B-194 Development and Validation of Rapid LC-MS/MS Method for Simultaneous Determination of 8-iso-PGF2α and 11-DHTXB2 in Human Urine

Abstract

Abstract Background Cardiovascular disease (CVD) is one of the leading causes of death globally, the prevalence ofCVD is expected to increase from 36.9% to 40.5% between 2010 and 2030 in the United States.Atherosclerosis (AS) is thought to be the primary cause of cardiovascular diseases (CVDs),however there are several others. About half of Americans between ages 45 and 84 haveatherosclerosis and don’t know it. Eicosanoids (8-iso-PGF2α and 11-Dehydrothromboxane B2) arethe most common biomarkers for predicting atherosclerosis. The objective of this study was todevelop a rapid LC-MS/MS method for simultaneous determination of 8-iso-PGF2α and 11-DehydrothromboxaneB2 (11-DHTXB2) in human urine. Methods One milliliter of human urine spiked with 0.5 µL of each internal standard (1 µg/mL) was mixedwith 1 mL of heptane, 1 mL of ethyl acetate, and 1 mL of Acetonitrile in a glass tube followed by theaddition of 1 mL of pure water. The mixture was vortexed for 1 min and then centrifuged (4000rpm) for f 5 min. The middle organic layer was transferred to another tube and dried undernitrogen and resuspended with 50 µL of the initial mobile phase composition (70:30 v/v H2O:ACN) for the analysis. One milliliter of human urine spiked with 0.5 µL of each internal standard(1 µg/mL) was loaded to a conditioned SPE cartridge, then washed with 1 mL of 10% ACN afterthat analytes eluted with 1 mL of pure ACN at the end eluent is collected, dried and reconstitutedin 50 µL of (70:30 v/v H2O: ACN). LC-MS/MS was performed using AB Sciex 5500 QTRAPtriple quadrupole tandem mass spectrometer coupled to Shimadzu HPLC with 10 minutesgradients for both samples. Results The method was optimized by using standard solutions of 8-iso-PGF2α and 11-dehydrothromboxane B2 and showed separation at 5.27 and 5.45 min respectively. The methodapplied on pooled urine sample from healthy individuals and showed No bias in retention time.Two purification methods were used to extract the two analytes from pooled urine samples, threephase liquid extraction (3PLE) and solid phase extraction (SPE). 3PLE showed a better resultsSPE method in eicosanoids extraction which results in higher intensity signals by 2-fold for 8-iso-PGF2α and 5-fold for 11-DHTXB2.The method was shown to be linear from 0.1 to 5 ng/mL forboth analytes (R2 ≥ 0.98). Conclusion The three-phase liquid extraction method extracted the two analytes better than the solid phaseextraction method, which is the most used purification method for eicosanoids. Furthermore, arapid and simple LC-MS/MS method for simultaneously measuring urinary 8-iso-PGF2α and 11-DHTXB2 was successfully developed.