Antibody-mediated extraction/negative-ion chemical ionization mass spectrometric measurement of thromboxane B2 and 2,3-dinor-thromboxane B2 in human and rat urine.
An antibody-mediated extraction method for gas chromatographic-mass spectrometric analysis of thromboxane A2 (TXA2) urinary metabolites is reported. An antibody (Ab) raised against thromboxane B2 (TXB2) (35% cross-reacting with 2,3-dinor-TXB2) was coupled to CNBr-activated Sepharose 4B (Se) and used as stationary phase for simultaneous extraction of both compounds from urine. After addition of deuterium-labeled TXB2 as internal standard, rat or human urine was percolated through a small Ab-Se column. After being washed, the eluate was directly derivatized to the pentafluorobenzyl ester, methyloxime, and trimethylsilyl ether. Quantitation was performed by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring the carboxylate anions. This method was applied to evaluate the urinary excretion of TXB2 and 2,3-dinor-TXB2 in humans and rats. We report on the excretion of 2,3-dinor-TXB2 in the rat. This novel approach to the extraction of urinary thromboxanes is more convenient than currently available methods in terms of simplicity, rapidity, and recovery. This method could be extended to any other prostanoid for which an antibody could be obtained.
D000818:Animals; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000096:biosynthesis; D008297:Male; D051381:Rats; D012685:Sepharose; D013929:Thromboxane B2 / Q000031:analogs & derivatives* / Q000276:immunology / Q000652:urine*
D007074:Immunoglobulin G; D013929:Thromboxane B2; C028965:2,3-dinor-thromboxane B2; D012685:Sepharose