線上微透析取樣技術配合高效能液相層析連線分析系統之研發及其生物醫學與環境分析應用

本研究主要目的為利用微透析取樣技術(microdialysis sampling)、流動注入系統(flow injection)配合高效能液相層析儀 (HPLC)發展可偵測氧化壓力指標物、抗生素與重金屬之多種連線分析方法，經由各項最佳分析條件之探討與分析方法可信度之驗證，並應用於分析生物檢體或環境樣品之分析研究，以確認連線分析方法之可行性。本研究內容主要分四個部分：第一部份，發展線上微透析取樣結合流動注入系統與高效能液相層析儀連線偵測血液中脂質過氧化傷害指標物丙二醛(malondialdehyde, MDA)之分析方法。並應用於連續監測給予鐵劑後活體動物血中MDA濃度變化之研究。第二部份，發展以高效能液相層析法同時偵測血液中MDA與抗生素ofloxacin (OFL)之分析方法，並利用此分析方法探討OFL對於血中脂質過氧化傷害的影響。第三部份，發展全自動化之線上微透析取樣系統、流動注入系統與高效能液相層析之連線分析方法，直接同時偵測全血中MDA與OFL，並利用此自動化之連線分析方法，應用於探討OFL在紫外線暴露下對於血中脂質過氧化傷害的影響。第四部份，發展線上微透析取樣、奈米金/二氧化鈦吸附管線、流動注入系統與高效能液相層析儀連線系統，以紫外線偵測器直接同時偵測水樣中鈷和鎳含量之分析方法。此方法使用微透析探針進行水樣透析，利用流動注入介面，將透析所得之透析液導入奈米金/二氧化鈦之吸附管中，進行線上前濃縮步驟。再將濃縮液導入高效能液相層析儀以紫外線偵測器同時進行鈷與鎳之連線分析。本研究研發的多種連線分析方法，將有助於進一步探討抗生素與氧化壓力的生物醫學角色；而且對於環境重金屬的檢測，將有莫大助益。

關鍵字

連線；微透析；液相層析；丙二醛；鈷；鎳

並列摘要

This study describes the development of analytical methods based upon hyphenated techniques coupled to on-line microdialysis (MD) sampling, high performance liquid chromatography (HPLC) with flow injection (FI) equipment for the detection of biomarker of oxidative stress, antibiotics and heavy metals. This dissertation explored on-line methods to continuous and dynamic monitoring of those analytes described above. Four developed methods are discussed in detail. In first investigation, an HPLC method with fluorescence detection was developed employing MD sampling to on-line detecting malondialdehyde (MDA) in living rabbit blood. We examined many conditions to optimize the measurements. MDA concentration found increasing from the basal concentration as a result of lipid peroxidation by Fe ion-induced oxidative stress. In the subsequent investigation, we established the applicability of using HPLC with fluorescence detection for the simultaneous determination of MDA and ofloxacin (OFL). This method afforded linear responses between the MDA and OFL concentrations and the HPLC peak areas. To the best of our knowledge, it is the first practical analytical approach for simultaneously monitoring the levels of MDA and OFL in plasma. In the third investigation, we have developed a system coupling an on-line MD system with flow injection HPLC-fluorescence detection for simultaneous measurement of the concentrations of MDA and OFL in whole blood samples. Validation experiments demonstrated good linearity, precision, accuracy, and recovery. The average recoveries of MDA and OFL were each close to 100%. The use of this on-line MD-HPLC system permitted continuous monitoring of MDA and OFL in OFL-treated whole blood subjected to UV-A irradiation. Based on the results, we found that the UV-A irradiation markedly increased the level of MDA in the OFL-treated whole blood. Finally, an on-line configuration of microdialysis (MD), Au/TiO2 nanoparticle tubing, and high performance liquid chromatography- ultraviolet (HPLC-UV) detection was developed for the simultaneous measurement of cobalt (Co) and nickel (Ni) concentrations in water. The proposed method offers a novel and reliable method to determine the levels of Co and Ni in environmental water samples. Moreover, the methodology described in this study adherent to the concept of green chemistry, including using no organic solvents in the MD sampling and in extraction processes. The proposed method is the first reported on-line connection of MD, Au/TiO2 nanoparticle tubing, and HPLC devices for the measurement of Co and Ni concentrations in water. These development of analytical methods described in the present study may benefit further researches in the biomedical and environmental science.