Akira K, Nakamura T, Shinohara Y, Baba S
Tokyo College of Pharmacy, Japan.
Lipids. 1993 Apr;28(4):361-4. doi: 10.1007/BF02536324.
A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane, O-methylhydroxylamine and dimethyl-isopropylsilylimidazole. The recovery of total 14C-radioactivity was 93.1 +/- 7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.
描述了一种通过放射性气相色谱法（GC）分析花生四烯酸代谢产物的方法。将兔血小板与[14C]花生四烯酸的孵育混合物在Sep-Pak C18柱上进行纯化，并用重氮甲烷、O-甲基羟胺和二甲基异丙基硅咪唑进行衍生化。14C总放射性的回收率为93.1±7.2%。衍生化过程中的放射性损失可忽略不计。使用同步累积放射性同位素检测器（GC/SARD）通过GC在45分钟内单次运行即可实现[14C]花生四烯酸及其代谢产物的基线分离。从GC柱中回收的放射性几乎为100%。代谢产物的化学结构通过GC/质谱法确认；通过将亚甲基单元值与GC/SARD分析中的放射性峰的亚甲基单元值进行比较来确定花生四烯酸代谢产物的峰。GC/SARD分析中的批内变异系数小于10%。该方法用于绘制在吲哚美辛、黄芩苷或谷胱甘肽存在下兔血小板形成的花生四烯酸代谢产物的图谱。
