Acta Chromatographica 25(2013)4, 755–764

DOI: 10.1556/AChrom.25.2013.4.13

Simultaneous Determination of Lycorine and

Galanthamine in Galanthus woronowii by
HPLC–DAD
A. EMIR, D. CICEK POLAT, G.I. KAYA, B. SARIKAYA, M.A. ONUR, AND
N. UNVER SOMER*
Department of Pharmacognosy, Faculty of Pharmacy, Ege University,
35100 Bornova, Izmir, Turkey
E-mail: nehir.somer@ege.edu.tr; nehirunver@hotmail.com

Summary. Galanthamine, used as a drug for the treatment of Alzheimer's disease and
lycorine possessing important biological properties, are widely distributed alkaloids in
the members of Amaryllidaceae family. In the present study, the contents of these alka-
loids have been quantitatively determined by high-performance liquid chromatography
(HPLC) coupled with diode array detector (DAD) in the aerial parts and bulbs of Galan-
thus woronowii Losinsk. collected from different localities in Northeastern Turkey.
A simple extraction method utilizing columns pre-packed with diatomaceous earth (Ex-
trelut®) was used. Chromatographic separation was achieved using an isocratic system
with a mobile phase of trifluoroacetic acid–water–acetonitrile (0.01:90:10) applied at a
flow rate of 1 mL min−1. Validation studies were also carried out to show that the
method is specific, accurate, and precise. In the tested specimens, the lycorine content
ranged between 0.008 and 0.364% whereas the galanthamine content was found to be be-
tween 0.003 and 0.506%.

Key Words: alkaloids, Amaryllidaceae, Galanthus, galanthamine, lycorine

Introduction
Galanthus L. species belong to the family Amaryllidaceae and occur natu-
rally throughout much of Central and Southern Europe and in parts of
Western Asia. Among the Galanthus species growing naturally in Turkey,
Galanthus woronowii Losinsk. is distributed in Northeastern Turkey and also
outside of Turkey mainly in Caucasus, Transcaucasus, southern Russia, and
Georgia [1].
Galanthus species are known to possess Amaryllidaceae alkaloids with
diverse structures and a wide spectrum of biological activities [2, 3]. Galan-
thamine, the most important and amply investigated alkaloid found in
Amaryllidaceae plants, is used for the treatment of mild and moderate cases
of Alzheimer's disease (AD). Today, total and stereoselective synthesis of
galanthamine has been achieved and form the basis of industrial production
of this alkaloid. However, plants are still regarded as an important source
for the production of this alkaloid [4]. Lycorine is also a common alkaloid
found in Amaryllidaceae species, and it has been shown to have interesting
0231–2522 © 2013 Akadémiai Kiadó, Budapest
756 A. Emir et al.

biological activities such as antitumor [5], antiviral [6], and antimalarial ac-
tivities [7].
Due to their important biological properties, there have been a quite
number of reports regarding the qualitative and quantitative determination
of lycorine and galanthamine in various parts of different Amaryllidaceae
species. A detailed literature search revealed that HPLC is the most em-
ployed method [8–12]. Also, in recent reports, gas chromatography–mass
spectrometry (GC–MS) [13–17], high-performance thin-layer chromatogra-
phy (HPTLC) [18, 19], and proton nuclear magnetic resonance (1H NMR)
[20] are other techniques used in the analysis of these alkaloids.
In the present study, aerial parts and bulbs of G. woronowii, collected
from different localities in Northeastern Turkey were quantitatively ana-
lyzed for their contents of lycorine and galanthamine by using high-
performance liquid chromatography (HPLC) coupled with a diode array
detector (DAD). Moreover, in the context of validation procedures, the line-
arity, precision, limits of detection and quantification, accuracy, and speci-
ficity of the method were displayed.

Experimental
Plant Material
Specimens of G. woronowii were collected from seven different localities in
Northeastern Turkey, during flowering time. The plants were identified by
Prof. M. Ali Onur from the Department of Pharmacognosy, Faculty of
Pharmacy, Ege University, Izmir, Turkey. Voucher samples of G. woronowii

Table I. G. woronowii specimens employed in the study

Sample ID Part used Collection site Voucher sample

GW-1B Bulbus
Çaykara, Trabzon 1358
GW-1H Herba
GW-2B Bulbus
Arhavi, Artvin 1375
GW-2H Herba
GW-3B Bulbus Aralık village, Borçka,
1392
GW-3H Herba Artvin
GW-4B Bulbus Near Aralık village, Borçka,
1393
GW-4H Herba Artvin
GW-5B Bulbus
Borçka, Artvin 1414
GW-5H Herba
GW-6B Bulbus
Kemalpaşa, Hopa, Artvin 1415
GW-6H Herba
GW-7B Bulbus
Derepazarı, Rize 1417
GW-7H Herba
Simultaneous Determination of Lycorine and Galanthamine 757

specimens are deposited in the Herbarium of the Department of Pharma-

cognosy, Faculty of Pharmacy, Ege University. A detailed list of these
specimens and their collection sites are found in Table I.

Chemicals and Solvents

Standard samples of lycorine and galanthamine were previously isolated in
our laboratory and authenticated by spectral analysis (UV, IR, 1D NMR,
MS) [21]. TFA (trifluoroacetic acid) (Merck), HPLC grade acetonitrile (Lab-
Scan Analytical Sciences), and chromatographic grade double-distilled wa-
ter were used for the HPLC analysis of the analytes. Other chemicals were
of analytical grade.

Sample Preparation
Lycorine and galanthamine extraction from the plant material were carried
out by the modification of a previously used method [15]. Two hundred
milligrams of powdered plant material were macerated with 5 mL 2% hy-
drochloric acid for 5 h in an ultrasonic bath at 40°C. Then, 1 mL of 26% am-
monia solution was added, and the volume was adjusted to 10 mL in a
volumetric flask with distilled water. After centrifugation at 5000 rpm for
10 min, aliquots of 3.0 mL were applied to the Extrelut® columns. The alka-
loids were eluted with (5 mL × 3) chloroform after 10 min. The organic sol-
vent was evaporated under vacuum to afford the alkaloidal extract. The ex-
tract was dissolved in 1 mL 0.1% TFA, filtered using a 0.45-μm filter (Grace
Davison, USA) and injected (20 μL) into the HPLC column.

Chromatography
The analysis of the samples and validation studies were performed on a liq-
uid chromatographic system (Agilent 1100 series), equipped with a quater-
nary pump, a vacuum degasser, a thermostatted column compartment, a
manual injector with 20 μL loop (Rheodyne 7725i) and a diode array detec-
tor (DAD) (Agilent 1200 series). Column temperature was set at 25°C. The
chromatographic separation was achieved isocratically using a mobile
phase consisting of TFA–water–acetonitrile (0.01:90:10) on a Hichrom C18
column (5 μm, 250 mm, 4.6 mm), and detection was carried out at 290 nm.
The flow rate was 1 mL min−1, and the injection volume was 20 μL. The
chromatographic run time was 45 min. Data analysis was performed using
Agilent Chem Station software. Quantitative determinations were carried
out by an external standard method based on peak areas.
758 A. Emir et al.

Method Validation
Linearity, accuracy, and precision studies were carried out according to the
ICH validation guidelines on the validation of analytical procedures [22].

Linearity

Stock standard solutions of lycorine and galanthamine (external standards)

were prepared by dissolving 5 mg in 10 mL 0.1% TFA. The linearity of the
method was shown by injecting nine known concentrations of the standards
in the range of 1–300 μg mL−1 and 1–400 μg mL−1 for lycorine and galan-
thamine, respectively. Each standard solution (20 μL) was injected into the
column in triplicate, and then the calibration curve of each analyte was ob-
tained by plotting the peak area versus the concentration.

Precision

The precision of the method was assessed by studying intra-day and inter-
day variation. Intra-day precision was determined by injecting solutions of
nine different concentrations of standard lycorine (1, 2.5, 5, 10, 25, 50, 100,
200, and 300 μg mL−1) and galanthamine (1, 5, 10, 25, 50, 100, 200, 300, and
400 μg mL−1) in triplicate on the same day. Inter-day precision was deter-
mined by performing the same procedure on two different days.

Limits of Detection (LOD) and Quantification (LOQ)

Limit of detection and limit of quantification were established at a signal-to-

noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ were experimen-
tally determined by 10 injections of lycorine and galanthamine.

Recovery

The recovery of the method was performed by standard addition analysis.

Three known amounts of individual standards were added to sample solu-
tions, and the mixtures were analyzed by the same method used in the
analysis of lycorine and galanthamine in the plant samples.

Specificity

Specificity is described as the ability to measure the analyte response in the

presence of components such as impurities, degredants, matrix, etc. [22].
Simultaneous Determination of Lycorine and Galanthamine 759

The specificity of the method for the analysis of lycorine and galanthamine
was determined in the presence of other constituents found in the extracts.

Results and Discussion

Method Validation
Linearity

The regression equation for lycorine was y = 15. 8423032x − 1.339608, and
for galanthamine, it was y = 11.451763x − 2.40657. Excellent linearity was
obtained for lycorine (r2 = 0.99985) and galanthamine (r2 = 0.9999) exhibiting
a good correlation between the alkaloid concentration and the peak area.
Moreover, standard deviation values of the slope and the intercept for ly-
corine were found as 0.167 and 8.421, and for galanthamine, 0.314 and 0.382,
respectively. Standard error of the regression for lycorine was found as
28.330 whereas, for galanthamine, it was calculated as 18.956. F-factors of
the calibration curves of lycorine and galanthamine were 0.971 and 0.927,
respectively.

Precision

Intra-day and inter-day variations were examined to determine the preci-

sion of the method. Variations were expressed by relative standard devia-
tion (RSD) for intra- and inter-day. These values were found to be less than
1.0% for the two compounds indicating the good reproducibility of the
method. The results are shown in Tables II and III.

Table II. Precision of lycorine in G. woronowii

Amount Intra-day precision Inter-day precision

(μg mL−1) (RSD, %) (RSD, %)

1 0.252 0.378
2.5 0.122 0.098
5 0.345 0.128
10 0.023 0.139
25 0.037 0.005
50 0.048 0.112
100 0.034 0.039
200 0.028 0.032
300 0.014 0.056
760 A. Emir et al.

Table III. Precision of galanthamine in G. woronowii

Amount Intra-day precision Inter-day precision

(μg mL−1) (RSD, %) (RSD, %)
1 0.095 0.495
5 0.040 0.589
10 0.020 0.107
25 0.051 0.027
50 0.122 0.511
100 0.467 0.051
200 0.251 0.149
300 0.094 0.122
400 0.080 0.080

Limits of Detection (LOD) and Quantification (LOQ)

The LOD and LOQ were calculated as 0.0492 μg mL−1 and 0.1641 μg mL−1
for lycorine, respectively. For galanthamine, the LOD was found to be
0.00745, and the LOQ was determined as 0.0279.

Recovery

Recovery assay was carried out by spiking three different known concentra-
tions of lycorine and galanthamine into sample solutions. The mean extrac-
tion recovery of lycorine was in the range of 98.5704–108.3902% whereas,
for galanthamine, the mean extraction recovery laid between 93.2038 and
119.3738%. The results of the experiments are given in Table IV.

Table IV. Recoveries for the determination of lycorine and galanthamine in G. woronowii

Mean
Concentration Amount
amount Mean recovery RSD
Analyte in sample spiked
found (%) ± SD (%)
(mg mL−1) (mg mL−1)
(mg mL−1)
0.03 0.048 108.390 ± 1.066 0.983
Lycorine 0.058 0.06 0.058 98.573 ± 1.087 1.102
0.12 0.092 103.210 ± 1.047 1.014
0.075 0.131 119.374 ± 0.502 0.421
Galan-
0.159 0.15 0.144 93.204 ± 1.848 1.983
thamine
0.30 0.231 100.56 ± 2.837 2.822
Simultaneous Determination of Lycorine and Galanthamine 761

Specificity

Peak purities of lycorine and galanthamine were evaluated by the acquisi-

tion of UV spectra with the DAD detector. The spectra of the analyzed com-
pounds are shown in Figs 1 and 2. In the analyzed extracts, the spectra of the
compounds were in good agreement with the spectra of the standards. The
values of spectra match for lycorine and galanthamine were found to be
999.775 and 999.982, respectively.

Fig. 1. Spectrum of lycorine

Fig. 2. Spectrum of galanthamine

762 A. Emir et al.

Sample Analysis
The method [8, 12] was applied to simultaneously identify and quantify ly-
corine and galanthamine in G. woronowii specimens. Detection of these alka-
loids was accomplished by comparison of the retention times and UV spec-
tra with those of standard compounds. Under the above-mentioned chro-
matographic conditions, lycorine and galanthamine were resolved within
approximately 9 min (Figs 3 and 4). Quantitative determination was carried
out by the external standard method based on peak areas. The results of the
mean values of three replicate injections of the two compounds were re-
ported in Table V. Significant variation in the contents of both alkaloids was
found in the analyzed samples. The lycorine content ranged between 0.008
and 0.364%. The highest content was detected in the bulbs of G. woronowii
collected from Kemalpasa, Hopa, Artvin. The galanthamine content in the
tested specimens ranged between 0.003 and 0.506%, and the highest amount
was found in the bulbs of G. woronowii collected from Arhavi, Artvin.

Table V. Contents of lycorine and galanthamine in G. woronowii

Sample ID Content of lycorine (%) Content of galanthamine (%)

GW-1B 0.069 ± 0.002 0.124 ± 0.004
GW-1H 0.028 ± 0.001 0.071 ± 0.002
GW-2B 0.077 ± 0.002 0.506 ± 0.014
GW-2H 0.008 ± 0.0004 0.128 ± 0.003
GW-3B 0.038 ± 0.001 0.244 ± 0.006
GW-3H 0.070 ± 0.002 0.326 ± 0.008
GW-4B 0.101 ± 0.001 0.197 ± 0.002
GW-4H 0.045 ± 0.001 0.089 ± 0.002
GW-5B 0.071 ± 0.002 0.094 ± 0.002
GW-5H 0.052 ± 0.001 0.087 ± 0.002
GW-6B 0.364 ± 0.007 0.017 ± 0.0005
GW-6H 0.234 ± 0.006 0.003 ± 0.0002
GW-7B 0.051 ± 0.002 0.160 ± 0.005
GW-7H 0.067 ± 0.001 0.266 ± 0.004

Fig. 3. HPLC chromatogram of mixed standards lycorine and galanthamine

Simultaneous Determination of Lycorine and Galanthamine 763

Fig. 4. HPLC chromatogram of an extract of G. woronowii

Conclusion
Quantitative determination of lycorine and galanthamine in G. woronowii
was achieved in the present study. The employed method has several ad-
vantages including simple and rapid sample preparation, requirement of a
small amount of plant material, and a simple mobile phase. The method
was also validated with respect to linearity, intra and inter-day precision,
recovery, and limits of detection and quantification. Validation studies
demonstrated that the method could simultaneously determine lycorine
and galanthamine in G. woronowii samples, which provided an efficient and
reliable method for the quality control of G. woronowii. Previously, the con-
tents of these two alkaloids in several Galanthus species [9, 16, 23, 24] have
been reported; however, to the best of our knowledge this is the first report
on the quantification of these alkaloids in G. woronowii of Turkish origin.

Acknowledgments
This study was financially supported by the Ege University Research Fund
(Project Nos. 09/ECZ/037 and 09/ECZ/008) and partially supported by
TUBITAK (104T272) and EBILTEM (2007/BIL/007).

References
[1] A.P. Davis, in: M. Bishop, A.P. Davis, and J. Grimshaw (eds) Snowdrops, A Mono-
graph of Cultivated Galanthus, Griffin Press Publishing Ltd., Cheltenham, 2006, pp.
9–63
[2] O. Hoshino, in: G.A. Cordell (ed) The Alkaloids Chemistry and Biology, vol. 51,
Academic Press Inc., New York, 1998, pp. 323–424
[3] N. Unver, Phytochem. Rev., 6, 125 (2007)
[4] M. Heinrich, in: G.A. Cordell (ed) The Alkaloids: Chemistry and Biology, vol. 68,
Elsevier Inc., San Diego, 2010, pp. 157–165
[5] D. Lamoral-Theys, A. Andolfi, G. Van Goietsenoven, A. Cimmino, B. Le Calvé,
N. Wauthoz, V. Mégalizzi, T. Gras, C. Bruyére, J. Dubois, V. Mathieu,
A. Kornienko, R. Kiss, and A. Evidente, J. Med. Chem., 52, 6244 (2009)
764 A. Emir et al.

[6] S. Li, C. Chen, H. Zhang, H. Guo, H. Wang, L. Wang, X. Zhang, S. Hua, J. Yu, P.
Xiao, R. Li, and X. Tan, Antiviral Res., 67, 18 (2005)
[7] S. van Dyk, S. Griffiths, R.L. van Zyl, and S.F. Malan, Afr. J. Biotechnol., 8, 5595
(2009)
[8] N.R. Mustafa, I.K. Rhee, and R. Verpoorte, J. Liq. Chromatogr. Relat. Technol., 26,
3217 (2003)
[9] G.I. Kaya, A. Fillik, Y. Hısıl, and N. Unver, Turkish J. Pharm. Sci., 1, 105 (2004)
[10] F.M. Diop, A. Ptak, F. Chretien, M. Henry, Y. Chapleur, and D. Laurain-Mattar,
Nat. Prod. Commun., 1, 475 (2006)
[11] G.S. Citoglu, B.S. Yılmaz, and O. Bahadır, Chem. Nat. Comp., 44, 826 (2008)
[12] G.I. Kaya, D. Cicek, B. Sarıkaya, M.A. Onur, and N. Unver Somer, Nat. Prod. Com-
mun., 5, 873 (2010)
[13] S. Berkov, A. Pavlov, M. Ilieva, M. Burrus, S. Popov, and M. Stanilova, Phytochem.
Anal., 16, 98 (2005)
[14] R. Gotti, J. Fiori, M. Bartolini, and V. Cavrini, J. Pharmaceut. Biomed., 42, 17 (2006)
[15] S. Berkov, J. Bastida, F. Viladomat, and C. Codina, Phytochem. Anal., 19, 285 (2008)
[16] F. Conforti, M.R. Loizzo, M. Marrelli, F. Menichini, G.A. Statti, D. Uzunov, and
F. Menichini, Pharm. Biol., 48, 2 (2010)
[17] S. Berkov, J. Bastida, F. Viladomat, and C. Codina, Talanta, 83, 1455 (2011)
[18] A.H. Abou-Donia, S.M. Toaima, H.M. Hammoda, and E. Shawky, Phytochem.
Anal., 19, 353 (2008)
[19] T. Mroczek and J. Mazurek, Anal. Chim. Acta, 633, 188 (2009)
[20] A. Lubbe, B. Pomahaćová, Y.H. Choi, and R. Verpoorte, Phytochem. Anal., 21, 66
(2010)
[21] A. Latvala, M.A. Onur, T. Gozler, A. Linden, B. Kivcak, and M. Hesse, Phytoche-
mistry, 39, 1229 (1995)
[22] ICH Guidelines Q2 (R1), Validation of Analytical Procedures: Text and Methodol-
ogy, Geneva, November 2005
[23] L. Kintsurashvili and V. Vachnadze, Pharm. Chem. J., 41, 492 (2007)
[24] B. Bozkurt Sarıkaya, N. Unver Somer, and M.A. Onur, Turkish J. Pharm. Sci., 9, 61
(2012)
Accepted by MWH