Quantitative Estimation of Eszopiclone in Bulk and in Formulation by Simple UV and Difference Spectroscopic Methods

 

K Anandakumar^*, G Kumaraswamy,T Ayyappan, ASK Sankar and D Nagavalli

Adhiparasakthi College of Pharmacy, Melmaruvathur - 603 319, Tamil Nadu, India.

*Corresponding Author E-mail: anandkarunakaran@gmail.com

ABSTRACT:

Two simple efficient, precise and accurate simple UV and difference spectroscopic methods have been developed for the estimation of Eszopiclone in bulk and in pharmaceutical dosage form. In method A, UV Spectra of Eszopiclone in 0.1M Hydrochloric acid exhibits the absorption maximum 304 nm. Method B is based on the measurement of difference in absorbance of Eszopiclone at maxima 307 nm and minima at 239 nm. The measured value is the amplitude of maxima minima between two equimolar concentrations of the analyte in different chemical forms, which exhibits different spectral characteristics.  Beers law limit the concentration range of 3-18 µg/ ml for method A, and method B, for 2-12 µg/ ml. The accuracy of the method was determined by recovery studies. The methods were validated statistically. The methods showed good reproducibility and recovery with % RSD less than 2. The methods were found to be simple, economical, accurate and reproducible and can be used for routine analysis of Eszopiclone in bulk drug and in formulation.

 

 

 

INTRODUCTION:

Eszopiclone (ESZ) ^1-8a new drug and it is used as hypnotic agent⁹ which is chemically [(7S)-6-(5-chloropyridin-2-yl-5-oxo-7H-pyrrolo [3, 4-b] pyrazin-7-yl] 4- methylpiperazine1-carboxylate (Fig.1). ESZ is not official in any pharmacopoeia.  The mechanism action of Eszopiclone is not completely understood but it thoughts that acts on benzodiazepine and interacts on GABA receptor complexes. Literature survey revealed that there are so many methods were reported for the pharmacokinetic study and pharmacological study of ESZ^10-13 and a GC – MS and UPLC¹⁴ methods were reported for the estimation of ESZ in bulk. But there is no method was reported for the estimation of ESZ in bulk drug and in formulation. Hence the present work aims to develop two simple, precise, accurate and validated difference spectroscopic method for the estimation of ESZ in bulk and in tablet dosage form by using simple UV method and difference spectroscopic method^15-16.Confirmation of the applicability of the developed method were validated according to the International Conference on Harmonization (ICH) ^17-18 for the determination of ESZ in bulk and in tablet dosage form.

 

MATERIALS:

Instruments:

Shimadzu UV-1700 UV/VIS spectrophotometer with 1cm matched quartz cells was used for spectral and absorbance measurements.

 

Drug Material and Chemicals:

ESZ was obtained as a gift sample from Dr. Reddy’s Laboratories, Hyderabad.

Hydrochloric acid and Sodium hydroxide were purchased from Qualigens fine Chemicals Mumbai. The tablet formulation Fulnite containing 3mg of Eszopiclone manufactured by Sun Pharmaceuticals, Mumbai was procured from the local market. All the Chemicals and reagents used were of AR grade.

 

Preparation of Reagents:¹⁹

0.1M Sodium hydroxide:

4 gms of sodium hydroxide was accurately weighed and dissolved in distilled water and the volume was made up to 1000 ml with distilled water.

 

0.1M Hydrochloric acid:

8.5 ml of hydrochloric acid was made up to 1000 ml with distilled water.

 

METHODS:

Simple UV Spectroscopic Method (Method A):

This method is based on the measurements of absorbance of ESZ at its λ max was found to be 304 nm. 0.1M HCl of analytical reagent grade was selected as solvent for developing spectral characteristics of drug. The selection was made after assessing the solubility of the drug in different solvents.

 

Preparation of standard stock solution:

15 mg of Eszopiclone raw material was accurately weighed and transferred into the 100 ml volumetric flask and dissolved in minimum quantity of 0.1M HCl and made up to 100 ml with 0.1M HCl.  The solution was observed to contain 150 mg/ ml.

 

Fig.1. Chemical structure of Eszopiclone

 

Fig 2   Simple UV spectrum of Eszopiclone containing 10 µg/ ml

 

The above stock solution (0.5–3.0 ml of 150 μg/ ml) was transferred into six 25 ml volumetric flasks and made up to mark with 0.1M Hydrochloric acid. The absorbances of resulting solutions were measured at 304nm using 0.1M Hydrochloric acid as blank. Calibration curve was plotted by using concentration versus absorbance. The curve was linear with the concentration range of 3-18 μg/ ml.

 

Difference Spectroscopic Method (Method B):^18-19

The essential future of a difference spectrophtometric assay is that the measured value is the difference in absorbance between two equimolar solutions of the analyte in different chemical forms which exhibit difference spectral characteristics.  This method is based on measurement of absorbance of Eszopiclone at maxima 307nm and minima 239nm.The measured value is the amplitude of absorbance maxima and minima between two equimolar solutions of the analyte in different chemical forms, which exhibits the different spectral characteristics.

 

Preparation of standard stock solution:

10 mg of ESZ was weighed accurately and dissolved in Acetonitrile and made up the volume to 100 ml in a volumetric flask. The solution was further diluted with 0.1M hydrochloric acid and 0.1M sodium hydroxide separately to get the concentration of 100µg/ ml (working standards). Different aliquots were taken from their working standards and diluted with 0.1M hydrochloric acid and 0.1M sodium hydroxide separately to get the of concentrations from 2-12 µg/ ml  for  both the series of solutions. Difference spectrum was recorded by placing ESZ in 0.1M sodium hydroxide in reference cell and 0.1M Hydrochloric acid in sample cell. Difference in absorbance between 239 nm and 307 nm was measured to find out the amplitude. The calibration curve was prepared by plotting amplitude versus concentration.

 

Fig 3. Difference UV Spectrum of Eszopiclone in 0.1M HCl against Esopiclone in 0.1M NaOH as a blank

 

Fig 4. Calibration Curve of Esopiclone by UV Method

 

Application of the proposed procedure for the determination of dosage form:

Marketed tablet formulation Fulnite was used for analysis. Twenty tablets were weighed and their average net weight was calculated. The tablet was made to a fine powder.

 

Method –A:

The powder equivalent to equivalent to 15 mg of Eszopiclone was transferred into a 100 ml volumetric flask, added 0.1M HCl to dissolve and made up to the volume. Then the solution was sonicated for 10 minutes. After sonication the solution was centrifuged at 100 rpm for 15 minutes. The solution was filtered through Whatmann filter paper No.41. From the clear solution, 2 ml of the solution was transferred into a 25 ml standard flask and made up to the mark with 0.1M HCl to produce 12 mg/ ml concentration the absorbance measurements were made six times for the formulation at 304 nm. The amount of Eszopiclone present in formulation was determined by using slope and intercept values from calibration graph.

 

Table 1: Optical characteristics

 

PARAMETERS	METHOD  A	METHOD B
λmax (nm) Beers Law Limit (µg/ ml) Molar absorptivity (mol.l-1cm-1) Régression equation (Y= mx+c) Slope (m) Intercept (c) Corrélation coefficient (r) Standard error LOD(µg/ml) LOQ(µg/ml)	304 3 - 18 1.5820 x 104 Y = 0.0398x+0.0090 0.0398 0.0090 0.9993 0.0103 0.0376 0.1139	307a   239b 2 - 12 2.0670 x 104 Y = 0.0554x+0.0031 0.0554 0.0031 0.9998 0.0004 0.1220 0.3697

^A and ^b are the maxima and minima, respectively.

 

Table 2: Assay of commercial formulation

Methods	Label amount mg/tablet)	Amount found*(mg/tablet)	%Label Claim	SD	% RSD	SE
A	3.0	3.01	100.08	0.4414	0.4410	0.1802
B	3.0	3.07	102.32	1.3467	1.3162	0.5498

* Mean + SD of six observations

 

 

Table 3: Intraday and interlay precision of the method

Methods	Amount Found (Percentage Obtained)		% RSD
	Intraday*	Interday*	Intraday*	Interday*
A	99.35	100.38	0.0355	0.01955
B	102.12	101.23	1.0236	1.8398

* Mean + SD of three observations

 

Method –B:

The powder equivalent to 10 mg of ESZ was weighed and transferred in to 100 ml volumetric flask. Dissolved in Acetonitrile and made up to the volume with the same the solution was filtered through Whatman filter paper No.41. From the stock solution, 8 µg/ ml solutions were prepared separately by using 0.1M hydrochloric acid and 0.1M sodium hydroxide. The amplitude was calculated by measuring the absorbance of the equimolar concentrations at maxima and minima in the difference spectrum. The amount of ESZ was calculated. The procedure was repeated for six times to perform precision.

 

Recovery studies:

The accuracy of the proposed methods was examined by determining the recovery of the drug by standard addition technique. To the preanalysed formulation a known amount of raw material added and it can be analyzed by proposed methods.

 

Method –A:

15 mg equivalent of Eszopiclone formulation was taken into a series of three 100 ml standard flasks. To that 2.5 mg, 5 mg and 7.5 mg of raw material were added in to series of standard flasks 1, 2 and 3, respectively. Dissolved with 0.1M HCl and made up to volume with 0.1M HCl .The solutions were sonicated for 10 minutes. After sonication, the solution was centrifuged at 100 rpm for 15 minutes. The solutions were filtered through Whatmann filter paper No. 41.  From each standard flask, 2 ml of the clear filtrate was transferred into a series of six 25 ml standard flasks and made up to volume with 0.1M HCl. The amount of drug recovered was calculated. Each concentration was repeated for three times.

 

Fig 5. Calibration Curve  of Esopiclone by Difference Spectroscopic Method

 

Method –B:

The powder equivalent to 10 mg equivalent of Eszopiclone formulation was taken into a series of three 100 ml standard flasks.  To that 2 mg, 3 mg and 4 mg of raw material were added in to series of standard flasks 1, 2 and 3, respectively dissolved with Acetonitrile and made up to volume with acetonitrile .The solutions were sonicated for 10 minutes. After sonication, the solution was centrifuged at 100 rpm for 15 minutes. The solutions were filtered through Whatmann filter paper No. 41.  From the above solution 2 ml was taken in two 25 ml volumetric flasks and one was made up to the volume with 0.1M sodium hydroxide and another one was made up with 0.1M hydrochloric acid. The amount of drug recovered was calculated. Each concentration was repeated for three times.

 

RESULTS AND DISCUSSION:

Two simple precise and accurate methods were developed for the estimation of ESZ in bulk and in formulations, Method A is simple UV simple spectroscopic method (Fig 2), and Method B is the Difference spectroscopic method (Fig 3). The linearity of the methods was found to be 3 -18 μg/ ml for method A and 2-12μg/ ml for method B. The optical characteristics such as correlation co-efficient, slope,

Table 4: Recovery studies

Methods	Amount Present (µg/ml)	Amount added* (µg/ml)	Amount found* (µg/ml)	Amount recovered* (µg/ml)	Percentage recovery* %	Average ± S.D	% RSD	S.E
A	12.06	2	14.13	2.07	102.98	101.49 ± 9424	0.9286	0.3847
	12.06	4	16.12	4.06	101.50
	12.06	6	18.06	6.00	100.00
B	8.05	1.6	9.69	1.64	102.50	100.20 ± 1.2713	1.2687	0.5190
	8.05	2.4	10.42	2.37	98.75
	8.05	3.2	11.23	3.18	99.37

* Mean + SD of six observations

 

 

intercept, molar obsorptivity, LOD and LOQ were calculated and are shown in Table 1. The correlation co-efficient was found to be 0.9993 and 0.9998 for method A and method B respectively. The calibration graph for both the methods are shown in figure 4 and 5.The absorbance was measured and the amount of ESZ in tablet formulation was found to be 100.08 ± 0.4414 mg for method A, and 102.32 ± 1.3467mg for method B (Table 2). To study the precision of the method the analysis of formulation was carried out for six times. The % RSD values were found to be 0.4410 for method A and 1.3162 for method B. By the low % RSD values, the precision of the methods were confirmed.  Further the precision was confirmed by intermediate precision. The analysis of formulation was carried out for three times in the same day and on three successive days. The % RSD values for interlay and intraday analyses of formulation was found to be less than 2% and are shown in table 3. The accuracy of method was confirmed by recovery studies. A known amount of standard drug material was added with pre-analyzed formulation in different levels. The amount of drug recovered was calculated and the percentage recovery was found to be in the range of 101.50% - 102.98% for method A, 99.37 –102.50% for method B. The procedure was repeated for 3 times for each concentration and the % RSD values were calculated. The low %RSD values ensure that the excipients used in formulation are not interfering in the analysis of ESZ. The results of recovery studies are shown in table 4.

 

CONCLUSION:

The proposed method is simple, accurate, precise and selective for the estimation of ESZ in bulk and in tablet dosage forms. The method is economical, rapid and do not require any sophisticated instruments contrast to chromatographic method. Hence it can be effectively applied for the routine analysis of ESZ in bulk and in tablet dosage forms.

 

ACKNOWLEDGEMENTS:

The authors wish to thank Dr. Reddy’s Laboratories, Hyderabad for providing the gift sample of ESZ. The authors are thankful to Arul Thiru Amma, Thirumati Amma, ACMEC TRUST and Dr. T. Vetrichelvan, Principal Adhiparashakthi College of Pharmacy for their kind help and providing all necessary facilities.

 

REFERENCE:

1.        Eszopiclone available at www.enwikipedia.com

2.        CIMS, CMP Medical India Private Limited; Bangalore. 2008, 13, 157.

3.        Budavari S.The Merck Index: An Encyclopedia of chemicals, Drugs and Biologicals. 14^th ed. Whitehouse station (NJ): Merck Research Lab. Division of Merck; 2006, 1096.

4.        Anthony Moffer C, David Osselton M and Brain Widdop. Clarke’s analysis of drugs and poisons; 3^rd ed. Royal pharmaceutical society of Great Britain, 2004, II, 1717.

5.        http:// www. rx list. com.

6.        http:// www.science direct com

7.        http:// www.ingentapharma.com

8.        http:// www.duke health.com

9.        Krystal AD, Lasaka E, Wessel TC et al., sustained efficacy of Eszopiclone, over six months of treatment of nightly treatment;results randomized,double-blind placebo controlled study in adults with chronic insomnia, U.S National health ins.med.sci. 2003, 26(7), 739.

10.     Walsh J K, Krystal A D, Amtho D A, Ruben Caron J, Wessel C, Schaeferk, Roach J, Wallen stain G, Roth T et al.,nightly treatment for pwith primary insomnia with Eszopiclone for six months; effect on sleep, quality of life and work limitations, U.S National health ins.med.sci. 2005, 30(8), 959-68.

11.     Boveley J Johnsen, Roach J, Robens R et.al.,Next day cognition, psychomotor function, and driving related skills following nighttimes admistration of Eszopiclone, U.S National health ins.med.sci.  23(5), 2008, 385-97.

12.     Zamith GK, Mc nabb LJ, Caron J, Amatho DA, et al., efficacy and safety of treatment for primary insomnia,U.S National health ins.med.sci..20(12), 2004, 1979-91.

13.     Rosenberg R, Roth T, et al., “an insomnia and safety of Eszopiclone in the treatment of transient insomnia in healthy adults”, U.S National health ins.med.sci.2005, 6(1), 15-22.

14.     Roxanne E. Franckowski M.S. et al., reported a GC – MS and an UPLC method, United States of Drug Enforcement Administration 2007, Vol.II, 192-195. (Electronic version. Available on http://www.pubmed.com)

15.     Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry.  4^th ed. Part II, New Delhi: CBS Publishers; 2002, 4, 293.

16.     Gnana Babu jose C et al., estimation of Nicordinil in tablet dosage form by differences spectrometry Indian j pharm edu.res.2007; 41(2):163

17.     International conference of Harmonisation, Guidance for Industry. In; Q2A Validation on Analytical Procedure; ethodology.  Switcherland: IFPMA 1994, 27 October 1- 5.

18.     International conference of Harmonisation, Guidance for Industry. In; Q2B Validation on Analytical procedures; ethodology.  Switcherland: IFPMA 1996; 1-8

19.     The Indian Pharmacopoeia, addendum, The Controller of Publication, New Delhi 1996,volume II, A-19 , A-169.

 

 

 

 

 

Received on 29.08.2009          Modified on 15.11.2009

Accepted on 24.12.2009         © RJPT All right reserved