Investigation of 6-O-methyl-scutellarein metabolites in rats by ultra-flow liquid chromatography/quadrupole-time-of-flight mass spectrometry

Figures & data

Figure 1. The chemical structures of scutellarin (1), scutellarein (2) and 6-O-methyl-scutellarein (3).

Scheme 1. Reagents and conditions: (a) HCl, EtOH, N₂, reflux, 36 h, 17%; (b) Ph₂CCl₂ (1.5 equiv.), Ph₂O, 175 °C, 30 min, 85%; (c) PhCH₂Br (1.5 equiv), K₂CO₃ (1.75 equiv), DMF, 25 °C, 12 h, 93%; (d) HAc–H₂O (4:1), reflux, 1.5 h, 95%; (e) PhCH₂Br (1.3 equiv), K₂CO₃ (1.5 equiv), DMF, 25 °C, 12 h, 93%; (f) CH₃I (1.3 equiv), K₂CO₃ (1.5 equiv), DMF, 25 °C, 4 h, 94%; (g) Pd/C (10 wt%), H₂ (atm), THF/EtOH, 8 h, 96%.

Figure 2. UFLC/MS chromatograms of rat urine, plasma, faeces and bile samples before and after oral administration of 6-O-methyl-scutellarein (3): (a) urine sample in ESI^-mode, (b) plasma sample in ESI⁻ mode, (c) faeces sample in ESI⁻ mode, (d) bile sample in ESI⁻ mode.

Table 1. UFLC/ESI-MS, retention time and fragment ions of 6-O-methyl-scuetellarin (3) and its metabolites in rats.

No.	tR (min)	Mass	Metabolite name	Formula	MS/MS fragment ions	Δ (ppm)	Source
3	7.19	300.0634	Parent	C16H12O6	299 [M − H]^−, 283 [M − H-CH2]^−, 183	0.8	Urine, blood,  bile, faeces
M1	6.21	476.0955	Glucuronide conjugation	C22H20O12	475 [M − H]^−, 356, 299 [M − H-GlcUA]^−	5.3	Urine, bile, blood
M2	5.72	476.0955	Glucuronide conjugation	C22H20O12	475 [M − H]^−, 356, 299 [M − H-GlcUA]^−	−4.1	Urine, bile
M3	4.73	652.1276	2 × Glucuronide conjugation	C28H28O18	651 [M − H]^−, 475 [M − H-GlcUA]^−, 356,  299 [M − 2GlcUA-H]^−,	−2.8	Urine, bile
M4	5.43	380.0202	Sulphation	C16H12O9S	379 [M − H]^−, 299[M − 1-SO3]^−, 263, 183	−2.7	Urine
M5	4.92	556.0523	Sulphation + glucuronide	C22H20O15S	555 [M − H]^−, 475 [M − 1-SO3]^−, 379 [M − GlcUA-H]^−,  299 [M − GlcUA-SO3−H]^−	−4.1	Urine
M6	8.22	314.0790	Methylation	C17H14O6	313 [M − H]^−, 299 [M − CH2−H]^−, 135	−0.8	Urine
M7	7.67	342.0740	Acetylation	C18H14O7	341 [M − H]^−, 299 [M − 2H-Ac]^−, 163	−0.3	Faeces

Figure 3. Representative MS/MS spectra: 3, m/z 299; M1, m/z 475; M2, m/z 475; M3, m/z 651; M4, m/z 379; M5, m/z 555; M6, m/z 313; M7, m/z 341.

Figure 4. Proposed fragmentation pathway of 3, M1, M4, M6, M7.

Figure 5. Structures of 6-O-methyl-scutellarein (3) and its metabolites and possible biotransformation pathways. GlcUA,  glucuronyl unit; Ac,  acetyl group. (a) Glucuronidation; (b) acetylation; (c) methylation; (d) sulphation.