B-5. 4-Bromomethyl-7-acetoxycoumarin (Br-Mac) を利用したProstaglamdinsの高速液体クロマトグラフィー

抄録

4-Bromomethyl-7-acetoxycoumarin (Br-Mac) reacted with prostaglandins (PGs) to form the ester derivatives in iacetone solvent in the presence of KHCO₃ and dibenzo-18-crown-6. The esters were separated by high-performance liquid chromatography (HPLC). Each lebeled cornpund eluted from a column was successively mixed with 0.1 N NaOH and hydrolyzed to the fluorescent coumarin derivative, and then introduced to a flow-through fluorometer (Ex 365, Em 460nm). In this system, whatever the kind of PG is, the fluorophore to be detected is common to all PGs. Therefore, the fluorescence quontum yield was hardly influenced by the PG moiety, compared with 4-bromomethyl-7-methoxycoumarin (Br-Mmc), which was previously reported as a fluorescence labeling reagent for HPLC of PGs. Gradient elution technique could be more effectively used in this system than Br-Mac method because the fluorescence intensity of this hydrolysate was not subject to the solvent effect. It was found that Br-Mac could react with all PGs and related compounds investigated in this work: PG F_1α, F_2α, E₁, E₂, D₁, D₂, B₁, B₂, A₁, A₂, 6-keto-F_1α, TX B₂ and arachidonic acid etc. Calibration graph of PGs showed good linearity at least from 1 nmol to 5 pmol and the detection limit was about 10 fmol. Attempts were made to apply this method to the determination of PGs in human seminal fluid. After deproteinization with methanol containing 16-methyl-PG F1α (internal standard), the supernatant was acidified with 0.1 N HCl (pH 3-4) and extracted with ethyl acetate. The organic phase was evaporated and then allowed to react with Br-Mac. An aliquot of the resulting solution was injected to a HPLC column. The peaks corresponding to PG F2α, E2 and E1 were found on the chromatogram, but any prominent peaks corresponding to PG B and A series (and their 19-OH analogs) could not be found, the occurence of which was previously reported. These results seem to agree with the suggestion of Jonsson et al. that some, if not all, of the PGs of the B and A series (and their 19-OH analogs) in human seminal fluid were artifacts.