液相層析螢光法對胺基酸相關化合物之藥物監測及疾病指標之分析

Drug Monitoring and Disease Biomarker Analysis of Amino Acid Related Compounds by Fluorescent Liquid Chromatographic Method

謝俊宇(Chun-Yu Hsieh)

指導教授 ： 吳信隆

高雄醫學大學/藥學院/藥學研究所/博士(2009年)

摘要

本論文所指胺基酸類化合物包括protein及non-protein胺基酸，常為具生物活性之藥物或指標成分；由於其結構多不具顯著呈色基團，不易以常用檢測器(如UV-Vis)進行生物檢體中該類化合物之微量分析。因此本研究主要以衍生試劑naproxen acyl chloride (NAC)及levofloxacin acyl chloride (LFAC)，對不具發色基團的胺基酸待測物加以衍生，所得之衍生物具有螢光性；以螢光偵測器加以檢測可得高感度及選擇性分析。因本研究之樣品皆為生物檢體，在複雜基質中，檢測微量待測物，所用方法須具良好分離功能，故本論文結合高效能液相層析及螢光偵測器，對γ-amino-n-butyric acid (GABA)、vigabatrin (VGB)及D-serine等進行微量分析，所得主要結果摘錄如下：

1. 癌症生物指標性成分 – γ-amino-n-butyric acid (GABA)
在癌症病人的尿液中，GABA的濃度會增加，因此GABA可以當做生物指標(biomarker)，作為癌症診斷及治療之參考。本研究開發一個簡單且靈敏的液相層析法，利用螢光衍生試劑NAC對GABA做衍生反應，使原本沒有螢光的GABA轉換為具有螢光性之衍生物，以螢光偵測器檢測(λex = 230 nm, λem = 350 nm)即可分析待測物。本法定量下限為100 nM，檢測極限可達10 nM (S/N = 3，注射量為20 μL)；並成功應用於卵巢癌及子宮癌病人尿液中生物指標性成分GABA之分析研究。
2. 抗癲癇藥物 – vigabatrin (VGB)
VGB是一個廣泛被運用的抗癲癇藥，一般市售的劑型是以消旋異構物(racemate)供應，但是其只有(S)-(+)-型異構物[(S)-(+)-VGB]具有藥理活性。本研究開發一個簡單且靈敏的螢光液相層析法(λex = 230 nm, λem = 350 nm)，配合具有光學選擇性的螢光衍生試劑NAC對VGB的消旋異構物行衍生反應，分析衍生後的非鏡像異構物(diastereomer)產物。此方法對於(S)-(+)-VGB與(R)-(-)-VGB的偵測，定量下限均為25 nM，檢測極限也都可達2.5 nM (S/N = 3，注射量為10 μL)。此方法也已應用於VGB投藥病人血清中VGB鏡像異構物之分離與檢測。

3. D-serine
長久以來，一直以為人類體內只有左旋胺基酸(L-amino acid)的存在，後來的研究發現，右旋胺基酸(D-amino acid)存在，還與疾病有著某種程度的關連。本研究開發一個具光學選擇性之衍生試劑LFAC搭配螢光液相層析法，對右旋性絲胺酸(D-serine)做衍生分析，並將應用於人類阿茲海默症(Alzheimer's disease)篩檢上。本法分析D-serine與L-serine之定量下限為50 nM，檢測極限為5 nM。

關鍵字

γ-Amino-n-butyric acid ； Vigabatrin ； D-serine ； 生物指標 ； 生物檢體 ； 衍生化液相層析 ； 卵巢癌 ； 子宮癌 ； 阿茲海默症

並列摘要

Amino acids, including protein or non-protein amino acids, are widely used as drugs or disease biomarkers. Most of amino acids lack a chromophore for being detected by common absorption spectrophotometry at practical UV range. In this study, we labeled some amino acids, γ-amino-n-butyric acid, vigabatrin and D-serine, with fluorescent reagent NAC or LFAC and analyzed the resulting derivatives with a fluorescent detector to improve the sensitivity and selectivity of the analytes. Quantification of amino acids in biological fluids such as plasma and urine found important application in biomedical studies. The main results of the study are summarized as follows:

1. GABA as a biomarker of ovarian and uterine cancer
A simple and sensitive liquid chromatographic method is described for the analysis of γ-amino-n-butyric acid (GABA) in human urine. GABA is increased in the urine of cancer patients and could be used as a biomarker in the diagnosis and treatment of related patients. The method is based on derivatizing GABA with a fluorescent reagent (naproxen acyl chloride) for transforming the non-chromophoric GABA to a derivative with chromophoric and fluorophoric properties. The resulting derivative is highly responsive to a fluorimetric detector (λex = 230 nm, λem = 350 nm). The lower quantitation of the method is attainable at 100 nM GABA with a detection limit of about 10 nM (S/N = 3 with 20 μl injected). Application of the method to the analysis of GABA in the urine of patients with ovarian and uterine cancer was demonstrated.

2. Drug monitoring of active vigabatrin
Vigabatrin is widely used as an anticonvulsant in the treatment of seizures. Vigabatrin is usually supplied as racemate in formulation, but only the (S)-(+)-enantiomer of vigabatrin is pharmacologically active. A simple and sensitive liquid chromatographic method is described for the separation and quantification of vigabatrin enantiomers. The method is based on derivatizing racemic vigabatrin with a fluorescent chiral reagent (naproxen acyl chloride). The resulting diastereomeric derivatives are highly responsive to a fluorimetric detector (λex = 230 nm, λem = 350 nm). The lower quantitation limit of the method is attainable at 25 nM for (S)-(+)-vigabatrin or (R)-(-)-vigabatrin with a detection limit of about 2.5 nM (S/N = 3 with 10 µl injected). Application of the method to the analysis of vigabatrin in serum of dosed patients proved feasible.

3. D-serine as a biomarker for disease
Amino acids (AA) are the building blocks of various peptides and proteins for essential life. In general, natural AA is existed as L-form for their functions. But some D-amino acids at trace levels found to be associated with human diseases such as D-serine in Alzheimer’s; therefore, a fluorimetric liquid chromatographic method (λex = 290 nm, λem = 460 nm) is being developed for the analysis of DL-serine in urine by using a chiral derivatization reagent LFAC. The lower quantitation limit of the method is 50 nM for D-serine or L-serine with a detection limit of about 5 nM (S/N = 3 with 10 µl injected). Application of the method to the analysis of urine in patients of Alzheimer’s is being developed.

並列關鍵字

γ-Amino-n-butyric acid ； Vigabatrin ； D-serine ； Biomarker ； Biosample ； Derivatization liquid chromatography ； Ovarian cancer ； Uterine cancer ； Alzheimer's disease

參考文獻

參考文獻

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