Measurement of the 15-keto-13,14-dihydro metabolite of prostaglandin E2 (PGE2) in plasma is a more reliable index of the entry of PGE2 into the circulation than direct quantitation of PGE2. This chapter presents the procedures for preparation of 3,3,4,4-tetradeutero- 15K-H2-PGE2 for use as an internal standard for gas chromatography–mass spectrometry (GC-MS) assay of the PGE2 metabolite, for minimization of loss of endogenous 15K-H2-PGE2 in blood samples prior to addition of the tetradeuterated internal standard, and for stabilization of 15K-H2-PGE2 via derivatization for prevention of additional loss during purification. 3,3,4,4-Tetradeutero-15K-H2-PGE2 and 5,6,8,11,12,14-hexatritiated- 15K-H2-PGE2 ([3H]15K-H2-PGE2) are simultaneously prepared from 3,3, 4,4-tetradeutero-PGE2 and 5,6,8,1 l, 12,14,15-heptatritiated-PGE2 ([3H]PGE2), respectively, via incubation with the 100,000 g subcellular fraction of either swine kidney or guinea pig liver. In partial derivatization, the plasma extract containing 15K-H2-PGE2 is oximized and esterified. Quantitation of 15K-H2-PGE2 in plasma employing GC-MS is performed by simultaneously monitoring characteristic fragment ions of the bismethyloxime-methyl ester-trimethylsilyl ether derivative of 15K-H2- PGE2 and of 3,3,4,4-tetradeutero-15K-H2-PGE2.
ASJC Scopus subject areas
- Molecular Biology