1987 Fiscal Year Final Research Report Summary
Sensitive Determination of Biologically Active Unsaturated Compounds by HPLC with Chemiluminescence Dewtection
| Project/Area Number |
61571036
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | Toho University |
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Principal Investigator |
YUKI Hidetaka Schoo. of Pharmaceutical Science, Toho University, 薬学部, 教授 (80028828)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASAKI Hideki School of Pharmaceutical Science, Toho University, 薬学部, 講師 (30120257)
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| Project Period (FY) |
1986 – 1987
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| 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 |
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| 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.
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