Microdetermination of 11-dehydrothromboxane B3 in human urine by gas chromatography--selected-ion monitoring using (18O2) analogue as an internal standard.

The microdetermination of 11-dehydrothromboxane B3 (11-dehydro-TXB3) in human urine is described. We prepared [18O2]11-dehydrothromboxane B3 for use as an internal standard. Samples containing an [18O2] analogue were extracted chromatographically using Sep Pak tC18 and a silica gel column. Conversion of the extracted 11-dehydro-TXB3 to 1-methyl ester-11-n-propylamide-9,12,15-dimethylisopropylsilyl ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using an MP-65HT column. Human urine was monitored using the ions of m/z 696.4511 (11-dehydro-TXB3), m/z 700.4597 (18O2]1l-dehydro-TXB3), m/z 698.4668 (11-dehydro-TXB3) and m/z 702.4918 ([2H4]11-dehydro-TXB2). 11-Dehydro-TXB3 and [18O2]11-dehydro-TXB3 appeared at the same retention time and were eluted 15 s after 11-dehydro-TXB2 on this capillary column. This difference in retention time was due to the double bond in the omega3 position in 11-dehydro-TXB3. A good linear response over the range of 10 pg to 10 ng/tube was demonstrated. We detected 11-dehydro-TXB3 in the range from 1.29 to 7.64 pg/mg creatinine in the human urine. In the healthy volunteers, the 11-dehydro-TXB3 level was less than 1% of that of the 11-dehydro-TXB2, but 11-dehydro-TXB3 was increased by dietary supplementation of eicosapentaenoic acid. This method can be applied to the determination of 11-dehydro-TXB3 in human urine.