INTRODUCTION:

Etizolam¹ known as 4-(2-Chlorophenyl)-2-ethyl-9-methyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4] diazepine is a thienodiazepine chemically related to benzodiazepine drug class; it differs from benzodiazepine in having a benzene ring replaced with a thiophene ring. It is an agonist at GABA-A receptors and possesses amnesic, anxiolytic, anticonvulsant, hypnotic, sedative and skeletal muscle relaxant properties. Literature survey revealed that very few analytical methods are available for determination of Etizolam in formulations such as UV-Visible spectroscopy² and HPLC³. Etizolam is also available in combination with other anxiolytic agents such as Escitalopram and some HPLC and spectroscopy methods are available in literature for different combinations^4-6. Hence an attempt has been made to develop a simple stability indicating RP-HPLC method for the determination of Etizolam in tablets.

Fig. 1: Chemical structure

MATERIAL AND METHODS:

Chemicals and reagents:

Etizolam was obtained as a gift sample from Dr. Reddy’s laboratories. HPLC grade solvents like acetonitrile and methanol were procured from Fisher Scientific. All the chemicals used in the study like ortho phosphoric acid, triethylamine, sodium hydroxide, hydrochloric acid and hydrogen peroxide (AR grade) were procured from E-Merck. Triple distilled water (SRL make) was used throughout the study. Marketed tablets of Etizolam (NITOCALM, 0.5 mg) were procured from the local pharmacy.

Instrumentation:

Chromatographic separation was achieved by using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with Agilent C18 column (150 x 4.6mm x 3.5µm).

Preparation of Buffer solution:

About 0.8 mL of triethylamine was pipetted into a 1000 mL volumetric flask, diluted with water, pH was adjusted to 3.2 using ortho phosphoric acid and finally made up with water. The solution was filtered through a membrane filter (Durapore Membrane, Miilipore GV 0.22 μm) and degassed by ultrasonication for 15 min.

Preparation of stock and working standard solution:

The stock solution was prepared by dissolving 25.0 mg of Etizolam in 25 mL methanol (1000 µg/mL). This solution was suitably diluted to obtain a solution of 100 µg/mL (working standard) and further dilutions were prepared from this solution.

Preparation of sample solution (Tablets):

Locally purchased tablets (NITOCALM, 0.5 mg) were weighed, powdered and powder equivalent to 2.5 mg of Etizolam was transferred to a 25 mL volumetric flask, dissolved in methanol, sonicated and the volume made up with the same solvent (100 µg/mL). The resulting solution was suitably centrifuged and further dilutions were made from this solution with methanol as per the requirement.

Table 1: Optimized chromatographic conditions for Etizolam

Parameter	Value
Column	Agilent C18 (150 x 4.6 mm x 3.5µm) column
Mobile phase	Acetonitrile and triethylamine solution, pH 3.2 (50:50, v/v)
Elution mode	Isocratic
Flow rate	1.0 mL/min.
Detection wave length	254 nm
Column temperature	25^oC
Volume of injection	20 µL
Run time	10.0 min
Retention time obtained	3.21 ± 0.24 min.

Fig. 2: Chromatogram for Etizolam standard (10 µg/mL)

METHOD VALIDATION:

The developed method was validated for linearity, precision, accuracy, robustness, specificity, LOD and LOQ as per the ICH⁷ regulatory requirements.

Linearity:

Accurately measured aliquots of working standard solution of Etizolam were transferred to a series of 10 ml volumetric flasks and diluted with methanol to obtain solutions in the range of 0.5-100 μg/mL of Etizolam. 20 µL of each solution was injected into the column twice and the peaks obtained were analyzed at 254 nm. A calibration curve was constructed at optimum experimental conditions by plotting peak area vs. concentration and regression analysis was done

Precision:

Precision was studied in terms of intra and inter - day precision. Intra-day precision was determined by analyzing three different concentrations (20, 40 and 60 µg/mL) of drug in triplicate on the same day. Inter-day precision was determined by analyzing the three different concentrations of the drug in triplicate on different days. The %RSD for the assay values obtained was calculated.

Accuracy:

The accuracy of the proposed method was established by recovery studies. Standard addition method was followed by adding known amount of drug to preanalyzed sample at three different levels (50%, 100% and 150%) in triplicate and the percentage recovery of Etizolam was calculated.

Limit of Detection:

It is the lowest amount of analyte in a sample that can be detected but not necessarily quantified under the stated experimental conditions. Limit of detection can be calculated from the regression data using following equation as per ICH guidelines.

LOD = 3.3 x σ/s

Where,

σ = Standard deviation of the y intercept; S=Slope of the calibration curve

Limit of Quantification:

It is the lowest concentration of analyte in a sample that can be determined with the acceptable precision and accuracy under stated experimental conditions. Limit of quantification can be calculated using following equation as per ICH guidelines.

LOQ = 10 x σ/s

Where, σ = Standard deviation of the y intercept; S=Slope of the calibration curve

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness of the method was studied by injecting standard and sample solutions in replicate with altered flow rate (±0.1mL/min), pH (±0.1) and mobile phase composition (± 10%). The changes in the response of Etizolam were studied in terms of system suitability parameters like tailing factor and theoretical plates. The assay was calculated for the altered peak areas for which the % RSD was computed.

System Suitability:

System suitability parameter is established to ensure that the validity of the analytical method is maintained whenever used. A standard solution of Etizolam was injected six times before each validation study and the parameters like theoretical plates, resolution, retention time and RSD of peak area for replicate injections were studied.

Specificity:

The specificity of the method was established in the presence of excipients in formulation and degradants from forced degradation study. The chromatogram obtained by injecting the tablet solution was compared with that of the blank to study the interference of the excipients.

Forced Degradation Study^8,9:

1. Acid hydrolysis:

Accurately measured 1.0 ml of Etizolam (100 μg/ml) and 1 ml 0.1N HCl was transferred in to a 10 ml volumetric flask, the solution was heated for 30 min. at 80˚ C for acid hydrolysis and neutralized with 0.1N NaOH. The volume was made up with methanol, filtered through 0.22 μm membrane filter paper and injected into HPLC system. The chromatograms were recorded and peak areas analysed to calculate the stability of the sample.

2. Base hydrolysis:

Accurately measured 1.0 ml of Etizolam (100 μg/ml) and 1 ml 0.1N NaOH was transferred in to a 10 ml volumetric flask, the solution was heated for 30 min. at 80˚ C for base hydrolysis, cooled and neutralized with 0.1N HCl. The volume was made up with methanol, filtered through 0.22 μm membrane filter paper and injected into HPLC system in replicates.

3. Oxidative hydrolysis:

Accurately measured 1.0 ml of Etizolam (100 μg/ml) and 1 ml of 3% H₂O₂ was transferred in to a 10 ml volumetric flask, the solution was heated for 30 min. at 80˚ C for oxidative hydrolysis, cooled and made up the volume with methanol. The solution was filtered through 0.22 μm membrane filter paper and injected twice into the HPLC system. The chromatograms were recorded and analysed for peak areas and peak purity.

4. Thermal Degradation:

Accurately measured 1.0 ml of Etizolam (100 μg/ml) was transferred in to a 10 ml volumetric flask and the solution was heated for 30 min. at 80˚ C for thermal stress studies. The solution was cooled, volume made up with methanol, filtered through 0.22 μm membrane filter paper and injected into HPLC system. The chromatograms were recorded and analysed.

5. Photo stability studies:

Accurately measured 1.0 ml of Etizolam (100 μg/ml) was transferred in to a 10 ml volumetric flask and the solution was exposed to short UV light in a UV Chamber for 30 mins. The exposed solution was diluted with methanol and injected into the system. The peak areas were calculated for the chromatograms obtained and compared with the standard.

Assay procedure for the commercial formulations (Tablets):

The optimized method was applied to determine the purity of locally available tablets (NITOCALM, 0.5 mg). The sample solution of Etizolam was suitably diluted to get a 10 µg/mL solution and 20 µL of this was injected thrice into the HPLC. The peak areas were noted and the assay was calculated.

RESULTS AND DISCUSSIONS:

A validated stability indicating RP-HPLC method (isocratic mode) for the determination of Etizolam has been developed after a series of trials. Initially the drug solution was analysed using a mixture of acetonitrile and triethylamine solution, pH 3.2 (20:80, v/v) with a flow rate of 1.0 mL/min in which a peak was obtained at 4.47 min. but the peak symmetry was not satisfactory. The mobile phase ratio was changed to 90:10 %v/v and the drug sample was injected in to the loop when a sharp peak was eluted at 1.29 min. with tailing. Finally, the mobile phase composition was modified as 50:50 %v/v and a sharp Etizolam peak was eluted at 3.21 ± 0.24 min. which satisfied the system suitability parameters. Several solvents have been tried in the method optimization to achieve better spectral characteristics for the drug. The maintained pH in the method was chosen based on the pKa of the drug around 2.76. The proposed method for determination of Etizolam showed good absorption in the present solvent as expressed from the peak absorbance and corresponding peak area. Etizolam exhibits maximum absorption at 254 nm and obeyed Beer’s law in the range of 0.5 - 100.0 μg/mL (Table 2) in the optimized mobile phase conditions with a correlation coefficient of 0.999 as given in the linearity plot (Fig. 3). The LOD and LOQ values were found to be 0.48 µg/mL and 1.46 µg/mL indicating the sensitivity of the method.

Table 2: Linearity data

Concentration (µg/mL)	*Peak area ± SD, %RSD
0.5	29192 ± 499.8, 1.7
1	43722 ± 554.6, 1.3
2	83109 ± 353.5, 0.4
4	152637 ± 2168.7, 1.4
5	236036 ± 2121.2, 0.9
8	356523 ± 5071.8, 1.4
10	442737 ± 5784.3, 1.3
20	910555 ± 3785.4, 0.4
50	2190914 ± 31421.8, 1.5
80	3430697 ± 55101.1, 1.6
100	4289412 ± 67640.3, 1.6

*Mean of two determinations

Fig. 3: Linearity plot at 254 nm

The method gave consistent results indicating the precision as observed from the % RSD for intraday (0.71-1.62) and interday (0.38 - 1.10) studies (Table 3). Accuracy of the method was ascertained by performing recovery studies in the commercially available formulations. The percentage recovery value in the range of 98.38-99.60 (Table 4) indicates that there is no interference from the excipients present in the formulation.

Table 5: Robustness study of Etizolam

Parameter	Condition	*Standard peak area	*Sample peak area	Tailing factor	Theoretical plates	*Assay (%)	#Assay ± SD, RSD
Flow rate (± 0.1mL/min)	0.9	456239	458392	1.147	5123.25	100.47	100.06 ± 0.36, 0.36
	1	448627	445815	1.149	5057.66	99.37
	1.1	435082	434841	1.148	5126.89	99.94
pH ± 0.1	3.1	439265	437693	1.147	4969.49	99.64	99.83 ± 0.22, 0.22
	3.2	448627	445815	1.149	5057.66	99.37
	3.3	435428	435707	1.15	5089.97	100.06
Mobile phase composition (± 10 %)	45-55	432358	438251	1.148	5059.37	101.36	100.29 ± 0.93, 0.93
	50-50	448627	445815	1.149	5057.66	99.37
	55-45	439544	438387	1.148	5168.39	99.74

*Mean of two determinations; # Mean of six determinations

Fig. 4: Chromatogram for Etizolam in formulation (15 µg/mL)

Table 6: Assay of Etizolam in Tablets

Formulation	Label claim (mg)	*Amount found (mg)	*Assay (% w/w) ± S. D
NITOCALM	0.5	0.49	99.6 ± 1.05

*Mean of three determinations

Forced degradation study was conducted by exposing the drug to various stress conditions to test for the specificity of the method in the presence of degradants and also to explore the stability indicating capabilities of the method. Etizolam was found to almost completely degrade in acidic (95.2%), basic (93.99 %) and oxidation environments (85.3 %). A considerable degradation was also observed in photolysis (4.66 %) and thermal (3.57 %) conditions. Additional peaks were observed in acidic and oxidation stress and yet the method could clearly resolve all the chromatographic peaks making the method highly specific for Etizolam. The blank (Fig. 5a) and various degradation chromatograms (Fig. 5b-5f) were compared and analyzed. The eluted peak for the drug in the presence of degradant peaks satisfied all the system suitability parameters (Table 7). The peak purity index was greater than the single point threshold values for the chromatograms obtained in various degradations indicating the peak purity.

Fig. 5a: Chromatogram for blank

Fig. 5b: Chromatogram for acidic stress study (10 µg/mL)

Fig. 5c: Chromatogram for basic stress study (10 µg/mL)

Fig. 5d: Chromatogram for oxidation stress study (10 µg/mL)

Fig. 5e: Chromatogram for photolytic stress study (10 µg/mL)

Fig. 5f: Chromatogram for thermal stress study (10 µg/mL)

Table 7: Forced degradation studies of Etizolam

Condition	Peak area	*Assay (%)	Drug Decomposed	Tailing factor	Theoretical plates
Control	447625	99.9	-	1.149	5057.67
Acid (0.1N HCl)	21094	4.80	95.20	1.275	5701.38
Base (0.1N NaOH)	26375	6.01	93.99	1.235	5543.17
Oxidation (3% H₂O₂)	64706	14.75	85.25	1.227	5898.71
Photolysis	426721	95.24	4.66	1.275	5971.83
Thermal	430429	96.33	3.57	1.318	6584.04

*Mean of two determinations

ACKNOWLEDGEMENT:

The authors are grateful to GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam for providing the necessary research facilities.

CONCLUSION:

The proposed method is simple, sensitive, precise, robust, accurate and free from interference due to common excipients of tablets. The RP-HPLC method could also indicate the stability of Etizolam under a series of stress conditions and so can be employed as a stability indicating method. Hence, it is concluded that the developed method can be conveniently used for the regular quality control analysis of Etizolam in API and tablets.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

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Received on 03.10.2018 Modified on 27.11.2018

Research J. Pharm. and Tech. 2019; 12(4):1637-1642.

DOI: 10.5958/0974-360X.2019.00273.7

Conc.(µg/mL)	Intraday		Interday
	Conc. found (µg/mL)	*Assay (% w/w) ± SD, % RSD	Conc. found (µg/mL)	*Assay (% w/w) ± SD, % RSD
20	19.93	100.60 ± 1.01,1.00	20.07	100.46 ± 0.28,0.28
	20.33		20.05
	20.11		20.16
40	39.37	100.09 ± 1.62.1.62	39.97	100.32 ± 0.74,0.73
	40.08		39.94
	40.66		40.47
60	60.28	100.44 ± 0.71,0.71	60.05	100.88 ± 1.10,1.09
	60.68		61.28
	59.83		60.25

Level (%)	Total amount of drug (µg/mL)	*Drug recovered (µg/mL)	Recovery (%) ± SD, % RSD*
50	15	14.85	98.99 ± 0.21,0.21
100	20	19.92	98.38 ± 0.40,0.41
150	25	24.59	98.38 ± 0.36,0.37