Quantitative profiling of 6-ketoprostaglandin F 1α, 2,3-dinor-6-ketoprostaglandin F 1α, thromboxane B 2 and 2,3-dinor-thromboxane B 2 in human and rat urine by immunoaffinity extraction with gas chromatography—mass spectrometry

Abstract

A rapid and simple method based on immunoaffinity extraction, stable isotope dilution and gas chromatography—mass spectrometry has been developed for profiling urinary metabolites of prostacyclin and thromboxane. 6-Ketoprostaglandin F 1α (6-keto-PGF 1α), 2,3-dinor-6-ketoprostaglandin F 1α (2,3-dinor-6-keto-PGF 1α), thromboxane B 2 (TXB 2) and 2,3-dinor-thromboxane B 2 (2,3-dinor-TXB 2) were quantitatively extracted from human or rat urine spiked with deuterated internal standards using mixed-bed columns containing immobilized anti-6-keto-PGF 1α and anti-TXB 2 antibodies (cross-reacting with 2,3-dinor-6-keto-PGF 1α and 2,3-dinor-TXB 2, respectively). The extract was directly derivatized to form pentafluorobenzyl ester, methyloxime, trimethylsilyl ether derivatives. Quantitation was performed by stable isotope dilution assay and high-resolution gas chromatography-negative ion chemical ionization mass spectrometry, by monitoring the carboxylate anions (M — 181) of the derivatized metabolites. The method was applied to evaluate the urinary excretion of 6-keto PGF 1α, 2,3-dinor-6-keto-PGF 1α, TXB 2 and 2,3-dinor-TXB 2 in humans and rats. Results were in accordance with previously reported data obtained by other methods. Novel data on the urinary excretion of 2,3-dinor-6-keto-PGF 1α in rats under basal conditions are presented. This sensitive and selective method represents a significant advance in terms of rapidity and simplicity over other immunoaffinity—gas chromatography-mass spectrometry methods for measuring single prostanoids, such as 6-keto-PGF 1α or TXB 2, since it allows profiling of a group of metabolites whose balance is important in several physiopathological conditions.