Sensitive Determination of Biologically Active Unsaturated Compounds by HPLC with Chemiluminescence Dewtection

Research Project

Project/Area Number	61571036
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Physical pharmacy
Research Institution	Toho University
Principal Investigator	YUKI Hidetaka Schoo. of Pharmaceutical Science, Toho University, 薬学部, 教授 (80028828)
Co-Investigator(Kenkyū-buntansha)	KAWASAKI Hideki School of Pharmaceutical Science, Toho University, 薬学部, 講師 (30120257)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥1,800,000 (Direct Cost: ¥1,800,000) Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000) Fiscal Year 1986: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywords	Chemiluminescence / Bioactive unsaturated fatty acid / Eicosapentaenoic acid / High-performance liquid chromatography / Unsaturated fatty acid / Luminol / Reversed phase / プロスタグランジン / アラキドン酸
Research Abstract	Luminol delivatives were separated by reversed-phase high performance liquid chromatography(HPLC). The eluate was mixed with 0.2 um mictoperoxidase(phosphate buffer solution, PH 8.6) and 90 mM bhdrogen perxide (phosphate buffer solution. PH7.4) with the aid of double-planger pumps successively. and the chemiluminescence was detected in a flow cell. The floe ratg of HPLC. microperoxidase and hydrogen peroxide were 0.5, 0.5 and 1.0 ml/min, respectively. In the frow-injection method, the calibration curve of isoluminol was lunear in the range of 1 to 500 fmol, and the detection limit of luminol was 100 amol. Eicosapentaenoic acis(EPA) and other fatty acids were labeled with N-(4-aminobutyl)-Nethyl-isoluminol (ABEI). The labeling reaction was carried out at 90゜C for 30 min by 3,4dihydro-2H-prrido(1,2-a)pyrimidine-2-on and 2-chloro-1-methyl-pyridinium iodide. The separation of the laberled compounds on HPLC was wchieved with 75% methamol-phosphate buffer solution(PH 6.5). Under thest conditions, unreacted ABEI and C14 to C20 saturated and unasaturated garry acids were well resolved and the reproducibility of chromatograms was excellent. This method applied for the analysis of fatty acids extracted from normal human serum. Margaric acis was used as a internal standard. The concentration of EPA determined was 0.5 unol/ml, and eleven fatty acids werte also detected. In the case of prostagrandins, the HPLC separation was carried out by the gradient elution from 40 to 90% methanol in phosphate buffer solution(PH 6.0). Prostagrandin F2x and prostagrandin B2 were labeled almost quantitatively at 50゜C for 30 min.