1. INTRODUCTION:

As no stability indication method was previously developed for Eszopiclone, there was found a need to develop an analytical method. In the current study, the analytical method was developed for Eszopiclone on HPTLC instrument, validated and stability testing was carried out by referring guideline Q1A(R2)¹ and Q1B² issued by the International Conference on Harmonization (ICH).

The proposed method was validated referring Q2(R1)³ guideline issued by the International Conference on Harmonization (ICH), FDA guidelines and as per USP general guidelines <1225> Validation of Compendial Procedures⁴.

Eszopiclone belongs to the category of nonbenzodiazepine. It is a hypnotic agent (viz., a sedative) used in the treatment of insomnia. It is the active dextrorotatory stereoisomer of Eszopiclone and belongs to the class of drugs known as cyclopyrrolones. Drugs belonging to the class of cyclopyrrolone display low toxicity and high efficacy. Eszopiclone is approved by FDA for long term use, in the treatment of insomnia. Chemically it is (S)-6-(5-Chloro-2-pyridinyl)-7-oxo-6,7-dihydro-5H-pyrrolo[3,4b]pyrazin5-yl-4-methyl-1-piperazinecarboxylate⁵.

Literature review reveals that some analytical methods have been reported for Eszopiclone by UV and difference spectroscopic methods⁶, in biological fluids using LCMSMS⁷, validated LC method for the estimation of Eszopiclone in bulk and tablet dosage form⁸, stability indicating RP-LC method for determination of Eszopiclone is reported^9,10 and only one method by HPTLC is reported for water-induced degradation kinetics¹¹.

In the current study, a new stability indicating HPTLC method for Eszopiclone is proposed that is simple and reliable. The aim of the work is to develop specific, accurate, repeatable and stability-indicating HPTLC method. The developed method can determine Eszopiclone, its degradation products and related impurities for assessment of purity and stability of bulk drug and dosage forms.

HPTLC has become a routine analytical technique because of high sample throughput, low operating costs, need for minimum sample preparation, and several samples can be analyzed at a time by using a small quantity of mobile phase, unlike HPLC. Thus, analysis time and cost per analysis is reduced.

2. EXPERIMENTAL:

2.1. Materials:

Wockhardt Pharmaceuticals Ltd. Aurangabad, India, kindly supplied pure Eszopiclone as drug sample of Batch No.: ES71228. No further purification was carried out and was certified to contain 99.8% (w/w) Eszopiclone on dry weight basis. All reagents and chemicals used were of analytical grade and were purchased from Merck Chemicals, India.

2.2. Instrumentation:

Aluminum plates precoated with Silica gel 60 _F–254 [20 cm × 10cm with 250µm thickness; E. Merck, Darmstadt, Germany)] were taken on which samples were applied in the form of bands of width 6mm and the space between two bands was kept 6mm. Camag 100 microlitre sample (Hamilton, Bonaduz, Switzerland) syringe was used for sample application using a CamagLinomat V (Switzerland) sample applicator. The prewashing of the plate was done with methanol and the washed plate was activated at 110^oC for 5 min prior to analysis. The application rate was kept constant i.e. 0.1µL/s. Twenty nm was set as the monochromatic bandwidth, scanning for each track was done thrice and baseline correction was used. The speed for scanning was 10mm/s and the slit dimension was kept at 5mm × 0.45mm. For each chromatographic run 12mL of mobile phase was taken using toluene: ethyl acetate: methanol (6: 4: 2 v/v/v). Twin trough glass chamber (Camag, Muttenz, Switzerland) of dimension 20cm × 10cm was saturated with the mobile phase using linear ascending development. The chamber saturation time for the mobile phase was optimized for 30 min at room temperature (25^oC ± 2) at relative humidity of 60% ± 5. The HPTLC plates were run till 8cm length and the plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed in the reflectance-absorbance mode at 304nm using a Camag TLC scanner III and operated by CATS software (V 3.15, Camag). The radiation source used was deuterium lamp emitting UV spectrum between 190 and 400nm.

2.3. Forced Degradation Studies:

Standard stock solutions of Eszopiclone was prepared by weighing 100mg of standard and dissolving in 100mL of methanol. The solution for sonicated for 15 min to obtain a concentration of 1000µg/mL. This prepared solution was used for forced degradation study for studying the specificity and stability-indicating property of the proposed method.

2.3.1. Acid and base-induced degradation studies:

For performing acid decomposition studies, the drug solution was refluxed in 0.1 M hydrochloric acid at 80°C for 30 min. Under alkaline conditions, the drug solution and 0.1M sodium hydroxide was refluxed at 80°C for 30 min. The prepared solutions were applied to precoated HPTLC plate so that final concentration applied was 1000ng/spot and the plate was run as described in section 2.2.

2.3.2. Oxidative degradation:

For studying oxidative degradation, drug was exposed to 3% hydrogen peroxide at room temperature for 30 min, the solution was then heated in a water bath for 5-10 min to remove excess of hydrogen peroxide. The solution was then applied on precoated HPTLC plate to get a final concentration of 1000ng/spot and the plate was run as described in section 2.2.

2.3.3. Photolytic degradation:

Photo stability of the drug was carried out by exposing the 1000µg/mL stock solution in direct sunlight (kept on terrace) for 45 min. The intensity of the exposed sunlight during the day was measured as 60,000–70,000 lux using a calibrated lux meter (Model ELM 201, Escorp, New Delhi, India).

The photo stability was also performed by keeping the 1000µg/mL stock solution in the stability chamber for 15 min.

For the HPTLC study, the resultant solutions were applied on the precoated HPTLC plate so that final concentration obtained was 1000 ng/spot and the plate was run as described in section 2.2.

2.3.4 Neutral hydrolysis:

The behavior of drug and formation of degradation product in neutral condition was studied by mixing drug solution in water and refluxing the solution at 80°C for 15 min.

2.4. Optimization of the stability-indicating TLC method:

The HPTLC method was optimized to develop stability-indicating assay method. Pure drug and degraded drug solutions were applied on to HPTLC plates and were run in various mobile phases. Initially, ethyl acetate and methanol was tried but the spots were overrun. For imparting non-polarity, toluene was added to the mobile phase. Finally, the mobile phase consisting of toluene: ethyl acetate: methanol inthe ratio of 6: 4: 1 v/v/v was found optimum with R_fvalue of 0.52 (Figure 2). To avoid variation in R_fvalue and neckless effect, the chamber was saturated using saturation pads for 20 min.

2.5. Validation of the method:

Validation of the developed HPTLC method was carried out as follows:

2.5.1. Linearity and range:

Eszopiclone standard solutions were prepared in a concentration range of 150-300µg/mL. From the above solutions, 1µL was applied on the HPTLC plate six times to get 150-300ng/spot concentrations. The development of the plate was done using the mobile phase described above and the calibration curve was obtained by plotting peak areas against the concentrations.

2.5.2. Precision:

The precision studies were performed by repeatability and intermediate precision studies. Repeatability studies were performed six times on the same day by analysis of three different concentrations (150, 210, 270ng/spot). Intermediate precision studies were performed by repeating the study on three different days.

2.5.3. Limit of detection and limit of quantitation:

Limits of detection (LOD) and limit of quantification (LOQ) represent that concentration of analyte which yields signal-to-noise ratios of 3 for LOD and 10 for LOQ, respectively. LOD and LOQ were determined by spotting a series of solutions of blank and Eszopiclone standards and calculating the signal-to-noise ratio to obtain S/N ratio. To obtain LOD and LOQ, serial dilutions from standard stock solution of Eszopiclone were made in the range of 100–200ng/spot. The samples were applied to HPTLC plate and the plates were run as described in Section 2.2 and the signals obtained from blank were compared with the samples.

2.5.4. Robustness of the method:

Small and deliberate changes in the composition of mobile phase were made (± 0.1mL for each component) and the changes on the results were studies. Different compositions of mobile phases, e.g. toluene: ethyl acetate: methanol (6.1: 4: 2 v/v/v), (6: 4.1: 2 v/v/v), (6: 4:2.1 v/v/v) were tried to examine the results. The quantity of mobile phase was changed by ± 5%. The prewashing of the plates was done with methanol and the activation was done for 2, 5, and 7 min at 60°C prior to chromatography. The time between spotting, chromatography and scanning was varied by 10 min. Three different concentration levels i.e. 150, 210 and 270ng/spot were taken for performing robustness studies.

2.5.5. Specificity:

The specificity studies were carried out by analyzing standard and test solution. The band for Eszopiclone in the sample solutions was confirmed by comparing the R_fand spectrum of the spot with that of a standard. The R_f value and spectrum of Eszopiclone in the sample solution was compared with the R_fand spectrum in standard solution. The peak purity of Eszopiclone at three regions of the spot i.e. peak start, peak apex and peak end was determined by comparing the three spectrum.

2.5.6. Accuracy:

The accuracy study of the optimized method was carried out on Eszopiclone tablets. To the powdered tablets, standard Eszopiclone of know amount, which is corresponding to 80, 100 and 120% of label claim was added, mixed, extracted and analyzed as mentioned in section 2.2.

2.6. Analysis of a formulation:

To find the content of Eszopiclone (Brand name: BEXOMER 1mg) tablets, twenty tablets were weighed; their mean weight was determined and was finely triturated. The powdered tablet equivalent to 1mg of Eszopiclone was transferred to a 25mL volumetric flask which contained around 15-20mL methanol; it was sonicated for 30 min and diluted upto 25mL with methanol (40μg/mL). The solution was centrifuged at 2000rpm for 5-7 min. From the above solution 0.025mL this solution (1000ng/spot) was spotted on TLC plate in triplicate, developed and scanned as described in Section 2.2. Interference, if any, with the excipients and sample was examined.

3. RESULTS AND DISCUSSION:

3.1. Stability-indicating property:

Stress testing studies were carried out on Eszopiclone standard under different conditions and were analyzed using toluene: ethyl acetate: methanol (6: 4: 1 v/v/v) as the mobile phase. The following degradation behavior was suggested:

3.1.1. Acid-induced degradation:

Initially, the drug was refluxed with 1M hydrochloric acid at 80°C but the degradation was found be more than 30%, hence the strength of acid was reduced to 0.1M hydrochloric acid. In the subsequent study, the drug was refluxed at 80°C in 0.1M hydrochloric acid for 30 min. The degradation was found to be around 25-30% of the drug. Densitogram for acid-degraded Eszopiclone showed R_fvalue at 0.38 (Figure 3).

3.1.2. Base-induced degradation:

The drug was refluxed with 1M sodium hydroxide at 80°C for 1 h but too much of degradation product was formed, hence, the strength of base was reduced. In the subsequent study, the drug was refluxed with 0.1M sodium hydroxide at 80°C for 30 min, the degradation was found to be around 25-30% of the drug. Densitogram for base-degraded Eszopiclone showed R_fat 0.33 and 0.70 (Figure 4).

3.1.3. Oxidative degradation:

After performing oxidative studies it was found that Eszopiclone was highly unstable to hydrogen peroxide induced degradation. The reaction in 3% H₂O₂ at room temperature was performed for 6 h and it was found that around 80% of the drug was degraded. Subsequently, studies were performed in 3% H₂O₂for 30 min and around 25% of the drug was degraded. Densitogram of oxidative-degraded Eszopiclone showed R_fat 0.82 (Figure 5).

3.1.4. Photochemical degradation

After performing photochemical degradation it was found that Eszopiclone was highly unstable to sunlight as more than 50% drug was degraded in 2 h. Subsequent degradation was performed for 45 min and around 20-25 % of the drug was degraded. Densitogram of photochemically degraded Eszopiclone showed R_fat 0.67 (Figure 6a).

After exposing the drug to photo stability chamber for 15 min around 25% drug was degraded. Densitogram of degraded Eszopiclone showed R_fat 0.40 (Figure 6b).

3.1.5. Neutral degradation:

The drug solution was refluxed in water at 80°C for 15 min to obtain degradation in the range of 25-30% of the drug. Densitogram of degraded Eszopiclone showed R_fat 0.68 (Figure 7).

3.2. Validation of the stability-indicating method:

The results of validation studies on the developed stability-indicating analytical method for Eszopiclone on the optimized mobile phase using toluene: ethyl acetate: methanol (6: 4: 2, v/v/v) are as below:

3.2.1. Linearity:

The response of the drug was found linear in the concentration range of 150-300ng/spot. The regression coefficient (R²) value is 0.9952 (± 1.02), mean (± RSD) values of the slope is 7.1571 (± 0.62) and intercept is 137.14 (± 0.23).

3.2.2. Precision:

The results of the repeatability and intermediate precision experiments are shown in Table 1. The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were < 2%, respectively as recommended by ICH guideline. Separation of the drug and different degradation products in stressed samples was found to be similar when analyses were performed using different chromatographic system on different days.

Table 1 shows the results of repeatability and intermediate precision experiments. The optimized method was found precise as the RSD value was < 2 % for repeatability and intermediate precision studies. Resolution between the drug and the degradation products was found similar even after analyzing the sample in different chromatographic conditions and also after analyzing the samples on different days.

Table 1 Precision studies

Concentration (ng/spot)	Repeatability (n= 6)			Intermediate precision (n= 6)
Concentration (ng/spot)	Measured concentration ± SD	RSD (%)	Recovery (%)	Measured concentration ± SD	RSD (%)	Recovery (%)
150	149.32 ± 0.24	1.22	99.54	147.97 ± 0.19	0.94	98.64
210	207.11 ± 0.41	0.98	98.62	206.37 ± 0.36	1.25	98.27
270	270.34 ± 0.85	1.46	100.12	269.83 ± 0.68	0.83	99.93

Table 2 Robustness testing (n = 3)

Parameter

SD of peak area

% RSD

Mobile phase composition (± 0.1 mL)

Amount of mobile phase (± 5%)

Time from spotting to chromatography (10 min)

Time from chromatography to scanning (10 min)

4.32

2.10

5.74

3.29

0.81

1.23

1.48

0.97

Table 3 Recovery studies (n = 6)

Label claim (mg/tablet)	Amount added (mg)	Total amount (mg)	Amount Recovered (mg) ± % RSD	% Recovery
1	0.8 (80%)	1.8	1.81 ± 0.51	100.55
1	1.0 (100%)	2.0	1.97 ± 0.88	98.50
1	1.2 (120%)	2.2	2.19 ± 1.09	99.54

3.2.3. LOD and LOQ:

Signal-to-noise ratios for LOD was 3:1 and for LOQ was 10:1. The LOD was found to be 130ng/spot and LOQ was found to be 150ng/spot.

3.2.4. Robustness of the method:

Each selected factor was altered at three levels i.e. - 1, 0 and 1. To estimate the effect of the change, one factor was changed at one time. Six replicate injections at three different levels were performed by performing small changes in chromatographic parameters. Insignificant variation in retention time, peak areas and resolution was observed (Table 2), which indicates that the method is rugged.

3.2.5. Specificity:

Eszopiclone peak purity was evaluated by comparison of peak at start (S), middle (M) and end position (E) i.e., r(S, M) at 0.9952 and r(M, E) at 0.9949. Standard and sample spectra of Eszopiclone obtained correlation coefficient (r) at 0.9950.

3.2.6. Recovery studies:

Good recoveries for Eszopiclone were obtained in the range of 98.50 to 100.55 % at different added concentrations (Table 3).

3.3. Analysis of a formulation:

The results obtained after analyzing tablet formulation suggested that there was no interference of excipients with Eszopiclone peak. Two different lots of Eszopiclone tablets were analyzed and the drug content was found to be 98.96 % ± 0.22 (Table 4).

Table 4 Analysis of commercial formulation

Eszopiclone (BEXOMER 1mg)	Eszopiclone found (mg per tablet)
Eszopiclone (BEXOMER 1mg)	Mean ± SD (n= 6)	Recovery (%)
1^st Lot	0.98 ± 0.62	98.91
2^nd Lot	0.99 ± 0.23	99.02

4. SUMMARY:

No stability indicating method is reported for Eszopiclone. Hence, stability indicating HPTLC method for the determination of the drug was developed. Linearity for Eszopiclone was found in the range of 150-300ng/spot with regression coefficient (R²) = 0.9952 demonstrating that the proposed method is linear. LOD and LOQ values were 130ng/spot and 150ng/spot, respectively. The RSD values were found to be less than 2% for intraday and interday precision studies. These low values of RSD indicate that projected method is precise. Significant degradation of Eszopiclone was found in acid, alkali, oxidative, photolytic and neutral stress conditions.

5. CONCLUSION:

The developed HPTLC method is specific, accurate, precise and stability- indicating assay method, as recommended by ICH guidelines. The results obtained showed that the method is best suited for the analysis of Eszopiclone as bulk drug and in pharmaceutical formulation it was observed that there was no interference of Eszopiclone drug with the excipients. This method can determine the peak purity of the drug. This work can further be extended for studying the degradation kinetics and for estimation of Eszopiclone in biological fluids and blood plasma. While analyzing the drug in industry, the proposed method can be used to analyze the drug and its degradation products.

6. CONFLICT OF INTEREST:

The authors do not have any conflict of interest.

7. ACKNOWLEDGEMENT:

The authors are thankful to Wockhardt Ltd. (Aurangabad, India) for providing standard Eszopiclone as a gift sample. The authors would also like to thank, Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Pune, India for providing necessary facilities to carry out the work. The authors would also like to thank AICTE for providing financial support for carrying out research work.

8. REFERENCES:

1. International Conference on Harmonization ICH, Q1A (R2) Stability Testing of New Drug Substances and Products, IFPMA, Geneva, Switzerland.

2. International Conference on Harmonization ICH, Q1B Photostability Testing of New Active Substances and Medicinal Products.

3. International Conference on Harmonization ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology.

4. USP 43 General Information 〈1225〉 Validation of Compendial Procedures

5. https://www.drugbank.ca/drugs/DB00402 (Accessed May 28, 2020).

6. Anandakumar K., Kumaraswamy G, Ayyappan T, Sankar ASK., Nagavalli, D. Estimation of Eszopiclone in Bulk and in Formulation by Simple UV and Difference Spectroscopic Methods. Research Journal of Pharmacy and Technology. 2010; 3(1): 202-205.

7. Meng M, Rohde L, Capka, V, Carter SJ, Bennett PK. Fast chiral chromatographic method development and validation for the quantitation of Eszopiclone in human plasma using LC/MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2010; 53(4): 973-982.

8. Anandakuma K., Kumaraswamy G, Ayyappan T, Sankar ASK, Nagavalli D. Validated RP-HPLC method for the estimation of Eszopiclone in bulk and tablet dosage form. Asian Journal of Research in Chemistry. 2010; 3(1): 63-66.

9. Dhaneshwar SR, Bhusari VK. Development of a validated stability-indicating HPLC assay method for Eszopiclone. International Journal of ChemTech Research. 2011; 3(2): 680-689.

10. Kumar NR, Rao NG, Naidu PY. Stability Indicating RP-LC Method for Determination of Eszopiclone in Bulk and Pharmaceutical Dosage Forms. Asian Journal of Research in Chemistry. 2010; 3(2): 374-379.

11. El-Yazbi AF, Youssef RM. An eco-friendly HPTLC method for assay of Eszopiclone in pharmaceutical preparation: investigation of its water-induced degradation kinetics. Analytical Methods. 2015; 7(18): 7590-7595.

Received on 10.12.2020 Modified on 24.04.2021

Asian J. Res. Pharm. Sci. 2021; 11(3):219-223.

DOI: 10.52711/2231-5659.2021.00035