IMPROVED HPLC METHOD FOR DETERMINATION OF

AMARYLLIDACEAE ALKALOIDS

I. Ivanov1, S. Berkov2 and A. Pavlov1

1
Department of Microbial Biosynthesis and Biotechnologies – Laboratory in Plovdiv, The Stephan Angeloff Institute of
Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria,
2
Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona,
Barcelona, Catalonia, Spain
Correspondence to: Ivan Ivanov
E-mail: lbpmbas@yahoo.com

ABSTRACT
An improved HPLC method for quantitative determination of galanthamine, lycorine and norgalanthamine was developed.
HPLC separation was achieved using a reversed phase C18 column Symmetry® and a gradient system with acetonitrile as an
organic phase and 1% (w/v) ammonium acetate buffer adjusted to pH 6.6 with acetic acid (flow rate 0.3 – 0.5 ml/min.). The
calibration curves were linear from 5 to 150 μg/ml (r2>0.99). The reliability of the proposed system was proved through
reproducibility test with alkaloids extracts from in vitro shoot-clumps culture obtained from Leucojum aestivum L. and
Pancratium maritimum L.

Keywords: Amaryllidaceae alkaloids, galanthamine, HPLC,
lycorine
Introduction
Amaryllidaceae are known as ornamental plants, furthermore
some species of this family contain alkaloids that are know to
exhibit a wide range of biological activities (6).
Galanthamine (1), an isoquinoline alkaloid, is long acting,
selective, reversible, and competitive acetylcholinesterase
inhibitor used for symptomatic treatment of senile dementia
of the Alzheimer’s type (8), poliomyelitis and other
neurological diseases (17). Lycorine (3), a The shoot-clumps were cultivated on sold MS medium
pyrrolophenanthridine alkaloid, displays a strong antiviral
effect (9) against poliovirus, measles, and Herpes simplex
type 1 viruses as well as high antiretroviral (19), strong
antimitotic (10), and cytotoxic activities (20). Different
analytical techniques have been described for the qualitative
and quantitative determination of Amaryllidaceae alkaloids in
various parts of different Amaryllidaceae plants including
CGC-MS (4), spectrophotometric (13), HPTLC (1, 2) and
enzyme immunoassay (15). HPLC methods were used for
determination of some Amaryllidaceae alkaloids in plant and
tissue-culture extracts of Narcissus ssp. (11, 12, 18) and
Leucojum aestivum L. (11, 14, 16). These methods differ
mainly in the column type, pH range of the mobile phase, and
analysed plants. However reported methods do not meet our
requirements for determination of galanthamine (3), lycorine
(2) and norgalanthamine (1) in extracts from in vitro L.
aestivum shoots. In the presented manuscript we describe an
improved HPLC method for determination of Amaryllidaceae
alkaloids mixtures from in vitro cultures.
Materials and methods
Plant material
In vitro shoot-clumps cultures were obtained from calli of
Leucojum aestovum L. and Pancratium maritimum L. (14).
supplemented with 3% sucrose, 5.5 g/l “Plant agar” (Duchefa
Biochemie, Netherlands), 2 mg/l Benzylamino purine and
1.15 mg/l α - Naphthaleneacetic acid at 26°C under
illumination 16h light/8h darkness photoperiod.
Alkaloid extraction
0.2-0.3 g dry biomass was extracted three times with 5 ml of
methanol in an ultrasonic bath for 15 min. combined extracts
were concentrated under vacuum and dissolved in 2 ml of 3%
sulfuric acid. The neutral compounds were removed by
extraction (tree times) with diethyl ether. The alkaloids were
fractionated after basification of the extracts with 1 ml of
25% ammonia and extraction (3 X 3 ml) with chloroform.

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The chloroform extracts were dried over anhydrous sodium (3) and galanthamine (2), the gradient profiles and mobile
sulphate and evaporated to dryness. phases were optimized in order to obtain better peak
resolution. Оptimizаtion was accomplished by a change of
Equipment
the pH of inorganic mobile phase (solvent B) - adjusted to pH
HPLC analysis system Waters 2487 Dual λ Absorbanc
6.6. As compared to the original method, the gradient starts
Detector and Waters 1525 Binary HPLC pump (Waters,
from 10% of solvent A instead 31% as in base system, and
Milford, USA). The chromatographic assay was performed
reaches up to 90% (18 min) instead of 70% (10 min) Table 1.
on a Symmetry® C18 column (150 x 4.6 mm) reversed phase
The identification and quantitative determination of the
matrix (5 μm) (Waters) and elution was carried out in a
compounds were accomplished both by a comparison of
gradient system with acetonitrile as the organic phase
retention time, peak heights and areas with standard
(solvent A) and 1% (w/v) ammonium acetate buffer adjusted
methanolic solutions of the alkaloids and by internal
to pH 6,6 with acetic acid (solvent B). UV detector was set at
standards (Fig. 1B). The linearity of the HPLC method was
287 nm and the volume of injection was 20 μl. The gradient
checked by injecting six mixtures of solutions containing 10,
system and flow rate are shown in Table 1.
25, 50, 75, 100 and 150 μg/ml of norgalanthamine (1),
lycorine (2) and galanthamine (3). The relationships between
Results and Discussion
peak height ratio (Y) and the concentration injected (X;
One of the most used HPLC method for the quantitative
μg/ml) is listed in Table 2. Calibration curves showed
determination of galanthamine and some other
linearity between 1 – 150 μg/ml with correlation coefficients
Amaryllidaceae alkaloids was developed by Sellés et al. (18).
higher than 0.999.
This method was successfully applied for galanthamine
The optimized method allowed successful identification
quantification in plants and tissue shoot-clump cultures from
and quantification of target compounds galanthamine (3),
Narcissus confusus Pugsley. Unfortunately, it was not
lycorine (2) and norgalanthamine (1) in the samples from
adapted to plants producing lycorine (3) and norgalanthamine
shoot-clumps cultures of L. aestivum and P. maritimum (Fig
(1) (3) because it was unable to separate efficiently a
2). Priority of the method was separation of these tree
combination of these compounds and galanthamine (Fig. 1A)
alkaloids from the mixtures of other the accompaning
therefore it was inapplicable for analyses of extracts from
Amaryllydaceae alkaloids. These samples were used to
L.aestivum and P.maritimum shoot in vitro cultures, which
determine the reproducibility of results. Mean values,
contain all these compounds in their alkaloids mixtures (7,
standard deviations and coefficient of variation of alkaloids
14). For this reason, an adaptation of the analytical method
concentration were presented in Table 3.
was performed, based on optimization of HPLC
chromatographic conditions.
For good separation of the alkaloids, especially lycorine
TABLE 1
Gradient elution scheme employed for the HPLC analysis of alkaloids
Gradient profile of origin method Gradient profile of improved method
Time, min Flow rate, %A %B Time, min Flow rate, %A %B
ml/min ml/min
1. 0,50 31,0 69,0 0,40 10,0 90,0
2. 6,00 0,50 31,0 69,0 11,00 0,30 31,0 69,0
3. 9,00 0,50 70,0 30,0 15,00 0,50 70,0 30,0
4. 10,00 0,50 70,0 30,0 16,00 0,50 90,0 10,0
5. 12,00 0,50 31,0 69,0 18,00 0,50 90,0 10,0
6. 20,00 0,50 31,0 69,0 20,00 0,40 31,0 69,0
7. - - - - 22,00 0,40 10,0 90,0
8. - - - - 31,00 0,40 10,0 90,0

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Fig 1. HPLC chromatogram of standarts (A) gradient system described from Sellés et al. (18) (B) optimized gradient system
1.- norgalanthamine, 2. – licorine and 3. – galanthamine.

Fig 2. HPLC chromatograms: (A) Shoot-clumps culture from L.aestivum, (B) Shoot-clumps culture from P. maritimum
1.- norgalanthamine, 2. - lycorine and 3. - galanthamine.

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 TABLE 2
Parameters of retention time and calibration curves for separated alkaloids
Alkaloid Retention time (min) Equation r2
Norgalanthamine (1) 10,600 Y= 2.41x104X + 8.17x103 0,9997
Lycorine (2) 14,430 Y= 3.62x104X + 4.35x104 0,9980
Galanthamine (3) 15,130 Y= 4,55x104X – 7.14x104 0,9998
TABLE 3
Reproducibility test: determination of norgalanthamine [1], lycorine [2] and galanthamine [3] in shoot-clumps culture samples
of L. aestivum and P. maritimum.
L.aestivum culture P. maritimum culture
Nor - Lycorine Galanthamine Nor -galanthamine Lycorine Galanthamine
galanthamine (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)
(μg/ml)
1 51.784 9.106 48.195 12.433 82.291 28.566
2 54.650 8.327 49.824 11.539 75.697 26.780
3 53.735 9.276 50.943 11.455 82.065 29.080
4 52.528 8.497 52.331 13.482 83.976 29.521
5 58.266 8,994 49.753 12.846 84.266 29.170
Mean 54.190 8.840 50.209 12.351 81.660 28.623
± SD ± 2.530 ± 0.408 ± 1.537 ± 0.865 ± 3.470 ± 1.086
CV% 4.670 4.610 3.060 7.000 4.250 3.790

Conclusion
The improved HPLC method is suitable for quantitative
determination of norgalanthamine (1), lycorine (2) and
galanthamine (3) from complex alkaloid mixtures in plants
extracts and shoot-clumps cultures obtained from
Amaryllidaceae species. Moreover this method may be used
by biotechnology industry for quality control of these
compounds.
Acknowledgment
The work was supported by the Bulgarian Science
Foundation, Bulgarian Ministry of Education and Science
(project TK – B – 1605/2006).
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