Acta Chromatographica 25(2013)2, 331–338

DOI: 10.1556/AChrom.25.2013.2.9

Quantitative Analysis of Lycorine in Endemic

Galanthus xvalentinei nothosubsp. subplicatus
by HPLC–DAD
N. UNVER SOMER, D. CICEK POLAT, A. EMIR, M.A. ONUR, AND G.I. KAYA*
Ege University, Faculty of Pharmacy, Department of Pharmacognosy,
35100, Bornova, Izmir, Turkey
E-mail: [email protected]

Summary. Galanthus xvalentinei (J. Allen) Beck nothosubsp. subplicatus (N. Zeybek) A. P.
Davis (Amaryllidaceae) is an endemic hybrid between G. nivalis L. and G. plicatus M.
Bieb. subsp. byzantinus (Baker) D. A. Webb. Lycorine, a common alkaloid found in Ama-
ryllidaceae plants, has been shown to display important biological activities. In the pre-
sent study, a reversed-phase high-performance liquid chromatographic method has been
used for the quantitative determination of lycorine in the aerial parts and bulbs of G. xva-
lentinei nothosubsp. subplicatus. A simple method for the extraction of lycorine in low-
mass plant samples was employed utilizing pre-packed columns with diatomaceous
earth (Extrelut®). The chromatographic separation was carried out using an isocratic
system with a mobile phase of trifluoroacetic acid–water–acetonitrile (0.01:95:5) applied
at a flow rate of 1 mL min−1 using diode array dedector. The content of lycorine in the
bulbs of G. xvalentinei nothosubsp. subplicatus was found to be 0.0028%, however, it was
not detected in the aerial parts of this hybrid.

Key Words: Galanthus xvalentinei nothosubsp. subplicatus, Amarylidaceae, lycorine,

HPLC

Introduction
Galanthus L. is represented by 14 taxa and one hybrid in Turkey [1]. Galan-
thus xvalentinei (J. Allen) Beck nothosubsp. subplicatus (N. Zeybek) A. P.
Davis (Amaryllidaceae) is a naturally occurring Galanthus hybrid between
G. nivalis L. and G. plicatus M. Bieb. subsp. byzantinus (Baker) D. A. Webb.
This hybrid is endemic, and it is distributed in north-western Turkey [2].
Plants of the Amaryllidaceae family are known to produce alkaloids with
diverse chemical structures possessing interesting biological activities [3].
Lycorine, the most common alkaloid found in Amaryllidaceae plants, dis-
plays different biological properties including antiviral [4], cytotoxic [5] and
antimalarial activities [6]. During the course of our ongoing studies on
Turkish Amaryllidaceae species, previously quantitative determination of
lycorine has been carried out in a number of Galanthus [7, 8], and Sternbergia

0231–2522 © 2012 Akadémiai Kiadó, Budapest

332 N. Unver Somer et al.

Waldst. & Kit. species [9, 10] plus in Pancratium maritimum L. [9]. In the pre-
sent study, quantification of lycorine in the wild hybrid, Galanthus xvalenti-
nei nothosubsp. subplicatus is described.
Pre-packed columns based on diatomaceous earth for liquid–liquid
sample purification were used for the extraction of lycorine and reversed-
phase high-performance liquid chromatography (RP-HPLC) coupled with
DAD detector was succesfully employed for the separation and quantitative
determination of lycorine in the plant material.

Experimental
Plant Material
Galanthus xvalentinei nothosubsp. subplicatus was collected from Begendik-
İgneada (Kırklareli). The plant was identified by Prof. M. Ali Onur (Ege
University) and Dr. Sırrı Yüzbasıoglu (Istanbul University). Voucher speci-
men (No: 1410) is deposited in the Herbarium of the Department of Phar-
macognosy, Faculty of Pharmacy, Ege University.

Chemicals and Solvents

Lycorine was isolated from several Galanthus species in our laboratory and
authenticated by means of spectroscopic methods (1H and 13C NMR, MS)
[11]. HPLC grade acetonitrile (Lab-Scan Analytical Sciences), TFA (trifluoro-
acetic acid) (Merck) and chromatographic grade, double-distilled water
were used for the HPLC analysis. The other reagents were of analytical pu-
rity.

Sample Preparation
Dried and powdered aerial parts and bulbs of the plant (200 mg) were mac-
erated with 5 mL of 2% hydrochloric acid for 5 h in an ultrasonic bath at
40 °C. The extract was basified with 1 mL of 26% ammonia solution and the
volume was adjusted to 10 mL in a volumetric flask with distilled water.
Then, it was centrifuged at 5000 rpm for 10 min and aliquots of 3.0 mL were
applied to the Extrelut® columns. After 10 min, the alkaloids were eluted
with (5 mL × 3) chloroform. The organic solvent was distilled in vacuo to
yield the alkaloidal extract. The extract was then dissolved in 1 mL 0.1%
TFA, filtered using a 0.45-μm filter (Alltech, Carnforth), and injected (20 μL)
into the HPLC system [9].
Quantitative Analysis of Lycorine 333

Chromatography
HPLC analysis was carried out using a liquid chromatographic system
(Agilent 1100 series), equipped with a quaternary pump, vacuum degasser,
a thermostated column compartment, a manual injector with 20 μL loop
(Rheodyne 7725i) and a DAD (Agilent 1200 series). The chromatographic
assay was performed on a Hichrom C18 column (5 μm, 250 mm, 4.6 mm) at
25°C. Data analysis was performed with Agilent ChemStation software. All
the calculations concerning the quantitative analysis were carried out by an
external standard method based on peak areas. HPLC analysis was
performed by isocratic elution with a flow rate of 1.0 mL min−1. The mobil
phase consisted of TFA–water–acetonitrile (0.01:95:5). Quantification of
lycorine was performed at 290 nm. The chromatographic run time was
45 min.
Validation of the Method
Linearity

Standard stock solution (0.2 mg mL−1) of lycorine was prepared in % 0.1

TFA. To prepare the stock solution, lycorine was accurately weighed into
10 mL volumetric flask, dissolved in % 0.1 TFA, and then the volume was
adjusted to 10 mL. This solution was further diluted with % 0.1 TFA to yield
solutions containing 0.5, 1.0, 2.0, 4.0, and 8 μg mL−1. The linearity of the
method was studied by injecting five known concentrations of the standard
in the range of 0.5–8 μg mL−1. Results from each analysis were averaged and
subjected to regression analysis.

Precision

Precision was assessed at five concentration levels in intra-day and inter-

day studies. Intra-day precision was determined by triplicate injection of
standard solutions of lycorine at five concentration levels (0.5, 1.0, 2.0, 4.0,
and 8.0 μg mL−1), on the same day. Inter-day precision was checked by re-
peating the studies on two different days.

Limits of Detection (LOD) and Quantification (LOQ)

Limit of detection (LOD) and limit of quantification (LOQ) were established at a

signal-to-noise ratio (S/N) of 3 and 10, respectively. The LOD and LOQ were
experimentally verified by ten injections of lycorine.
334 N. Unver Somer et al.

Recovery

Pre-analyzed samples were spiked with 1.50, 3.00, 6.00 μg/mL−1 of lycorine
standard and the mixtures were analysed by using the same method em-
ployed in the quantitative determination of lycorine in the plant samples.
The experiment was conducted three times.

Specificity

Specificity is the suitability of the method for measurement of analyte re-

sponse in the presence of potential impurities. The specificity of the method
was assessed by detection of lycorine in the presence of other constituents
present in the extracts.

Results and Discussion

Validation of the Method
Linearity
The calibration plots for lycorine were found to be linear within the range of
0.5 to 8 μg mL−1. The regression equation was y = 14.9668622x + 0.7717199
(r2 = 0.999).

Precision

The results of the precision analysis of lycorine are summarized in Table I.

RSD was found to be always less than 4.50% when the analysis was per-
formed at five different concentrations (0.5, 1.0, 2.0, 4.0, 8.0 μg/mL−1).

Table I. Intra-day and inter-day precision of the method

Amount Intra-day precision Inter-day precision

Compound
(µg/mL−1) (RSD, %) (RSD, %)
0.5 4.29 2.24
1.0 2.07 4.41
Lycorine 2.0 4.50 2.93
4.0 2.29 0.85
8.0 2.23 1.18
Quantitative Analysis of Lycorine 335

Limits of Detection (LOD) and Quantification (LOQ)

The LOD (signal /noise ratio of 3:1) was calculated as 0.071 μg mL−1 and the
LOQ (signal/noise ratio of 10:1) was determined as 0.238 µg mL−1. At the
LOD and LOQ levels RSD% was determined to be 4.95 for lycorine.

Recovery

Mean extraction recovery for lycorine was found to be between 100.41 and
116.23% following the analysis of this alkaloid in samples spiked with 1.5,
3.0, and 6 µg/mL−1 of lycorine standard (Table II).

Table II. Statistical data showing recovery studies of lycorine (n = 3)

Concentration Amount Mean amount

Mean re-
Compound in sample spiked found in mix- % RSD
covery %
(µg/mL−1) (µg/mL−1) ture (µg/mL−1)
1.50 1.50 1.62 116.23 1.58
Lycorine 1.50 3.00 2.32 105.05 1.54
1.50 6.00 3.76 100.41 1.86

Specificity

UV spectra acquired with the photodiode-array detector (DAD) were used

for the evaluation of the peak purity of lycorine.

Sample Analysis
The results of the quantitative analysis of lycorine in the aerial parts and
bulbs of Galanthus xvalentinei nothosubsp. subplicatus are listed in Table III.
The composition of the mobile phase was selected after several trials with
TFA–water–acetonitrile [12], in varying proportions. Finally, a mobile phase
consisting of TFA–water–acetonitrile (0.01:95:5) was chosen to achieve
maximum resolution and sensitivity. A flow rate of 1 mL min−1 gave an op-
timal signal-to-noise ratio. Under the described chromatographic condi-
tions, lycorine was resolved within approximately 17 min. The chroma-
tograms of the standard lycorine and Galanthus xvalentinei nothosubsp. sub-
plicatus extract were given in Figs 1 and 2. The identification of lycorine in
the plant samples was accomplished by comparison of the retention time
and also the UV spectrum of standard lycorine. The whole analysis, includ-
ing sample preparation, was repeated three times. The content of lycorine in
336 N. Unver Somer et al.

the bulbs of G. xvalentinei nothosubsp. subplicatus was found to be 0.0028 ±

0.0004%. Lycorine was not detected in the aerial parts of this hybrid. G. xva-
lentinei nothosubsp. subplicatus samples were also investigated for their gal-
anthamine content and it was not detected in the aerial parts and bulbs of
this plant.

Fig. 1. HPLC chromatogram of standard lycorine

Fig 2. HPLC chromatogram obtained from G. xvalentinei nothosubsp. subplicatus extract

Table III. Lycorine content of some Galanthus species

Plant species Plant parts Lycorine, %

Bulbs 0.0028
Galanthus xvalentinei nothosubsp. subplicatus*
Aerial parts ND
Bulbs 0.0021
G. gracilis [7]
Aerial parts ND
G. trojanus (previously named as G. nivalis subsp. Bulbs 0.0036
cilicicus) [7] Aerial parts 0.0014
Bulbs 0.2063
G. rizehensis [8]
Aerial parts 0.0132

*The results of the present study, ND: not detected

Quantitative Analysis of Lycorine 337

Conclusion
To the best of our knowledge, Galanthus xvalentinei nothosubsp. subplicatus
has not been investigated for its alkaloidal content prior to this study.
Therefore, the present study is the first report on the alkaloids of this natu-
rally occurring hybrid. Regarding the HPLC method used in this study, it
was validated by determination of linearity, intra and inter-day precision,
recovery and limits of quantification and detection. Validation procedures
showed that the method was specific, accurate and precise. Appropriate lit-
erature data are available concerning the use of pre-packed columns based
on diatomaceous earth for liquid–liquid sample purification for the extrac-
tion of the Amaryllidaceae alkaloids [13]. This type of column enables a
rapid quantitative determination of alkaloids and also allows to work with
small quantities of plant material which makes this method simple and effi-
cient. A high percentage of recovery also displayed that the extraction
method was successful.
In our previous investigations, a number of Galanthus species has been
found to contain lycorine, but not galanthamine (Table III). The results of the
present study also indicated that similar to other previously investigated
Galanthus species [7,8], the bulbs and aerial parts of Galanthus xvalentinei
nothosubsp. subplicatus were not found to contain galanthamine.

Acknowledgments
This research was supported by the Ege University Research Fund (no.
09/ECZ/037) and partially supported by TUBİTAK (TBAG-104T272) and
EBİLTEM (2007-BİL-007).

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