微乳液電動層析及其線上濃縮技術於非類固醇抗發炎性藥物之應用

Abstract

本研究利用微乳液電動層析，在低pH值抑制電滲流的條件下，分離flurbiprofen、fenoprofen、naproxen、ketoprofen、suprofen、indoprofen等六種具有異丙酸共同結構，pKa相近，一般電泳不易分離的低水溶性非類固醇抗發炎藥物。由研究發現，微乳液在通電壓進行預平衡一段時間後，吸收度會突然劇烈下降，最後達一穩定狀態。以吸收度發生轉折前、後兩區段分別對分析物進行電泳分析，結果發現以吸收度發生轉折後的穩定態區段進行電泳分析時，實驗的再現性及靈敏度皆較佳。而吸收度發生轉折的時間與經由微乳液標記物dodecylbenzene所測得的微乳液遷移時間(tME)相符。研究中藉由改變緩衝液種類、pH值及有機修飾的添加，發現以微乳液組成為0.8% (w/w) n-octane、3.3% (w/w) SDS、6.6% (w/w) 1-butanol、5% (w/w) methanol及84.3% (w/w) phosphate buffer (25 mM, pH 5.5)，分析電壓-10 kV的條件下，可在40分鐘內使六種分析物達基線分離，平均理論板數為9100 m-1，滯留時間、譜峰高度及面積的RSD值皆＜5％(n=3)。由分析物的流析順序顯示微乳液與分析物間的疏水性作用力為主要的分離機制。為降低此方法的偵測極限，本研究進而以微乳液組成為0.8% (w/w) di-n-butyl-L-tartrate、3.3% SDS (w/w)、6.6% (w/w) 1-butanol、15 % (w/w) acetonitrile及74.3 % (w/w) phosphate buffer (25 mM, pH 2.5)作為分離條件，在分析電壓-17.5 kV下，首先以ASEI- sweeping MEEKC對分析物進行線上濃縮，發現與normal MEEKC相比，flurbiprofen、fenoprofen及naproxen的譜峰面積平均增大約280。為同時偵測六種分析物，改以SRMP (stacking with reverse migrating pseudostationary phase)進行線上濃縮，30分鐘內可使所有分析物達基線分離，且譜峰面積平均增大約170倍，偵測極限為2~45 μg/L。本研究藉由改變微乳液中油相、水相緩衝溶液的組成及有機修飾劑的添加，配合線上濃縮技術，提供了一種快速、簡易及高靈敏度的分析方法。

In this study, microemulsion electrokinetic chromatography under pH-suppressed EOF was investigated for the separation and on-line preconcentration of six non-steroidal anti-inflammatory drugs, including flurbiprofen, fenoprofen, naproxen, ketoprofen, suprofen and indoprofen. In the precondition process, the absorbance dropped suddenly after applying voltage for a period of time and finally reached a steady state. The absorbance inflection time consisted with the migration time of the microemulsion. Reproducibility and sensitivity were better when the analytes were injected after the absorbance drop. Selectivity and resolution were studied by changing the pH over the range from 2.5 to 5.5 and the variation of the organic modifier, such as acetonitrile, methanol and 1-propanol. The optimum microemulsion background electrolyte solution consisted of 0.8% (w/w) octane, 3.3% (w/w) SDS, 6.6% (w/w) 1-butanol, 5% (w/w) methanol and 84.3% (w/w) phosphate buffer (25 mM, pH 5.5). With an applied voltage of -10 kV, a baseline separation of the drug mixture could be achieved within 40 minutes. Under the optimized conditions, the reproducibility of the retention time, peak height and peak area were less than 5%. The elution order was mainly determined by the hydrophobility of these analytes. In order to enhance the sensitivity, two on-line preconcentration techniques were applied, including ASEI-sweeping and SRMP. Used microemulsion was composed of 0.8% (w/w) di-n-butyl-L-tartrate, 3.3% (w/w) SDS, 6.6% (w/w) 1-butanol, 15% acetonitrile and 74.3% (w/w) phosphate buffer (25 mM, pH 2.5). When ASEI-sweeping was used, the sensitivity which was evaluated by the peak area of flurbiprofen, fenoprofen and naproxen, was enhanced about 280-fold in comparision with normal MEEKC. When SRMP was used, all analytes could be detected simultaneously and baseline separation could be achieved within 30 minutes. The sensitivity which was evaluated by the peak area of all analytes except for indoprofen, was enhanced about 170-fold and the limits of detection were 2~45 μg/L.