Abstract
No useful profiling analyses of prostaglandins (PGs) in biological matrix have been reported. We present here a quantitative profiling analysis of urinary PGs determined by GC or GC-MS using a new simple extraction method. In both cases ODS silica column (Sep-Paks, Waters) was used for extraction of PGs from human urine. This column was pretreated with 20 mlethanol followed by 20 mlwater before use. The 20 mlsample was acidified with formic acid to pH 3.0 and passed through a ODS silica column using a glass syringe. The column was eluted successively with 20 mlethanol/water (15/85), 20 mlpetroleum ether and 10 mlmethyl formate. The recovery rate estimated using3H-PGs in methyl formate fraction was as follows. 6-keto-PGF1α: 68%, PGE2: 60%, PGF2α: 65%. The samples were derevatized to 6-keto-PGF1α-MOX-TMS-Bz, PGE2-MOX-TMS-Bz and PGF2α-TMS-Bz after extractive alkylation using THAH and benzyl bromide. GC peaks of the PG derivatives appeared at the same retention time as external standards. The quantitative determination of endogenous PGs were thus achieved by GC. d7-6-keto-PGF1α, d7-PGE2and d8-PGF2αwere added as internal standards to the human urine, which was treated with the same procedure as above. PGs in the methyl formate fraction were derivatized to 6-keto-PGF1αMOX-Me-TMS, PGE2-Me-TMS and PGF2α-Me-TMS. The sample was analysed by GC-MS (JEOL D-300) using selected ion monitoring.
In conclusion, we have developed a new simple extraction method for PGs using a convenient ODS silica column chromatography in combination with the extractive alkylation. It seems to be very useful for the quantitative profiling analysis of urinary PGs.
