Isolation of prostacyclin from whole blood

A method was developed 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 F_1α (6-K-PGF_1α). Prostacyclin was stabilized in whole blood by rapidly raising the pH to 10 with Na₂CO₃ and cooling the samples to 0°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-PGF_1α from the alkaline plasma with recoveries of greater than 95% using an acetonitrile/2mM Na₂B₄O₇, pH 10, elution solvent mixture. An additional 1.7% hydrolysis was found during the column extraction procedure. Final separation of PGI₂ and 6-K-PGF_1α was performed with HPLC using an alkaline solvent system. This method is capable of rapidly and efficiently extracting and separating PGI₂ and 6-K-PGF_1α from whole blood or plasma. It introduces less than 5% hydrolysis of PGI₂, thus providing a means of applying highly sensitive 6-K-PGF_1α assays to the determination of PGI₂ levels in physiological samples.