Narcissus and Daffodil

Analysis of Amaryllidaceae alkaloids 291
High performance liquid chromatography (HPLC) for the
determination of galanthamine and its metabolites in
biological fluids and tissues
High performance liquid chromatography (HPLC)
In addition to the analysis of plant material, several HPLC methods were developed
to screen galanthamine and its metabolites in biological fluids. Claessens et al.
(1983) initially reported an HPLC method for the quantitative determination of
galanthamine in biological fluids using a normal phase silica column with hexanedichloromethane
containing ethanolamine as the mobile phase. The minimum
detectable concentration was 5 ng/ml, and the standard deviation varied between
18.9 and 2.5% for the concentration range 10–100 ng/ml. Subsequently, a reversedphase
HPLC method was described, using a C-18 column and acetonitrilepentanesulfonic
acid buffer in water as the solvent (Claessens et al., 1988). In this
method, body fluids were pre-processed by a preparative isotachophoresis prior to
chromatographic analysis. Satisfactory recoveries, excellent resolution and good
sensitivity were achieved. In both these procedures, a UV detector operating at
235 nm was used to detect the test compounds.
Tencheva et al. (1987) and Tencheva and Budevski (1987) developed a
reversed-phase HPLC method for the quantitative determination of galanthamine
and its metabolites, epigalanthamine and galanthaminone, in human body fluids.
They used a C-8 column and a methanol:water (40:60) mobile phase modified
with dibutylamine adjusted to pH 7 with 85% phosphoric acid. Codeine was used
as an internal standard. The test compounds were detected by fixed-wavelength
UV detector (280 nm). The detection limit was 0.05 µg/ml. This method was also
subsequently used to quantify galanthamine in several native populations of
Leucojum aestivum (Gorinova et al., 1993).
A somewhat similar HPLC method was reported by Bickel et al. (1991a) to
determine galanthamine and epigalanthamine in plasma and tissues of mice.
A reversed-phase C-8 column and a mobile phase containing acetonitrile, tetrahydrofuran,
water and di-n-butylamine at pH 7, adjusted with 85% phosphoric
acid, were employed in this procedure. Codeine was used as an internal standard,
and a fluorescence detector, set to 290 and 320 nm for excitation and emission
frequencies, respectively, was used to detect the test compounds.
High performance liquid chromatography – mass spectrometry (HPLC-MS)
An HPLC-MS procedure for the determination of galanthamine and its metabolite,
6-O-demethylgalanthamine in biological samples was described by Bores
et al. (1996). In this procedure, a reversed-phase HPLC system was utilised with a
mass spectrometer operating both in the multiple reaction monitoring mode (using
a heated nebuliser interface) and in the selected ion monitoring mode (with an
electrospray interface).
Thin layer (TLC) and paper chromatography (PC)
Several thin layer and paper chromatographic methods have been developed
for the quantitative and qualitative analysis of Amaryllidaceae alkaloids.