HIGHLY SENSITIVE METHOD FOR THE DETERMINATION OF ARYLHALIDES BY USING A FLUORESCENT ARYLBORONIC ACID AS A LABELING REAGENT

以荧光芳基硼酸为标记试剂的高灵敏度测定芳基卤化物的方法

• 批准号: 13672256
• 负责人: KURODA Naotaka
• 金额: $ 0.7万
• 依托单位: NAGASAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672256/
• 关键词: FLUORESCENT LABELING REAGENT; HPLC; ARYLBORONIC ACID; ARYL HALIDES; FLUORESCENCE DETECTION; CLOFIBRATE

Derivatization of aryl halides with a fluorescent arylboronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl) phenylboronic acid (DPA), based on Suzuki coupling reaction was investigated. Several bromobenzenes were chosen as analytes and their derivatization conditions with DPA were optimized. This reaction proceeded well in the presence of palladium (II) acetate, 2-(dicyclohexylphosphino) biphenyl and potassium fluoride by the use of dioxin as a solvent. Resultant fluorescent derivatives were separated on a reversed-phase HPLC and detected at 410 nm wife excitation at 320 nm. Calibration curves were linear up to 100 pool/injection with detection limits of sub and lower pmol levels. Labeling efficiencies were investigated using various aryl iodides and chlorides in addition to aryl bromides. Effects of subsistents position of aryl bromides on the reactivity with DPA were also examined using bromotoluene and bromoanisole.To evaluate the scope and limitation of analytical performance of the derivatization method, applicability to biological samples was examined with clofibrate in human plasma. Clofibtare spiked to plasma sample (50 μl) was extracted with liquid-liquid extraction using ethyl acetate, and then applied to the labeling reaction, followed by HPLC separation. For the efficient label of clofibrate, a mixture of water and N,N-dimethylformamide, and potassium hydroxide were used in place of dioxane and potassium fluoride, respectively, for the labeling of bromobenzenes. The calibration curve of clofibrate spiked into human plasma were linear (r=0.998) over the range of 1-400 mol/ml and the detection limit was 170 pmol/ml (280 fool/injection) at a signal to noise ratio of 3.The method for the determination of clofibrate was highly sensitive and the proposed method should be useful for the analysis of other pharmaceuticals involving aryl halide moiety.

基于Suzuki偶联反应，研究了荧光芳基硼酸4-（4，5-二苯基-1H-咪唑-2-基）苯基硼酸（DPA）对卤代芳烃的衍生化反应。选择了几种溴苯类化合物作为待测物，优化了它们与DPA的衍生化条件。该反应在乙酸钯（II）、2-（二环己基膦基）联苯和氟化钾存在下通过使用二恶英作为溶剂进行得很好。在反相HPLC上分离所得荧光衍生物，并在410 nm和320 nm激发下检测。校准曲线线性高达100池/进样，检测限低于和低于pmol水平。除了芳基溴之外，还使用各种芳基碘和氯化物研究了标记效率。用溴甲苯和溴苯甲醚考察了溴代芳烃的取代位置对衍生化反应的影响，并以人血浆中的氯贝特为例考察了衍生化方法对生物样品的适用性。加标到血浆样品（50 μl）中的氯吡格雷用乙酸乙酯进行液-液萃取，然后用于标记反应，随后进行HPLC分离。对于氯贝特的有效标记，水和N，N-二甲基甲酰胺的混合物，和氢氧化钾被用来代替二氧六环和氟化钾，分别用于溴苯的标记。在1-400 mol/ml范围内，线性关系良好（r=0.998），检出限为170 pmol/ml（280 μ mol/注射），信噪比为3。该方法灵敏度高，可用于其它含卤代芳烃药物的分析。

项目成果

Mitsuhiro Wada, et al.: "A simple HPLC-fluorescence detection of nitric oxide in cultivated plant cells by in situ derivatization with 2,3-diaminonaphthalene"Analytical Sciences. 18. 631-634 (2002)

Mitsuhiro Wada 等人：“通过使用 2,3-二氨基萘进行原位衍生化，对培养植物细胞中的一氧化氮进行简单的 HPLC 荧光检测”分析科学。

N.Kuroda, et al.: "Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent"J.Pharm.Biomed.Anal.. 30. 1743-1749 (2003)

N.Kuroda 等：“使用荧光标记试剂通过 HPLC 测量人血清和腹水中的双酚 A 水平”J.Pharm.Biomed.Anal.. 30. 1743-1749 (2003)

S. Sugihara, et al.: "Derivatization of aryl halides with a newly developed fluorescent arylboronic acid"Analytical Sciences. 17 Supplement. i1261-i1262 (2001)

S. Sugihara 等人：“用新开发的荧光芳基硼酸衍生化芳基卤化物”分析科学。

N. Kuroda: "Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent"J. Pharm. Biomed. Anal.. 30. 1743-1749 (2003)

N. Kuroda：“使用荧光标记试剂通过 HPLC 测定人血清和腹水中的双酚 A 水平”J。

Naotaka Kuroda, et al.: "Chemiluminescence Detection in Liquid Chromatography in "Chemiluminescence in Analytical Chemistry" ed.By W.R.G.Baeyens, et al."Marcel Dekker, Inc.. 34 (2001)

Naotaka Kuroda 等人：W.R.G.Baeyens 等人编着的“分析化学中的化学发光”中的“液相色谱中的化学发光检测”Marcel Dekker, Inc.. 34 (2001)