衍生化液相層析螢光法對人體尿液中Sarcosine之分析研究

Determination of Sarcosine in Human Urine by Derivatization and Fluorimetric Liquid Chromatography

李建德(Jian-De Li)

指導教授 ： 柯黃盛

高雄醫學大學/藥學院/藥學研究所/碩士(2012年)

摘要

Sarcosine 具有潛力成為前列腺癌之腫瘤標誌，為進一步探討此代謝物於疾病診斷上之確切角色，解決文獻上爭議，需具備準確的sarcosine 測定方法，因此本研究利用高效能液相層析螢光偵測法 (HPLC-FLU) 建立一簡單、快速、靈敏並符合經濟效益之分析方法，對尿液中sarcosine 進行定量分析。待測物以本實驗室自行開發之螢光試劑levofloxacin acyl chloride (L-OAC) 進行衍生反應，所得產物於激發波長290 nm╱放射波長460 nm 偵測下具有強烈螢光訊號，固定相採用hexyl-phenyl 層析管柱，在移動相醋酸鈉緩衝溶液 (50 mM, pH 3.8) 與tetrahydrofuran 梯度沖提下具有良好分離效果。本分析方法以sarcosine 標準品評估檢量線線性範圍，於0.5-20 μM 之線性關系良好 (相關係數r=0.999)，偵測極限 (limit of detection, LOD) 為0.1 μM (S/N=3，注射體積為20 μL)，同日內 (intra-day) 與異日間 (inter-day) 之相對標準偏差 (relative standard deviation, RSD) 與相對誤差 (relative error, RE) 均小於4.8%，顯示本方法具備良好之精密度與準確度。目前此方法已成功應用於健康男性尿液中之sarcosine 含量測定，未來將進一步應用於臨床分析，探討sarcosine 與前列腺癌之相關性。

關鍵字

Sarcosine ； 前列腺癌 ； 高效能液相層析螢光偵測法 ； Levofloxacin acyl chloride ； 衍生反應

並列摘要

Sarcosine, an N-methyl derivative of glycine, is reported to be a potential biomarker for prostate cancer with conflicting results. To accurately assess the diagnostic efficiency of sarcosine for prostate cancer, there is a need of a sensitive and reliable analytical method to determine sarcosine. A high performance liquid chromatography-fluorescence detection (HPLC-FLU) method was studied for the analysis of urinary sarcosine at trace levels. Pre-column derivatization of sarcosine with a novel fluorescent reagent levofloxacin acyl chloride (L-OAC) was performed, and the resulting derivative was highly responsive to a fluorescence detector (excitation at 290 nm and emission at 460 nm). The derivatized sarcosine was separated on a hexyl-phenyl column with the mobile phase of sodium acetate buffer solution (50 mM, pH 3.8) and tetrahydrofuran by gradient elution. The linear range of sarcosine in water was over 0.5-20 μM, and the limit of detection (LOD) was 0.1 μM (S/N = 3). Intra-day and inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) were all less than 4.8%, indicating this method was precise and accurate. This simple, rapid, sensitive and eco-friendly method has been successfully applied to the determination of sarcosine in urine samples from healthy male volunteers, which proved this HPLC-FLU technique is feasible for clinical and biochemical research. Application of this method to study the correlation between sarcosine and prostate cancer is in progression.

並列關鍵字

Sarcosine ； Prostate cancer ； High performance liquid chromatography-fluorescence detection ； Levofloxacin acyl chloride ； Derivatization

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