Quantitative profiling of 6-ketoprostaglandin F1α, 2,3-dinor-6-ketoprostaglandin F1α, thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine by immunoaffinity extraction with gas chromatography-mass spectrometry

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 B₂ (TXB₂) and 2,3-dinor-thromboxane B₂ (2,3-dinor-TXB₂) 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-TXB₂ antibodies (cross-reacting with 2,3-dinor-6-keto-PGF_1α and 2,3-dinor-TXB₂, 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, TXB₂ and 2,3-dinor-TXB₂ 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 TXB₂, since it allows profiling of a group of metabolites whose balance is important in several physiopathological conditions.