Development of a gas chromatographic-mass spectrometric method using a stable isotope internal standard for quantitation of thromboxane B2.

Tetradeuterated 19, 19',20,20'-2H4-thromboxane B2 was synthesized and used as a stable isotope internal standard for the development of a gas chromatographic/mass spectrometric (GC/MS) method to quantitate thromboxane B2. Quantitation was based on the detection of fragment ions at m/z 301 for thromboxane B2 and m/z 305 for the corresponding tetradeuterated internal standard. At m/z 301/305 a response equivalent to a protium/deuterium ratio of 0.2% thromboxane B2 could be measured with a standard deviation of 15% when 100 nanograms of the tetradeuterated internal standard was analyzed. For measurement of cellular biosynthesis of thromboxane B2 in whole blood or platelet rich plasma, the internal standard and naturally occurring thromboxane B2 were i) isolated by solvent extraction, or sequestration to XAD-2 columns; ii) purified by reversed phase high performance liquid chromatography; and iii) converted to methoxime methyl ester trimethylsilyl ethers prior to analysis. A limited comparison was made using both radioimmunological and mass spectrometric quantitation of thromboxane B2.