第一部分：開發毛細管電泳方法同時分析人體血漿中Triazole 類抗黴菌藥物濃度 第二部分：開發超高效液相層析結合串聯式質譜儀方法定量柴胡與生物樣品中柴胡皂苷A、柴胡皂苷B2和柴胡皂苷D之濃度

Abstract

第一部分：開發毛細管電泳方法同時分析人體血漿中Triazole 類抗黴菌藥物濃度 侵入性黴菌感染發生率在過去二十年顯著增加，此種全身性感染易危及生命。Voriconazole為強效之三環類抗黴菌藥物，其為侵入性麴菌感染症（invasive Aspergillosis）治療藥物的首選。本研究首先開發且確效一靈敏且有效之掃集－微胞電動層析（sweeping-MEKC）方法以定量病患血漿中voriconazole 濃度。方析方法包括以下幾個步驟：（1）將血漿樣品利用10 M 尿素進行去蛋白之樣品前處理，（2）利用OASIS HLB cartridge進行固相萃取以淨化血漿樣品，（3）利用掃集－微胞電動層析電泳方法分析樣品，背景電解質的組成為40 mM磷酸、110 mM 十二烷基硫酸鈉和20％乙腈，voriconazole可在10.5分鐘內和血液中的內生性物質達基線分離。峰面積和遷移時間一天之內相對標準差分別低於2.8％和5.5％（n = 6），峰面積和遷移時間天與天之間的相對標準差分別低於4.1％和6.3％（n = 3），準確度介於96.5％到107.4％之間。Voriconazole校正曲線的線性範圍介於0.25 μg mL-1到15 μg mL-1之間，涵蓋voriconazole臨床有效治療濃度範圍，最低偵測濃度低於0.075 μg mL-1。 三環類抗黴菌藥物itraconazole和posaconazole同樣為常用來治療廣泛性黴菌感染的藥物，它們與voriconazole皆於個體間藥動差異大，故有療劑監測之必要性。本研究進一步開發可同時定量人體血漿中之itraconazole、voriconazole和posaconazole濃度之掃集－微胞電動層析（sweeping-MEKC）方法。分析方法中的樣品前處理條件與voriconazole前處理條件相同。最適化sweeping-MEKC條件的分析緩衝液組成為25 mM磷酸、100 mM 十二烷基硫酸鈉、13％乙腈和13%四氫呋喃，本分析方法可在13分鐘內將分析物和血液中的內生性物質達基線分離。遷移時間和峰面積同日內的相對標準差分別小於6.1％和9.6％（n = 5），峰面積和遷移時間異日間的相對標準差低於6.5％和10.9％（n = 3），itraconazole、posaconazole和voriconazole之準確度介於90.5％到111.9％之間。最低偵測濃度分別為0.033 μg mL-1、0.016 μg mL-1和0.041 μg mL-1。本方法可用於定量血漿中itraconazole、voriconazole和posaconazole的濃度以進行療劑監測與臨床研究。 第二部分：開發超高效液相層析結合串聯式質譜儀方法定量柴胡與生物樣品中柴胡皂苷A、柴胡皂苷B2和柴胡皂苷D之濃度 柴胡皂苷為從柴胡根部取得之活性成分，柴胡皂苷之藥理活性包含抗發炎、抗肝炎、抗肝癌、抗腎炎、抗感染和免疫調節。眾多柴胡皂苷中，柴胡皂苷A和柴胡皂苷D為主要產生藥理活性之成分，其中柴胡皂苷B2為柴胡皂苷D於胃酸中的代謝物。本研究採用超高效液相層析結合串聯式質譜儀（UHPLC-MS/MS）建立柴胡皂苷之分析方法，並用以定量柴胡及生物樣品中柴胡皂苷含量。於最適化層析條件之下，柴胡皂苷A、柴胡皂苷B2和柴胡皂苷D可於二十分鐘內達基線分離。研究中針對質譜儀參數做最適化調整，其中包括：毛細管高電壓、樣品錐電壓、撞擊能量、去溶劑氣體流速、離子源溫度、去溶劑氣體溫度等，定量方面使用選擇反應監測模式（selective reaction monitoring，SRM）進行柴胡皂苷A、柴胡皂苷B2和柴胡皂苷D之分析。本分析方法分析物遷移時間同日與異日之相對標準差小於 5.9 %，分析物峰面積同日與異日之相對標準差小於 5.6 %，柴胡皂苷A、柴胡皂苷B2和柴胡皂苷 D標準品之定量極限分別為0.91 ng mL-1、1.58 ng mL-1與0.78 ng mL-1，最低偵測極限分別為0.22 ng mL-1、0.31 ng mL-1與0.33 ng mL-1。本研究已建立快速、有效與靈敏之UHPLC-MS/MS方法可同時分析三種柴胡皂苷，所開發方法的實際樣品應用性以分析植物萃取物、大鼠血清樣品與人體血漿樣品驗證。

Part I : Simultaneous determination of three triazole antifungal drugs in human plasma by using sweeping-micellar electrokinetic chromatography Invasive fungal infection is a life-threatening condition; its occurrence has increased significantly over the past 20 years. Voriconazole is a high potency triazole antifungal drugs, and it’s the drug of choice of invasive Aspergillosis. We have developed a sensitive and efficient sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method to quantify voriconazole, a potent triazole antifungal drug, in patient plasma. Solid phase extraction (SPE) conditions were first optimized to minimize plasma interference while maintaining a high recovery; the sweeping-MEKC conditions were then systematically optimized to obtain a high sweeping efficiency with good selectivity. Under the optimal analytical conditions, voriconazole was baseline-separated from endogenous materials within 10.5 min with a limit of detection of 0.075 μg mL-1. The background electrolyte comprised 40 mM phosphoric acid, 110 mM sodium dodecyl sulfate, and 20% acetonitrile. In terms of method repeatability, the relative standard deviations (RSDs) of the migration time and the peak area (intra-day; n = 3) were both less than 5.5%; in terms of intermediate precision, and the RSDs of the peak area and the migration time (inter-day; n = 3) were both less than 6.3%. We successfully applied this developed method to the quantitative determination of plasma voriconazole levels in 16 patients; the results correlated well with those obtained through analyses using high-performance liquid chromatography. Like voriconazole, itraconazole and posaconazole are triazole antifungal drugs, and used to treat invasive fungal infection. These drugs show great individual difference of pharmacokinetic behaviors, so therapeutic drug monitoring of these drugs is recommended to improve outcome of treatment. Therefore, we further developed a sensitive and efficient sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method to quantify itraconazole, voriconazole and posaconazole in human plasma. The sweeping-MEKC conditions were modified and then systematically optimized. Under the optimal analytical conditions, itraconazole, voriconazole and posaconazole were baseline-separated from endogenous materials within 13 min with a limit of detection of 0.033 μg mL-1、0.016 μg mL-1、0.041 μg mL-1, respectively. The background electrolyte comprised 25 mM phosphoric acid, 100 mM sodium dodecyl sulfate, 13% acetonitrile and 13% THF. In terms of method repeatability, the relative standard deviations (RSDs) of the migration time and the peak area (intra-day; n = 5) were both less than 9.6％; in terms of intermediate precision, and the RSDs of the migration time and the peak area (inter-day; n = 3) were both less than 10.9％. This sweeping-MEKC method is accurate and efficient and appears to be applicable to therapeutic drug monitoring and clinical research. IXAbstract ( II ) Part II：Determination of saikosaponin A, B2 and D in Bupleuri radix and biological samples by ultra-high-pressure liquid chromatography–tandem mass spectrometry Saikosaponins are bioactive oleanane saponins derived from the Chinese medicinal herb Bupleuri radix. Pharmacological activities of saikosaponins include anti-inflammation, antihepatitis, antihepatoma, antinephritis, antibacterial effects and immunomodulation. Among various saikosaponins, saikosaponin A(SSa) and saikosaponin D(SSd) are reported to play the major roles in producing these pharmacological activities whereas saikosaponin B2(SSb2) is the metabolite of saikosaponin D in gastric juice. HPLC-UV was the most frequently used technique for saikosaponins determination, but it’s not sensitive enough to analyze plasma samples. Recently, the LC-MS technique was applied to analyze saikosaponins in Chinese multiherb remedy, but it takes long analytical time and seldom LC-MS methods were applied to analyze saikosaponins in animal biological fluid. In the present study, an ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established to analyze saikosaponin A, B2 and D. Using 0.1% acetic acid and acetonitrile as mobile phase with gradient elution, saikosaponin A, B2 and D can be baseline separated within 20 minutes. The detection was performed with triple quadruple mass spectrometer using electrospray ionization in positive-ion mode and selective ion monitoring. To reach the best sensitivity, several mass spectrometry parameters were systemically optimized. Relative standard deviation(RSD) of the run-to-run repeatability and intermediate precision of the retention time were both within 0.9% RSD. Run-to-run repeatability and intermediate precision of the retention time were both within 5.9% RSD. The detection limit of saikosaponin A, B2 and D are 0.22 ng mL-1、0.31 ng mL-1and 0.33 ng mL-1 respectively. The developed UHPLC-MS/MS method is sensitive and efficient, and it could be applied to quantify saikosaponin A, B2 and D in Bupleuri radix and biological samples.