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.^10-13 Literature survey revealed that there are so many methods were reported for the pharmacokinetic study and pharmacological study of ESZ 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.

The aim of the present work was to develop and validate a simple, fast and reliable isocratic RP-HPLC method with UV detection for the determination of ESZ in bulk and in tablet dosage forms. The important features and novelty of the proposed method included simple sample treatment with sonication of small amount of powder sample at ambient temperature, short elution time (less than 5 min) ESZ, good precision (R. S. D. less than 2%) and high recovery (greater than 98%).

Confirmation of the applicability of the developed method validated according to the International Conference on Harmonization (ICH)^15-16 for the determination of ESZ in bulk and in tablet dosage form.

EXPERIMENTAL:

Chemicals and reagents:

HPLC grade Acetonitrile and water was purchased from Loba fine Chemicals (Mumbai, India). Eszopiclone standard sample was provided by Dr. Reddy's Laboratories (Hyderabad, India). Fulniteâ commercial formulation (Sun Pharmaceutical (Mumbai) was procured from local market. The tablet dosage forms containing obtained was 3 mg of ESZ for oral administration. The molecular weight is 388.80 for ESZ.

Instrumentation and analytical conditions:

The HPLC system (Shimadzu, Japan) consisted of a pump (LC-10 ATVP series pump) equipped with a Rheodyne model -7161 injection valve with a 20ml loop (Rheodyne Inc., Cotati, CA, USA), an UV-visible detector (type SPD 10 AVP) set at 304 nm. The Analytical column, a Phenomenax Luna C₁₈ (150mm ´ 4.6 mm i.d., 5m particle size) was operated at ambient temperature (20 ± 1°c). Isocratic elution with Acetonitrile: Phosphate buffer (25:75% v/v pH2.5) was used at a flow rate of 1ml/ min. The mobile phase was prepared freshly and degassed by sonicating for 5 min before use (Soltec, Soluzioni tecnologiche, Luglio, Italy). The UV spectrum of ESZ for selecting the working wavelength of detection was taken using a Shimadzu UV-1700, UV -Visible spectrophotometer (Shimadzu, Kyoto, Japan).

Fig.1. Chemical structure of Eszopiclone

Fig 2. UV absorbance spectrum for Eszopiclone

Fig 3. Typical chromatogram obtained from the analysis of ESZ standard solution. Retention time of Eszopiclone was 3.92 min.

Fig 4. Calibration curve of Eszopiclone

Stock and working standard solutions:

Stock standard solution of 1000mg/ ml of ESZ was prepared freshly by accurately weighing 25mg of ESZ into 25ml volumetric flask. Dissolved and made up to the volume with Phosphate buffer (pH 2.5). The solution was diluted by pipetting 1ml into 25ml volumetric flask to obtain 40 mg/ ml solution. The solution was further diluted with mobile phase in 10 ml volumetric flask to obtain six working standards in the concentration range 4-24 mg/ ml of ESZ. All the solutions were prepared in triplicates. Before being subjected to analysis, all the working standard solutions were filtered through 13mm membrane syringe filter (Pore size 0.2 mm).

Table 1: Statistical analysis of calibration curves in the HPLC determination of Eszopiclone (n=6)

Parameters	Values
l_max(nm)	304
Beers Law Limit (mg/ ml)	4 – 24
Correlation coefficient (r)	0.9995
Regression equation (y = mx + c)	Y=616148.34x+121955.67
Slope (m)	616148.34
Intercept (c)	121955.67
LOD (mg/ ml)	0.6531
LOQ (mg/ ml)	1.9793
Standard Error	404384.6958

Before injecting solutions, the column was equilibrated for at least 60 min with the mobile phase flowing through the system. The calibration curve was plotted with the six concentrations of the 4 - 24 mg/ ml working standard solutions. Chromatogram was recorded thrice for each dilution. Calibration solutions were prepared daily and analyzed immediately after preparation.

Assay of sample preparation:

The contents of thirty commercial tablets (labeled concentration 3 mg of ESZ) were weighed and their mean mass was determined. After grinding the tablets into a fine powder in a glass mortar, an accurately weighed quantity of the tablet powder equivalent to 25 mg of ESZ was quantitatively transfer into a 25 ml volumetric flask with about 20 ml of phosphate buffer pH 2.5. The solution was sonicated for 10 min, brought to the volume with phosphate buffer, mixed well and filtered through 13mm membrane syringe filter (pore size 0.2 mm). 1 ml filtered test solution was transferred into 25 ml volumetric flask and made up to the volume with mobile phase (40 mg/ ml). 3.0 ml aliquot was transferred into a 10 ml volumetric flask. The theoretical ESZ concentration after dilution was 12 mg/ ml (100% of ESZ). An aliquot of this solution was filtered through a 13mm membrane syringe filter (Pore size 0.2 mm) prior to the injection into the HPLC system. Peak area of Eszopiclone was measured for the determinations.

Validation procedure:

The objective of method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision (repeatability and intermediate precision), accuracy, specificity, short term stability and system suitability.

Table 2: Repeatability of Eszopiclone

S. No	Labelled amount (mg/tab)	Amount found (mg/ tab)	Percentage obtained	Average (%)	S.D.	% RSD	S.E
1	3	2.97	99.00
2	3	2.95	98.33
3	3	2.95	98.33	99.60	1.5389	1.5450	0.0427
4	3	2.98	99.33
5	3	2.97	99.00
6	3	3.08	102.66

* Mean of six observations

Table 3: Intraday and inter day precision of the method

Amount Found (Percentage Obtained)		% RSD
Intraday*	Inter day*	Intraday*	Inter day*
99.35	100.38	0.0355	0.01955

* Mean + SD of Three observations

Table 4: Accuracy study for Eszopiclone (n =9)

S. No	Amount Present (mg/ ml)	Amount Added (mg/ ml)	Amount *Found (**mg/ ml)	Amount Recovered (mg/ ml)	Percentage Recovery*	S.D	% RSD	S.E
1.	11.91	3.6	15.59	3.59	99.72	0.3195	0.3207	0.1304
2.	11.91	7.2	19.11	7.19	99.86
3.	11.91	10.8	22.65	10.72	99.25
3.	11.91	10.8	22.65	10.72	99.82

* Mean of three observations

Standard plots were constructed with six concentrations in the range of 4 - 24 mg/ ml prepared in triplicates to test linearity. The peak area of ESZ was plotted against the concentration to obtain the calibration graph. The linearity was evaluated by linear regression analysis that was calculated by the least square regression method.

The precision of the assay was studied with respect to both repeatability and intermediate precision. Repeatability was calculated from six replicate injections of freshly prepared ESZ test solution in the same equipment at a concentration of 100% (12mg/ ml) of the intended test concentration value on the same day. The experiment was repeated by assaying freshly prepared solution at the same concentration additionally on two consecutive days to determine intermediate precision. Peak area of ESZ was determined and precision was reported as % R.S.D.

Method accuracy was tested (% recovery and % R.S.D. of individual measurements) by analyzing samples of ESZ at three different levels in pure solutions using three preparations for each level. The results were expressed as the percentage of ESZ recovered in the samples.

Sample solution short term stability was tested at ambient temperature (20 ± 1°C) for three days. In order to confirm the stability of both standard solutions at 100% level and tablet sample solutions, both solutions protected from light were re injected after 24 and 48 hrs at ambient temperature and compared with freshly prepared solutions.

RESULTS AND DISCUSSION:

Selection of the detection wavelength

The UV spectra of ESZ in 75:25 v/v mixture of phosphate buffer and Acetonitrile in the region between 200 and 400 nm are shown in Fig 2. It shows that at 304 nm, ESZ have maximum absorbance. Hence l max of ESZ in mobile phase was selected as an optimum detection wavelength for the quantification of ESZ.

Table 5: System suitability study of Eszopiclone

S. NO.	Parameters	Eszopiclone
1.	Tailing factor	1.11
2.	Asymmetrical factor	1.20
3.	Theoretical plates	5622
4.	Capacity factor	1.50
5.	HETP	0.0266

Optimization of the chromatographic conditions:

Proper selection of the stationary phase depends upon the nature of the sample, molecular weight and solubility. The drug ESZ is non polar. Non polar compounds preferably analyzed by reverse phase columns. Among C₈ and C₁₈, C₁₈ column was selected. Non polar compound is very attractive with reverse phase columns. So the elution of the compound from the column was influenced by polar mobile phase.

Mixture of Phosphate buffer and Acetonitrle was selected as mobile phase and the effect of composition of mobile phase on the retention time of ESZ was thoroughly investigated. The concentration of acetonitrile (20-25%v/v) and water (70-75%v/v) were optimized to give symmetric peak with short run time (Fig 3). A short run time and the stability of peak asymmetry were observed in the ratio of 75:25 % v/v of phosphate buffer and acetonitrile. It was found to be the optimum mobile phase concentration.

Validation of methods:

Linearity:

Six point’s calibration graphs were constructed covering a concentration range 4-24 mg/ ml (Three independent determinations were performed at each concentration. Linear relationships between the of peak area signal of ESZ the corresponding drug concentration was observed as shown in Fig 4. The standard deviations of the slope and intercept were low. The determination coefficient (r²) exceeded 0.999.The statistical analysis of calibration is shown in table 1.

Precision:

The validated method was applied for the assay of commercial tablet Fulnite containing 3 mg of ESZ. Sample was analyzed in for six times after extracting the drug as mentioned in assay sample preparation of the experimental section. The results presented in good agreement with the labeled content. Assay results, expressed as the percentage of label claim, was found to be 99.60 ± 1.538 for Fulnite showing that the content of ESZ in tablet formulations confirmed to the content requirements (95 - 105 %) of the label claim. Low values of standard deviation denoted very good repeatability of the measurement. Thus it was showing that the equipment used for the study was working correctly and hence the developed analytical method is highly repetitive.

For the intermediate precision a study carried out by the same analyst working on the same day and on three consecutive days (n=3) indicated a R.S.D of 0.0355 and 0.01955% respectively. Both values were far below to 2%, the limit percentage indicated a good method precision. The results of analysis are shown in table 2 and table 3.

Accuracy:

The data for accuracy were expressed in terms of percentage recoveries of ESZ in the real samples. These results are summarized in table 4. The mean recovery data of ESZ in real sample were within the range of 98.72 and 99.82 %. The Mean % R.S.D. was 1.348 %, satisfying the acceptance criteria for the study. It proved that there is no interference due to excipients used in tablet formulation .Hence the accuracy of the method was conformed.

Stability:

The stability of ESZ in standard and sample solutions containing determined by storing the solutions at ambient temperature (20 ± 1°C). The solutions were checked in triplicate after 3 successive days of storage and the data were compared with freshly prepared samples. In each case, it could be noticed that solutions were stable for 48 hrs, as during this time the results did not decrease below 98%. This denotes that ESZ is stable in standard and sample solutions for at least 2 days at ambient temperature.

System suitability:

The system suitability parameter like capacity factor, asymmetric factor, tailing factor, HETP and No. of theoretical plates also calculated. It was observed that all the values are within the limits (table 5).

The statistical evaluation of the proposed method revealed its good linearity, reproducibility and its validation for different parameters and let us to the conclusion that it could be used for the rapid and reliable determination of ESZ in tablet formulation.

CONCLUSION:

A validated isocratic HPLC - UV method has been developed for the determination of Eszopiclone in tablet dosage form. The proposed method is simple, rapid, accurate, precise, and specific. Its chromatographic run time of 6 min allows the analysis of a large number of samples in a short period of time. There fore, it is suitable for the routine analysis of Eszopiclone in pharmaceutical dosage form. The simplicity of the method allows for application in laboratories that lack sophisticated analytical instruments such as GC-MS that is complicated, costly and time consuming rather than a simple HPLC-UV method. Hence the proposed method could be useful for the national quality control laboratories in developing countries.

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.

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Received on 28.08.2009 Modified on 09.10.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 63-66