電泳淌度差異於液相層析與毛細電泳電灑法質譜訊號提升之研究

Signal Enhancement by Electrophoretic Mobility Difference in LC/ESI-MS and CE/ESI-MS

王南絢(Nan-Hsuan Wang)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.03230

摘要

液相分離電灑質譜為一常見的分析技術，然而一般液相分離的溶液組成中常有非揮非性鹽類或是添加劑，對於電灑質譜有相當程度的干擾和訊號抑制。本研究利用分析物和鹽類之間的電泳淌度差異，選擇性地單只將分析物送往質譜端進行偵測，以提升分析物在液相層析電灑質譜與毛細電泳電灑質譜的訊號。本研究提出一電泳淌度控制裝置銜接於液相層析管柱出口處，進而減緩離子對試劑於液相層析電灑質譜抑制。探討胜肽於含有三氟醋酸添加動相下毛細液相層析質譜訊號及氨基醣苷類化合物於七氟丁酸添加下液相層析質譜訊號。毛細電泳質譜部份提出一一體式壓力輔助雙槽式介面，以克服十六烷基三甲基溴化銨動態塗佈毛細電泳電灑質譜分析除草劑訊號抑制之研究。
使用逆相液相層析分離帶電荷的物質時，常會加入少量的離子對試劑以提升分離解析度，但在電灑游離中卻使分析物無法有效的拆解造成分析物的質譜訊號抑制。本研究將電泳淌度差異的概念運用於液相層析質譜上，提出電泳淌度控制之概念，電泳淌度控制裝置為一簡單的電場裝置，包括一低流速鞘流介面、一個液體接合槽及一3公分的銜接管，銜接於液相層析管柱出口處。在液相層析管柱後施加一正向電場，將帶正電荷的分析物與帶負電荷的離子對試劑成功地拆解。帶正電荷的胜肽會往質譜方向前進，而帶負電荷的三氟醋酸根則停留在液體接合槽中，有效地避免進入質譜造成電灑訊號的抑制，訊號可提升9-35倍。亦使用蛋白質水解胜肽進行分析，可得到和甲酸環境相同的訊號強度，亦能保有三氟酸酸下的分離效果。
氨基醣苷類化合物為一高極性的化合物，文獻中常添加七氟丁酸以使用逆相液相層析進行分離。本研究中亦使用電泳淌度控制裝置，以帶正電荷分析物和離子對試劑進行拆解進而提升電灑訊號。比較經過電泳淌度控制裝置和未經過電泳淌度控制裝置的訊號強度，在一次質譜的觀測下，可以有較好的偵測極限，且二次質譜下也有較佳的訊號靈敏度。此外此介面亦嘗試銜接較高流速的層析條件 (分析級液相層析管柱約需要250 μL/min)，利用流動式注入分析進行簡單的探討。，利用分流的方式可以順利的銜接高流速分析於電泳淌度控制裝置。移除了離子對試劑後，可得到不錯的訊號提升。
本研究於毛細電泳質譜研究中開發一新介面，一體式壓力輔助雙槽式毛細電泳介面，結合壓力和電場作用力克服非揮發性的十六烷基三甲基溴化銨正離子鹽類於正電荷的除草劑毛細電泳質譜分析中所造成的質譜訊號抑制情形。一體式壓力輔助雙槽式毛細電泳介面為聚二甲基矽氧烷聚合而成，包括1公分的毛細銜接管連接兩個液體接合槽。一為液體接合電噴灑介面之液體槽，另一液體槽則用於銜接毛細電泳之分離管。銜接毛細電泳分離管之液體槽利用高分子管塞住形成進行密閉腔體，利用針式幫浦於液體槽加壓。由針式幫浦加壓導入甲醇溶液可於銜接管產生推往質譜端的壓力流，同時也增加分析物和十六烷基三甲基溴化銨正離子的電泳淌度的差異。於銜接管施加一反向電場，此電泳淌度較大的CTA+受到反向電場的控制停留在後方的液體槽中時，而電泳淌度較小的分析物則可隨壓力流推動方向進到質譜端進行偵測。成功地減緩正電荷鹽類CTA+於質譜訊號抑制情形和質譜儀離子源汙染的問題，分析物的訊號約有20-60倍提升。

關鍵字

液相層析電灑法質譜 ； 毛細電泳電灑法質譜 ； 電泳淌度控制 ； 壓力輔助雙槽式毛細電泳介面 ； 訊號抑制 ； 離子對試劑 ； 十六烷基三甲基溴化銨

並列摘要

On line coupling of separation technique with mass spectrometry is a common analytical approach for the analysis of trace or complicate samples. However, the common solution compositions used in liquid chromatography or capillary electrophoresis generally contain nonvolatile salts or additives, which produce interferences and ion suppressions to ESI-MS. In this study, the concept of electrophoretic mobility difference among analytes and salts was utilized. The analytes were selectively transported to MS and signals of analytes in LC-ESI-MS and CE-ESI-MS were elevated. A strategy of postcolumn electrophoretic mobility control (EMC) was proposed to alleviate ion suppression caused by ion pairing reagents in LC-ESI-MS. Peptides analysis using LC-ESI-MS with trifluoroacetic acid (TFA) containing mobile phase, as well as aminoglycans analysis with heptafluorobutyric acid (HFBA) containing mobile phase were discussed. With regard to studies of signal suppression in CE-ESI-MS, the pressure assisted double junction interface was developed to overcome the adverse effect of nonvolatile cation salt of cetyltrimethylammonium bromide (CTAB) on triazine analysis using CTAB dynamic coating CE-ESI-MS..
In analysis of charged analytes using reversed phase liquid chromatography, a small amount of ion pairing reagent was usually added in the mobile phase to neutralize the analytes , increase the hydrophobicity of analytes and thus improve chomatographic performance . However, the strong ion pairs between ion pairing reagents and protonated analytes can not be dissociated during the electrospray ionization process and thus result in serious signal suppression in ESI-MS. The EMC was used to alliveate the problem. The EMC device was a simple electric field setup comprising a low sheath flow interface, a liquid junction and a connecting capillary (3 cm), which was located at the terminus of LC column. An electric filed was created across the connecting capillary. Due to the electric field, the positive charged peptides migrated toward the ESI sprayer, whereas the ion pairing reagent TFA anions remained in the junction reservoir and did not enter the ESI source. Ion pairs were successively dissociated.and ion suppression from TFA was alleviated. The signals for the peptides were enhanced 9-35 times. The optimized conditions were also applied to the LC-MS analysis of a tryptic digests of bovine serum albumin. Using the EMC, good chromatographic performance from TFA was maintained.
Aminoglycans are highly polar compounds, and thus are usually analyzed using reversed phase liquid chromatography with the addition of HFBA ion pairing reagents. In this study, the EMC device was utilized to dissociate ion pairs between HFBA anions and positive charged aminoglycoside cations in capLC-ESI-MS. Based on the comparison of signal intensities with and without EMC, a lower detection limit in full scan in ESI-MS as well as a better signal sensitivity in MS/MS can be achieved with EMC under optimized conditions. In addition, we also attempted to apply the EMC concept on a higher flow rate analysis (e.g., 250 μL/min). The flow injection analysis (FIA) was used to demonstrate the possibility of a high flow rate separation system coupling with EMC. The coupling of EMC with a higher flow rate system was successful after splitting flow rate to the several hundred nanoliters per minute.The signal of analytes increased due to the removement of HFBA.
A new interface for CE-ESI-MS, pressure assisted double junction interface, was proposed by the combination of pressure and electric field to alleviate the MS signal suppressions resulted by non-volatile CTAB when analyzing positive charged triazines by CTAB dynamic coating CE-ESI-MS. The pressure assisted double junction interface was constructed by the polymerization of poly(dimethylsilane)(PDMS), including a short connecting capillary(1 cm) to connect two liquid junction reservoirs. One reservoir was used as an ESI-MS interface while the other was used to connect CE separation column. The liquid reservoir connected to the separation column was sealed by a poly(ethylene ketone)(PEEK) tubing with an inserted Pt wire, and a pressure was introduced using by a syringe pump. Due to (1) the pressure flow introduced from a syringe pump toward ESI sprayer, (2) the larger electrophoretic mobility difference among analytes and CTA+ by the addition of organic phase, and (3) a reversed electric field applied across the connecting capillary, only the analytes of lower electrophoretic mobilities can be carried toward MS following the direction of pressure flow, whereas of the higher electrophoretic mobility CTA+ was controlled by the electric field to remain in the junction reservoir and did not enter the ESI source. In this way, the problems of signal suppressions caused by positive charged salt CTA+ and contamination of ion source can be successfully solved and the signals of analytes were elevated for 20-60 fold.

並列關鍵字

LC-ESI-MS ； CE-ESI-MS ； electrophoretic mobility control (EMC) ； pressure assisted double junction interface ； ion suppression ； ion pair reagent ； cetyltrimethylammonium bromide (CTAB)

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