Narcissus and Daffodil

Immunoassays
Analysis of Amaryllidaceae alkaloids 293
Immunoassay provides a sensitive and specific tool for many analytical tasks.
Tanahashi et al. (1990) developed a radioimmunoassay procedure for quantifying
galanthamine. The antiserum was raised in rabbits against a conjugate of galanthamine-2-O-hemisuccinate-bovine
serum albumin. This was highly specific for
galanthamine, and showed practically no cross reactivity against other common
Amaryllidaceae alkaloids. However, other minor cross-reactive materials were
present in a crude extract of Leucojum aestivum. This procedure was very sensitive
(measuring range 0.5–100 ng), and was used to determine the galanthamine
content of crude extracts of several L. aestivum samples, as well as of a number of
South African Amaryllidaceae.
The same group subsequently replaced the radio-labelled antigen with enzymelabelled
antigen, thereby avoiding the use of radioactive material (Poulev et al.,
1993). This enzyme immunoassay procedure was easier to perform and more
sensitive by a factor of 100, and required only a very small amount (1–12 mg) of
plant material. By using this method, galanthamine contents of 1000 individual
Leucojum aestivum plants and of more than 130 herbarium samples of Amaryllidaceae
and closely related plant families were determined. Preliminary investigation
showed that this method could be applied to analyse galanthamine in
biological fluids, and the results generated by this method were in good agreement
with those obtained by an HPLC method. This method was later used to
determine the galanthamine content in bulbs and callus of two Pancratium species
(Sarg et al., 1996).
Quantitative determination of galanthamine by
acetylcholinesterase inhibition
Ghous and Townshend (1998) reported a method to determine galanthamine
quantitatively by measuring its inhibition of acetylcholinesterase immobilised on
controlled pore glass. The determination was carried out by a flow injection
procedure where galanthamine and substrate, acetylthiocholine, were injected to
coincide in the buffer stream, which then passed through the immobilised acetylcholinesterase
column. Active enzyme cleaves the substrate to a chromogen
reactive product. The eluent was mixed with a chromogen (5,5′-dithiobis-(2nitrobenzoic
acid)) solution and the absorbance was measured at 405 nm. Under
optimised conditions, the response was linear over the range 5 × 10 –7 to 6 × 10 –6 M.
The limit of detection was 5 × 10 –7 M. Another group (Nikol’skaya et al., 1989;
Kugusheva et al., 1992) also reported a procedure to determine galanthamine
based on the same principle, using enzyme-containing membranes.
Spectrophotometric and fluorometric determination
Several simple spectrophotometric procedures have been developed to quantify
galanthamine. These methods involved the measurement of absorbance in the UV
range of galanthamine as a free base (λ 290 nm) (Bagdasarova, 1984) or in the
visible range as a complex with other reagents (Kuznetsov et al., 1969; Pavlov and
Ponomarev, 1981; Tokhtabaeva, 1987). Kolusheva and Vulkova (1966) studied the