Quantitative profiling of 6-ketoprostaglandin F1 alpha, 2,3-dinor-6-ketoprostaglandin F1 alpha, 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 F1 alpha (6-keto-PGF1 alpha), 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha), 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-PGF1 alpha and anti-TXB2 antibodies (cross-reacting with 2,3-dinor-6-keto-PGF1 alpha 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 PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha, 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-PGF1 alpha 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-PGF1 alpha or TXB2, since it allows profiling of a group of metabolites whose balance is important in several physiopathological conditions.
Chiabrando
C
C
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Pinciroli
V
V
Campoleoni
A
A
Benigni
A
A
Piccinelli
A
A
Fanelli
R
R
D015121:6-Ketoprostaglandin F1 alpha / Q000031:analogs & derivatives* / Q000652:urine*; D000818:Animals; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013929:Thromboxane B2 / Q000031:analogs & derivatives* / Q000652:urine*
D013929:Thromboxane B2; D015121:6-Ketoprostaglandin F1 alpha; C028965:2,3-dinor-thromboxane B2; C028290:2,3-dinor-6-ketoprostaglandin F1alpha