Analysis of prostaglandins in aqueous solutions by supercritical fluid extraction and chromatography.

Trace amounts of prostaglandins (PGs) were selectively analysed without derivatization using supercritical fluid extraction (SFE) and open tubular column supercritical fluid chromatography (SFC). The specific compounds studied were prostaglandin F2 alpha (PGF2 alpha), esters of PGF2 alpha, prostaglandin F1 alpha) and prostaglandin E2 (PGE2). An open tubular column was used with carbon dioxide as the mobile phase and with universal flame ionization detection. Samples were introduced into the column by direct injection using a 1-microliter sample loop or by SFE with solute focusing. The 11 standard compounds were effectively separated within 35 min using a density program at constant temperature. The minimum detectable quantity (signal-to-noise ratio = 3) using the direct injection method was 9 ng for 15-propionate PGF2 alpha isopropyl ester. Using the extraction method, the sample size in the extraction cell was increased to 100 microliters, which made it possible to analyse compounds that were present in low concentrations. Aqueous PG samples were extracted from adsorbents onto which the samples had been loaded.

Dioxygen transfer during vitamin K dependent carboxylase catalysis.

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.