Isolation of prostacyclin from whole blood.
A method was development for the isolation of prostacyclin (PGI2) from whole blood in a fraction suitable for high pressure liquid chromatography (HPLC) separation of PGI2 and 6-keto-prostaglandin F1 alpha (6-K-PGF1 alpha). Prostacyclin was stabilized in whole blood by rapidly raising the pH to 10 with Na2CO3 and cooling the samples to 0 degree C. Under these conditions, 2.9% hydrolysis was observed after 20 min. Reverse phase extraction columns were used to directly extract both PGI2 and 6-K-PGF1 alpha from the alkaline plasma with recoveries of greater than 95% using an acetonitrile/2mM Na2B4O7, pH 10, 40/60 elution solvent mixture. An additional 1.7% hydrolysis was found during the column extraction procedure. Final separation of PGI2 and 6-K-PGF1 alpha was performed with HPLC using an alkaline solvent system. This method is capable of rapidly and efficiently extracting and separating PGI2 and 6-K-PGF1 alpha from whole blood or plasma. It introduces less than 5% hydrolysis of PGI2, thus providing a means of applying highly sensitive 6-K-PGF1 alpha assays to the determination of PGI2 levels in physiological samples.
D015121:6-Ketoprostaglandin F1 alpha / Q000097:blood; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D011464:Epoprostenol / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011453:Prostaglandins / Q000097:blood*; D011817:Rabbits
D011453:Prostaglandins; D015121:6-Ketoprostaglandin F1 alpha; D011464:Epoprostenol