TABLE 2

Urinary profiles of AF metabolites in wild-type (m1a2+/+) and Cyp1a2-null (m1a2−/−) and CYP1A2-humanized (h1A2+/+, m1a2−/−) mice

Twenty-four-hour urine samples (n = 4 for each mouse line) after oral dosing with 50 mg/kg AF were collected and analyzed as described under Materials and Methods. Screening and identification of major metabolites were performed by using MetaboLynx software based on accurate mass measurement. After structural elucidation of major metabolites by MS2 fragmentation (Fig. 3), the abundance of each metabolite was represented as a relative peak area (area percent ± S.D.) by calculating its percentage in the total peak area of AF and its urinary metabolites.


Identification

Formula

Accurate Mass [M + 1]+

Reaction

RT

M1a2+/+

m1a2−/−

h1A2+/+ m1a2−/−

MS2 Fragments
min area %
I C16H11F3 N2O2 321.0851 Parent 6.17 12.8 ± 2.16 13.7 ± 3.48 9.20 ± 4.75 321.084
186.038
136.057
II C16H11F3 N2O3 337.0800 N5-OH 5.15 4.21 ± 1.33* 13.2 ± 4.56*,*** 5.62 ± 1.83*** 337.079
320.080
202.035
136.059
VII C16H11F3 N2O6S 417.0378 N5-OH+sulfation 4.25 0.31 ± 0.62* 4.19 ± 0.70*,*** 0.35 ± 0.70*** 337.082
202.038
136.059
VIII C16H11F3 N2O6S 417.0368 3-OH + sulfation 5.70 3.33 ± 0.57*,** 1.71 ± 0.36*,*** 24.3 ± 5.67*,** 337.082
186.038
152.053
IX C16H11F3 N2O7S 433.0317 3-OH + N5-OH + sulfation 4.80 43.9 ± 4.94* 3.82 ± 1.13*,*** 35.3 ± 5.37*** 353.076
336.076
202.036
152.054
X C22H19F3 N2O9 497.1172 N5-gluc 5.21 6.26 ± 0.26 7.04 ± 1.25 5.66 ± 0.29 497.117
393.112
363.093
321.085
186.038
136.054
XI C22H19F3 N2O9 513.1121 N5-OH + N-gluc 4.74 14.0 ± 4.96*,** 44.3 ± 3.26*,*** 2.04 ± 0.53**,*** 513.115
337.079
319.071
291.075
202.033
136.057
XII C22H19F3 N2O9 513.1121 N4′-OH + N-gluc 5.30 6.46 ± 1.34 7.31 ± 3.27 9.11 ± 2.19 513.115
337.079
320.078
293.075
186.038
XIII C22H19F3 N2O10 529.1070 N5-OH + N4′-OH + N-gluc 3.88 1.75 ± 0.32 1.48 ± 0.17 2.31 ± 0.39 529.101
395.079
353.070
336.070
309.059
202.039
XIV C22H19F3 N2O10 529.1070 N5-OH + 3-OH + N-glu 4.45 7.02 ± 1.42* 3.29 ± 0.61*,*** 6.14 ± 0.94*** 529.110
353.076
336.066
202.035








152.051
  • * Significant difference between wild-type and Cyp1a2-null mice (P < 0.01).

  • ** Significant difference between wild-type and CYP1A2-humanized mice (P < 0.01).

  • *** Significant difference between Cyp1a2-null and CYP1A2-humanized mice (P < 0.01).