Stability Indicating RP-LC Method for Determination of Eszopiclone in Bulk and Pharmaceutical Dosage Forms

 

R Narendra Kumar*, G Nageswara Rao and PY Naidu

*Corresponding Author E-mail: narenrokkam@yahoo.co.in

 

ABSTRACT:

An isocratic stability indicating liquid chromatographic method has been developed and validated for the determination of Eszopiclone in bulk drug and its pharmaceutical dosage form. Separation of the drug with degradation products was achieved using Peerless HT, C8, 50 x 4.6 mm; 1.8 m column as stationary phase and pH 4.5(0.05) buffer: Acetonitrile: Tetrahydrofuran (81:18:1,v/v) as mobile phase at a flow rate of 1.0 mL/min. UV detection was performed at 304 nm. The method is linear over the range of 10 - 150 g/mL. The percent recovery of drug in dosage forms was ranged from 97.7 to 100.5. The method is simple, rapid, precise, selective and stability indicating and can be used for the assay in quality control and stability studies samples.

 

KEYWORDS: Bird flu, Chemsketch, Docking, Hex, Rasmol.

 


1.0 INTRODUCTION:

Eszopiclone (Figure 1) film coated tablets are non-benzodiazepine hypnotic agent for oral administration. Eszopiclone (ESZ) is a compound related to the Pyrrolopyrazine derivative of the cyclopyrrolone class with an empirical formula C17H17ClN6O3. Its chemical name is (+)-(5S)-6-(5-Chloropyridin-2-yl)-7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl-4-methylpiperazine-1-carboxy-late. It is a white to light yellow crystalline solid with a molecular weight of 388.81. Eszopiclone is slightly soluble in water and ethanol and soluble in Phosphate buffer (pH 3.2). It has a single chiral center with an (S)- configuration1-6.

 

It is not official in any pharmacopoeia and till now, few liquid chromatographic (LC) procedures have been reported for the determination of Eszopiclone and its metabolites in biological fluids. However, there are no reports on the LC analysis of Eszopiclone in bulk and pharmaceutical dosage forms7-20. Hence, an LC procedure was developed to serve as a rapid and reliable method for the determination of Eszopiclone in the presence of related impurities in bulk and pharmaceutical dosage forms. In the proposed method, related impurities were well separated and eluted with in 10 min. Finally the method was thoroughly validated for the assay of Eszopiclone.

 

2.0 EXPERIMENTAL:22

2.1 Instrumentation:

The Waters LC system equipped with 2489 pump and 2996 Photodiode array (PDA) detector was used. The output signal was monitored and integrated using Waters Empower 2 software.

 

2.2 Solutions:

2.2.1 pH 4.5 buffer solution:

3.5 g of anhydrous sodium salt of N-hexane sulphonic acid and 7.0 g of sodium dihydrogen orthophosphate dihydrate were dissolved in 1000mL of milli-Q water and pH of the solution was adjusted to 4.500.05 with orthophosphoric acid / Triethylamine.

 

2.2.2 Mobile phase:

A mixture of pH 4.5 buffer, acetonitrile and Tetrahydrofuran in the ratio 81:18:1(v/v) was prepared and filtered through 0.2 m nylon membrane filter and degassed for about 10 min. prior to use as mobile phase.

 

2.2.3 Diluent (0.1N HCl):

8.7 ml of Concentrated HCl were diluted to 1000 ml with milli-Q water and filtered through 0.45m nylon membrane filter.

 

2.2.4 Standard solution (100g/ml):

50mg of Eszopiclone working standard was transferred in to a 100 mL volumetric flask and dissolved in 70 ml of diluent and sonicated to dissolve and diluted to volume with the diluent. Further 5 mL of the resulting solution was taken into 25 mL volumetric flask and made up to volume with the diluent. Solution was filtered through 0.45m nylon membrane filter prior to use.

 

2.2.5 Test Solution:

The number of tablets equivalent to 10 mg of Eszopiclone were weighed and transferred in to a 100 mL volumetric flask and about 70 ml of the diluent was added and swirled the flask to disintegrate, sonicated for 10 min and diluted to the volume with the diluent. The solution was filtered through 0.45m nylon membrane filter prior to use.

 

2.2.6 Preparation of Samples for Specificity Study:

For Acid degradation Eszopiclone sample was stressed with 0.1N HCl on water bath for 60 min at 70C and then neutralized by adjusting pH to 7.0 with 0.1 N NaOH. The solution was further diluted to the required concentration with the diluent.

 

For Alkali degradation Eszopiclone sample was stressed with 0.1 N NaOH on bench top for 1 min and then neutralized by adjusting pH to 7.0 with 0.1 N HCl. The solution was further diluted to required concentration with the diluent.

 

For Oxidative degradation Eszopiclone sample was stressed with 1% H2O2 by heating on water bath at 40C for 60 min. The solution was further diluted to required concentration with the diluent.

 

For Water degradation Eszopiclone sample was stressed with water by heating on water bath at 70C for 60 min. The solution was further diluted to required concentration with the diluent.

 

For Photolytic stress the samples were exposed to UV light at 254 nm for 56 h 30 min and visible light for 264 h meeting the specification of ICH i.e. UV (200 watt/m2) and Visible (1.2 million Lux hours).

 

For Thermal degradation samples were exposed to temperature at 105C for 24hrs.

The Photolytic and Thermal stress sample solutions were prepared to required concentration with the diluent.

 

2.2.7          Chromatographic Conditions:

A Peerless HT C8 (50 x 4.6 mm; 1.8 m packing) column was used for analysis at column temperature 40C. The mobile phase was pumped through the column at a flow rate of 1.0 mL/min (flow rate adjusted to get Eszopiclone retention time (RT) about 5 min). The sample injection volume was 5L. The photodiode array detector was set to a wavelength of 304 nm for the detection.

 

RESULTS AND DISCUSSION:

3.1 Method development:21

3.1.1 Separation of known degradation impurities:

To develop a suitable rapid and robust LC method for the determination of Eszopiclone, different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a C8 column using a mobile phase containing pH 4.5 buffer, Acetonitrile and Tertahydrofuran in the ratio 70:28:2, where elution was found to be very broad. Early elution with slight separation was observed with mobile phase consisting of above components in the ratio 78:21:1. Finally the mobile phase with the ratio 81:18:1 was found to be appropriate with good separation and symmetrical peak shape by adjusting the flow rate to get Eszopiclone peak RT about 5 min (1.0 ml/min) using Peerless HT C8 (50 x 4.6 mm;1.8 m packing) column. Under the last condition all related compounds were eluted with in 10 min and well separated. The chromatogram of Eszopiclone sample spiked with the related compounds (impurities) using the proposed method is shown in Figure 1. In the proposed method the resolution is more than 2 between the ESZ and Desmethyl Eszopiclone. System suitability results of the method are presented in Table 1. Eszopiclone and its related compounds show significant UV absorbance at wavelength 304 nm. Hence this wavelength has been chosen for detection in the analysis of Eszopiclone.

 

Figure 1: TYPICAL LC CHROMATOGRAM OF FORMULATED ESZOPICLONE (3mg)

1) 2 Amino -5 chloropyridine, 2) Zopiclone N-Oxide, 3) Imino alcohol, 4) Desmethyl Eszopiclone, 5) Dehydroxy Imino alcohol

 

TABLE 1: SYSTEM SUITABILITY REPORT

Compound

Tailing Factor a

Theoretical platesa

Resolutiona

%RSDa

Eszopiclone

1.1

7937

----

0.3

Related compound-4

1.0

7897

2.4

1.1

a Number of samples analyzed are six

3.1.2         Column Selectin:

Based on the retention and separation of the compounds Peerless HT C8 (50 x 4.6 mm; 1.8 m packing) column was selected as suitable column for the analysis of Eszopiclone.

 

3.2          Method Validation:23-25

The developed LC method was extensively validated for assay of Eszopiclone using the following parameters.

 

3.2.1 Specificity:

Placebo Interference:

A study to establish the interference of placebo was conducted. Assay was performed on placebo in triplicate equivalent to about the weight of placebo in portion of test preparation as per test method. Chromatograms of placebo solutions showed no peaks at the retention time of Eszopiclone peak. This indicates that the excipients used in the formulation do not interfere in the estimation of Eszopiclone in Eszopiclone tablets.

 

Interference from degradation products:

A study was conducted to demonstrate the effective separation of degradants from Eszopiclone peak. Separate portions of Drug product, Drug substance and Placebo were exposed to the following stress conditions to induce degradation. Stressed samples were injected into the HPLC system with PDA detector by following test method conditions. All degradant peaks were resolved from Eszopiclone peak in the chromatograms of all samples. The chromatograms of the stressed samples were evaluated for peak purity of Eszopiclone using Empower 2 software. In all the forced degradation samples, Eszopiclone peak purity angle was less than purity threshold. From the above results it is clear that the method can be used for determining the stability of Eszopiclone as bulk and pharmaceutical formulations. Figure 2 shows the separation of Eszopiclone from its degradation products.

 

Figure 2: HPLC CHROMATOGRAMS OF ESZOPICLONE AND ITS DEGRADATION PRODUCTS

 

ALKALI DEGRADATION

 

OXIDATIVE DEGRADATION

 

VISIBLE LIGHT DEGRADATION

 

ON UV LIGHT DEGRADATION

 

HEAT DEGRADATION

 

WATER DEGRADATION

 

ACID DEGRADATION

TABLE 2: RESULTS FOR PRECISION OF TEST METHOD

Sample No.

%Assay

1

100.4

2

100.0

3

100.1

4

100.9

5

101.3

6

101.0

MEAN

100.6

%RSD

0.5

 

3.2.2         Linearity of Detector Response:

Linearity of detector response was established by plotting a graph to concentration versus average area and determining the correlation coefficient. A series of solutions of Eszopiclone standard were prepared in the concentration range of 10.0293 g/mL to 150.4395 g/mL. A graph was plotted to concentration in g/mL on the abscissa versus response on the ordinate. The detector response was found to be linear (Figure 3) with a correlation coefficient of 0.999.

 

 


TABLE 3: ACCURACY IN THE ASSAY DETERMINATION OF ESZOPICLONE

Sample No.

Spike level

mg added

mg found (recovered)

% Recovery

Average %recovery

1.        

25%

5.97

6.00

100.5

100.5

2.        

5.97

6.00

100.5

3.        

5.97

6.00

100.5

4.        

50%

11.87

11.61

97.8

97.9

5.        

11.87

11.62

97.9

6.        

11.86

11.62

98.0

7.        

75%

17.83

17.62

98.8

98.9

8.        

17.83

17.65

99.0

9.        

17.83

17.65

99.0

10.      

100%

23.84

23.30

97.7

97.7

11.      

23.84

23.31

97.8

12.      

23.83

23.27

97.7

13.      

150%

35.79

35.11

98.1

97.9

14.      

35.78

35.01

97.8

15.      

35.78

35.02

97.9

 

TABLE 4: STABILITY DATA OF ESZOPICLONE IN STANDARD AND TEST SOLUTIONS

BENCH TOP STABILITY

Time in Days

% Assay of Standard preparation

Difference from Initial

% Assay of test preparation

Difference from Initial

Test - 1

Test - 2

Test - 1

Test 2

Initial

99.3*

NA

100.5

100.8

NA

NA

1

97.7

1.6

99.4

99.5

1.1

1.3

2

96.5

2.8

98.6

99.0

1.9

1.8

REFRIGERTOR STABILITY

Time in Days

% Assay of Standard preparation

Difference from Initial

% Assay of test preparation

Difference from Initial

Test - 1

Test - 2

Test - 1

Test 2

Initial

99.3*

NA

100.5

100.8

NA

NA

1

99.3

0.0

101.3

100.6

0.8

0.2

2

98.4

0.9

99.5

99.9

1.0

0.9

* Potency of Eszopiclone on as is basis

 

TABLE 5: ROBUSTNESS DATA OF ESZOPICLONE IN TEST SOLUTIONS

TABLE: 5A

Effect of variation Mobile phase composition(Acetonitrile)

System Suitability

Organic phase ratio (Acetonitrile)

100%

90%

110%

Acceptance criteria

USP Tailing factor for Eszopiclone Peak

1.3

1.2

1.2

NMT 2.0

%RSD of Eszopiclone peak area from five replicate injections of standard solution

0.4

0.4

0.4

NMT 2.0

Effect of variation Mobile phase composition(Acetonitrile)

Organic phase ratio (Acetonitrile)

% Assay of test preparation

Avg.

Assay

Difference

Trail - 1

Trail - 2

100%(Organic phase)

101.6

101.4

101.5

NA

90%(Organic phase)

101.7

101.2

101.5

0.0

110%(Organic phase)

101.9

100.9

101.4

0.1

 

TABLE: 5B

Effect of variation Mobile phase composition(Tetrahydrofuran)

System Suitability

Tetrahydrofuran ratio in mobile phase composition

100%

90%

110%

Acceptance criteria

USP Tailing factor for Eszopiclone Peak

1.4

1.4

1.4

NMT 2.0

%RSD of Eszopiclone peak area from five replicate injections of standard solution

0.1

0.1

0.1

NMT 2.0

Effect of variation Mobile phase composition(Tetrahydrofuran)

Tetrahydrofuran ratio

% Assay of test preparation

Avg.

Assay

Difference

Trail - 1

Trail - 2

100 % ( Tetrahydrofuran )

101.5

102.4

102.0

NA

90 % ( Tetrahydrofuran )

101.2

102.3

101.8

0.2

110 % ( Tetrahydrofuran )

101.5

102.3

101.9

0.1

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE: 5C

Effect of variation in pH of Mobile phase

System Suitability

Variation in pH of Mobile phase

pH 4.5

pH 4.3

pH 4.7

Acceptance criteria

USP Tailing factor for Eszopiclone Peak

1.4

1.4

1.4

NMT 2.0

%RSD of Eszopiclone peak area from five replicate injections of standard solution

0.0

0.2

0.1

NMT 2.0

Effect of variation in pH of Mobile phase

Variation in pH of Mobile phase

% Assay of test preparation

Avg.

Assay

Difference

Trail - 1

Trail - 2

pH 4.5

102.8

101.6

102.2

NA

pH 4.3

102.2

102.1

102.2

0.0

pH 4.7

102.2

101.7

102.0

0.2

 


3.3.3) Precision of test Method:

The precision of the test method was conducted by assaying six samples of Eszopiclone tablets. The Average % assay of Eszopiclone in Eszopiclone tablets was found to be 100.6% with an RSD of 0.5%. The results are given in Table 2.

 

3.3.4) Accuracy:

A study of recovery of Eszopiclone from spiked placebo was conducted at six different spike levels i.e. 25%, 50%, 75%, 100% and 150%. Samples were prepared by mixing placebo with Eszopiclone raw material equivalent to that of the target initial concentration of Eszopiclone. Sample solutions were prepared in triplicate for each spike level and assayed as per proposed method. The % recovery and correlation coefficient were calculated and given in Table 3. The mean recoveries of Eszopiclone from spiked were found to be in the range of 97.7-100.5%.

 

3.3.5) Ruggedness

A study to establish the stability of Eszopiclone in standard and test solutions were conducted on bench top and Refrigerator at Initial, 1 day and 2 days. The assay of Eszopiclone in standard and test solutions was estimated against freshly prepared standard each time. The difference in % assay of standard and test solutions from initial to 1 day and 2 days was calculated and given in Table 4. From this study, it was established that the test preparation and standard are stable for a period of 2 days on bench top and Refrigerator.

 

3.3.6) Robustness

A study to establish the effect of variation in mobile phase composition, temperature and pH of buffer in mobile phase was conducted. Standard and test solutions prepared as per proposed method were injected into HPLC system. The system suitability parameters and % assay were evaluated and given in Table 5. From the above study the proposed method was found to be Robust.

 

ACKNOWLEDGEMENTS:

The authors wish to thank the Orchid Healthcare for providing the samples of Eszopiclone.

 

REFERENCES:

1.       Indian Pharmacopeia, Vol I, II, 1996 & Addendum 2000 and Vet, Government of India, Ministry of Health and Family Welfare, Controller of Publications, Delhi, 2000.

2.       British Pharmacopeia, Vol. I & II and Vet 1993, HMSO, London, 1999.

3.       United States Pharmacopeia USP XXIV & NF IXX, 2000 and 4 Addendum, USP convention Inc., Rockville, 2002.

4.       .European Pharmacopeia, 3rd Edition, 1997 and Supplement, Council of Europe, Strasbourg, 1999.

5.       Japan Pharmacopoeia, XIIIth Edition, 1996.

6.       The Merck Index, 12th Edition, Merck & Co Inc, New York, 1996.

7.       H. N. Mistri, A. G. Jangid, A. Pudage and P. Shrivastav, J. Chromatog. B, 864, 2008, 137-148.

8.       N. Brunello, P. Bettica, D. Amato, G. Maier and D. Nutt, Eur. Neuropsychopharmacol., 18, 2008, S581-S582

9.       The Merck index, 13th edition, Merck Research Laboratories., Division of Merck and CO, INC, White House station, NJ, 2003

10.     C.S.P. Sastry, D.G. Sankar, M.N. Reddy and M. Aruna, Indian J. Pharm. Sci., 50, 1998, 178

11.     Wolff, M.E., Ed., Burgers Medicinal Chemistry, Part IV, 4th Edn., Wiley Interscience, New York, 1981.

12.     Deorge, R.F., (Ed), Wilson and Gisvoldss Text book of Organic and Medicinal and Pharmaceutical Chemistry, 8th Edn., Lippincott Company, 1982.

13.     Korolkovas, A., Essentials of Medicinal Chemistry, 2nd Edn., Wiley Interscience, New York, 1988.

14.     Topliss, J.G., Ed., Quantitative Structure activity relationships of Drugs, Vol 19, Academic Press, London, 1983.

15.     William O, Foye, (Ed), Principles of Medicinal Chemistry, 3rd Edn., Varghese, Bombay, 1989.

16.     Melentyeva, G., Antonova, L., Pharmaceutical Chemistry, Mir Publishers, Moscow, 1988.

17.     L.M. Atherden, (Ed), Bentley and Drivers Text Book of Pharmaceutical Chemistry, 8th Edn., Oxford University Press, 1996, 4th Impression.

18.     The Drugs and Cosmetics Act and Rules, Government of India Publications, 1984.

19.     Reynolds, J.E.F.; Martindale, The Complete Drug Reference (Extra Pharmacopeia) 30th Edition, The Pharmaceutical Press, London, 1993.

20.     Chattan, L.G., Ed., Pharmaceutical Chemistry, Vol I & II, Moral Dekker Inc, New York, 1966.

21.     Higuchi, T., Brochman Hansen, E., Edt., Pharmaceutical Analysis, Interscience, London, 1961.

22.     Lachman, L., Liberman, H.A., and Kaning, J.L., The Theory and Practice of Industrial Pharmacy, 2nd Edn. London.

23.     ICH stability testing of new drug substances and products (Q1AR2), International Conference on Harmonization, IFPMA, Geneva,2003

24.     ICH Draft Guidelines on Validation of Analytical Procedures: Text and methodology (Q2R1), IFPMA, Switzerland, 1995

25.     Goodman, L.S. and Gilman, A.G., The Pharmacological Basis of Therapeutics, 9th Edn By Hardman, J.G.Limbard, L.E.; Editors in Chief, McGraw Hill, 1996.

 

 

 

Received on 25.12.2009 Modified on 18.02.2010

Accepted on 14.03.2010 AJRC All right reserved

Asian J. Research Chem. 3(2): April- June 2010; Page 374-379