ABSTRACT
A novel method for the simultaneous determination of 3-nitrotyrosine (NT) and 3-chlorotyrosine (CT) in human plasma has been developed based on direct analysis in real time-tandem mass spectrometry (DART-MS/MS). Analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) of the ion transitions at m/z 216.2/170.1 for CT, m/z 227.2/181.1 for NT and m/z 230.2/184.2 for the internal standard, d (3)-NT. The assay was linear in the ranges 0.5-100 μg/mL for CT and 4-100 μg/mL for NT with corresponding limits of detection of 0.2 and 2 μg/mL. Intra- and inter-day precisions and accuracies were respectively <15% and ±15%. Matrix effects were also evaluated. The method is potentially useful for high throughput analysis although sensitivity needs to be improved before it can be applied in clinical research.
ABSTRACT
The inter-species differences of estradiol metabolism were investigated in human, Beagle dog and rat liver microsomes by comparing enzyme kinetics of parent drug and the formation of its major metabolites. The incubation systems of estradiol with liver microsomes of the three species were optimized in terms of estradiol concentration, microsomal protein content and incubation time. The concentrations of estradiol and its metabolites were measured by LC-MS/MS method. The t1/2, CLint, CLh, Km and Vmax of estradiol incubated with male human liver microsomes were 40.02 +/- 8.32 min, 41.39 +/- 6.57 mL x min(-1) x kg(-1), 13.81 +/- 12.36 mL x min(-1) x kg(-1), 26.8 +/- 6.99 micromol x L(-1) and 0.75 +/- 0.92 micromol x L(-1) x min(-1), respectively. The corresponding parameters of female human were 44.71 +/- 10.21 min, 29.85 +/- 8.97 mL x min(-1) x kg(-1), 0.01 +/- 0.68 mL x min(-1) x kg(-1), 44.2 +/- 7.73 micromol x L(-1) and 1.27 +/- 4.41 micromol x L(-1) x min(-1), that of male dog were 21 +/- 7.33 min, 165.53 +/- 29.33 mL x min(-1) xkg(-1), 26.01 +/- 8.39 mL x min(-1) x kg(-1), 19.5 +/- 7.34 micromol x L(-1) and 1.6 +/- 0.65 micromol x L(-1) x min(-1), that of female dog were 25.5 +/- 5.32 min, 135.11 +/- 42.34 mL x min(-1) x kg(-1), 0.24 +/- 3.18 mL x min(-1) x kg(-1), 8.33 +/- 6.32 micromol x L(-1) and 0.51 +/- 2.15 micromol x L(-1) x min(-1), that of male rat were 5.11 +/- 3.84 min, 485.63 +/- 36.52 mL x min(-1) x kg(-1), 49.57 +/- 15.29 mL x min(-1) x kg(-1), 62 +/- 13.74 micromol x L(-1) and 19.16 +/- 9.67 micromol x L(-1) x min(-1), and that of female rat were 7.0 +/- 3.69 min, 354.82 +/- 33.33 mL x min(-1) x kg(-1), 8.04 +/- 3.23 mL x min(-1) x kg(-1), 35.38 +/- 7.65 micromol x L(-1) and 8.39 +/- 4.91 micromol x L(-1) min(-1), respectively. There were nine metabolites detected from all the three species, but the relative amounts of the metabolites generated were different in three species. The results indicted that the major phase I metabolic pathway of estradiol was similar in the liver microsomes from all the three species. However, the inter-species differences were found in the view of relative amounts of the metabolites as well as the metabolic characteristics of estradiol in liver microsomal incubation.