 612, 204-211.
(17) Hwang, P. L.; Wei, S. Y.; Yeh, H. H.; Ko, J. Y.; Chang, C. C.; Chen, S. H.,
"Simultaneous determination of aripiprazole and its active metabolite,
dehydroaripiprazole, in plasma by capillary electrophoresis combining
on-column field-amplified sample injection and application in
schizophrenia" Electrophoresis 2010, 31, 2778-2786.
(18) Saracino, M. A.; Mercolini, L.; Flotta, G.; Albers, L. J.; Merli, R.; Raggi, M.
A., "Simultaneous determination of fluvoxamine isomers and quetiapine in
human plasma by means of high-performance liquid chromatography" J.
Chromatogr. B 2006, 843, 227-233.
(19) Morgan, P. E.; Manwaring, V.; Flanagan, R. J., "HPLC of basic drugs using
non-aqueous ionic eluents: evaluation of a 3 mu m strong cation-exchange
material" Biomed. Chromatogr. 2010, 24, 318-323.
(20) Bao, J. Q.; Potts, B. D., "Quantitative determination of olanzapine in rat
brain tissue by high-performance liquid chromatography with
electrochemical detection" J. Chromatogr. B 2001, 752, 61-67.
(21) Chiu, J. A.; Franklin, R. B., "Analysis and pharmacokinetics of olanzapine
(LY170053) and two metabolites in rat plasma using reversed-phase HPLC
with electrochemical detection" J. Pharm. Biomed. Anal. 1996, 14,
609-615.
(22) Sabbioni, C.; Saracino, M. A.; Mandrioli, R.; Albers, L.; Boncompagni, G.;
Raggi, M. A., "Rapid analysis of olanzapine and desmethylolanzapine in
human plasma using high-performance liquid chromatography with
coulometric detection" Anal. Chim. Acta 2004, 516, 111-117.
(23) Raggi, M. A.; Mandrioli, R.; Sabbioni, C.; Ghedini, N.; Fanali, S.; Volterra,
V., "Determination of olanzapine and desmethylolanzapine in the plasma of
64
schizophrenic patients by means of an improved HPLC method with
amperometric detection" Chromatographia 2001, 54, 203-207.
(24) Berna, M.; Shugert, R.; Mullen, J., "Determination of olanzapine in human
plasma and serum by liquid chromatography tandem mass spectrometry" J.
Mass Spectrom. 1998, 33, 1003-1008.
(25) Bogusz, M. J.; Kruger, K. D.; Maier, R. D.; Erkwoh, R.; Tuchtenhagen, F.,
"Monitoring of olanzapine in serum by liquid chromatography-atmospheric
pressure chemical ionization mass spectrometry" J. Chromatogr. B 1999,
732, 257-269.
(26) Gervasini, G.; Vizcaino, S.; Herraiz, A. G.; Benitez, J.; Carrillo, J. A.,
"Applicability of an assay for routine monitoring of highly variable
concentrations of olanzapine based on HPLC with mass spectrometric
detection" Clin. Chem. 2003, 49, 2088-2091.
(27) Nirogi, R. V. S.; Kandikere, V. N.; Shukla, M.; Mudigonda, K.; Maurya, S.;
Boosi, R.; Yeffamilli, A., "Development and validation of a sensitive liquid
chromatography/electrospray tandem mass spectrometry assay for the
quantification of olanzapine in human plasma" J. Pharm. Biomed. Anal.
2006, 41, 935-942.
(28) Choong, E.; Rudaz, S.; Kottelat, A.; Guillarme, D.; Veuthey, J. L.; Eap, C.
B., "Therapeutic drug monitoring of seven psychotropic drugs and four
metabolites in human plasma by HPLC-MS" J. Pharm. Biomed. Anal. 2009,
50, 1000-1008.
(29) Kirchherr, H.; Kuhn-Velten, W. N., "Quantitative determination of
forty-eight antidepressants and antipsychotics in human serum by HPLC
tandem mass spectrometry: A multi-level, single-sample approach" J.
Chromatogr. B 2006, 843, 100-113.
(30) Barrett, B.; Holcapek, M.; Huclova, J.; Borek-Dohalsky, V.; Fejt, P.;
Memec, B.; Jelinek, I., "Validated HPLC-MS/MS method for determination
of quetiapine in human plasma" J. Pharm. Biomed. Anal. 2007, 44,
498-505.
(31) Zuo, X. C.; Wang, F.; Xu, P.; Zhu, R. H.; Li, H. D., "LC-ESI-MS for rapid
and sensitive determination of aripiprazole in human plasma"
Chromatographia 2006, 64, 387-391.
(32) Bogusz, M. J., "Liquid chromatography-mass spectrometry as a routine
method in forensic sciences: a proof of maturity" J. Chromatogr. B 2000,
748, 3-19.
(33) Limited, E. L. a. C., 金菩薩膜衣錠仿單.
(34) de la Torre, C. S.; Martinez, M. A.; Almarza, E., "Determination of several
psychiatric drugs in whole blood using capillary gas-liquid chromatography
with nitrogen phosphorus detection: comparison of two solid phase
extraction procedures" Forensic Sci. Int. 2005, 155, 193-204.
(35) Saracino, M. A.; Gandolfi, O.; Dall'Olio, R.; Albers, L.; Kenndler, E.;
65
Raggi, M. A., "Determination of Olanzapine in rat brain using liquid
chromatography with coulometric detection and a rapid solid-phase
extraction procedure" J. Chromatogr. 2006, 1122, 21-27.
(36) Zhang, G.; Terry, A. V.; Bartlett, M. G., "Simultaneous determination of
five antipsychotic drugs in rat plasma by high performance liquid
chromatography with ultraviolet detection" J. Chromatogr. B 2007, 856,
20-28.
(37) Limited, A. U., 思樂康膜衣錠仿單.
(38) Mandrioli, R.; Fanali, S.; Ferranti, A.; Raggi, M. A., "HPLC analysis of the
novel antipsychotic drug quetiapine in human plasma" J. Pharm. Biomed.
Anal. 2002, 30, 969-977.
(39) Hasselstrom, J.; Linnet, K., "Fully automated on-line quantification of
quetiapine in human serum by solid phase extraction and liquid
chromatography" J. Chromatogr. B 2003, 798, 9-16.
(40) Otsuka Pharmaceutical Co., L. T. I. F., 大塚安立復R 錠仿單.
(41) Lancelin, F.; Kayssa, D.; Tabaouti, K.; Linda, K.; Sophie, B. A.; Paubel, P.;
Piketty, M. L., "Development and validation of a high-performance liquid
chromatography method using diode array detection for the simultaneous
quantification of aripiprazole and dehydro-aripiprazole in human plasma" J.
Chromatogr. B 2008, 867, 15-19.