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Detection of tetracyclines at low pars per billion level using non-aqueous capillary electrophoresis with laser-induced fluorescence detector
|關鍵字:||capillary electrophoresis;毛細管電泳;tetracyclines;laser-induced fluorescence;CE;LIF;TC;四環黴素;雷射螢光偵測||出版社:||獸醫學系暨研究所||引用:||Aga DS, O'Connor S, Ensley S, Payero JO, Snow D, Tarkalson D. Determination of the persistence of tetracycline antibiotics and their degradates in manure-amended soli using enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry. J Agric Food Chem 53: 7165-7171, 2005. Agwuh KN, MacGowan A. Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother 58: 256-265, 2006. Akhter MS, Alawi SM. The effect of organic additives on critical micelle concentration of non-aqueous micellar solutions. Colloids Surf A: Physicochem Eng Aspects 175: 311-320, 2000. Andersen CR, Rupp HS, Wu WH. Complexities in tetracycline analysis-chemistry, matrix extraction, cleanup, and liquid chromatography. J Chromatogr A 1075: 23-32, 2005. Andersen WC, Roybal JE, Gonzales SA, Turnipseed SB, Pfenning AP, Kuck LR. 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本研究主要目的為建立一個簡單有效且敏感的四環黴素類毛細管電泳（CE）偵測方法。研究採用非水相毛細管電泳法搭配雷射螢光偵測器（LIF detector）進行四環黴素類抗生素之偵測，不僅分析方法簡便且較傳統紫外光偵測方法敏感。為求進一步降低偵測極限，因而針對樣品注射方法、樣品注射時間、分析用毛細管內徑及分析緩衝液中添加界面活性劑等方向進行改良。所得之結果突破了以往毛細管電泳分析方法敏感度較差的缺點，將偵測極限降至4-40 ppb。最佳分析條件如下：10 kV電壓注射樣品90秒進入有效長度為20公分之100μm內徑毛細管中，並以含有500 mM meganesium acetate tetrahydrate及100 mM SDS之N-methylformamide溶液作為分析緩衝液，給予15 kV電壓進行分離，以發射波長488 nm之氬氣雷射，於激發波長520 nm進行偵測。依此條件，30分鐘內可完全分離3種四環黴素類波峰，其波峰出現順序分別為四環黴素（Tetracycline; TC）、羫四環黴素（Oxytetracycline; OTC）（或脫氧羥四環黴素（Doxycycline; DC））及氯四環黴素（Chlortetracycline; CTC）。波峰面積與濃度之對應關係於0.05 ppm至50 ppm間之線性關係達0.999。日內變異度及日間變異度試驗結果其標準偏差分別為1至6％及7.7至12.2％。血漿、尿液、飼料及牛奶內之四環黴素類抗生素經固相萃取步驟後則可以此方法進行殘留檢測。固相萃取管之基質回收率範圍在72.8-111％之間。若結合固相萃取之樣品濃縮，則混和四環黴素類抗生素總偵測極限（LOD）可達0.2 ppb至8 ppb之間；單一四環黴素類抗生素總偵測極限可達0.27ppb至2.67 ppb之間，為現有方法中最佳者。此方法已成功應用於偵測脫氧羥四環黴素在豬隻及雞隻之血漿內藥物濃度變化，並用以探討脫氧羥四環黴素之吸收率以及排除速率。此外，本方法亦成功應用於ELISA疑陽性動物組織中四環黴素類殘留量確認。此最佳化之CE-LIF方法不僅簡單，且具極佳的偵測極限，為一方便且可常規使用之四環黴素類抗生素檢測方法。
A capillary electrophoresis method with laser induced fluorescence detector was developed for sensitive detection of tetracyclines(TCs)in biological samples including plasma, urine, feed and milk. CE is a powerful technique for analysis of TCs because of its high efficiency, high resolution and ability to analyze high ionizable compounds. Laser induced fluorescence (LIF) is a very sensitive detection method for CE that has become very applicable due to the availability of a wide variety of fluorescent tags and lasers . Non-aqueous capillary electrophoresis (NACE) is one kind of CE modes that not only can provide sample stacking during analysis but can also enhance the LIF signal by organic solvent. In order to improve the limit of detections(LODs)of TCs, injection mode, injection times, inner diameter of analytic capillary and surfactant in analytic buffer were studied. TCs were injected at 10 kV for 90 s and separated in a 31 cm × 100 μm uncoated capillary at 15 kV and detected at 520 nm with Argon laser emitting light at wavelength of 488 nm. The run buffer contained 500 mM magnesium acetate tetrahydrate and 100 mM lauryl sulfate(SDS)dissolved in N-methylformamide. With optimized coditions, the LODs of mixed TCs could reach 4 ppb to 40 ppb, and the LODs of single TC could reach 1.33 ppb to 13.3 ppb. To purify and further improve LODs from biological samples, solid phase extraction(SPE)procedure was developed with the recoveries ranging from 72.8-111％ in plasma, urine, feed and milk. Taken together the instrumental LOD and SPE concentration, the overall LODs for mixed TCs were 0.8 ppb to 8 ppb in plasma and urine, and 0.2 ppb to 2 ppb in feed and milk, with working ranges linear from 0.05 to 50 ppm. The developed CE-LIF method was proved to be less complicated and more sensitive than the best method at present. This analytic method has been successfully applied to plasma samples from swine and chicken receiving Doxycycline and to confirm suspected ELISA-positive bovine serum samples.
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