開發具高靈敏度毛細管電泳及基質校正之串聯式液相層析質譜分析方法於生醫檢體應用

Abstract

由於近幾年來生醫樣品分析的需求量逐漸增加，例如藥物治療監測、藥物動力學研究、篩選生物標誌、以及個人化醫療的發展，因此如何開發快速並準確的生醫樣品分析方法成為重要課題之一。液相層析搭配電噴灑電離子化質譜儀（liquid chromatography-electrospray ionization mass spectrometry）和毛細管電泳（capillary electrophoresis）是兩種常用於生醫樣品的分析平台。液相層析搭配電噴灑離子化質譜儀具有較高的靈敏度和選擇性的優點，常被用來作為生物樣品分析平台。毛細管電泳是一種具有高解析度、高分析速度、高度自動化、低成本且低汙染的分析技術。本論文採用此兩種平台發展生醫檢體的分析技術。 在生醫檢體分析中常常遇到的兩個重要問題，例如隨著樣品的高複雜度而來的嚴重基質效應（matrix effect），以及分析物濃度過低所造成的無法定量或較大的誤差都會影響到分析品質。基質效應常被指出會嚴重影響液相層析搭配電噴灑電離子化質譜儀的精密度（precision）及準確性（accuracy）。因此我們發展柱後注入內部標準品（postcolumn infused-internal standard）的校正方法來校正基質效應所造成的定量誤差，我們使用這個方法定量25 個尿液檢體中的6個benzodiazepines 藥物濃度，超過90%結果的定量誤差小於20%，且所有分析結果的定量誤差皆小於30%。由於單純使用柱後注入內部標準品技術對於校正基質組成差異過大的樣品有其限制，為此，我們另外提出合併基質標準化參數(matrix normalization factors) 與柱後注入內部標準品的方法校正不同體液中顯著不同程度的基質效應，並且簡化定量方式。我們使用這個方法定量血液及腦脊液中的etoposide 與etoposide catechol濃度，確效結果顯示大於93%分析結果之定量誤差小於20%，且99% 的分析結果定量誤差小於30%。我們另以此方法應用於定量內生性代謝物（endogenous metabolites），解決過去正確定量內生性物質只能用標準品添加法(standard addition method)或同位素內標法(isotopically labeled internal standard (SIL-IS))的限制，我們以此方法定量血液中之androstenedione 與testosterone濃度，該方法定量結果與同位素內標法定量結果之相關係數高達0.98。 靈敏度是生醫檢體分析另一個常見的問題，為了解決這個問題，我們使用了線上濃縮（on-line concentration）的技術來提高毛細管電泳的檢測極限，以最適化分析條件定量血液中的 posacnazole濃度，定量極限可達到10 ng mL-1。另外，我們也建立了一個毛細管膠電泳（capillary gel electrophoresis）的分析平台來同時檢測單核苷酸多態性（single nucleotide polymorphism）及拷貝數變異（copy number variation），簡化基因檢測的步驟，我們以多重聚合媒鏈鎖反應結合毛細管電泳法偵測50個DNA檢體CYP2D6基因的單核苷酸多態性及拷貝數變異，並將分析結果與DNA定序法及長聚合媒鏈鎖反應法所得結果比較，各方法分析結果之相關技術大於90%。 本研究對於所發展的柱後注入內部標準品的校正方法，線上濃縮技術和基因檢測方法新技術皆以實際的臨床案例評估其可行性，我們證明所發展的技術具有高準確度、經濟、靈敏的優點，期望未來能透過這些技術應用於更多的臨床實例，提供準確的醫療數據，改善治療成效。

Nowadays, bio-pharmaceutical analysis gains growing importance due to the increasing needs in medication care such as therapeutic drug monitoring, biomarker discovery, and genotype testing. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) and capillary electrophoresis (CE) are two of the commonly used platforms for bio-pharmaceutical analysis. LC-ESI-MS is a versatile analytical tool and has been applied for many quantitative purposes because of its excellent sensitivity and selectivity. CE is an environmental friendly analytical technique which shows advantages in high resolution, high analysis rate, automation and low cost. This thesis used both platforms to develop analytical methods for bio-pharmaceutical analysis. Two main challenges encountered in bio-pharmaceutical analysis include the serious matrix effects (MEs) come along with high sample complexity, and the low analyte concentration. MEs have been regarded as the “Achilles heel” of LC-ESI-MS because MEs cause poor precision and quantification accuracy. To overcome this problem, we proposed a postcolumn-infused internal standard (PCI-IS) strategy for universal correction of MEs in biospecimens. When the PCI-IS method was used to correct the 6 benzodiazepines in 25 real human urine samples, over 90% of the test results exhibited quantification errors of less than 20%, and all of the test results had quantification errors of less than 30%. As PCI-IS method could not provide good correction efficiency for different biofluids that exhibited distinct MEs, we additionally introduced matrix normalization factors (MNFs) combined with PCI-IS method to improve quantification accuracy. When using the PCI-IS method in combination with MNFs, the calibration curve generated from standard solutions can be applied to quantify the target analytes in various biofluids. We applied this new approach to quantify etoposide and etoposide catechol in plasma and CSF. The accuracy test showed that over 93% of the data have quantification errors less than 20%, and 99% of the data have quantification errors less than 30%. We further applied the MNFs combined with PCI-IS method to quantify endogenous metabolites. In order to acquire the MNF values in the specific sample matrix without interfering by the endogenous metabolites, excessive amount of analyte was spiked in to the specific sample matrix. This method provides a new economic and effective approach to quantify endogenous metabolites. We used the MNFs combined with PCI-IS method to quantify androstenedione and testosterone. The result showed a correlation coefficient of 0.98 for both compounds compared to the results acquired by the stable isotope labeled-internal standard method. The sensitivity requirement is another challenge for bio-pharmaceutical analysis, especially when using CE for measurement. To solve this problem, on-line concentration technique was used to improve the detection limit of CE. We used the field-amplified sample stacking (FASS) technique to quantify posaconazole concentration in plasma samples, and the limit of quantification could reach 10 ng mL-1. Moreover, considering that genotype testing gains high attentions for personalized medicine, we built a genotype determination method for simultaneous identification of both single nucleotide polymorphism and copy number variation by capillary gel electrophoresis. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90 %. This study developed the PCI-IS correction method, the on-line concentration method, and genotype testing method to solve problems in bio-pharmaceutical analysis. All of these methods had been applied to real cases to demonstrate their feasibility for clinical measurement. We anticipate these methods can provide accurate and sensitive quantitation for other clinical applications to improve the quality of medication care.