臺灣博碩士論文加值系統

尿液中的有機酸（organic acid）幾乎包含所有醣類、胺基酸、脂肪、維生素、及藥物等代謝後產生的水溶性產物或中間產物，能夠反映出人體生理的情況。其中苯甲酸（Benzoic acid, BA）、馬尿酸（Hippuric acid, HA）、苯乙酸（Phenylacetic acid, PAA）、苯丙酸（Phenylpropanoic acid, PPA）、對羥基苯甲酸（4-Hydroxybenzoic acid, 4-HBA）、4-羥基苯乙酸（4-Hydroxyphenylacetic acid, 4-HPAA）、吲哚乙酸（Indoleacetic acid, IAA）、二羥基苯丙酸（3,4-Dihydroxyphenyl propionic acid, 3,4-DHPPA）、3-羥基苯丙酸（3-Hydroxyphenylpropionic acid, 3-HPPA），這 9 種有機酸被認為與腸道菌叢失衡有關，可經由尿液檢測，作為腸道菌叢代謝異常的指標。因此，建立一種簡單、快速、可靠且精確，能夠同時監測以上有機酸的分析方法是有其必要性。本研究主要目的在開發一 HPLC 及 LC-MS/MS 分析方法，期能同時測定尿液中九種主要有機酸，再經由生物性方法確效來驗證其適用性。所開發的方法具最低可定量濃度 10 ~ 40 ng/mL，r > 0.99 呈現良好線性，測定尿液檢品後，良好選擇性且無干擾，同批間與異批間的準確度（Accuracy）在 85.8 ~ 109.7%，精確度（Precision）在 1.4 ~ 13.3%；相關安定性結果皆符合生物性方法確效規範。

Urine organic acid contains water-soluble metabolites and/or metabolites derived from sugar, amino acid, lipid, vitamin, and drug, which could reveal the human physiological condition. Among those urine organic acids, the benzoic acid, hippuric acid, phenylacetic acid, phenylpropionic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid, indoleacetic acid, 3,4-dihydroxyphenyl propionic acid, and 3-Hydroxyphenylpropionic acid were the eligible candidates for the dysbiosis of gut microbiota. The aim of this proposal is to develop and validate a high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalysis method for the nine organic acids in urine. The method for selectivity of urine sample no interference, and lower limit of quantification 10 ~ 40 ppb, r2 > 0.99 showed good linearity, accuracy in 85.8 ~ 109.7%, precision in 1.4 ~ 13.3%. All results were met the requirement of bioanalytical method validation.

中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 viii
表目錄 x
第一章　緒論 1
第一節　研究背景 1
第二節　液相層析串聯質譜儀介紹 2
壹、 高效液相層析儀 3
貳、 電噴灑游離法（electrospray ionization, ESI） 5
參、 串聯質譜（tandem mass spectrometer, MS/MS） 7
第三節　有機酸簡介 9
壹、 Benzoic acid（BA）苯甲酸 11
貳、 Hippuric acid（HA）馬尿酸 11
參、 Phenylacetic acid（PAA）苯乙酸 12
肆、 Phenylpropanoic acid（PPA）苯丙酸 12
伍、 4-Hydroxybenzoic acid（4-HBA）4-羥基苯甲酸 13
陸、 4-Hydroxyphenylacetic acid（4-HPAA）4-羥基苯乙酸 13
柒、 Indoleacetic acid（IAA）吲哚乙酸 13
捌、 3,4-Dihydroxyphenyl propionic acid（3,4-DHPPA）3,4-二羥基苯丙酸 14
玖、 3-Hydroxyphenylpropionic acid（3-HPPA）3-羥基苯丙酸 14
壹拾、Creatinine 肌酐酸 14
第四節　目前與此 9 種有機酸在 LC-MS/MS 分析之現況 17
第五節　移動相pH值與有機酸的相關性 19
第六節　生物性分析方法確效 20
壹、 選擇性（Selectivity） 23
貳、 線性與最低可定量濃度（Linear & Lower Limit of Quantification，LLOQ） 23
參、 殘留評估（Carry-over） 23
肆、 稀釋的完整性評估（Dilution integrity） 23
伍、 準確度與精確度（Accuracy & Precision） 24
陸、 基質效應（Matrix effect） 24
柒、 穩定性（Stability） 25
第二章　研究動機與實驗架構 27
第一節　研究動機 27
第二節　實驗架構 28
第三章　材料與方法 29
第一節　儀器設備 29
壹、 高效液相層析儀系統 29
貳、 液相層析串聯質譜儀系統 29
參、 離心機 29
肆、 去離子水製造機 30
第二節　實驗藥品及試藥 30
第三節　試劑和試藥配製 30
壹、 試劑配製 30
貳、 HPLC 標準溶液配製 31
參、 LC-MS/MS 標準溶液配製 31
肆、 HPLC 標準曲線配製 33
伍、 LC-MS/MS 標準曲線配製 34
陸、 LC-MS/MS 添加基質標準曲線配製 35
柒、 移動相配製 36
第四節　器具 36
第五節　檢體來源 36
第六節　高效液相層析儀條件 37
第七節　液相層析串聯質譜儀條件 38
第八節　實驗步驟 42
壹、 尿液前處理的實驗步驟探討 42
貳、 9 種有機酸之高效液相層析儀分析方法開發與確效 45
參、 液相層析串聯質譜儀條件探討 46
肆、 9 種有機酸液相層析串聯質譜儀分析方法開發與確效 46
第四章　實驗結果與討論 53
第一節　9 種有機酸之高效液相層析儀分析方法開發與確效 53
壹、 選擇性（Selectivity） 53
貳、 線性與最低可定量濃度（Linear & Lower Limit of Quantification，LLOQ） 53
參、 準確度與精確度（Accuracy & Precision） 53
第二節　液相層析串聯質譜儀條件探討 54
壹、 移動相添加甲酸與乙酸比較 54
第三節　9 種有機酸液相層析串聯質譜儀分析方法開發與確效 55
壹、 選擇性 55
貳、 線性與最低可定量濃度（Linear & Lower Limit of Quantification，LLOQ） 55
參、 殘留評估（Carry-over） 56
肆、 稀釋的完整性評估（Dilution integrity） 56
伍、 準確度與精確度（Accuracy & Precision） 56
陸、 基質效應（Matrix effect） 57
柒、 穩定性（Stability） 59
第五章　結論 62
第六章　圖表 63
第七章　參考文獻 96

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