雷射脫附游離法結合液相層析與傅立葉轉換式質譜儀之介面開發

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雷射脫附游離法結合液相層析與傅立葉轉換式質譜儀之介面開發

Introducing Internal Calibrants for High Performance Liquid Chromatography/ Fourier Transform-Ion Cyclotron Resonance (HPLC/FT-ICR) by Ambient Laser Desorption

黃于珉 , Masters　　Advisor：謝建台　　

繁體中文

雷射脫附游離法；高解析度質譜；傅立葉轉換離子迴旋共振質譜儀；融合微滴電噴灑游離法；內校正法；四極柱-飛行時間質譜儀； Fourier Transform-Ion Cyclotron Resonance ； Quadrupole Time-of-Flight ； Internal Calibration ； High Resolution Mass Spectrometry ； Laser Desorption Ionization Mass Spectrometry ； Fused-Droplet Electrospray Mass Spectrometry

Abstract │ Reference (44) │ Altmetrics

Abstract 〈TOP〉

傅立葉轉換式質譜儀( Fourier transform-ion cyclotron resonance mass spectrometry, FT-ICR-MS )是利用離子在低溫超導磁鐵所產生之磁場下進行迴旋運動，由於各離子在運行間所產生特異性的感應電流頻率，再藉由傅立葉轉換的方式將各分析物離子的電流頻率轉換成具有質量資訊，其具超高質量解析能力。由於高解析度質譜具有的質量準確度易受外在環境影響，使準確質荷比的解析能力隨著時間、溫度造成偏差，因此必須經過內校正 ( internal calibration )，使質量準確度 (mass accuracy) 可以小於 1 ppm，提供準確分子量測定及化合物元素鑑定的指標，近年高解析度質譜已成為最可信的蛋白質和代謝物鑑定工具。然而，在液相層析的長時間分離，更加難以獲得各個分離出的分析物離子的準確的質荷比，因而造成後續軟體比對上的困難，因此，本研究中利用大氣壓力質譜法結合液相層析與高解析度質譜儀進行內校正，來提升質量準確度。研究分三部分，分別敘述如下：
一、	探討液相層析結合大氣壓力質譜法進行內校正的介面開發
    由於液相層析在長時間的層析條件改變，造成質譜分析過程容易因為游離條件改變，難以獲得各個分離出的分析物離子的準確質荷比，又因無法持續進行校正，造成後續軟體比對上的困難，因此，本研究中探討了傳統分析方法和利用三向閥進行液液融合(liquid-liquid junction using three-way tee)、融合微滴電噴灑 (fused-droplet electrospray) 及雷射脫附 (laser desorption) 的方式進行質量校正，由實驗結果可發現，雷射脫附的方式不僅可同時在質譜圖上獲得校正溶液和分析物的訊號，也可在不受液相層析之移動相游離效率的干擾下穩定提供質譜訊號。
    在實驗過程中，將脈衝雷射裝載於系統平台上，將透過注射幫浦不斷流出的校正溶液進行雷射脫附；同一時間，由液相層析分離出的分析物溶液會透過電噴灑游離源進行游離，而所產生帶電分析物液滴會與雷射所脫附的校正溶液液滴結合，並發生離子-分子反應 ( Ion-molecule reactions, IMRs)，使校正溶液也帶有電荷，並一同進入質譜偵測，可同時得到分析物與校正溶液的高解析度質譜圖。

二、	雷射脫附游離法結合液相層析與高解析度質譜儀之應用
    利用雷射脫附結合液相層析與傅立葉轉換式質譜儀進行內校正，來提升質量準確度。實驗結果顯示，本研究方法既可在不影響分離結果的前提下，同時得到分析物與校正溶液的離子訊號，也能夠將不同滯留時間所沖提之分析物進行離子訊號的校正與分析，進而得到準確分子量以利於元素組成的鑑定。將上述系統應用在 β-blocker 的藥物分析上，其校正後的準確質量範圍落在-0.821到0.241 ppm之間，以獲得準確度較高的物種鑑定；而在 Triglyceride 的脂質學分析上，在 Non-aqueous 的移動相及不易游離的條件下，訊號較複雜且易形成[M+Na]+，藉由此方法仍可得到落在 -0.511 到 0.159 ppm 之間的準確質量範圍，亦可將鑑定物種的可能性從 44-61 種降低到 4-7 種。最後利用此方法鑑定出老鼠尿液中的檳榔鹼及其代謝物。

Parallel Abstract 〈TOP〉

Accurate mass measurement is crucial in unambiguously identifying an organic compound. Fourier transform ion cyclotron resonance (FT-ICR) is capable to provide such an accurate mass measurement with a resolving power greater than 1,000,000 and an error lower than 1 ppm. Obtaining a mass spectrum containing the ion signals of analyte and calibrant is necessary to perform the internal calibration. This is usually achieved by mixing the analyte with the calibrant solution followed by electrospray ionization. However, when the analytes are introduced into the ESI source through high performance liquid chromatography (HPLC), it is difficult to mix the calibrant with the analyte for accurate mass measurement. In this study, an interface was developed to efficiently mix the calibrants with the analytes for HPLC/ESI/FT-ICR analysis. 
The interface for introducing the calibrant into the HPLC/ESI/FT-ICR is comprised of: (1) pulsed UV laser beam – to desorb calibrant from the solution flowing out of a syringe, (2) three-way tee - to connect the solution flowing out of a gradient HPLC column (4.6 × 250 mm) with a high voltage, and (3) high voltage power supply – to induce ESI from the solution flowing from one arm of the three-way tee. The high voltage (4 kV) was applied to the sample solution in the three-way tee through an electrode. 
Electrospray ionization was induced from the sample solution flowing out of the three-way tee. The calibrant solution (i.e., tuning mix or sodium formate) flowing out of a syringe was continuously desorbed by irradiating the solution with a pulse laser beam. The desorbed calibrant moved upward and joined in the ESI plume.The calibrant was ionized by interacting with the charged solvent species in the ESI plume. The analyte together with the calibrant ions were subsequently detected by the FT-ICR attached after the ESI source. The calibrant used for internal calibration was introduced into the HPLC/ESI/FT-ICR through laser desorption followed by post-ionization in the ESI plume. The system was applied for high resolution analysis of a mixture of triglycerides and phospholipids. The mass spectrum containing the ion signals of analyte and calibrants was useful for accurate mass measurement of the analyte ion. Although the mass range covered by the tuning mix is high enough, such a calibrant did not provide enough ion peaks to perform internal calibration on all of the analytes. It was found that sodium formate ions contain known masses over a wide mass rang and provide enough ion peaks for internal calibration of all analytes. The compound was then chosen as the calibrant for high resolution mass measurement. The proposed method showed a mass measurement error ranging from -0.85 to 0.24 ppm for all lipid analytes.

Reference ( 44 ) 〈TOP〉

1. Thomson, J. J., Cathode Rays. Philosophical Magazine 1897, 44 (293).
連結：
3. Yamashita, M.; Fenn, J. B., Negative ion production with the electrospray ion source. The Journal of Physical Chemistry 1984, 88 (20), 4671-4675.
連結：
4. Yamashita, M.; Fenn, J. B., Electrospray ion source. Another variation on the free-jet theme. The Journal of Physical Chemistry 1984, 88 (20), 4451-4459.
連結：
5. Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T., Protein and polymer analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 1988, 2 (8), 151-153.
連結：
6. Karas, M.; Hillenkamp, F., Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical chemistry 1988, 60 (20), 2299-2301.
連結：

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