毛細管氣相層析及毛細管電泳用衍生試劑對trans,trans-muconic acid、碘化物及胺基藥物之分析應用

Application of Derivatization Reagents to Capillary Gas Chromatography and Capillary Electrophoresis Analysis of trans,trans-Muconic Acid, Iodide and Amino Drugs

林楓閔(Feng-Min Lin)

指導教授 ： 吳信隆

高雄醫學大學/藥學院/藥學研究所/博士(2004年)

摘要

微量分析為生物分析之基礎，生物分析常對複雜檢體，偵測其所含微量或超微量之成分。基本上使用的方法需兼具高解析度及高感度，現行microscale techniques具有高解析度之特性者，包括毛細管氣相層析、高壓液相層析及毛細管電泳等，但由此分離所得成分，直接對偵測器呈現高感度者並不多。因此利用衍試劑將待測物標示呈高感度之衍生物，成為微量分析可行方法之一；除了改善分析感度外，常亦能提升待測物與基質之分離效果，本研究主要結果如下：
1、利用本研究室前開發之ECD (electron-capture detector) 導向之氣相層析用衍生試劑2-(pentafluorophenoxy)ethyl 2-(piperidino)ethanesulfonate (PFPES)，對暴露苯 (benzene, C6H6) 之生物指標成分 (biomarker) trans,trans-muconic acid進行超微量分析。研究方法將指標成分trans,trans-muconic acid溶於氰甲烷中，碳酸鉀為鹼活化劑，18-crown-6為催化劑，於70℃下進行衍生反應，所得衍生物配合電子捕獲型氣相層析儀 (GC-ECD) 進行分析之定量下限為0.3 μM，偵測極限可達60 nM (S/N=3) (60 fmol, 注入體積1.0 μL)；本方法可以應用到抽煙者及高危險群工作者 (使用苯為分離溶媒之實驗室工作人員)，其尿液中trans,trans-muconic acid含量之監控。
2、利用氣相層析用衍生試劑PFPES進行海苔中微量碘離子 (iodide, I-) 之分析。本研究利用簡單之前處理步驟，抽提海苔中的碘離子，在甲苯及水的兩相反應中，碘離子由水層經相間轉移催化劑tetra-n-hexylammonium bromide (THAB) 轉移至甲苯層，於70℃下與甲苯層中之PFPES進行衍生反應，所得衍生物以GC-ECD進行分析之偵測極限可達2.7 nM (2.7 fmol, 注入體積1.0 μL)；此分析方法已成功的應用到海苔中碘離子及全碘質之含量偵測。
3、本論文另探討具有高呈色團 (chromophore) 之毛細管電泳用新衍生試劑ofloxacin acyl chloride (OAC)，以供具生物活性胺基化合物之衍生化分析。OAC之合成，是利用ofloxacin與thionyl chloride，在80℃下反應，加熱迴流2小時，再將反應液濃縮，所得殘渣直接製備OAC衍生試劑溶液，針對不具有呈色團之一級胺基 (RNH2) 化合物，如amantadine (amino drug)、tranexamic acid (amino carboxylic acid)、glycine 及 methionine (amino acids) 加以衍生；這些胺基待測物在常溫下可與OAC快速反應，所得衍生物之三級胺的結構，經質子化可呈陽離子銨鹽，利用簡單的毛細管區帶電泳 (CZE, capillary zone electrophoresis) 加以分析，使用紫外光檢出器 (UV detector) 在300 nm下偵測，偵測極限分別為1.0 μM (methionine)、2.0 μM (tranexamic acid及glycine) 及2.5 μM (amantadine) (S/N=3, 注射時間3秒)，此方法之精密度 (precision, RSD) 和準確度 (accuracy, RE) 在同日間或異日間分析皆分別小於4.5 %及3.9 %；此分析方法已應用到人體血漿中tranexamic acid之含量分析。
4、利用毛細管電泳用衍生試劑OAC，對gabapentin 及vigabatrin等抗癲癇類新型藥物建立製劑分析法。gabapentin 及vigabatrin為不具發色團之胺基化合物，gabapentin或vigabatrin在溫和的條件下可與OAC進行衍生反應，所得衍生物，可以CZE-UV (300 nm) 同時進行分析，偵測極限在5.0 μM (S/N=3, 注射時間3秒)，此分析方法已經可以成功應用到製劑中gabapentin及vigabatrin之含量分析，本分析方法在製劑分析之精密度 (precision, RSD) 和準確度 (accuracy, RE) 在同日間或異日間分析皆分別小於3.1 %及4.8 %。

關鍵字

衍生 ； 氣相層析 ； 毛細管電泳 ； muconic acid ； 碘化物 ； 胺基藥物

並列摘要

Trace or ultra-trace analysis is essential for bioanalysis. In biosamples, analytes usually coexist with various components from complicated matrices. Therefore, effective methods for the analysis of analytes at sub-trace levels have to be highly resolutive and highly sensitive. A number of methods with microscale techniques have the merit of high resolution such as capillary gas chromatography (GC), high-pressure liquid chromatography (HPLC) and capillary electrophoresis (CE). Unfortunately, a lot of analytes thus separated can not afford enough response for sensitive detection. Therefore, detection-oriented derivatization coupled with GC, LC or CE finds a wide-spread application. The main results of this work were summarized as follows:
1. Wu’s group has developed a highly responsive reagent for electron-capture detection (ECD), 2(pentafluorophenoxy)ethyl 2-(piperidino)ethanesulfonate (PFPES). PFPES is a strong electrophore and it was used for GC-ECD of trans,trans-muconic acid at ultratrace levels. trans,trans-Muconic acid is a biomarker of human exposed to carcinogenic benzene (C6H6) from certain environment. The method is based on the derivatization of trans,trans-muconic acid with PFPES using potassium carbonate and 18-crown-6 ether as reaction activators. The resulting pentafluorophenoxy derivative of trans,trans-muconic acid was analyzed by capillary GC with ECD. The lower quantitation limit of the method is attainable at 0.3 μM of trans,trans-muconic acid with a detection limit of about 60 nM (S/N=3) (60 fmol per 1.0 μL injection). Application of the method to the analysis of trans,trans-muconic acid in urine proved feasible.
2. Further, application of PFPES to the analysis of iodide anion (iodide, I-) in processed seaweed was performed. The method is based on the derivatization of aqueous iodide extracted from seaweed with PFPES in toluene using tetra-n-hexylammonium bromide (THAB) as a phase-transfer catalyst. The resulting pentafluorophenoxyethyl iodide is highly responsive to an ECD and was analyzed by GC-ECD, giving a low detection limit of about 2.7 nM (2.7 fmol per 1.0 μL injection). Interferences of common anions in the analysis of iodide were studied and proved to be minimal.
3. We found that ofloxacin acyl chloride (OAC) is a potential chromophoric reagent for labeling amino analytes for CE. OAC was prepared by reacting ofloxacin with thionyl chloride and not isolated from the reacted solution due to its high reactivity. The reacted solution was concentrated in a rotary evaporator. The resulting residue dissolved with acetonitrile as OAC solution for derivatization. OAC has tertiary amino function in structure and the derivatives from OAC reacting with amino analytes can be ionized by an acid treatment and analyzed by simple capillary zone electrophoresis (CZE). OAC was used to derivatize model analytes (without chromophore) of amantadine (amino drug), tranexamic acid (amino carboxylic acid), glycine and methionine (amino acids). The resulting derivatives were analyzed by CZE with UV detection (300 nm). The detection limits of the analytes were in the range of 1.0-2.5 μM (S/N=3, injection 3 s). The precision (relative standard deviation) and accuracy (relative error) of the method for intra- and inter-day analyses of the analytes were respectively below 4.5 % and 3.9 %. Application of the method to the analysis of tranexamic acid in plasma proved feasible.
4. Further application of OAC to the analysis of gabapentin and vigabatrin was studied. Gabapentin and vigabatrin are widely used as anticonvulsants. In structure, they are amino carboxylic compounds which lack practical chromophoric groups for detection by absorption spectrophotometry. Derivatization of gabapentin and vigabatrin with chromophoric OAC was performed and the OAC derivatives of analytes can be analyzed by simple CZE-UV (300 nm). The detection limit (S/N=3, injection 3 s) of the method for the gabapentin or vigabatrin was 5.0 μM. Application of the method to the analysis of gabapentin and vigabatrin in pharmaceutical preparations proved simple and feasible. The precision (relative standard deviation) and accuracy (relative error) of the method for intra- and inter-day analyses of both drugs in pharmaceuticals were below 3.1 % and 4.8 %, respectively.

並列關鍵字

gas chromatography ； iodide ； muconic acid ； capillary electrophoresis ； amino drugs ； derivatization

參考文獻

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