|Budget Amount *help
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
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.