Selective Solid-Phase Extraction of Urinary 2,3-Dinor-6-ketoprostaglandin F1α for Determination with Radioimmunoassay

Abstract

This paper describes a method for selective two-step solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F1α for reliable determination with radioimmunoassay. In the immunoreactivity profile of nonselectively extracted urine after HPLC separation, over 90% of the total 2,3-dinor-6-ketoprostaglandin F1α immunoreactivity consisted of interfering material coeluting with 6-ketoprostaglandin F1α and 2,3-dinor-6-ketoprostaglandin F1α. Among the alkyl silica sorbents studied (methyl, butyl, octyl, and octadecyl), an efficient separation of 2,3-dinor-6-ketoprostaglandin F1α from 6-ketoprostaglandin F1α and the lowest immunoreactive concentration of analyte were achieved in extraction on the methyl silica sorbent by elution of 2,3-dinor-6-ketoprostaglandin F1α with chloroform:hexane (85:15, v/v) from the cartridge. The proportion of specific immunoreactivity could be further increased by two-step extraction of sample on methyl silica cartridges, first at pH 3 and then at pH 10 using diethyl ether:hexane (85:15, v/v) and chloroform as eluent, respectively. After this, a high correlation was found with concentrations of samples determined by radioimmunoassay using three different antisera. A significant correlation of values was also observed between samples measured by radioimmunoassay and those measured by GC-MS. The values of 12-h excretion of 2,3-dinor-6-ketoprostaglandin F1α in eight volunteers (268 ± 204 ng/g creatinine, mean ± SD) as well as the inhibitory effect of acetylsalicylic acid (74 ± 12%) are in accordance with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring subsequent TLC or HPLC purification.