1. No category

RP TLC Analysis of New Antidepressants
in Pharmaceutical Preparations
Taťána Gondová* and Ibrahim A. Amar
Key Words
Citalopram
Fluoxetine
RP TLC
Drug analysis
Summary
A simple and precise reversed-phase thin-layer chromatographic
(RP TLC) method for simultaneous separation of fluoxetine and
citalopram in pharmaceutical preparations has been developed and
validated. Separation was performed on RP-18 F254 TLC plates with
methanol–0.05 M phosphate buffer (pH 5)–triethylamine 68:27:5
(v/v) as mobile phase. Densitometric detection was performed at
230 nm. The method was validated for linearity, accuracy, precision,
selectivity, and robustness. Calibration plots showed the response,
as peak area, was a linear (r2 > 0.9988) function of the amounts of
the compounds in the concentration range 500–5000 ng per spot.
Statistical analysis proved the method was both precise and accurate. The method was successfully used for analysis of citalopram
and fluoxetine in their pharmaceutical preparations, with recovery
of the compounds ranging from 99.10 to 101.70% of the labeled
amount.
1 Introduction
Selective serotonin reuptake inhibitors (SSRI) are a new generation of antidepressants used for treatment of depressive disorders and other indications [1]. These substances can regulate the
concentration of serotonin (a neurotransmitter affecting mood,
cognition, sleep, appetite, hormone secretion, etc.) in the central
nervous system. Over the past decade, SSRI, which include fluoxetine, fluvoxamine, sertraline, citalopram, and paroxetine,
have become the most widely used group of antidepressants,
because they are regarded as both safe and well-tolerated [2].
The various analytical methods developed for each drug individually have been described in a review [3]. Methods which
enable simultaneous analysis of several SSRI antidepressants in
a single step are useful for practical and economic reasons, however. Several methods for simultaneous analysis of selective
serotonin reuptake inhibitors in biological samples or in pharmaceutical formulations have been reported, and include highperformance liquid chromatography (HPLC) [4, 5], gas chro-
matography (GC) with mass spectrometry (MS) [6, 7], and thinlayer chromatography (TLC) [8, 9]. Use of capillary electrophoresis for assay of SSRI in pharmaceutical preparations and
biological fluids has recently been reported [10].
Although TLC is less sensitive than GC–MS or HPLC, it has
several advantages, including simplicity of use, the ability to
repeat detection and quantification with changed conditions,
cost effectiveness, and minimum sample cleanup. The development of HPTLC instruments for automated sample application
and densitometric evaluation in situ has made it possible to
obtain results that are comparable with those obtained by
HPLC [11].
There are few literature reports of simultaneous analysis of
SSRI antidepressants by thin-layer chromatography. Misztal and
Skibiñski [8] reported simultaneous analysis of six new antidepressants by reversed-phase and normal-phase TLC. Only qualitative analysis of the drugs has been performed on RP-18 TLC
plates with a variety of aqueous–organic mobile phases. The
best separation was achieved with methanol–0.09 M phosphate
buffer (pH 1.98) 60:40 (v/v) as mobile phase. Videodensitometric detection was performed at 254 nm. There is also a report of
the use of normal-phase TLC with densitometric detection for
analysis of four SSRI antidepressants, and analysis of citalopram and fluoxetine in pharmaceutical preparations [9].
As far as we are aware, there is no reported reversed-phase TLC
method for simultaneous quantitative analysis of fluoxetine and
citalopram in pharmaceutical formulations. In this paper we present a simple and reliable TLC method with densitometric
detection which enables analysis of the two most commonly
prescribed SSRI antidepressants fluoxetine and citalopram
(Figure 1) in pharmaceuticals.
2 Experimental
2.1 Chemicals and Standards
T. Gondová and I.A. Amar, Department of Analytical Chemistry, Faculty of
Science, P.J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic.
E-mail: [email protected]
40
Journal of Planar Chromatography 24 (2011) 1, 40–43
0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
Fluoxetine hydrochloride and citalopram hydrobromide were
supplied by Slovakofarma (Hlohovec, Slovak Republic). The
DOI: 10.1556/JPC.24.2011.1.7
RP TLC of Antidepressants
Table 1
Validation data for the TLC method for quantification of fluoxetine
and citalopram.
Fluoxetine
Citalopram
Linear range
[μg per spot]
500–5000
500–5000
Figure 1
Regression equation
y = 32.089x + 19000
y = 53.733x + 9201
The chemical structures of fluoxetine and citalopram.
Correlation coefficient
0.9994
0.9988
Limit of detection
[ng per spot]
100
80
Limit of quantitation
[ng per spot]
250
200
Precision, RSD
[%] (n = 6)
1.00
0.79
Different days, RSD
[%] (n = 3)
0.37
0.47
Specificity
Specific
Specific
Fluoxetine
Citalopram
methanol for 10 min with the aid of sonication. An appropriate
volume of the supernatant was diluted with methanol so that the
final concentration of each antidepressant was within the working range of the calibration plot.
2.3 Chromatography
Figure 2
Representative TLC densitogram obtained from fluoxetine and
citalopram at 230 nm.
commercially available pharmaceutical preparations used were:
Deprenon (20 mg fluoxetine per capsule; Slovakofarma Hlohovec, Slovak Republic), Floxet (20 mg fluoxetine per capsule;
Egis Pharmaceuticals, Hungary), Citalec (20 mg citalopram per
tablet; Zentiva, Czech Republic), and Cipralex (10 mg escitalopram per tablet; Lundbeck, Denmark).
Methanol, potassium dihydrogen phosphate, orthophosphoric
acid, triethylamine and potassium hydroxide, all analytical
grade, were obtained from Lachema (Brno, Czech Republic).
Redistilled deionized water was used for preparation of buffer.
Stock standard solutions (1 mg mL–1) of the antidepressants
were prepared individually in methanol and stored at 4°C.
Separate calibration standards for each antidepressant were
prepared daily by diluting the stock solutions with methanol
to yield concentrations of 100, 200, 400, 600, 800, and
1000 μg mL–1. These concentrations were used to construct calibration plots.
2.2 Sample Preparation
Ten citalopram tablets or ten fluoxetine capsules were weighed
and finely pulverized. A portion of the powder corresponding to
20 mg of the antidepressant was accurately weighed, quantitatively transferred to a 25-mL volumetric flask and dissolved in
Journal of Planar Chromatography 24 (2011) 1
Chromatographic analysis was performed on 10 cm × 10 cm
aluminum foil-backed RP-18 F254s TLC plates (Merck, Darmstadt, Germany). Standard and sample solutions (5 μL) were
applied to the plates by use of a CAMAG (Muttenz, Switzerland) Nanomat II sample applicator.
The mobile phase was methanol–0.05 M phosphate buffer
(pH 5)–triethylamine 68:27:5 (v/v). Linear ascending development, at ambient temperature, to a distance of 8.0 cm, was
accomplished in a glass flat-bottom chamber (20 cm × 15 cm ×
5 cm; Labora, Bratislava, Slovak Republic) previously saturated
with mobile phase vapor for 15 min. After development, plates
were dried in the air and the spots were detected under a UV
lamp (254 nm). Densitometric scanning was performed at
230 nm with a Shimadzu CS-930 dual-wavelength TLC scanner
used in reflectance–absorbance mode. This wavelength was
selected after acquiring the UV spectra of both analytes. Peak
areas were used for quantitative analysis.
2.4 Method Validation
Method validation was conducted in accordance with published
guidelines (ICH) [12]. The method was validated by establishing linearity, limits of detection and quantitation, precision,
accuracy, and specificity.
Linearity was determined from six replicate applications of each
of six different amounts of each antidepressant. Six-point calibration plots in the range 500–5000 ng per spot were constructed by plotting peak area against the corresponding amounts of
the analytes by means of the least-squares method.
The limits of detection (LOD) and quantification (LOQ) were
established on the basis of signal to noise ratios of 3:1 and 10:1,
respectively.
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RP TLC of Antidepressants
Table 2
Recovery of fluoxetine and citalopram by the TLC method (n = 3).
Compound
Fluoxetine
Citalopram
a)Matrix
Amount addeda) [%]
Theoretical content [ng]
Recovery [%]
RSD [%]
50
1500
100.86
1.45
100
2000
101.05
1.52
150
2500
100.93
1.23
50
1500
99.75
0.67
100
2000
100.94
0.76
150
2500
99.90
0.78
containing 1000 ng drug
Table 3
Results from TLC quantification of fluoxetine and citalopram in tablets (n = 5).
Compound
Fluoxetine
Citalopram
Preparation, content [mg]
Amount found [mg] (mean ± SD)
RSD [%]
Deprenon, 20
19.82 ± 0.51
2.56
99.10
Floxet, 20
20.32 ± 0.46
2.26
101.60
Citalec, 20
19.94 ± 0.21
1.03
99.70
Cipralex, 10
10.17 ± 0.04
0.38
101.70
The repeatability of sample application and measurement of
peak area were determined on the same day by repeated (n = 6)
application of the drugs extracted from tablet samples and calculation of relative standard deviation (RSD, [%]). Intermediate
precision was evaluated by comparison of assays performed on
three different days.
The accuracy of the method was studied by determination of the
recovery [%] of known amounts of fluoxetine and citalopram
standards added to solutions of the corresponding commercial
product within the linear range. Previously analyzed samples
were spiked with an extra 50, 100, and 150% of fluoxetine or
citalopram standards.
The selectivity of the method was studied in relation to possible
matrix interferences from the with pharmaceutical formulations.
No interference peaks or matrix effects from excipients were
observed in the chromatograms obtained from the formulations,
thus confirming the selectivity of the method.
To test robustness, the mobile phase pH was varied by ±0.2 units
and the concentration of the buffer by ±0.01 M to determine the
effect on the results obtained.
2.5 Analysis of Fluoxetine and Citalopram in Pharmaceutical
Preparations
Standard solutions and sample solutions were analyzed as
described above. Four pharmaceutical products commercially
available in Slovakia were analyzed.
3 Results and Discussion
To optimize the TLC separation, several mobile phases of different composition were tried. Satisfactory separation of the
compounds was achieved with methanol–phosphate buffer–tri-
42
Recovery [%]
ethylamine as mobile phase. Use of triethylamine enabled us to
separate the mixture of the basic compounds fluoxetine and
citalopram and to obtain more symmetrical and oval spots of
both separated compounds with minimum tailing.
In the next step the composition of the mobile phase for efficient
separation of the antidepressants was determined by studying an
effects of organic modifier content, pH, and buffer concentration
on the retention factors, RF, and resolution, RS, of the drugs.
The mobile phase methanol–phosphate buffer (pH 5; 0.05 M)–
triethylamine 68:27:5 (v/v) enabled baseline separation of the
drugs on RP-18 F254s TLC plates. The RF values, at 230 nm,
were 0.12 for fluoxetine and 0.28 for citalopram (with a standard deviation of less than 0.02 in all cases). A representative
densitogram obtained from the antidepressants is shown in
Figure 2.
Calibration curves were linear over the concentration range of
500–5000 μg per spot for both antidepressants; the correlation
coefficients were 0.9994 and 0.9988 for fluoxetine and citalopram, respectively, confirming good linearity for both calibration plots (Table 1).
Recovery of the drugs from the sample matrix was in the ranges
100.86–101.05% and 99.75–100.94% for fluoxetine and citalopram, respectively (Table 2). Corresponding RSD values were
less than 1.6% for fluoxetine and less than 0.8% for citalopram,
indicating the accuracy of the method. No interference peaks or
matrix effects from the excipients were observed in the chromatograms obtained from the pharmaceuticals, confirming the
selectivity of the method.
The mean amounts of the antidepressants in the pharmaceutical
preparations investigated were 99.10% and 101.60% of the label
claim for fluoxetine and 99.70% and 101.70% of the label claim
for citalopram (Table 3). Low values of relative standard deviation (below 2.6%) for both citalopram and fluoxetine indicate
Journal of Planar Chromatography 24 (2011) 1
RP TLC of Antidepressants
the precision of the method is sufficient. The method was therefore suitable for quantification of these antidepressants in the
pharmaceuticals.
[5] C. Frahnert, M.L. Rao, K. Grasmäder, J. Chromatogr. B 794
(2003) 35–47.
Acknowledgment
[7] J.J. Berzas, C.M.J. Villasenor, V. Rodriguez, Anal. Chim. Acta 519
(2004) 219–230.
This work was supported by the Grant Agency of the Slovak
Republic, grant No. 1/4461/07.
[8] G. Misztal, R. Skibinski, J. Planar Chromatogr. 14 (2001) 300–304.
[6] S.M.R. Wille, K.E. Maudens, C.H. van Peteghem, W.E.E. Lambert,
J. Chromatogr. A 1098 (2005) 19–29.
[9] T. Gondová, D. Halamová, K. Špacayová, J. Liq. Chromatogr.
Relat. Technol. 31 (2008) 2429–2441.
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Ms received: November 19, 2009
Accepted: June 14, 2010
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