負離子大氣壓離子化法質譜儀於藥物之分析

Analysis of Synthesized Drugs by Negative Ion Atmospheric Pressure Ionization Mass Spectrometry

沈振峰(Chen-Feng Shen)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.01797

摘要

本研究主要是針對一些不同類型的藥物，在負離子大氣壓下化學離子化法(atmospheric pressure chemical ionization, APCI)或電灑法(electrospray ionization, ESI)中透過衍生化反應或是加成離子生成的方式改進其離子化效率，以提高其訊號反應，並將所開發的技術應用於人類血漿中藥物的分析。

我們以pentafluorobenzylbromide (PFB)為衍生試劑，針對帶有酸性質子的氯酚及苯氧乙酸進行PFB-衍生化，證實PFB-衍生物可在負離子APCI下進行類似傳統低壓下負離子化學離子化法的解離式電子捕捉反應生成衍生物[M-PFB]-的離子，並接著將此PFB-衍生物應用到三個含酸性質子的藥物分子，其在負離子APCI下均獲得明顯的靈敏度提昇(10-25倍)，同時其偵測極限約在1-4.5 pg左右(SIM模式)。

中性的羰基(carbonyl)化合物通常在大氣壓下離子化法上表現出較差的靈敏度。我們以pentafluorophenyl hydrazine (PFPH) 對所選的兩個含有羰基的中性藥物，nabumetone 及 testosterone進行衍生化生成其PFPH-衍生物(hydrazones)，此PFPH-衍生物在負、正離子APCI下可進行電子捕捉及質子化反應生成訊號不錯之[M-HF]-離子及[M+H]+ 離子。在進行碰撞引致裂解時，PFPH-衍生物的[MH]+離子生成相關於分析物部分結構的裂解離子，而PFPH-衍生物的[M-HF]-離子生成相關於衍生劑部分結構的裂解離子。和未衍生分析物相比，此PFPH-衍生物在正、負離子APCI均可獲得顯著的靈敏度提昇(nabumetone:2500倍及testosterone : 36倍)。以PFPH-衍生化法分析血中nabumetone和testoserone，無論在正、負離子APCI下，其靈敏度皆可清楚偵測血中0.1 ng/ml之nabumetone及女性空白血漿中內生性testosterone訊號。同時在0.1-25 ng/ml的範圍均可得良好的線性關係。

我們針對一些含氫氧基的中性藥物以氟、氯、溴三種鹵素陰離子加成的方式來提高分析物在負離子ESI下的靈敏度。其中溴、氯離子和藥物雖可生成清楚的[M+Br]-及[M+Cl]-加成離子，但在CID下卻生成不具特異性的Br-及Cl-子代離子，相對來說，氟離子加成法不僅可生成訊號不錯的[M+F]-加成離子，亦可生成高靈敏度且具特異性的子代離子([M-H]-及其裂解離子)。我們以氟離子加成法分析於血漿中藥物，方法偵測極限可達0.025-0.05 ng/ml，同時在0.05-50 ng/ml的範圍內亦有不錯的線性關係。

最後我們嘗試在商業化ESI探針的前端直接加以修飾，透過掛勾形式之放電針端，直接引用ESI之高電壓在離子源中將APCI引入ESI介面之中，以同時進行ESI/APCI反應增加介面對不同分析物的偵測能力。此外，我們亦嘗試藉套件對的ESI抑制，透過套筒形式之放電針端，將ESI探針轉換為以進行APCI為主的探針，除可獲得較”純粹” APCI的質譜訊號之外，亦能改善生化基質對分析物的基質效應。

關鍵字

陰離子加成 ； 電子捕捉 ； 電灑法 ； 大氣壓下化學離子化法

並列摘要

Abstract

The ionization efficiency of several drugs have been improved by using chemical derivatization methods in negative ion atmospheric pressure chemical ionization (APCI) mode and anionic adduct ion formation methods in negative ion electrospray ionization (ESI) mode.

The analytes with acidic proton were derivatized with pentafluorobenzyl bromide ( PFB) for the electron capture capability under negative ion APCI conditions. The synthesized PFB-derivatives performed dissociative electron capture reaction and gave intense [M-181]- ions. The PFB-derivatization have been applied for three acidic drugs and the resulted [M-PFB]- ions expressed 10-25 fold higher sensitivity than the underivatized-drugs.

Two carbonyl compounds, nabumetone and testosterone, were derivatized with pentafluorophenyl hydrazine (PFPH) and analyzed by negative ion APCI. The PFPH-derivatives performed dissociative electron capture ionization and gave intense [M-HF]- ions in the mass spectra. In positive ion APCI, the PFPH-derivatives also performed efficient protonation and gave intense [M+H]+ ions in the mass spectra. In CID, the major product ions of the [M-20]- in negative ion APCI corresponded to the partial moiety of PFPH. On the contrary, the major product ions of [M+H]+ corresponded to the partial moiety of the analyte. In comparison with the detection limits of the underivatized analytes, the PFPH-derivatives expressed 2500-fold and 35-fold sensitivity enhancements for nabumetone and testosterone, respectively. By using the selected reaction monitoring (SRM) detection, low pg of nabumetone (1 pg) and testosterone (7 pg) could be detected in both ECAPCI and positive ion APCI. The PFPH-derivatives were applied to the analysis of nabumetone and testosterone in human plasma by both negative ion APCI and positive ion APCI. The PFPH-derivatives in both ECAPCI and positive ion APCI were found to be capable of detecting 0.1 ng/ml of nabumetone in spiked plasma. For testosterone, endogenous testosterone in female plasma was detected in both negative ion APCI and positive ion APCI.

For several neutral drugs, mephenesin, guaifenesin, simvastatin, podophyllotoxin and inositol, the analysis was accomplished by negative ion ESI-MS using adduct formation with three different halide ions. The fluoride, chloride and bromide adducts of the selected drugs exhibited intense signals in negative ion ESI. Under collision-induced dissociation, the major product ions of bromide and chloride adducts were the nonspecific bromide and chloride anions, respectively. In contrast, fluoride adducts produced strong [M-H]- ions as well as product ions with good intensity. Fluoride attachment LC/ESI-MS/MS was applied to the analysis of the selected neutral drugs in human plasma. Detection limits in the range of 0.025-0.05 ng/mL were achieved using 0.5 mL plasma. Good linearity was observed for each of the drugs examined in human plasma over the range of 0.05-50 ng/mL.

Finally, two types of corona discharge adaptor were attached on the ESI probe tip for modifying or switching the ionization modes. By using the metal hanger to add a platinum discharge needle on the ESI probe tip, both the ESI and APCI can be performed simultaneously. On the other hand, by changing the metal hanger into a square metal casing, the ESI could be reduced form the mixed ESI/APCI source. The metal casing- discharge interface expressed almost the same ionization properties with the standard APCI interface. Good resistance to the plasma extracts was also observed by using the metal casing- discharge interface.

並列關鍵字

ESI ； Electron capture ； anionic adduct formation ； APCI

參考文獻

22. Vasudevan, M.; Ravisankar, S.; Sathiyanarayanan, A.; Chandan, R. S. J. Pharm. Biomed. Anal. 2000, 24, 25-31.

7. Villa, J. S.; Cass, R. T.; Karr, D. E.; Adams, S. M.; Shaw, J. P.; Schmidt Jr, D. E. Rapid Connun. Mass Spectrom. 2004, 18, 1066-1072.

24. Tagawa, N.; Tamanaka, J.; Fujinami, A.; Kobayashi, Y.; Takano, T.; Fukata, S.; Kuma, K.; Tada, H.; Amino, N. Clinical. Chem. 2000, 46:4, 523-528.

4. Zhao, J. J.; Yang, A. Y.; Rogers, J. D. J. Mass Spectrom. 2002, 37, 421-433.

1. Lee, M. S.; Kerns, E. H. Mass Spectrom. Rev. 1999, 18, 187-279.