第一部份：以液相層析質譜儀結合柱後注入內標法定量採血卡上的新型抗凝血藥物 第二部分：建立液相層析質譜儀結合柱後注入內標法定量磷脂醯乙醇胺以測量採血卡上的血比容

Part I: Using the Postcolumn Infused-Internal Standard Method to Quantify Novel Oral Anticoagulants in Dried Blood Spots by LC-ESI-MS Part II: Development of a Postcolumn Infused-Internal Standard Method to Quantify Phosphatidylethanolamine for the Estimation of Hematocrit in Dried Blood Spots by LC-ESI-MS

張人祥(Ren-Shiang Jhang)

指導教授 ： 郭錦樺

國立臺灣大學/醫學院/藥學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803315

摘要

採血卡取樣技術在疾病篩檢上已有著悠久的歷史。由於個人化醫療的觀念亦趨重要，此採樣方法用作為一個普遍的醫療評估工具已於現今得到很高的重視。儘管採血卡取樣技術有很多的優點，但將其應用在臨床上例行的生物檢測仍然存在很多挑戰。本論文中所建立的分析方法為運用液相層析串聯式質譜儀並結合管柱後灌注內標法以分析採血卡上的化合物。
本論文第一部分，我們執行方法開發及確效以定量採血卡上三種新型口服抗凝血藥物，包括dabigatran、rivaroxaban、apixaban。利用管柱後灌注內標法校正在質譜儀內的基質效應和估計採血卡上的血液體積。在最佳化的分析條件下，此方法能在4分鐘內完成三種抗凝血藥物的定量分析。當使用0.1%甲酸並搭配乙腈(3:7 v/v)作為萃取液時，三個藥物萃取率可高達90%以上。選用[13C6]-rivaroxaban作為管柱後灌注法的內標準品，其估計體積的準確度介於87.1-119.8% (n=7). 此外，三個藥物沒有顯著受到5個不同血比溶的影響。在全部測試濃度的確效結果均顯示準確度介在88.9-104.3%，且同日內之重覆性及異日間之再現性精密度的相對標準差皆小於10.0%。本分析方法所使用的三種藥物在採血卡檢體建立線性的相關係數皆大於0.99，定量極限皆小於0.6 ng mL-1。三種藥物在採血卡檢體內於室溫、4°C及-20°C的條件下存放一個月後的安定性良好(91.0-111.4%)。我們成功地將管柱後灌注內標法用於定量採血卡中三種新型口服抗凝血藥物，研究結果指出此方法可以有效地用在測量採血卡上的藥物濃度。
第二部分我們進一步評估本實驗室先前找到的兩種磷脂醯乙醇胺用來估計血比容的準確度。先使用水浸潤並搭配Folch萃取法，結果顯示磷脂醯乙醇胺16:0/20:4及16:0/18:1在採血卡檢體的萃取率分別是84.6%及93.4%。我們選擇外生性的磷脂醯乙醇胺17:1作為管柱後灌注法的內標準品以校正基質效應，並在質譜端使用負離子模式以增加選擇性。在分析方法的最適化條件下，我們定量採血卡中的兩個磷脂醯乙醇胺並發現其兩者的濃度與血比容值呈現高度線性相關性(R2>0.98)。因此進一步使用此兩個內標估測60個臨床採血卡檢體的血比容值，結果顯示全部的樣品的估計偏差皆小於15%。帶入布蘭德-奧特曼差異分析方法後顯示超過90%的檢體量落在95%信賴區間內，從山形圖可知磷脂醯乙醇胺16:0/20:4及16:0/18:1的平均偏差分別為0.3及-2.5。我們的結果顯示此二個磷脂醯乙醇胺極具潛力作為估計採血卡檢體中的血比容值的標誌。
總結本研究為使用液相層析串聯式質譜儀建立方法；分別用於定量採血卡檢體內的三種新型抗凝血藥物，及估計採血卡檢體內血比容。此兩個簡易且高效的分析方法可提供作為療劑監測的一個新方法，以協助個人化醫療並可以更廣泛將採血卡應用在臨床上。

關鍵字

採血卡 ； 新型口服抗凝血藥 ； 血液體積 ； 血比容 ； 柱後注入內標 ； 質譜儀

並列摘要

Dried blood spots (DBSs) sampling technique has had a long history in disease screening. Recently, it has gained high attention as a common medical assessment tool because of the growing importance of personalized medicine. In spite of numerous advantages respecting the DBS sampling technique, this powerful sampling tool still faces challenges in the application of routine clinical bioanalysis. In this study, we develop analytical methods for DBS analysis by a liquid chromatography-tandem mass spectrometry combined with postcolumn infused-internal standard (PCI-IS) approach.
In the first part of this thesis, we developed and validated a method for quantification of three NOACs including dabigatran, rivaroxaban and apixaban in DBS. The PCI-IS method was adapted for correction of matrix effects in LC-ESI-MS and estimation of blood volumes on DBS cards. Under optimal conditions, three NOACs could be quantified in a single run within 5min. Over 90% of the recovery could be achieved when using 0.1% formic acid in water with acetonitrile (3:7 v/v) as the extraction solution. [13C6]-rivaroxaban was selected as the PCI-IS and the accuracy for volume estimation was between 87.1% and 119.8% (n=7). Furthermore, the result from hematocrit effect of three NOACs showed no significant variation for 5 different hematocrit (HCT) levels. Validation results demonstrated that the accuracies were among 88.9-104.3%, and intra-day and inter-day precisions were all below 10.0% for all test concentrations. The calibration curves of DBS samples for the analytes had coefficients of determination higher than 0.99, and the limits of detection were all below 0.6 ng mL-1. The stability of three NOACs revealed good stability (91.0-111.4%) in the DBS samples after one months of storage at room temperature, 4 °C and -20 °C. We successfully applied the PCI-IS method for quantification of NOACs in DBS, and the result revealed PCI-IS is an effective strategy for measuring drug concentrations in DBS.
In the second part, we further evaluated the accuracies of two potential phosphatidylethanolamine (PE) markers identified in our lab for estimation of HCT values. Using water pre-rinsed in combination with Folch extraction showed good recoveries with 84.6% and 93.4% for PE 16:0/20:4 and PE 16:0/18:1, respectively. We selected the exogenous lysoPE 17:1 as the PCI-IS for correction of matrix effect, and applied negative mode LC-ESI-MS to enhance selectivity. Under the optimized condition, we quantified these two PEs in the DBS samples and found the high correlation between the concentration of PEs and the HCT values of DBS samples (R2>0.98). Thus, PE 16:0/18:1 and PE 16:0/20:4 were further applied to estimate the HCT values of 60 DBS samples obtained from patients. Finally, the results showed that all of samples were presented less than 15% estimation error. The Bland and Altman analysis showed more than 90% of DBS samples within 95% CI. The mean biases from mountain plot were 0.3 and -2.5 for PE 16:0/18:1 and PE 16:0/20:4, respectively. Our results indicated that PE 16:0/18:1 and PE 16:0/20:4 exhibited high potential to serve as makers to estimate HCT values for the DBS samples.
In conclusion, we established a analytical methods for accurate quantification of NOACs in DBS samples and estimation of HCT levels by LC-ESI-MS. The developed two simple and efficient methods can offer a new strategy for routine therapeutic drug monitoring (TDM) to improve personalized medicine and expand applications of DBS technique in clinical use.

並列關鍵字

dried blood spot (DBS) ； novel oral anticoagulants (NOACs) ； blood volume ； hematocrit effect ； post-column infused internal standard (PCI-IS) ； mass spectrometry

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