Narcissus and Daffodil

290 N.P.D. Nanayakkara and J.K. Bastos
excitation wavelength of 290 nm and an emission wavelength of 320 nm. The
detection limit was less than 5 ng/ml.
Könükol and Sener (1992) described a reversed-phase HPLC procedure to
quantify lycorine and crinine simultaneously in bulbs of Pancratium maritimum.
A reversed-phase C-18 column and a mobile phase containing a mixture of
chloroform:methanol (90:10), with UV detection at 292nm, were used in this
procedure.
Another reversed-phase method was reported by Sellés et al. (1997), who quantitatively
evaluated galanthamine, N-formylgalanthamine, haemanthamine and
tazettine in wild populations and tissue cultures of Narcissus confusus. They used a
C-18 column and a mobile phase consisting of water (containing phosphoric acid,
pH 3) (solvent A) and acetonitrile (solvent B) in a 60:40 ratio. Peak broadening
and tailing were minimised by the addition of 10 and 12 mM of octanesulphonic
acid to solvents A and B, respectively. Compounds were detected using a diode
array UV detector operating at 280 nm.
High Performance Liquid Chromatography (HPLC) – Thin Layer
Chromatography (TLC)
Queckenberg and Frahm (1993) developed a procedure wherein reversed-phase
HPLC was coupled with automated multiple development (AMD) on normal
phase silica to detect and quantify Amaryllidaceae alkaloids in natural sources. At
first, conditions such as stationary phase, base solvent, modifier, buffer (pH and
concentration), gradient profile, flow rate, temperature and sample size and solvent
were optimised to achieve the best HPLC resolution for 20 reference Amaryllidaceae
alkaloids. Under optimum HPLC conditions, the eluent from the HPLC
column was applied to silica TLC plates and subjected to AMD. Compounds on
the developed TLC plate were detected and quantified by different spectroscopic
and chemical methods. The UV spectra of individual bands were used in combination
with retention data to confirm the identity of each compound. Densitometric
evaluation by a TLC scanner in the absorption/reflectance mode was used to
quantify individual bands. The detection limit for tazettine by this method was
0.2 ng. These authors applied the method to re-investigate the alkaloid extract of
Amaryllis belladona (Queckenberg et al., 1996). This led to the identification of nine
alkaloids, in addition to five previously known from this species, and three more
were tentatively identified.
High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS)
By coupling MS and HPLC, Eckers et al. (1980) analysed an alkaloid fraction of
Crinum glaucum. The extract was separated by reversed-phase HPLC using an
ODS 5 µ Spheresorb ® column and water:acetonitrile:ammonia (20:79.7:0.3) as
the mobile phase. The eluent was subjected to MS with a moving belt interface,
and total ion current in electron impact (EI) mode was recorded. Two alkaloids,
lycorine and ambelline, were identified on the basis of their EI spectra and
retention times. Another compound, criglaucine, of uncertain structure, was also
detected.