INTRODUCTION:

Zolpidem Tartrate is chemically N,N,6-Trimethyl-2-(4-methylphenyl)-imidazo[1,2-a]pyridine-3-acetamide as shown in Fig-1. Zolpidem is a prescription short-acting nonbenzodiazepine hypnotic that potentiates gamma-amino butyric acid (GABA), an inhibitory neurotransmitter, by binding to benzodiazepine receptors which are located on the gamma-amino butyric acid receptors.

A survey of literature^[1-12] reveals that good analytical methods are not available for the drugs like Zolpidem Tartrate. Even though very few methods of estimation of are available, many of them suffer from one disadvantage or the other, such as low sensitivity, lack of selectivity and simplicity etc. The existing physicochemical methods are inadequate to meet the requirements; hence it is proposed to improve the existing methods and to develop new methods for the assay of Zolpidem Tartrate, in pharmaceutical dosage forms adapting different available analytical techniques like RP-HPLC with a main objective to reduce the retention time of Zolpidem Tartrate. So according to ICH guidelines an attempt was made to create and validate a basic simple, accurate, precise and efficient RP-HPLC technique which could be easily and conveniently adopted for routine analysis of Zolpidem Tartrate in pure form and its dosage form.

Fig 1: Structure of Zolpidem Tartrate

MATERIALS AND METHODS:

Instrumentation:

HPLC (Make: WATERS, Model: Alliance 2695, Detector 2487 with Empower 2 software), UV Spectrophotometer (Make: Lab India, Model: UV-3000⁺with UV win 5 software), Weighing Balance (Make: Ascoset, Model:ER200A), Sonicator (Make: Enertech, Model: SE60US), pH Meter (Make: ADWA, Model: AD102U), Heating Mantle (Make: Bio Technics India, Model: BTI), Filter Paper 0.45 microns (Make: Milli Pore)

Reagents and chemicals:

Ammonium Acetate, Glacial Acetic Acid, HPLC Grade Methanol, HPLC Grade Acetonitrile, Double Distilled Water, Zolpidem Tartrate [ZOLFRESH (Abbott Pharmaceuticals), 10.0mg].

Chromatographic conditions:

Parameters	Method
Column (Stationary Phase)	Symmetry C18 (4.6 x 150mm, 5 mm, Make: XTerra) or equivalent
Mobile Phase	Ammonium Acetate Buffer (4.5 pH): Methanol (40:60%v/v)
Flow rate (ml/min)	0.8
Mode	Isocratic
Run time (min)	6
Column temperature(°C)	Ambient
Volume of injection loop (ml)	20
Detection wavelength (nm)	300
Drug RT (min)	3.14

METHOD DEVELOPMENT AND OPTIMISATION:

The detection wavelength was ascertained at 300nm from the UV spectrum of Zolpidem Tartrate (20µg/ml) in the mobile phase against reagent blank.

To develop a suitable and robust method for the determination of Zolpidem Tartrate, different mobile phases like Potassium dihydrogen ortho phosphate-Methanol, Ammonium acetate buffer-Methanol, Ammonium acetate buffer-Acetonitrile, Methanol-Water solution in various ratios (70:30, 40:60, 50:50, 60:40 V/V) were tried at flow rate 0.8ml/min. Ammonium Acetate buffer-Methanol solution in the ratio 40:60 at a flow rate of 0.8ml/min was selected of all since it gave the best result. The peak was symmetric and the drug was eluted at 3.14 minutes. The run time was thus fixed at 6 minutes.

METHOD:

Preparation of mobile phase:

Weigh 7.0grams of Ammonium Acetate in to a 1000ml beaker, dissolve and diluted to 1000ml with HPLC water. Adjusted the pH to 4.5 with Glacial Acetic acid. Mix a mixture of above buffer 400mL (40%) and 600 mL of Methanol HPLC (60%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45µm filter under vacuum filtration. 20µl of the solvent was injected after column equilibration.

Preparation of drug solutions:

10mg of Zolpidem Tartrate pure drug was weighed and dissolved in the mobile phase in a 10ml volumetric flask to get the stock solution (1000µg/ml). From this further aliquots of 0.2-0.6ml from the stock solution were taken and diluted with the mobile phase to get solutions in the concentration range of 20µg to 60µg/ml.

Preparation of sample drug solution from pharmaceutical dosage form:

Five tablets containing the drug were taken and powdered. The powder equivalent to 10mg of the active ingredient was accurately weighed and taken in a 10ml volumetric flask and mobile phase was added to make up to volume. The volumetric flask was sonicated for 30 minutes to effect complete dissolution of drug and the solution was made up to volume with mobile phase and filtered through Whatman filter paper (0.45µm) made up of cellulose nitrate. Aliquots solutions were prepared by taking 0.4ml of the filtered solution into 10ml volumetric flasks, separately and made up to volume with mobile phase to yield concentrations of drug in range of linearity previously described.

Estimation of Zolpidem Tartrate in the pharmaceutical dosage forms:

The amount of drug present in each pharmaceutical formulation was calculated by using the regression equation of the linearity plot. Percentage recovery was found to be 99.0±0.33 (each value is average of three determinations ± standard deviation) with an % RSD of 0.317. A typical chromatogram of Zolpidem Tartrate (30mg/ml) (Formulation) is as shown in Figure-2.

Fig 2: Typical chromatogram for the tablet formulation (10mg/ml)

METHOD VALIDATION PARAMETERS:

Linearity:

For all methods, 6-point calibration curve were prepared on single day. The results obtained were used to calculate the equation of the line by using linear regression by the least square method. An injection volume of 20ml of each of standard and sample solutions were injected into the HPLC system to get the chromatograms. The retention time and peak areas of the drug were recorded. A graph was plotted by taking concentration of the drug on x-axis and peak area on y-axis. The linearity range was found to be in between 20-60 mg/ml for Zolpidem Tartrate. The linearity table and calibration curve are as shown in Table 1 and Figure 4, respectively. A typical chromatogram of Zolpidem Tartrate (40mg/ml) (Pure drug) is as shown in Figure-3.

Table No. 1: Linearity range of proposed RP-HPLC method

S. No	Linearity Level	Concentration	Area
1	I	20µg/ml	926213
2	II	30µg/ml	1402091
3	III	40µg/ml	1862724
4	IV	50µg/ml	2352834
5	V	60µg/ml	2844035
Linearity range (µg/ml)			20-60
Regression equation
Slope			47336
Intercept			13206
Correlation coefficient			0.999

Fig 3: Typical chromatogram of pure drug (40µg/ml)

Fig 4: Linearity plot of Zolpidem Tartrate

Precision: (System precision)

The repeatability of the method was ascertained by precision (intra-day and inter-day) of the method by estimation of one replicates at single intermediate level (40µg/ml) on the same day and next day, respectively. The results along with the statistical calculations are as presented in the Table-2.

Method precision:

The repeatability of the method was ascertained by precision (intra-day and inter-day) of the method by estimation of one replicates at single intermediate level (40µg/ml) (Formulation) on the same day and next day, respectively. The results along with the statistical calculations are as presented in the Table-2.

Table No. 2: System and Method Precision (Intra-day and Inter-day)

System Precision
Intra-day		Inter-day
Injection	Area	Injection	Area
Injection-1	1892556	Injection-1	1858787
Injection-2	1898440	Injection-2	1851176
Injection-3	1896326	Injection-3	1851848
Injection-4	1896644	Injection-4	1851874
Injection-5	1899562	Injection-5	1851123
Average	1896705	Average	1852962
Standard Deviation	2670.8	Standard Deviation	3275.6
%RSD	0.14	%RSD	0.18
Method Precision
Intra-day		Inter-day
Injection	Area	Injection	Area
Injection-1	1910172	Injection-1	1882144
Injection-2	1912204	Injection-2	1881694
Injection-3	1918943	Injection-3	1880161
Injection-4	1920794	Injection-4	1882126
Injection-5	1928479	Injection-5	1886945
Injection-6	1932712	Injection-6	1879498
Average	1920551	Average	1882095
Standard Deviation	8840.8	Standard Deviation	2614.2
%RSD	0.46	%RSD	0.14

Accuracy:

To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (50%, 100%, and 150%) of bulk samples of Zolpidem Tartrate to pre-analyzed amount of formulation of concentration 40mg/ml. From this percentage recovery values were calculated. The results were shown in Table-3.

Robustness:

Robustness of the method reflects the reliability of an analysis with respect to deliberate variations in the method parameters. Here, the flow rate and mobile phase composition were slightly changed to lower and higher sides of the actual values to find if the change in the peak area and retention time were within limits. The results obtained with changes in the parameters on a 40µg/ml solution are as shown in Table-4.

System suitability parameters:

System suitability parameters can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The requirements for system suitability are usually developed after method development and validation has been completed. The USP (2000) defines parameters that can be used to determine system suitability prior to analysis. The system suitability parameters like Theoretical plates (N), Asymmetry (A), LOD (mg/ml) and LOQ(mg/ml) were calculated and found to be 2405.28, 1.65, 0.036 and 0.12 respectively.

FORCED DEGRADATION STUDIES OF ZOLPIDEM TARTRATE:

The specificity of the method was demonstrated through forced degradation studies conducted on the sample using acid, alkaline, oxidative and thermal degradations. The sample was exposed to these conditions and the drug peak was studied to find whether the degradation products could be clearly separated from the pure drug peak.

Initially, after equilibration of the column, 20µl of the solvent was injected. A typical chromatogram of the blank hence obtained is shown in Figure-5A.

The untreated sample, i.e. a sample not subjected to any stress condition was then estimated thrice by the established method, so as to form the basis of the percentage of degradation on exposure to the stress conditions, like acid degradation, alkaline degradation, oxidation and thermal degradation. The chromatogram for untreated sample obtained is shown in Figure-5B and values were shown in Table-5. A similar set of samples were subjected to the various stress conditions as illustrated in the following sections.

Acid Degradation:

About 10mg of the pure drug was accurately weighed and transferred to 10ml volumetric flask. 3ml of 0.1N HCl was added and kept for 24 hour and 12 hours of heating in water bath at 70ºC. It was then cooled, neutralized using 0.1N NaOH and the volume was made up to 10ml with mobile phase. Then from this 40µg/ml solution was prepared and 20µl of it was injected into the HPLC system to obtain the chromatogram. The chromatogram thus obtained is as shown in Figure 5C and values were given in Table-5.

Alkaline Degradation:

About 10mg of the pure drug was accurately weighed and transferred to a 10ml volumetric flask. 3 ml of 0.1N NaOH was added and kept for 24 hour and 12 hours of heating in water bath at 70ºC. It was then cooled, neutralized using 0.1N HCl and the volume was made up to 10ml with mobile phase. Then from this 40µg/ml solution was prepared and 20µl of it was injected into the HPLC system to obtain the chromatogram. The chromatogram thus obtained is as shown in Figure-5D and values were given in Table-5.

Oxidative Degradation:

About 10mg of the drug was accurately weighed and transferred to a 10ml volumetric flask and 3 ml of 3% w/v of hydrogen peroxide solution was added and kept for 24 hour heating in water bath at 70ºC. It was then cooled and made up to 10ml with mobile phase. Then from this 40µg/ml solution was prepared and 20µl of it was injected into the HPLC system to obtain the chromatogram. The chromatogram of treated sample thus obtained is as shown in Figure-5E and values were given in Table-5.

Thermal Degradation:

About 10mg of the pure drug was accurately weighed and transferred to a 10ml volumetric flask and kept in the hot air oven for 12 hours at a temperature of 70ºC and diluted with mobile phase to 10ml. From this 0.4 ml was diluted to 10 ml so as to obtain a concentration of 40µg/ml. It was then allowed to cool and then 20µL of this was injected into the HPLC system. The chromatogram obtained is as shown in Figure-5F and values were given in Table-5.

RESULTS AND DISCUSSION:

The Results shown that Zolpidem Tartrate can effectively be analysed by the HPLC method with Ammonium Acetate Buffer-Methanol as the mobile phase in the ratio 40:60 at a flow rate of 0.8ml/minute and detection wavelength of 300nm. The retention time of the drug was 3.141 minute. The linearity range was found to be 20-60µg/ml. The regression equation obtained for the linearity plot was y = 47336x - 13206, with correlation coefficient 0.999. In the System precision study, %RSD was found to be less than 2% intra day (0.14) inter day (0.18) and the Method precision study, %RSD was found to be less than 2% intra day (0.46) inter day (0.14) which indicates that the method has good reproducibility. The accuracy studies showed % recovery in the range 98.90 – 100.87% (% RSD = 0.185– 0.318), which indicates that the method was accurate and also revealed that the commonly used excipients present in the pharmaceutical formulations do not interfere in the proposed method. Robustness studies reveal that the method was reliable. The system suitability parameters were found to be within the specified limits for the proposed method.

The forced degradation studies showed that degradation was obtained in the range of 0.97 – 9.96% and that the degraded products were effectively separated from that of the drug peak and the amount of the drug remaining intact was specifically eluted at its characteristic retention time, thereby indicating that the method was specific.

CONCLUSION:

The proposed method was found to be simple, precise, accurate, rapid and specific for determination of Zolpidem Tartrate from pure and its dosage forms. The mobile phase is simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence, this method can be easily and conveniently adopted for routine analysis of Zolpidem Tartrate in pure form and its dosage form and also can be used for dissolution or similar studies.

ACKNOWLEDGMENT:

The authors are thankful to the Vels Institute of Science Technology and Advanced Studies and Management, VISTAS for providing us the necessary facilities and constant encouragement. Author also expresses sincere thanks to Aurobindo Pharmaceuticals, Hyderabad for providing the drugs.

REFERENCES:

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Received on 03.11.2019 Modified on 31.01.2020

Research J. Pharm. and Tech. 2021; 14(2):757-762.

DOI: 10.5958/0974-360X.2021.00132.3

Sample ID	Concentration (mg/ml)		% Recovery	Statistical Analysis
Sample ID	Pure drug	Formulation	% Recovery	Statistical Analysis
S₁ : 50 %	20	40	100.84	Mean = 100.87 SD = 0.1868 %RSD = 0.185
S₂ : 50 %	20	40	100.70
S₃ : 50 %	20	40	101.07
S₄: 100 %	40	40	98.47	Mean = 99.65 SD = 0.3175 %RSD = 0.318
S₅ : 100 %	40	40	99.02
S₆ : 100 %	40	40	98.47
S₇: 150 %	60	40	99.09	Mean = 98.90 SD = 0.2753 %RSD = 0.278
S₈ : 150 %	60	40	98.59
S₉ : 150 %	60	40	99.04

S. No.	Parameter	Condition	Peak area	Statistical analysis	Retention time	Statistical analysis
1	Flow rate (ml/min)	0.7	1942204	Mean= 1921616 SD= 18550 %RSD= 0.965	3.156	Mean= 3.128 SD= 0.032 %RSD=1.023
		0.8	1916444		3.137
		0.9	1906202		3.092
2	Mobile phase ratio	45:55	1975874	Mean= 1950589 SD= 30689 %RSD= 1.573	3.159	Mean= 3.130 SD= 0.031 %RSD=0.990
		40:60	1916444		3.137
		35:65	1959450		3.096

Conditions applied	Peak area	% drug recovered	RT of the analyte (min)	RT of major degradants (min)
Untreated	1959609	100	3.203	-
Acid degradation	1856871	94.75	3.265	2.694
Alkaline degradation	1841058	93.95	3.205	2.616
Oxidative degradation	1764441	90.04	3.273	10.270
Thermal degradation	1946063	99.03	3.202	11.01