Spectrophotometric determination of eszopiclone in pure and pharmaceutical forms

Article abstract of DOI:10.4067/S0717-97072012000400025

Six spectrophotometric methods for determination of eszopiclone have been developed, validated and applied for the assay of the drug in pharmaceuticals. Methods A, B, C and D are based on ion pair complexation of drug, in acidic buffers, with triphenylmethane dyes viz., Bromothymol blue(BTB), Bromophenol blue(BPB), Bromocresol green(BCG) and Bromocresol purple(BCP). The complexes are extracted into chloroform and absorbance is measured around at 415 nm as function of concentration of the drug. The stoichiometry of the complex is found 1:1 in each case. Method E depends upon charge transfer complexation of neutralized drug with iodine which produces iodide ion whose absorbance at 366 nm is measured as function of concentration of the drug. This complex, too, has 1:1 composition as determined by Job's method. Method F is developed on the basis of oxidation of the drug with alkaline KMnO₄ which generates green colored manganate ion with l_max 610 nm. As the intensity of green color increased with increasing time kinetics of the reaction is followed and calibration curves are constructed by using initial rate and fixed time methods. Excellent recovery studies with high accuracy and precession indicate that the methods can be successfully used in industries for the assay of drug in pure form and pharmaceuticals.

Full text of DOI:10.4067/S0717-97072012000400025

J. Chil. Chem. Soc., 57, Nº 4 (2012)
SPECTROPHOTOMETRIC DETERMINATION OF ESZOPICLONE IN PURE AND
PHARMACEUTICAL FORMS
CHENNAIAH M1, VEERAIAH T2 AND VENKATESHWARLU G*1
1Department of Chemistry, Nizam College (O.U.), Hyderabad, 500001, India
2Department of Chemistry, SAP College Vikarabad, Ranga Reddy, Andhra Pradesh, 501101, India
ABSTRACT
Six spectrophotometric methods for determination of eszopiclone have been developed, validated and applied for the assay of the drug in pharmaceuticals.
Methods A, B, C and D are based on ion pair complexation of drug, in acidic buffers, with triphenylmethane dyes viz., Bromothymol blue(BTB), Bromophenol
blue(BPB), Bromocresol green(BCG) and Bromocresol purple(BCP). The complexes are extracted into chloroform and absorbance is measured around at 415
nm as function of concentration of the drug. The stoichiometry of the complex is found 1:1 in each case. Method E depends upon charge transfer complexation
of neutralized drug with iodine which produces iodide ion whose absorbance at 366 nm is measured as function of concentration of the drug. This complex, too,
has 1:1 composition as determined by Job’s method. Method F is developed on the basis of oxidation of the drug with alkaline KMnO4 which generates green
colored manganate ion with l 610 nm. As the intensity of green color increased with increasing time kinetics of the reaction is followed and calibration curves
are constructed by using inimtiaax l rate and fixed time methods. Excellent recovery studies with high accuracy and precession indicate that the methods can be
successfully used in industries for the assay of drug in pure form and pharmaceuticals.
Key-words: Spectrophotometry, Eszopiclone, Bromothymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Iodine, Alkaline KMnO4
METHODS A, B, C and D
INTRODUCTION
The methods A,B,C and D are based on the interaction of the drug with
Bromothymol blue (BTB), Bromophenol blue (BPB), Bromocresol green
(BCG) and Bromocresol purple (BCP) respectively, to form chloroform
extractable ion pair complexes (Scheme1) which absorb around 415 nm. The
absorbance of this band increases with increasing the concentration of the drug
and formed a basis for the quantification of the drug. The dyestuffs were used
as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric
acid buffers of pH 2.8,2.5,3.5 and 2.5 were prepared by mixing 50ml of 1.0M
sodium acetate solution with 49.50 ml, 50.50ml ,46.25ml and 50.50 ml of 1.0
M HCl solution respectively and diluted to 250 ml with doubly distilled water.
The pH of each solution was adjusted to an appropriate value with the aid of
a pH meter.
Eszopiclone chemically (S)-6-(5-Chloro-2-pyridinyl)-7-oxo-6,7-dihydro-
5H-pyrrolo [3,4b]pyrazin5-yl-4-methyl-1-piperazinecarboxylate (I) , is a non-
benzodiazepine hypnotic agent used as a treatment for insomnia.
Eszopiclone is the active dextrorotatory stereoisomer of zopiclone, and
belongs to the class of drugs known as cyclopyrrolones. Eszopiclone is a short
acting nonbenzodiazepine sedative hypnotic[1]. Eszopiclone is a hypnotic
agent with a chemical structure unrelated to benzodiazepines, barbiturates,
or other drugs with known hypnotic properties, it interacts with the gamma-
aminobutyric acid-benzodiazepine (GABABZ) receptor complex. Subunit
modulation of the GABABZ receptor chloride channel macromolecular complex
is hypothesized to be responsible for some of the pharmacological properties
of benzodiazepines, which include sedative, anxiolytic, muscle relaxant, and
anticonvulsive effects in animal models. Eszopiclone binds selectively to the
brain alpha subunit of the GABA A omega-1 receptor[2]. Literature review
reveals that some analytical methods have been reported for Eszopiclone by UV
and difference spectroscopic methods [3], in biological fluids using LCMSMS
[4], validated LC method for the estimation of Eszopiclone in bulk and tablet
dosage form[5]. Recently stability indicating RP-LC method for determination
of Eszopiclone has been reported which is either tedious or expensive method
[6]. The LC method uses acetonitrile as the mobile phase which is expensive
than methanol as a mobile phase. Recently EZP has also been quantified and
validated by Stability-Indicating HPLC assay method [7].
In the present short communication we report six quantification methods
viz., A,B,C.D,E and F have been developed and validated for quantification of
the Eszopiclone (EZP) both in pure and pharmaceutical forms.
EXPERIMENTAL
Sheme 1
INSTRUMENTS
METHOD E
The spectra were recorded on SHIMADZU 140 double beam
spectrophotometer, Thermo Nicolet 1000 and also on ELICO 159 UV-Visible
single beam spectrophotometers using quartz cells of 10 mm path length. An
Elico model Li-120 pH meter was used for pH measurement.
The method depends up on the interaction of drug with Iodine that
generates iodide ion having an absorption band at 366 nm. The absorbance of
this band increases with increasing the concentration of the drug and formed a
basis for the quantification of the drug. Mixing the solution of iodine prepared
in DCE with EZP resulted in a change of violet color of iodine into light brown
to pale yellow color and as a consequence, absorption spectra exhibited a band
of 366nm. This is attributed due to I3- ion formed by the interaction of iodine
with drugs and the same is shown in Scheme 2.
MATERIALS
HPLC grade chloroform and Analytucal grade (AR) dyes viz., a) BTB b)
BPB c) BCG d) BCP were used in the study. Other chemicals such as HCl ,
Sodium acetate, KMnO and Sodium hydroxide are AR grade and are supplied
by Sd Fine Chemicals, 4Mumbai were used in the study. Iodine (BDH, Poole,
UK) was twice sublimed and preserved in vacuum desiccators (mp 113.6 oC).
Iodine in 1,2 dichloroethane was freshly prepared (daily) by dissolving 254mg
solute in 50ml of solvent (4.0×10-3 M ). The drug was procured from Hetero
drugs private limited, Hyderabad, as gift sample.
D + I → D. I2 ( outer complex )
fast ….
1
2
3
D. I2 (2outer complex )
→ (D.I +) I-(inner complex) slow..
(D.I +)I-(inner complex) + I2 → (D.I +) + I-3
,
fast….
Scheme 2
e-mail: venkateshwarlugoud@yahoo.com
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J. Chil. Chem. Soc., 57, Nº 4 (2012)
METHOD F
Kinetic method: The method depends on the oxidation of the drug with
alkaline KMnO (1x10-2 M) to produce Manganate ion which absorbs at 610
nm and formed 4a basis for quantification of drug. A solution of 0.45M NaOH
is used to produce required alkalinity. Mixing the solutions of permanganate
and the drug slowly developed green colour and hence kinetics of the reaction
was followed spectrophotometrically with a view to develop a method for the
quantitative determination of the drug. The initial rate and fixed time methods
are followed for the determination of Eszopiclone. The reaction is shown in
Scheme 3.
RESULTS AND DISCUSSION
METHODS A, B, C and D
EZP forms ion-pair complexes in acidic buffer with dyestuffs viz.,,
BTB, BPB ,BCG and BCP These complexes are quantitatively extracted into
chloroform. Ion-pair complexes of drug with dyes absorbed maximally at 415
nm (Fig. 1). The reagent blank under similar conditions showed no absorption.
EZP contains six nitrogen atoms to act as donor sites. But three of them are
secondary and formed part of the heterocyclic ring Out of the remaining three
, two nitrogens are adjacent to carbonyl and have less basicity. The nitrogen in
pyrazine ring is only potential donor site. Hence we propose the protonation of
tertiary nitrogen of pyrazine ring in acidic medium, while sulphonic acid group
is present in any of the dyes that is the only group undergoing dissociation in the
pH range 1-5. The colour of such dyes is due to the opening of lactoid ring and
subsequent formation of quinoid group. It is supposed that the two tautomers
are present in equilibrium but due to strong acidic nature of the sulphonic acid
group, the quinoid body must predominate. Finally the protonated EZP forms
ion-pairs with the dyestuff which is quantitatively extracted into chloroform.
Scheme 3
Calibration curves for method A,B,C and D
Different aliquots of drug solution were transferred into 125 ml separating
funnel. To this 5 ml of buffer, 5 ml of dye were added and total volume was
made up to 20 ml with water. 10 ml of chloroform was added and the contents
were shaken for 5 min. The two layers were allowed to separate for 5 min. The
organic layer was separated and absorbance of yellow colored solution which is
stable at least for 3 hrs is measured at 415 nm against blank similarly prepared.
The same procedure of analysis is followed either for assay of pure drug or for
dosage form. The calibration graphs are linear for all the dyes analysed using
these methods.
Calibration curve for method E
Into separate 10ml of volumetric flasks different aliquots of EZP solution
was transferred followed by the addition of 1ml of iodine solution prepared in
1,2-dichloroethane ( 4.0×10-3 M ). The volume was completed using the same
solvent and the absorbance was measured against reagent blank at 366nm.
Stoichiometry
In order to establish molar ratio between EZP and dyestuffs used, the Job’s
method of continuous variation has been applied [8]. In this method, solutions
of drug and dyestuff with identical molar concentrations [8 x 10-5M] were
mixed in varying volume ratios in such a way that the total volume of each
mixture was the same. The absorbance of each solution was measured and
plotted against the mole fraction of the drug, [drug]/[drug]+[dyestuff] . This
measurement showed that 1:1 complex was formed.
Calibration curve for method F
A) Initial rate method
Aliquots of 1 to 8 ml of Eszopiclone solution containing 2.5 mg ml-1 of
drug were pipetted into a series of 10 ml standard flasks. To each flask, 1.0 ml
of 0.45 M NaOH and 1 ml of 1.00 x10-2 M potassium permanganate were
added successively and then diluted with distilled water at 25 ±100C. The
contents of each flask were mixed well, and the increase in absorbance as a
function of time was measured at 610 nm. The initial rate of the reaction (ν) at
different concentrations was evaluated by measuring the slope of the tangent to
the absorbance-time plot. The calibration graphs were obtained by plotting the
initial rate of reaction (ν) versus the molar concentration of the Eszopiclone.
The Job’s method of stoichiometry is also applied for Iodine with EZP
which indicated the charge transfer complex formed is of 1:1 composition (Fig
2).
B) Fixed time method
A fixed time of 15 min was selected for the fixed time method. At this time
the absorbance of reaction mixture was measured at 610 nm against a reagent
blank prepared similarly without the drug. The calibration curve was obtained
by plotting the absorbance against the initial concentration of Eszopiclone (
Fig1).
Procedure for the assay of pure drug
Four different solutions of pure drug in the range of calibration curve were
selected and the recovery experiments were performed. The recoveries and
their relative standard deviation is tabulated Table 2.
Procedure for the assay of dosage forms
Ten tablets of Lunesta 3mg are powdered and dissolved in doubly distilled
water and stirred thoroughly, filtered through a Whatman No. 42 filter paper.
This solution was transferred into 100 ml standard volumetric flask and
diluted with doubly distilled water as required. Different solutions of drug in
the range of calibration curve were chosen and the assay was estimated using
the calibration curve. The results of the recovery experiments are tabulated in
Table 3.
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J. Chil. Chem. Soc., 57, Nº 4 (2012)
Table 1: Spectral and Analytical parameters for the determination of EZP using different methods.
Parameter/Method
λmax (nm)
A
414
B
411
C
D
E
F
366
417
405
610
Beer’s law limits (µg ml-1)
Molar Absorptivity (L Mol-1 cm-1)
Formation Constant (K, M-1)
Sandell Sensitivity (µg cm-2)
Slope (a)
2.2-25
19168
1.724x106
0.02
2.5-25
13725
1.436x106
0.0283
0.0353
0.0363
0.999
0.27
2.5-25
18079
1.68x106
0.0215
0.0465
0.0587
0.999
0.27
2.5-25
18001
1.57x106
0.0216
0.0463
0.0415
0.999
0.27
2.5-17.5
9603
18-162
2216
1.12x106
0.175
0.0057
0.0044
0.999
2.0
0.04
0.0247
0.0684
0.999
0.27
0.049
0.0804
0.9996
0.27
Intercept (b)
Correlation coefficient (r)
Limit of detection (µg ml-1)
Limit of quantification (µg ml-1)
0.83
0.83
0.83
0.83
0.83
6
Y=0.08+
0.0493x
Y=0.0363+ Y=0.0587+ Y=0.0415+ Y=0.0684+
Y=0.004+
0.0057x
Regression equation Y=b+ax
0.0353x
0.0465x
0.0463x
0.0247x
x is Concentration of drug (mg ml-1)
Table 2: Precision and Accuracy studies of the different methods developed for the essay of EZP.
A
4
8
12
16
B
4
8
12
16
C
4
8
12
16
D
4
8
12
16
E
4
8
12
16
F
20
30
40
50
Amount Taken
(µg ml-1)
3.997
8.01
12.007
16.04
4.011
7.98
12.002
15.94
3.97
8.005
12.08
15.96
3.99
8.001
12.08
15.97
4.004
7.92
11.92
16.13
4.02
7.94
11.93
16.08
Amount Found
(µg ml-1)
99.93
100.13
100.06
100.25
100.28
99.78
100.02
99.65
99.3
100.07
100.7
99.78
99.69
100.02
100.65
99.83
100.12
99.02
99.34
100.5
99.2
99.4
% Recovery
100.82
100.55
RSD%
0.135
0.278
0.58
0.424
0.81
0.71
Mean ± SD
100.1±0.13
99.93±0.28
99.96±0.58
100.05±0.4
99.82±0.8
99.9±0.88
Ref [7]Mean± SD
99.49±0.88
99.49±0.88
99.49±0.88
99.49±0.88
99.49±0.88
99.49±0.88
t-test
1.17
0.84
0.81
0.43
1.02
0.23
0.51
0.84
0.68
0.66
F-test
0.024
0.0998
Table 3 : Application of proposed method for the analysis of the EZP in pharmaceutical form.
Lunesta 3 mg/Tab.
A
B
C
D
E
F
4
8
12
16
4
8
12
16
4
8
12
16
4
8
12
16
4
8
12
16
4
8
12
16
AmountTaken
(µg ml-1)
3.98
7.98
12.007
16.06
3.96
8.03
11.95
15.94
4.005
8.005
11.96
15.96
4.005
7.98
11.98
16.01
3.98
7.98
12.02
15.96
3.986
7.98
12.03
16.14
Amount Found
(µg ml-1)
99.57
99.705
100.06
100.4
99.15
100.41
99.58
99.65
100.12
100.07
99.72
100.13
99.78
99.81
99.52
99.77
100.16
99.74
99.66
99.77
100.26
100.9
Recovery %
99.78
100.09
RSD %
0.36
0.52
0.2
0.182
0.27
0.56
99.92±
0.36
99.7±
0.52
99.93±
0.199
99.95±
0.181
99.8±
0.27
100.15±
0.56
Mean ± SD
99.49±
0.88
99.49±
0.88
99.49±
0.88
99.49±
0.88
99.49±
0.88
99.49±
0.88
Ref Mean ± SD[7]
t-Test
0.806
0.168
0.367
0.35
0.84
0.9
0.59
0.09
1.13
0.41
F-Test
0.051
0.04
The stoichiometric ratio between Eszopiclone and potassium permanganate was evaluated by limiting logarithmic method [9]. In this method two sets
of experiments were performed. In the first set the concentration of EZP was varied keeping a constant concentration of KMnO4, while in the second set,
the concentration of EZP was kept constant and the KMnO4 concentration was varied. Log absorbance versus log [EZP] or [KMnO ] (Fig. 3) was plotted to
evaluate the slopes of the respective lines. The slope was found to be unity in each case thus indicating the molar combining ratio of 41:1 between Eszopiclone
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J. Chil. Chem. Soc., 57, Nº 4 (2012)
and potassium permanganate. The literature survey reveals that Eszopiclone
undergoes oxidation at methylated N of piperizine ring, 4- N(CH3), to its N
oxide viz., 4- N(CH3)®O [10].
8. P. Job, Anal. Chim., 9, 113 (1928).
9. N. Rehman, M. Kashif, Japan Soc. Anal. Chem, 19, 907 (2003).
10. Yu Sha, Lei Zhang, Gui-Jie Du, Jian Ren, Mao-Sheng Cheng, Molecules,
13, 1817, (2008).
Formation constants
The formation constant was also estimated from Job’s plot by following
method described by Likussa and Boltz [11], and Momoki etal [12].The
method involves drawing the tangents at the origin of Job’s plot from both side
and the absorbance at intersection point is taken for 100% complexation. The
absorbance at peak height of Job’s plot is taken for (100-x)% where x is the %
degree of dissociation of the complex. The instability constant, K’ = Cx2/(100-
x) is calculated, where C is concentration of drug used for Job’s method. The
reciprocal of K’ is the required stability constant K.
11. W. Likussar D.F. Boltz, Anal. Chem., 43, 1265, (1971).
12. K. Momoki, J. Sekino, H. Sato, N. Yamaguchi, Anal. Chem., 41, 1286,
(1969).
13. H A Benesi and J R Hildebrand, J Am Chem Soc, 71, 2703 (1949)
14. International Conference on Harmonization (ICH) of Technical
Requirement for the Registration of Pharmaceuticals for Human use,
Validation of analytical procedures: definitions and Terminology Genera
(1996).
Formation constant for method E
Formation constant (K) has been evaluated by using Benesi-Hikdebrand
equation[13]
[A0]/d =1/K[D0]e +1/e
where d is absorbance, e is the molar absorbtivity, A0 and D0 are initial
concentrations of acceptor [I2] and donor[drug] respectively. A plot of [A0]/d
Vs 1/[D0] yields a straight line whose slope and intercept gives the value of K.
Optimization of the factors effecting the absorbance.
The factors effecting the absorbance of ion pair complexes like pH and
volume of the dye, in methods A,B,C and D, have been optimized. 1.8 ml of
BTB and buffer of pH 2.8, 1.6 ml of BPB and buffer of pH 2.5, 1.8 ml of
BCG and buffer of pH 3.5 and 1.7 ml of BCP and buffer of pH 2.5 are found to
ne optimal for methods A.B,C and D respectively. However 5 ml of each dye
is used, at optimal pH, in the study to ensure complete extraction of the drug.
Similarly the 1 ml of iodine for method E and 1 ml of KMnO 1 ml of 0.45
NaOH are found to be optimal and hence are used in the study.4 ,
Validation of the proposed methods
The proposed method have been validated in terms of guideline proposed
by ICH [14] viz., selectivity, specificity, accuracy, precision, limits of
calibration curve, LOD, LOQ, robustness, ruggedness and regression equation.
The student t-test and variance F-test have been performed in comparison with
a reference method. Table 1A summarizes the values for Beer’s law limits,
molar absorptivity, regression equation, correlation coefficients, relative
standard deviation and recoveries. To test the reproducibility of the proposed
methods, six replicate determinations of 12.5µg ml-1 of EZP were made. The
coefficient of variation was found to be less than 1.2% for all the procedures.
The proposed methods have been successfully applied to the determination
of EZP in pharmaceutical preparations. The result obtained and shown in Table
1C were compared to those obtained by a reference method [7] by means of
t-test at 95% confidence level. In all cases, the average results obtained by
proposed methods and reference method were statistically identical, as the
difference between the average values had no significance at 95% confidence
level.
The proposed methods are simple, sensitive and reproducible and can be
used for routine analysis of EZP in pure form and in formulation.
ACKNOWLEDGEMENTS
The authors are grateful to the Head of the Department Chemistry and to
the Principal, Nizam College for providing facilities. MC is thankful to UGC
for FDP fellowship. TV is thankful to Sri M. Ravindra Reddy, Chairman,
Managing Committee SAP College, Vikarabad for providing facilities and to
the UGC for financial assistance under Major Research Project.
REFERENCES
1. C.S. Mc. Crae, A. Ross, A. Stripling, N.D. Dautovich, Clin Interv Aging,
2, 313, (2007).
2. G.S.Jufe, Vertex, 16, 294, (2007).
3. K. Anandakumar, G. Kumaraswamy, T. Ayyappan, A.S.K. Sankar, D.
Nagavalli, Res. J. Pharm.Tech, 3, 202, (2010).
4. M. Meng, L. Rohde, V. Capka, P. Bennett, Chiral Chromatographic
Method, 1, (2007).
5. K. Anandakumar, G. Kumaraswamy, T. Ayyappan, A.S.K. Sankar, D.
Nagavalli, Asian J.Res.Chem, 3, 63, (2010).
6. R.N. Kumar, G.N. Rao, P.Y. Naidu, Asian J.Res.Chem, 3, 374, (2010).
7. R. Sunil, Dhaneshwar, K. Vidhya, Bhusari, Int. J. ChemTech. Res, 3. 680,
(2011).
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