連續注入噴灑游離質譜法之開發與應用

Abstract

大氣壓力下游離質譜法在近10年來進入了另一個黃金時期，科學家們開發出各種游離法可將分析物直接在大氣壓下進行氣化游離，最終的目標為樣品製備及游離源的設置可以愈簡便愈好，本論文也以這個方向為目標發展連續注入噴灑游離法。本論文第一部分的主題為研究超音波輔助噴灑游離法之特性及應用，超音波輔助噴灑游離法為本實驗室在2010年開發出的一種大氣壓力游離法，此游離法是藉著超音波輔助及毛細現象可以進行樣品在毛細管中之連續進樣，並在毛細管外另一端產生游離，裝置架構相當簡單，不需要另外提供電壓在前端樣品出口處，只需要一般實驗室常用的低頻率超音波清洗器及拉尖的毛細管即可把液體樣品氣化游離得到分析物氣相離子。在本論文我們發現到超音波輔助噴灑游離法和電噴灑游離法兩者在分析含有羥基的分析物如醣類時質譜分析結果有所差別，電噴灑游離法所得到的醣類分子是以鈉、鉀的離子加成物為主，而超音波輔助噴灑游離法所得到的醣類分子則是以加上18的假分子離子為主要離子峰。經過實驗證明醣類分子加上18的離子為來自於加成上NH4+的加成物離子訊號，此特性讓超音波輔助噴灑游離法在進行醣類樣品的二次碎裂質譜的時候具有優勢，可以較容易得到二次碎裂質譜。並且根據此特性也將超音波輔助噴灑游離法應用到未經處理的高鹽類樣品中，在實驗結果中我們發現傳統電噴灑游離法分析物離子訊號會受鹽類的影響很大，但是利用超音波輔助噴灑游離法可以在未經任何前處理的狀況下直接分析高鹽類的樣品。第二部分的主題為開發另一個新的連續注入噴灑游離法，由於上述之游離法不易控制流速，而此游離法的裝置設計是毛細管和注射針幫浦的結合並且直接連結質譜進行分析，這種設計可以直接使用注射針幫浦控制流速，由於連續注入噴灑游離法是使用具有拉尖開口的毛細管，使之靠近質譜前端電壓，可以在不外加高電壓的情況下得到分析物的氣相離子，設計簡單且方便。本論文探討了可能影響分析物訊雜比強度的參數，將此系統最佳化。在本論文的實驗結果中，已證實了此游離法可以用來分析胺基酸、胜肽至蛋白質等等，即從分子量由小至大的分析物。最終，我們使用氧化鐵/二氧化鈦(核/殼)磁性奈米粒子為吸附材料，將奈米粒子利用強力磁鐵固定在連續注入噴灑游離法的毛細管中央，藉著奈米粒子表面的二氧化鈦與磷酸根的親和力讓流過毛細管的含磷酸根分子可以暫時鍵結在磁性奈米粒子表面因而可和不含磷酸根的分子之流出毛細管尖端時間有所不同，因而可以達到初步分離的目的。此初步結果證明此游離法有潛力結合分離方法進行線上濃縮、分離與質譜分析，應可更進一步研究應用此方法在各式樣品之分析。

Over the past 10 years, atmospheric pressure ionization mass spectrometry has reached a golden era. Scientists have developed a variety of ionization methods for the generation of gas-phase ions. The goal is to make the pretreatment and ion source setup as simple as possible. This thesis will achieve this goal by developing continuous infusion spray mass spectrometry. The first part of this thesis will discuss the features and application of ultrasonication-assisted spray ionization mass spectrometry (UASI-MS) which had been explored in 2010 by our laboratory. In UASI-MS, the sample is continuously directed to the capillary outlet by ultrasonication-assisted capillary action and to the outlet of the tapered capillary, leading to the generation of gas-phase ions. UASI setup is rather simple and it does not require additional voltage at the end of the capillary outlet. UASI only requires an inexpensive low-frequency (~40 kHz) ultrasound source and a tapered capillary for the generation of gas-phase ions from liquid samples at atmospheric pressure. In this study, we found that there are some differences between electrospray ionization mass spectrometry (ESI-MS) and UASI-MS in the analysis of compounds containing hydroxyl groups. For the analysis of neutral saccharides, ESI-MS tends to form sodium or potassium ion adducts while UASI-MS tends to form M+18 ion adducts. The experiments show that the M+18 ion adducts are the NH4+ ion adducts, which gives UASI-MS an advantage for doing MS/MS analysis. Also, UASI-MS can direct analysis of high-salt samples without any sample pretreatment. The second part involves developing a new continuous infusion spray mass spectrometry (CIS-MS). Capillary and syringe pump can directly link to mass spectrometer to analysis. No external voltage is applied on the CIS capillary emitter, which is close (~1 mm) to the orifice of a mass spectrometer. Syringe pump plays a role by driving the liquid sample from the inlet to the capillary outlet, which generates gas-phase ions for MS analysis. The setup is quite simple. In this study, CIS-MS is suitable for the analysis of a range of analytes from small molecules, such as amino acids, to large molecules, such as peptides and proteins. Additionally, the parameters that may affect CIS-MS performance have been studied in this thesis. Furthermore, the Fe3O4@TiO2 magnetic nanoparticles, which has specific interactions with phosphate is fixed by magnet in the capillary. Because TiO2 can trap the phosphate group, phosphorylated and non-phosphorylated species reach the mass spectrometer at different times. Fe3O4@TiO2 magnetic nanoparticles can successfully separate the phosphate from the non-phosphate species. Preliminary results show that this method can potentially be combined with mass spectrometer for online enrichment and separation. It should be further studied so that this method can be utilized for the analysis of various types of samples.Over the past 10 years, atmospheric pressure ionization mass spectrometry has reached a golden era. Scientists have developed a variety of ionization methods for the generation of gas-phase ions. The goal is to make the pretreatment and ion source setup as simple as possible. This thesis will achieve this goal by developing continuous infusion spray mass spectrometry. The first part of this thesis will discuss the features and application of ultrasonication-assisted spray ionization mass spectrometry (UASI-MS) which had been explored in 2010 by our laboratory. In UASI-MS, the sample is continuously directed to the capillary outlet by ultrasonication-assisted capillary action and to the outlet of the tapered capillary, leading to the generation of gas-phase ions. UASI setup is rather simple and it does not require additional voltage at the end of the capillary outlet. UASI only requires an inexpensive low-frequency (~40 kHz) ultrasound source and a tapered capillary for the generation of gas-phase ions from liquid samples at atmospheric pressure. In this study, we found that there are some differences between electrospray ionization mass spectrometry (ESI-MS) and UASI-MS in the analysis of compounds containing hydroxyl groups. For the analysis of neutral saccharides, ESI-MS tends to form sodium or potassium ion adducts while UASI-MS tends to form M+18 ion adducts. The experiments show that the M+18 ion adducts are the NH4+ ion adducts, which gives UASI-MS an advantage for doing MS/MS analysis. Also, UASI-MS can direct analysis of high-salt samples without any sample pretreatment. The second part involves developing a new continuous infusion spray mass spectrometry (CIS-MS). Capillary and syringe pump can directly link to mass spectrometer to analysis. No external voltage is applied on the CIS capillary emitter, which is close (~1 mm) to the orifice of a mass spectrometer. Syringe pump plays a role by driving the liquid sample from the inlet to the capillary outlet, which generates gas-phase ions for MS analysis. The setup is quite simple. In this study, CIS-MS is suitable for the analysis of a range of analytes from small molecules, such as amino acids, to large molecules, such as peptides and proteins. Additionally, the parameters that may affect CIS-MS performance have been studied in this thesis. Furthermore, the Fe3O4@TiO2 magnetic nanoparticles, which has specific interactions with phosphate is fixed by magnet in the capillary. Because TiO2 can trap the phosphate group, phosphorylated and non-phosphorylated species reach the mass spectrometer at different times. Fe3O4@TiO2 magnetic nanoparticles can successfully separate the phosphate from the non-phosphate species. Preliminary results show that this method can potentially be combined with mass spectrometer for online enrichment and separation. It should be further studied so that this method can be utilized for the analysis of various types of samples.