Improved Assay For 6-Keto Prostaglandin F1α In Biological Fluids By Reversed-Phase High-Performance Liquid Chromatography In Conjunction With Radioimmunoassay

Prostacyclin production has been proposed as a fundamental mechanism by which intact vascular endothelium maintains a non-thrombogenic surface. Interest in a reliable assay for its stable breakdown product 6-keto prostaglandin F1α (6-KPGF1α) is, therefore, considerable. Previous studies have emphasised the need to reduce sample complexity prior to the quantification of PG’s by radioimmunoassay (RIA). This has been achieved by extraction of PG’s into organic solvents or onto resin columns followed by separation of PG metabolites by reversed-phase high-performance liquid chromatography (HPLC), but retention times are long if adequate resolution is to be obtained. By using an 8-min gradient elution programme with acetonitrile/water/acetic acid and a 6 ml/min flow rate through Waters reversed-phase (Radial Pak A) column under radial compression good separation of 6-KPGF1α from its closely-related metabolites 13,14-dihydro 6-KPGF1α 6, 15-diketo PGF1α as well as from other PG species was achieved as detected by ultra-violet absorbance spectrometry. In applying the method to the measurement of 6-KPGF1α in urine, PG’s were extracted onto a resin column, eluted in methanol and the extract subjected to HPLC as described. Good resolution of immunoassayable PG metabolites and known cross-reacting species was achieved. This method provides a significant improvement on existing PG assays by reducing sample-processing time dramatically, and is suitable for routine and repetitive clinical use.