利用奈米粒子固相萃取程序結合毛細管電泳線上濃縮技術於菸草葉、洗髮精與藥膏中水楊酸之定量分析

A Nanoparticle-Based Solid-Phase Extraction Procedure Followed by On-Line Concentration Technique in Capillary Electrophoresis to Determine Salicylic Acid in Tobacco Leaves, Shampoo and Ointment Samples

張育瑄(Yu-Hsuan Chang)

指導教授 ： 林泱蔚

國立彰化師範大學/理學院/化學系/碩士(2016年)

摘要

水楊酸(Salicylic acid，SA)為普遍植物中常見的的激素，又因為有良好的生物相容性、抗菌力、消炎等功效，SA被大量應用在藥物與生活用品中。首先，本研究針對SA、水楊酸甲酯(methyl salicylate, MeSA)、茉莉酸(jasmonic acid, JA)等目標物分子，探討毛細管電泳(Capillary electrophoresis, CE)連接紫外光偵測器(ultraviolet detector, UV)之最佳化分離參數（前導溶液：400 mM Tris Borate buffer (pH 10.0), 100 mM SDS, 15% Acetonitrile，分離溶液：100 mM Tris borate buffer (pH 9.0), 100 mM sodium dodecyl sulfate (SDS), 15% Acetonitrile, 0.3% poly(ethylene oxide) (MW: 8M)，分離電壓為10 kV）。同時利用毛細管電泳線上濃縮機制降低此三種物質的偵測極限，分別為SA: 3.2310-8 M ；MeSA: 9.8410-8 M；JA: 6.3510-7 M。其濃縮倍率最高可提升至131.4倍。其濃縮機制為大體積之分析物下於高黏度聚合物之界面進行堆積作用，使濃縮倍率隨之提升。為了更進一步提高偵測靈敏度，在本研究的前處理階段，針對目標分析物SA開發出一套前處理模式，以氧化鐵奈米粒子對SA獨特的選擇性，成功於真實樣品中純化並濃縮SA。結合此前處理方法，其濃縮倍率可至1120倍，SA之偵測極限為3.7910-9 M。利用此分析方法可偵測正常的圓葉菸草葉片(Nicotiana benthamiana)與受台灣番茄捲葉病毒(Nicotiana benthamiana-ToLCTWV)感染的圓葉菸草葉片上的SA含量。初步結果發現，受台灣番茄捲葉病毒感染的菸草其SA濃度較正常的菸草葉片低(81.5%)。本方法也可應用於治療疣症的市售藥膏與市售洗髮精中SA含量分析，其回收率為93.6%-102.7%。

關鍵字

水楊酸 ； 毛細管電泳 ； 氧化鐵奈米粒子 ； 圓葉菸草 ； 菸草 ； 洗髮精 ； 藥膏

並列摘要

Salicylic acid (SA) is a plant hormone which possesses biocompatibility, antibacterial activity, anti-inflammation ability, thus SA was used widely in our living life. My study is focused on determination of SA, methyl salicylate (MeSA), jasmonic acid (JA) by using capillary electrophoresis (CE) with ultraviolet detection. Under optimum conditions (leading buffer: 400 mM Tris Borate (TB) buffer (pH 10), 100 mM SDS, 15% ACN; running buffer: 100 mM TB buffer (pH 9), 100 mM sodium dodecyl sulfate (SDS), 15% acetonitrile(ACN), 0.3% poly(ethylene oxide, PEO) (MW: 8M)), the limit of detection (LOD, S/N =3) for JA, SA, and MeSA was 0.635, 0.0323 and 0.0986 µM, respectively. The enhance factors were achieved to 36.2 to 131 folds comparing to separation without stacking technique. The stacking mechanism by the proposed method is based on the mobility of analyst slowed down in the interface between high viscosity of PEO solution and background electrolyte, thus analyst got concentration. Further to improve detection sensitivity of SA, magnetic Fe3O4 nanoparticles were used to selectively extract SA. As we known, the enediol group of the SA coordinated to Fe3+ ions on the Fe3O4 nanoparticles surface, leading to the formation of Fe3+-SA coordination compounds. Combining CE on-line concentration and Fe3O4 nanoparticle based extraction, sensitivity enhancement for SA was achieved to 1119 folds with LOD (S/N =3) as low as 3.79 nM. This method has the potential to be an effective analytical tool for SA in different tobacco leaves, shampoo and ointment. The recoveries for real samples (tobacco leaves, shampoo and ointment) were ranged from 92.8-105.2%.

並列關鍵字

Salicylic acid ； capillary electrophoresis ； magnetic Fe3O4 nanoparticles ； tobacco leaves ； shampoo ； ointment

參考文獻

2. WEI-SHENG HUANG, Y.-P.C., CHYN-LIANG HUANG, and M.-C.H.A.M.-C. TZOU, Quality Investigation of Salicylic Acid in Cosmetics in Taiwan Area. 藥物食品檢驗局調查研究年報, 2008. 26: p. 19-23.

3. Carr, S.C.a.J.P., Cyanide Restores N Gene–Mediated Resistance to Tobacco Mosaic Virus in Transgenic Tobacco Expressing Salicylic Acid Hydroxylase. Stephen Chivasa and John P. Carr, 1998. 10: p. 1489–1498.

4. Leslie Friedrich, B.V., Tom Gaffney, Alison Morse, John Ryals, Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene. Plant Molecular Biology, 1995. 29: p. 959-968.

5. Xiong, X.J., et al., Ultrasensitive determination of jasmonic acid in plant tissues using high-performance liquid chromatography with fluorescence detection. J Agric Food Chem, 2012. 60: p. 5107-11.

7. Tian, M., et al., Efficient capillary electrophoresis separation and determination of free amino acids in beer samples. Electrophoresis, 2014. 35: p. 577-84.