Resolution of prostaglandin p-nitrophenacyl esters by liquid chromatography and conditions for rapid, quantitative p-nitrophenacylation

High-Performance Liquid Chromatographic Analysis of Prostaglandins Formed during In Vitro Incubations with Prostaglandin 15-Dehydrogenase

“…Chromatography-The principle of forming para-substituted phenacyl esters of prostaglandins to improve the detection limit with a 254-nm UV detector was described previously, and separations were reported by adsorption chromatography on microparticulate silica gel (11). The rationale for using reversed-phase, liquid chromatography as the basis for a quantitative assay on the in vitro enzyme system is due to the fact that the 15-keto metabolites are less polar than their parent compounds and elute close to the chromatographic void volume with only partial resolution on a microparticulate silica gel adsorption column, thereby presenting difficulties for quantitative analysis.…”

“…Procedure-Deriuatization-Prostaglandin p-bromophenacyl esters were prepared by a modification of the procedure of Morozowich and Douglas (11). Briefly, the prostaglandin (500 pg or greater) was dissolved in 1.0 ml of anhydrous acetonitrile containing a threefold molar excess of a,p-dibromoacetophenone.…”

Determination of 5-trans-prostaglandin E2 in prostaglandin E2 via high performance liquid chromatography of their p-nitrophenacyl esters on a silver ion-loaded cation exchange resin

“…Resolution was not adequate to separate completely the ester of 5- irons-PGE2 from those of ll-ep¿-PGE2, PGEi, and 8-iso-PGE2; other work had previously established the absence of these latter compounds in the PGE2 samples examined. Morozowich and Douglas have demonstrated that the base-catalyzed esterification conditions used in our procedure yield quantitative conversion of the prostaglandins to p-nitrophenacyl esters (6). Our modification of that procedure with the addition of silver nitrate was necessary to remove bromide generated in the reaction and excess reagent.…”

“…In Situ Preparation of p-Nitrophenacyl Esters. The p-nitrophenacyl esters of the prostaglandins were prepared for chromatography on the silver ion-loaded Vydac column by a modification of the procedure of Morozowich and Douglas (6). A 10-mg portion of the prostaglandin sample was weighed accurately into a 2-ml volumetric flask; 1 ml of a stock acetonitrile solution containing 15 mg/ml of 2bromo-4'-nitroacetopherione was added, followed by 5 µ of N,Ndiisopropylethylamine.…”

“…Chromatographic separation of a large number of closely related prostaglandins of the E-series as their p-nitrophenacyl esters on microparticulate silica gels has been recently reported (6). Formation of the uv-absorbing ester derivatives was necessary for detection of the prostaglandins in liquid chromatography with the commonly used 254-nm uv detector.…”