Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
46, Number 1, January 2008, pp. 74-80
Liquid Chromatography–Electrospray Ionization
Ion Trap Mass Spectrometry for Analysis of in Vivo and in Vitro
Metabolites of Scopolamine in Rats
Huaixia Chen[1], Yong Chen[2], Peng Du[2], and
Fengmei Han[2]
[1]Ministry-of-Education Key Laboratory for the Synthesis and
Application of Organic Functional Molecules & College of
Chemistry and Chemical Engineering, Hubei University, Wuhan 430062,
China and
[2]College of Life Science, Hubei University, 430062,
China
In vivo and in vitro metabolism of scopolamine
is investigated using a highly specific and sensitive liquid
chromatography–mass spectrometry (LC–MSn) method.
Feces, urine, and plasma samples are collected individually after
ingestion of 55 mg/kg scopolamine by healthy rats. Rat feces
and urine samples are cleaned up by a liquid–liquid extraction
and a solid-phase extraction procedure (C18 cartridges), respectively.
Methanol is added to rat plasma samples to precipitate plasma
proteins. Scopolamine is incubated with homogenized liver and
intestinal flora of rats in vitro, respectively. The metabolites
in the incubating solution are extracted with ethyl acetate.
Then these pretreated samples are injected into a reversed-phase
C18 column with mobile phase of methanol–ammonium acetate
(2mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected
by an on-line MSn system. Identification and structural elucidation
of the metabolites are performed by comparing their changes in
molecular masses (DM), retention-times and full scan MSn spectra
with those of the parent drug. The results reveal that at least
8 metabolites (norscopine, scopine, tropic acid, aponorscopolamine,
aposcopolamine, norscopolamine, hydroxyscopolamine, and hydroxyscopolamine
N-oxide) and the parent drug exist in feces after administering
55 mg/kg scopolamine to healthy rats. Three new metabolites (tetrahydroxyscopolamine,
trihydroxy-methoxyscopolamine, and dihydroxy-dimethoxyscopolamine)
are identified in rat urine. Seven metabolites (norscopine, scopine,
tropic acid, aponorscopolamine, aposcopolamine, norscopolamine,
and hydroxyscopolamine) and the parent drug are detected in rat
plasma. Only 1 hydrolyzed metabolite (scopine) is found in the
rat intestinal flora incubation mixture, and 2 metabolites (aposcopolamine
and norscopolamine) are identified in the homogenized liver incubation
mixture.
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