Accepted Manuscript

Title: DEVELOPMENT AND VALIDATION OF A

CAPILLARY ZONE ELECTROPHORETIC METHOD FOR
RAPID AND SENSITIVE DETERMINATION OF
GALANTHAMINE: APPLICATION IN PLANT AND
PHARMACEUTICALS

Author: Nurullah Sanli Ibrahim Bulduk Hatice Ozkurt Senem

Sanli Sibel A. Ozkan

PII: S0731-7085(16)30471-X
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2016.08.026
Reference: PBA 10822

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 18-4-2016

Revised date: 12-8-2016
Accepted date: 27-8-2016

Please cite this article as: Nurullah Sanli, Ibrahim Bulduk, Hatice Ozkurt,
Senem Sanli, Sibel A.Ozkan, DEVELOPMENT AND VALIDATION OF A
CAPILLARY ZONE ELECTROPHORETIC METHOD FOR RAPID AND
SENSITIVE DETERMINATION OF GALANTHAMINE: APPLICATION IN
PLANT AND PHARMACEUTICALS, Journal of Pharmaceutical and Biomedical
Analysis http://dx.doi.org/10.1016/j.jpba.2016.08.026

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DEVELOPMENT AND VALIDATION OF A CAPILLARY ZONE
ELECTROPHORETIC METHOD FOR RAPID AND SENSITIVE
DETERMINATION OF GALANTHAMINE: APPLICATION IN PLANT
AND PHARMACEUTICALS

Nurullah ANLI1, Ibrahim BULDUK2, Hatice OZKURT1, Senem ANLI3, Sibel A. OZKAN4*

1
Faculty of Engineering, Uak University, UAK, TURKEY
2
Health CareEducation, Researchand Application Center, Uak University, UAK, TURKEY
3
Department of Chemistry, Faculty of Science and Arts, Uak University, UAK, TURKEY
4
Department of Analytical Chemistry,Faculty of Pharmacy, Ankara University, ANKARA,
TURKEY

Address for Correspondence:

*Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100,

Tandoan, Ankara, Turkey; Fax: 0090 312 2238243 ; E-mail: ozkan@pharmacy.ankara.edu.tr

1
Graphical abstract

Highlights

Sensitive, simple, rapid, environmentally friendly, convenient, cost-effective and high-

throughput approach was developed for determination of Galanthamine in plant
extracts and pharmaceutical dosage forms.
First report on the quantification of Galanthamine in Leucojum aestivum L. of Turkish
origin collected from Black Sea Region.
Successful validation according to strictest criteria of ICH regulatory guidelines.
The method would have great value when applied in pharmaceutical preparations,
plants and may be in biological fluids, due to the lower LOD values with short
analysis time.

2
Abstract

Galanthamine is a second-generation cholinesterase inhibitor that has begun to be used in the

treatment of Alzheimer's disease. In the presented research, a simple, accurate, precise and
cost-effective capillary zone electrophoretic (CZE) method was used for the qualitative and
quantitative estimation of Galanthamine from bulbs, leaves and fringes of Leucojum aestivum
L. (summer snowflake) grown in Turkish habitats (Black Sea Region) and pharmaceutical
dosage forms by capillary zone electrophoresis with direct UV detection form. Ultrasonic
assisted extraction (UAE) and response surface methodology (RSM) were used to estimate
optimum experimental conditions on the content extraction of Leucojum aestivum L.
Metoprolol was used as a suitable internal standard. A linear relationship between the ratio
and concentrations of Galanthamine in the range of 0.25 g.mL-1-15.00 g.mL-1was
determined with a regression coefficient of 0.9996, for which the limit of detection (LOD)
and limit of quantitation (LOQ) were 0.027 and 0.081g.mL-1, respectively. The high
percentage recovery results showed that the matrix effect did not influence the developed
method for analysis of pharmaceutical preparations. Validation parameters were carried out
according to the guidelines of the International Conference for Harmonization (ICH). This
method also allows for a number of cost- and time-saving benefits and can be readily
employed for the analysis of plants and pharmaceutical formulations. The method can be used
in industries for the determination of Galanthamine to analyze the quality of extraction and
formulation without interference.

Keywords: Galanthamine, Alzheimer's disease, Leucojum aestivum L., Response Surface

Methodology, Capillary electrophoresis.

3
1. Introduction
Since Alzheimer's disease is associated with a decline in cholinergic
neutrotransmission, therapies designed to enhance acetylcholine levels have received the most
attention. Galanthamine (GA, (4aS,6R,8aS)-5,6,9,10,11,12-Hexahydro-3-methoxy-11-methyl-
4aH-[1]benzofuro[3a,3,2-ef][2]benzazepin-6-ol) is a selective inhibitor of
acetylcholinesterase, the enzyme responsible for the degradation of acetylcholine at
cholinergic synapses. Preliminary results from experiments on patients with Alzheimer's
disease have reported GA to be associated with a reduction in cognitive deterioration on some
neuropsychiatric rating scales [1]. Since the GA is an allosteric modulator for the nicotinic
receptor of the acetylcholine neurons, it could also be used as the cure for neuromuscular
diseases such as neuromuscular blockade, myasthenia, and drug-induced respiratory
depression [2]. In February of 2001, the FDA approved it as Reminyl [3].
Although, the synthesis of GA has been accomplished [4], the production of GA from
a sustainable natural source has still been considered important. This has resulted in studies on
several aspects including the isolation, qualitative and quantitative analysis, biosynthesis and
biotechnological production of this compound. The botanical sources of the Amaryllidaceae
alkaloids have been reviewed [5]. The GA content in some Amaryllidaceae species, which has
been determined by isolation, is for example: 0.00565% fresh weight (FW) in Leucojum
aestivum L. [6]. Also, the galanthamine content in the leaves of Leucojum aestivum, referred
to as dried weight (DW), was found to vary from trace amounts to 0.5% (generally 0.1-0.3%)
depending on the geographical location of the population [7].
Leucojum aestivum L. is a plant species widely cultivated as an ornamental. It is
considered native to Turkey, Iran, and the Caucasus [8]. A simple and rapid ultrasound-
assisted extraction (UAE) procedure was developed and optimized for the extraction of GA
from the bulbs, leaves and fringes of Leucojum aestivum L. grown in Turkish habitats (Black
Sea Region).
There are many methods by which to determine GA content, such as UV-visible
spectrometry [9], fluorescence spectrometry [10], micellar electrokinetic chromatography
[11], UHPLC [12], GC-MS [13], radioimmunoassay [14], and so on. There are limiting
methods related to the CE technique, such as experimental design and artificial neural
network optimization [15], capillary electrophoresis with an electrochemiluminescence
method [16], and enantiomeric separation of GA [17]. Although these techniques are
sufficiently sensitive, most of them are either highly costly or time consuming. So it is quite
meaningful to develop a rapid, simple, accurate, precise and cost-effective capillary zone

4
electrophoresis (CZE) method for the qualitative and quantitative estimation of GA in plant
extracts and pharmaceutical dosage forms.
Although GA is indicated for the treatment of mild to moderate vascular dementia and
Alzheimer's disease, to the best of our knowledge there are no previously published methods
for determining GA from bulbs, leaves, and fringes of Leucojum aestivum L.(summer
snowflake) grown in Turkish habitats (Black Sea Region) and pharmaceutical dosage forms
by capillary zone electrophoresis with direct UV detection. Thus, we have reported the
development of a simple, accurate, precise, and fully validated capillary electrophoresis
method for the qualitative and quantitative determination of GA in various samples.
Validation parameters were carried out as per the guidelines of the International Conference
for Harmonization (ICH) [18].

2. Materials and Methods

2.1. Chemicals

All chemicals and solvents were of analytical reagent grade and used without further
purification. GA, metoprolol, sodium hydroxide (NaOH), hydrochloric acid (HCl), methanol
(MeOH), sodium phosphate, phosphoric acid, sodium tetraborate (borate), and boric acid were
of analytical-grade purity and obtained from Sigma-Aldrich (St Louis MO, USA).
Stock standard solutions of GA and metoprolol were prepared in ultrapure water to a
concentration of 100 g.mL-1. Working solutions were diluted with water to a concentration
of 10 g.mL-1. Metoprolol was chosen as the internal standard for GA analysis. All stock and
working solutions were protected from light and stored in a fridge at about the temperature of
4C when not in use. All the solutions were first filtered with Whatman filter paper in order to
prevent capillary blockage, then filtered with a 0.22 micron membrane filter and then treated
with ultrasound (Bandelin, Sonorex, Germany) to remove the air dissolved in the solution.
2.2. Apparatus
Identification and quantitative determination of GA was established by Agilent 7100
Capillary Electrophoresis System (Waldbronn, Germany), equipped with a photodiode array
(PDA) detector. Instrument control, peak integrations, and peak purity calculations were done
by Agilent ChemStation software. A Mettler Toledo Seven multi MA 235 pH/ion analyzer
with a Mettler Toledo InLab Expert Pro combined glass electrode was used for the pH
measurements. The pH measurements were performed in triplicate to ensure the stability and

5
reproducibility of the potentiometric system. A 55 cm 50 m ID fused silica capillary
(Agilent Technologies) with an effective length of 45 cm was used for the separation.
2.3. Capillary Electrophoresis Procedure
The separation capillary was activated in the first use by flushing it sequentially with
MeOH (5 min), 1.0 M HCl (2 min), water (2 min), 1.0 M NaOH (20 min), water (2 min), and
then with the buffer (20 min). The capillary was conditioned daily by washing it with 1.0M
NaOH (20 min), water (2 min), and finally with the running buffer (20 min). Between each
analysis, the capillary was flushed with 1.0 M NaOH (3 min), water (2 min) and then the
running buffer (3 min). Prior to use, buffer solutions were filtered through 0.25 m membrane
filters and degassed by ultrasonication for 10 min. A 10 mM tetraborate buffer was used as a
background electrolyte (BGE) at pH 9.2. The capillary cassette temperature was set to 25oC
during all experiments. The applied voltage was 30 kV, the hydrodynamic injection was at
100 mbar for 3 s, and the detection was performed at 210 nm for GA and 267 nm for
metoprolol, respectively.
2.4. Ultrasound Assisted Extraction (UAE) of Leucojum aestivumL. (Summer snowflake)
A 0.250 g sample of the well-mixed, ground and dried bulbs was weighed and
transferred to an Erlenmeyer flask, and extraction was undertaken with 25 mL of solvent
(MeOH+ 0.1 M HCl) changing according to an experiment schedule (Table 3) in an ultrasonic
bath. An ultrasound assistant extraction was carried out using a Bandelin Sonorex brand
ultrasonic bath with a 50 kHz frequency. For the standard ultrasonic conditions, Erlenmeyer
flasks were placed inside the ultrasonic bath. The solvent level in the Erlenmeyer flask and
water level in the ultrasonic bath were kept the same. The temperature and time value of the
ultrasonic bath was set and the extraction was carried out. After the extraction process had
been completed, the mixture was first filtered with Whatman filter paper in order to prevent
capillary blockage and then filtered with a 0.22 m membrane filter.
2.5. Experimental Design

A three-level, three-factorial BoxBehnken experimental design [19] was used to

determine the best combination of extraction variables and the influence of three independent
parameters (temperature, time and MeOH concentration) on the extraction of GA. The ranges
of temperature (30-70 0C), time (15-45 min), and MeOH concentration (25-75%) were
determined based on preliminary experiments. The independent variables and their code
levels are shown in Table 1.
<Table 1 about here>

6
 Table 2 represents the non-coded values of the experimental variables and 17
experimental points. To express the GA content as a function of the independent variables, a
second order polynomial equation was used as follows;

= 0 + 4=1 + 4=1 2 + 4=1 4=1+1 + (1)

<Table 2 about here>

2.6. Analysis of Pharmaceutical Preparations
Ten tablets of Reminyle (containing 8 mg GA, Janssen Cilag, Turkey), were
accurately weighed and powdered. The required amount of this powder, equivalent to a stock
solution of 10 g mL-1 GA, was weighed and transferred into a 100 mL volumetric flask and
diluted with ultrapure water. The prepared solution was sonicated for 10 min to complete the
dissolution. After filtration, appropriate solutions were prepared by taking suitable aliquots of
clear filtrate and adding the appropriate IS solution, then diluting them with BGE in order to
obtain a final solution. The nominal content of the tablet amounts was calculated from the
corresponding regression equations of previously plotted calibration plots from the raw
material.
To confirm the accuracy of the developed assay methods and to study the interference
of formulation additives, recovery experiments were carried out. For this purpose, a known
amount of the pure standard substance of GA was added to the pre-analyzed tablets
formulation.
After five repeated measurements, the recovery results were calculated by using the
related calibration equation for all compounds.

3. Results and Discussions

3.1. Optimization of the Separation Method
The absorption spectra of GA (aqueous solutions) at different pH values were
measured. The absorbance maximum was observed at 210 nm. Several compounds were
tested as an internal standard to use in the validation process, such as Oseltamivir,
Lavumidine, Valganciclovir and Metoprolol. Among these compounds, Metoprolol had a
shorter analysis time, a good peak shape, and selectivity. The maximum absorbance of
Metoprolol (IS) occurred at 226 nm.
To find out the optimum conditions for the electrophoretic separation of the drugs, the
pH of the run buffer, buffer concentration, applied potential and injection time, and pressure

7
parameters were investigated. Several background electrolytes such as phosphate, tris, borate,
etc., were tested for the CE analysis. For the electrophoretic separations, compounds should
be ionized. GA is a weak base, and pKa value of this compound can be associated with
nitrogen atom in azepine. (pKa = 8.49-8.65) [20]. The buffering capacity of a borate buffer
was suitable for this pH range. The highest peaks and a shorter analysis time were obtained at
alkaline pH using a borate buffer, and therefore a borate buffer was used as the run buffer.
Buffer concentration was tested in the concentration range of 5-20 mM. Due to Joule
heating, higher buffer concentrations cause a high current. It is best to select the minimum
buffer concentration with less generated current and efficient separation. The total run time
increased as the run buffer concentration increased. A 10 mM borate concentration gave the
best separation of the drugs. The pH of the run buffer was investigated in the range of pH 9.0-
9.50. Well-shaped peaks with reasonable peak areas and peak resolution appeared at pH 9.2.
The applied potential was examined in the range of 18, 24, 26 and 30 kV. Sharp peaks
and less migration time were obtained by increasing the applied voltage. Since the maximum
potential is preferred in capillary electrophoresis, 30 kV was applied for the optimum results.
The capillary cassette temperature was set to 25oC. Several injection pressures and times were
tested and the longer times and/or the higher pressures caused zone broadening, resulting in
current generation and poor resolution. 100 mbar for 3 seconds was chosen as the optimal
injection condition. The capillary with total length of 45 cm was chosen for further analysis
because the shorter capillaries gave a shorter migration time.
The optimum separation was obtained with a 10 mM borate buffer, pH 9.2, as the run
buffer under the applied potential of 30 kV and the standards were injected at 100 mbar
pressure for 3 seconds. Under these conditions the migration times were 1.910.05 and
2.200.06 minutes for IS and GA, respectively. An electropherogram of the optimum
conditions is given in Fig. 1 (A).
<Figure 1 about here>

The method was validated in accordance with ICH [18] requirements. The calibration
curves were obtained by linear least squares regression. The concentration of IS was
maintained at a constant level of 10 g.mL-1in all validation experiments. The linearity was
calculated by plotting the peak area ratio of the drug to IS in relation to the concentration of
the compound. Linearity was investigated in the range between 0.25 and 15.00 g.mL-1, and
the method exhibited good linearity based on a correlation coefficient > 0.999 for the

8
compound studied. The obtained linear equation was y = 0.0624 0.0051 + 0.3672
0.0386x. LOD and LOQ were 0.0268 g.mL-1and 0.0812 g.mL-1 respectively and calculated
as LOD = 3.3 s x m-1 and LOQ = 10 s x m-1, where s is the standard deviation of response and
m is the slope of the corresponding calibration curve. Comparing the sensitivity of reported
methods, our strategy has better sensitivity, resolution, and wider linear range except coupled
with electrochemiluminescence method [9-12-16].

Precision and reproducibility of the method were evaluated by a replicate analysis of

the standard solutions containing 1, 5, 10 g mL-1 GA and IS were analyzed in consecutive
days with five replicates. Repeatability and reproducibility were characterized by mean
recovery and %RSD, and the results were between 99.142 0.478 and 100.937 0.149. As
can be deduced from obtained results, there was no significant difference for the assay, as
tested within-day (intra-day) and between-days (inter-day). The results suggest that the
method was precise.
4. Application of the method

4.1. Application of Leucojum aestivum L.

4.1.1. Effect of process variables on the UAE performance

Table 2 shows the experiment conditions of Box-Behnken design runs designed with
Design Expert 10 program. This table also displays the effects of MeOH concentration,
extraction time, and extraction temperature on the extraction efficiency obtained by
Ultrasound Assisted Extraction (UAE).

4.1.2. Effect of extraction time on the UAE performance

The influence of the extraction time on the extraction efficiency of GA was examined
over a range of 15-45 min, and Table 2 shows the results. The experiment results showed that
30 min is the optimum GA extraction time. When extraction time increased, the cell walls of
the leaves of Leucojum aestivum L. fully fell apart and GA experienced material liquid
diffusion so that the extraction yield was relatively rapid. During the long extraction time,
overheating of the Leucojum aestivum L. bulbs was prone to cause the thermal decomposition
of GA because of the unstable chemical bonds of GA molecules, such as unsaturated bonds,
after which GA content was decreased. Therefore, 30 min is favorable for extracting the GA.

9
4.1.3. Effect of extraction temperature on the UAE performance

An extraction process was carried out using an extraction temperature between 30 and
70 C. The extraction temperature has significant effects on the efficiency of GA. When the
extraction temperature was increased, the extraction efficiency increased rapidly up to a
maximum of 50 C. In general, higher temperatures in extractions are directly proportional to
the rates of mass transfer and extraction performance because of enhanced solute desorption
from the active sites of plant matrix [19]. When the extraction temperature went above 51 C,
the extraction yield started to decrease. Initially, the extraction yield increased with the rising
temperature, maybe because the elevated temperature accelerated GA chemical bond rupture
and sped up molecular motion, so that a large number of GA dissolved into the cell solution.
When the heating temperature was greater than 51C, the high temperature caused the
destruction of the GA structure, accelerated the degradation reaction, and lost GA activity so
that GA content was rapidly reduced. High temperatures could also decompose already
extracted GA or even involve the breakdown of those molecules still remaining in the plant
matrix [19]. Therefore, 51 C is favorable for extracting the GA.

4.1.4. Effect of methanol concentration on the UAE performance

An extraction process was carried out using a MeOH concentration between 25% and
75% (v/v). In the initial stage, the extraction yield of GA increased rapidly when the MeOH
concentration was increased from 25% to a peak of 50% (v/v). However, with a MeOH
concentration greater than the 50% (v/v) GA extraction yield showed a slowly decreasing
trend. This is because increasing the MeOH concentration leads to enhanced mass transfer
dynamics, solvents, and Leucojum aestivum L. getting full access, and then an increase in the
dissolution of the contents of GA. When the MeOH concentration reached a certain level, it
was difficult for all of the GA to be dissolved by the high concentration of MeOH, which also
led to an increase in the alcohol-soluble impurity content, resulting in a loss of GA content.
Moreover, the greater the MeOH concentrations, the more difficult it is to refine GA, which
will cause waste and increase the cost of production. Therefore, a MeOH concentration of
50% (v/v) is good for the GA extraction. Fig.2 shows the interactive effect of different
parameters for GA yield. The corresponding contour plots have also been depicted in Fig. 2.

4.1.5. Response Surface Analysis and Process Optimization

10
 Fixing one of the three factors at zero, the 3-dimensional response surface and contour
map was schematized as in Fig. 2. Thus we can determine the optimal factor levels through
analyzing and evaluating the interactive influence of any two factors on the rate of extraction
of GA.
According to Fig. 2 (A), the mutual effects of extraction temperature and time
displayed a remarkable effect on the response. Similarly, the extraction rate of GA increased
gradually along with the temperature, rising up to 51 0C, at which point the extraction yield of
GA was at its maximum. Also, we can conclude that temperature, rather than extraction time,
influenced the yield most efficiently.
According to Fig. 2 (B), we can see a significant influence on the extraction of GA by
the mutual effect of the MeOH concentration and extraction temperature. At the experimental
level range, the MeOH concentration has little effect on extraction yield, while temperature
did more. The extraction of GA first increased and then decreased, and reached the maximum
point when the temperature was 51 0C.

<Figure 2 about here>

4.1.6. Statistical Analysis

An Anova test was applied to identify the interaction between the variables and the
response using a Design-Expert 10 program. The statistical testing of this model was carried
out in the form of analysis of variance (ANOVA). The ANOVA for the fitted quadratic
polynomial model of extraction of GA is shown in Table 3. The quadratic regression model
indicated that the determination coefficient (R2) value was 0.9921, which suggested that
99.21% of the total variations could be illustrated by the fitted quadratic polynomial model.
The value of adjusted R2 was 0.9820, which implied that in fact less than 2.0% of the total
variations were not illustrated by this model. It also showed a high degree of correlation
between the predicted and observed values. A low value of the coefficient of variation (CV =
3.02%) explained a good deal about the reliability of the conducted experiment values. These
variables can be more significant if the F-value becomes higher and the p-value becomes
smaller. The p-value of less than 0.05 indicated that the quadratic model terms were
significant. Therefore, the F-value (F= 97.95) and p-value (p = < 0.0001) suggested that the
quadratic model was significant. Also, the significance of the quadratic model was
represented by the lack-of-fit test. The result is shown in Table 3: the F-value and p-value of

11
the lack-of-fit were 4.80 and 0.0818, respectively, which suggested that it was not significant
and a 21.6% margin of error could occur because of noise.

<Table 3 about here>

4.1.7. Optimization of UAE by Response surface methodology (RSM)
The individual effects of process variables, also known as the one-factor at-a-time
approach, were applied in the previous section. This classical approach ignores the possible
interactions of process variables with each other, which may result in misleading conclusions.
Response surface methodology (RSM) considers the probable interactions between operation
parameters [19]. Table 2 shows the three parameters (MeOH concentration, time, and
temperature) including minimum, median and maximum points. Seventeen experiments were
run and chosen randomly by the design expert software and the responses were recorded
(Table 3). Using response surface methodology with the software, a quadratic model applying
both forward stepwise and backward elimination regressions for GA was obtained. An
empirical relationship between the response and the independent variables has been expressed
by the following quadratic polynomial equation model:

GA Yield % = 0.20194 + 0.00583 X1 0.00548 X2 + 0.00500 X3 + 0.000008 X1 X2 +

0.000017 X1 X3 0.000015 X2 X3 0.000068 X12 0.000085 X22 0.000054 X32 (2)

X1, X2, X3, X1X2 are not significant effects for the model. After excluding their regression
coefficients, a new model may be given for a better explanation of the new condition.

GA Yield % = 0.20194 + 0.000017 X1 X3 0.000015 X2 X3 0.000068 X12 0.000085

X22 0.000054 X32 (3)

Theoretical recovery values for GA were calculated from this equation and plotted against
practical ones. These relationships are shown in Fig. 2(D). The linear correlation between the
experimentally obtained values of the extraction yields versus the calculated values using the
model equation was observed ( = 1.009 0.026 0.001 0.003; 2 = 0.991).
The optimal extraction conditions were found by using optimization choice in design
expert software to maximize the response. This value was measured at 50.61 of MeOH
concentration, 29.94 min of extraction time, 50.77 0C of extraction temperature. The
maximum response was found to be (0.154 %) under these operating conditions.

12
 After the completion of the method optimization, GA analysis was made in the leaves,
bulbs, and fringes of Leucojum aestivum L. under optimal conditions. The results of GA
analysis in the bulbs, fringes, and leaves of Leucojum aestivum L. as percentages of dry
weight were 0.154 0.041, 0.195 0.034 and 0.298 0.022 respectively. Obtained data were
the averages of five replications with the standard deviations. As comparison to the other
geographical location of the populations, the galanthamine content in the leaves of Leucojum
aestivum L., referred to dry weight, was found to be good (0.3%) in Turkish habitats (Black
Sea Region) because it could be vary from generally 0.1-0.3%.
A CE analysis of Leucojum aestivum L. in bulb, fringes, and leaf extracts in the
presence of the internal standard Metoprolol and GA obtained using a diode-array detector, is
given in Fig. 1(B).

4.2. Application of Pharmaceutical Dosage Forms

The proposed method was successfully applied to tablets containing 8.0 mg GA

(Reminyl). The tablet solutions were prepared as described previously and injected into the
system. There was no interference originating from the tablet matrixes, and the peaks carried
the characteristics of standard peaks and electropherogram of tablet samples were given in
Fig. 1 (A) and (C).
The concentration of the drugs determined in tablets using the CE method is reported
in Table 4. The mean results for the determination of GA were found to be very close to the
declared value of 8 mg. Recovery studies were realized from the tablets for accuracy and
precision of the proposed techniques. The recovery of the procedure was carried out by
spiking the already analyzed samples of tablets with known concentrations of standard
solutions of GA and IS. The results of the recovery analysis are also shown in Table 4. The
statistical evaluation of the analyses resulted in good percent recoveries of almost 100%.
Likewise, the RSD% values were below 2%, which is within acceptable limits [15]. It is
concluded that the proposed method is sufficiently accurate and precise to be applied to
pharmaceutical dosage forms.
<Table 4 about here>
5. Conclusions
The present study has demonstrated the viability of this sensitive, simple, rapid,
environmentally friendly, experimentally convenient, cost-effective and high-throughput
approach for the determination of GA in plant extracts and pharmaceutical dosage forms,

13
without the necessity for sample pretreatment or any time-consuming evaporation steps prior
to analysis. The amount of studied compounds can be determined within about 2.5 min after
setting instrument parameters. To the best of our knowledge, this is the first report on the
quantification of GA in Leucojum aestivum L. of Turkish origin collected from the Black Sea
Region.
The developed CE method might serve as a versatile analytical tool suitable for the
assay of GA for quality control and for therapeutic drug monitoring laboratories. Therefore,
the method developed here could be used in pharmaceutical preparations and plants, and
maybe in biological fluids, due to the lower LOD values and short analysis time.

Acknowledgements

This study was supported by the Uak University Scientific Research Projects Commission
(BAP) under the grant no: 2015/TP001.

14
References

[1] B. Fulton, P. Benfiled, Galanthamine, Drugs & Aging. 9 (1996) 60-65.

[2] D.A. Cozanitis, E. Toivakka, Treatment of Respiratory Depression with the

Anticholinesterase Drug Galanthamine Hydrobromide, Anaesthesia. 29 (1974) 581584.

[3] Food and Drug Administration (FDA). FDA Drug Approvals for Reminyl ,

http://www.fda.gov/drugs/developmentapprovalprocess/howdrugsaredevelopedandapproved/d

rugandbiologicapprovalreports/ucm081684.htm (accessed January 2016).

[4] D.H.R. Barton, G.W. Kirby, Phenol Oxidation and Biosynthesis. Part V. The Synthesis of

Galanthamine. J. Chem. Soc. 1962, 806817.

[5] O. Hoshino, The Amaryllidaceae Alkaloids. The Alkaloids, Chemistry and Biology;

Cordell, G.A., Ed.; Academic Press: San Diego, 1998; Vol. 51, 324342.

[6] S. Kobayashi, M. Kihara, K. Yuasa, Y. Imakura, T.Shingu, A.Kato, T. Hashimoto,

Alkaloidal Constituents of Leucojum asetivum L. (Amaryllidaceae), Chem. Pharm. Bull. 33

(1985) 52595263.

[7]. Stefanov J. (1990) Ecological, biological and phytochemical studies on natural

populations and introduced origins of the summer snowflake (Leucojum aestivum L.) in

Bulgaria. D.Sc. Thesis, Sofia.

[8] P.H. Davis, (ed.) Flora of Turkey and the East Aegean Islands. Edinburgh University

Press, Edinburgh, 1984, pp. 1-632.

[9] K. Mittal, R. Kaushal, R. Mashru, A. Thakkar, Estimation of Galanthamine Using

Derivative Spectrophotometry in Bulk Drug and Formulation. J. Biomed.Sci. Eng. 3 (2010)

439441.

[10] M.J. Culzoni, R. Q.Aucelio, G.M. Escandar, Spectrofluorimetry in Organized Media

Coupled to Second-Order Multivariate Calibration for the Determination of Galantamine in

the Presence of Uncalibrated Interferences, Talanta 82 (2010) 325332.

15
[11] I.N. Nicolaou, C.P. Kapnissi-Christodoulou, Simultaneous Determination of Nine

Acetylcholinesterase Inhibitors Using Micellar Electrokinetic Chromatography, J.

Chromatogr. Sci. 49 (2011) 265271.

[12] R.G. Deshpande, A.K. Roy, N.S. Rao, B.M. Rao, J.R. Reddy, Rapid Screening of

Volatile Ion-Pair Reagents Using UHPLC and Robust Analytical Method Development Using

DoE for an Acetyl Cholinesterase Inhibitor: GA HBr, Chromatographia, 73 (2011) 639-648.

[13] R.Gotti, J.Fiori, M.Bartolini, V.Cavrini, Analysis of Amaryllidaceae Alkaloids from

Narcissus by GCMS and Capillary Electrophoresis, J. Pharm. Biomed. Anal. 42(2006) 17

24.

[14] T. Tanahashi, A. Poulev,M.H. Zenk, Radioimmunoassay for the Quantitative

Determination of Galanthamine, Planta Medica. 56 (1990) 7781.

[15] L. Pokorna, A. Revilla, J. Havela, J. Patocka. Capillary Zone Electrophoresis

Determination of Galanthamine in Biological Fluids and Pharmaceutical Preparatives:

Experimental Design and Artificial Neural Network Optimization. Electrophoresis, 20 (1999)

1993-1997.

[16] Y. Wang, G. Zhu, X. Li, Z. Hao, Simultaneous Determination of Galanthamine and

Lycorine in Lycoris Radiata by a Capillary Electrophoresis with an

Electrochemiluminescence Method, J. Sep. Sci. 37 (2014) 30073012.

[17] V. Ravinder, S. Ashok, A.V.S.S. Prasad, G. Balaswamy, Y.R. Kumar, B.V. Bhaskar, A

Validated Chiral LC Method for the Enantiomeric Separation of Galanthamine,

Chromatographia, 67 (2008) 331-334.

[18] Guideline, ICH Harmonized Tripartite. "Validation of Analytical Procedures: Text and

Methodology." Q2 (R1) 1 (2005). Available from: URL:

http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html.

16
[19] J. Saha, A. Biswas, A. Chhetri, P.K. Sarkar, Response Surface Optimization of

Antioxidant Extraction from Kinema, a Bacillus-Fermented Soybean Food. Food Chemistry

129 (2011) 507513.

[20] Y. D. Daldal, E. . Demiralay, S. A, Ozkan Effect of Organic Solvent Composition on

Dissociation Constants of Some Reversible Acetylcholinesterase Inhibitors, J. Braz. Chem.

Soc., 27 (2016) 493-499.

17
Figure Captions

Figure 1. The electropherogram of A) 10 g.mL-1 standard Metoprolol (IS) and GA

monitored at 226 nm absorbance under optimum conditions, B) analysis of Leucojum
aestivum L. in bulb, fringes and leaves extracts in the presence of the internal standard
Metoprolol (M) and GA (G) obtained using a diode-array detector, C) analysis of tablet
dosage forms (Reminyl) at 210 nm absorbance and 226 nm absorbance. (1) Metoprolol (IS);
(2) GA.

Figure 2. Three-dimensional contour and response surface plots for GA extraction showing
the interactive effects of the A) methanol concentration and extraction time, B) methanol
concentration and extraction temperature, C) methanol concentration and extraction time.

18
Figure 1.

19
Figure 2.

20
Table 1. The variables and their coded levels used for optimization of GA extraction from
Leucojum aestivum L. by using the Box-Behnken design.

Independent Units Symbols of Coded Levels

Variables the variables
-1 0 1

0
Extraction Temp. C (X1) 30 50 70
Extraction Time min (X2) 15 30 45
Methanol Conc. % (X3) 25 50 75

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Table 2. A Box-Behnken Design of the independent variables (X1, X2, X3) and experimental
results for the GA
Ext. Methanol
Run Temperature Ext. Time Concentration GA Content
0
C min % %
1 30 15 50 0.109
2 50 30 50 0.156
3 30 30 25 0.102
4 30 30 75 0.083
5 70 15 50 0.110
6 50 15 75 0.109
7 50 30 50 0.155
8 70 30 25 0.087
9 50 45 75 0.101
10 50 30 50 0.153
11 70 30 75 0.102
12 50 30 50 0.156
13 70 45 50 0.111
14 50 45 25 0.105
15 50 15 25 0.091
16 30 45 50 0.101
17 50 30 50 0.131

22
Table 3. The analysis of variance (ANOVA) for the Response Surface Quadratic Model.

Sum of Mean F p-value

Source df
Squares Square Value Prob > F
Model 0.011 9 1.211E-003 97.95 < 0.0001 Significant
X1-Ext.
2.813E-005 1 2.813E-005 2.27 0.1752 Not sign.
Temperature
X2-Ext. Time 1.250E-007 1 1.250E-007 0.010 0.9227 Not sign.
X3-Methanol
1.250E-005 1 1.250E-005 1.01 0.3482 Not sign.
Concentration
X1X2 2.025E-005 1 2.025E-005 1.64 0.2414 Not sign.
X1X3 2.890E-004 1 2.890E-004 23.37 0.0019 Significant
X2X3 1.210E-004 1 1.210E-004 9.79 0.0166 Significant
X12 3.121E-003 1 3.121E-003 252.41 < 0.0001 Significant
X22 1.556E-003 1 1.556E-003 125.86 < 0.0001 Significant
X32 4.718E-003 1 4.718E-003 381.60 < 0.0001 Significant
Residual 8.655E-005 7 1.236E-005
Lack of Fit 6.775E-005 3 2.258E-005 4.80 0.0818 Not sign.
Pure Error 1.880E-005 4 4.700E-006

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Table 4.The GA assay results and the recovery analysis in pharmaceutical dosage forms
GA
Labeled claim (mg) 8.00
Amount found (mg)a 8.073
RSD (%) 1.672
Bias (%) -0.913
Added (mg) 4.00
Found (mg)a 4.021
Recovery (%) 100.53
RSD% of recovery 1.244
Bias (%) -0.525
a
Each value of the mean five experiments

24