1.0 INTRODUCTION:

Aripiprazole (Fig.1) is an atypical antipsychotic agent belonging to the chemical class of benzisoxazole derivatives and is indicated for the treatment of schizophrenia. Aripiprazole is a selective monoaminergic antagonist with high affinity for the serotonin Type 2 (5HT2), dopamine Type 2 (D2), 1 and 2 adrenergic, and H1 histaminergic receptors. Aripiprazole appears to mediate its antipsychotic effects primarily by partial agonism at the D2 receptor. In addition to partial agonist activity at the D2 receptor, Aripiprazole is also a partial agonist at the 5-HT1A receptor, and like the other atypical antipsychotics, Aripiprazole displays an antagonist profile at the 5-HT2A receptor. Aripiprazole has moderate affinity for histamine and alpha adrenergic receptors, and no appreciable affinity for cholinergic muscarinic receptors^1-2.

A through literature survey has revealed the following reported methods for the estimation of the Aripiprazole. The reported methods include estimation of Aripiprazole in biological samples using LC-UV and LC-MS^3-13. The reported methods also include HPLC, UPLC, and Spectroscopy for the estimation of Aripiprazole in bulk and pharmaceutical formulations^12-27.

In the present study attempts were made to develop a rapid, economical, precise and accurate stability indicating method for estimation of Aripiprazole in tablet formulations.

2.0 EXPERIMENTAL:

2.1 Chemicals and Reagents:

All the reagents were of analytical and HPLC grade unless stated otherwise. Milli-Q-Water was used throughout the study. Potassium dihydrogen phosphate, Hydrochloric acid, Sodium acetate, Sodium Hydroxide, Ethanol absolute, Acetonitrile, Hydrogen Peroxide, Formic acid (Merck, Mumbai, India) were used. Aripiprazole Standard was obtained as a gift sample from Dr. Reddy’s Laboratories, Hyderabad, India and the Aripiprazole tablets were purchased Form Local Pharmacy.

2.2 Instrumentation:

The HPLC system used was a Waters 2695 separation module with an auto injector and waters 2996 PDA Detector. The output signal was monitored and integrated using Empower software. Phenomenex Luna C18, 150 x 4.6mm, 5µm column was used.

2.3 Buffer Preparation:

0.05M Phosphate buffer was prepared by dissolving Potassium dihydrogen phosphate in water and pH of the resultant solution was adjusted to 3.2 with formic acid.

2.4 Mobile phase:

A filtered and degassed mixture of acetonitrile and 0.05 M phosphate buffer, pH 3.2 in the ratio of 40:60 v/v was used.

Fig.1: Molecular Structure of Aripiprazole

Fig.2a: A typical chromatogram of Aripiprazole.

Fig.2b: Typical Chromatogram showing separation of Aripiprazole from its depredation products.

2.5 Standard Preparations:

Standard stock solution was prepared by dissolving 25 mg of Aripiprazole working standard in 25 mL of mobile phase. From the above stock solution, a series of solutions were prepared at concentration levels ranging from 80% to 120% of target concentration. Measured the peak area responses of solutions at all levels in duplicate.

2.6 Sample solution:

20 tablets were accurately weighed and grounded to fine powder. Aripiprazole tablet powder equivalent to the label claim was accurately weighed and transferred into a 100 mL volumetric flask, few mL of mobile phase was added and sonicated to dissolve and made up the volume with mobile phase. The above solution was filtered and diluted to get a final concentration of 100 µg mL^-1.

2.7 Chromatographic conditions:

The chromatographic conditions used for the analysis were given as below.

Detection wavelength : 227 nm

Column : Phenomenex Luna C18, 150 x 4.6mm, 5µm

Flow rate : 1.0 mL min-1

Injection volume : 20µL

2.8 Stress degradation study:

To determine whether the analytical method was stability indicating, Aripiprazole API was stressed under various conditions includes photolytic degradation (Exposed to 1, 00,000 Lux.), acid hydrolysis (1N Hydrochloric acid), base hydrolysis (1N sodium hydroxide), Oxidative degradation (10% Hydrogen peroxide) and Thermal degradation (at 125ºC).

2.8.1 Photolytic degradation:

The sample solution was exposed to Exposed to 1, 00,000 Lux hours of white light to determine the effects of light irradiation on the stability of Aripiprazole.

2.8.2 Acid, alkaline, oxidative and thermal degradation:

Forced degradation in acidic media, alkaline media, H2O2 was performed with 1mg mL-1 concentration to determine the effects of chemical irradiation on the stability of Aripiprazole.

3.0. RESULTS AND DISCUSSION:

3.1 Optimization of the chromatographic conditions:

To optimize the method, initially a 10% Acetonitrile was used. However, this 10% Acetonitrile was unable to elute Aripiprazole. To elute Aripiprazole peak, mixtures of different ratios of ACN and 0.05 M Phosphate buffer have been used and lead to a better elution. The different flow rates from 0.8 to 1.2mL/min at the intervals of 0.1 mL/min have been tried. Sharp peak with good resolution were attained using 1.0 mL/min. A successful isocratic run (mentioned in the experimental section) was employed to resolve all the known degradation products which were formed during degradation. The optimized injection volume and detection wavelength were 20 μL and 227 nm, respectively. To achieve the separation of degradation products, stationary phase of C18 and a combination of mobile phase phosphate buffer and acetonitrile were used. The chromatographic behaviour of Aripiprazole in different mobile phases and stationary phases were shown Table 1and2. The separation of degradation products and Aripiprazole was achieved on a Phenomenex Luna C18, 150 x 4.6mm, 5µm column and a mobile phase comprised of acetonitrile and 0.05M phosphate buffer, pH 3.2 (40:60 v/v). The developed method was found to be specific and validated as per ICH guide lines.

Table 1: The chromatographic behaviour of Aripiprazole in different mobile phases.

Mobile phase	Retention time (min)
Methanol: Water (30:70 V/V)	13.2
Acetonitrile: Buffer (pH 3.2, 0.05M, potassium phosphate) (25:75 V/V)	9.2
Acetonitrile: Buffer (pH 3.2, 0.05M, potassium phosphate) (40:60 V/V)	5.7
Acetonitrile: Buffer (pH 3.2, 0.05M, potassium phosphate) (35:65 V/V)	6.2

Table 2: The chromatographic behaviour of Aripiprazole in different Columns.

Column	Retention time (min)
Inertsil ODS C18; 15cm × 4.6 mm i.d., 5 μm particle	9.8
Zorbax SB C18; 15cm × 4.6 mm i.d., 3.5 μm particle	4.2
Phenomenex Luna C18; 15cm × 4.6 mm i.d., 5 μm particle	5.6
Kromosil C18; 15cm × 4.6 mm i.d., 5 μm particle	7.1
Waters Symmetry C18; 15cm × 4.6 mm i.d., 5 μm particle	6.7

3.3 Method Validation:

The optimized chromatographic conditions were validated by evaluating Linearity, Precision, Accuracy, LOD and LOQ, robustness in accordance with ICH guidelines^28-29.

3.3.1 Linearity:

The curve proved to be Linear over a concentration range of 25 µg mL^-1 to 200 µg mL^-1 (Fig 3). Standard solution s were Prepared at five concentrations (25, 50,100,150 and 200 µg mL^-1) were injected in triplicate. The linear regression of concentration vs peak area resulted in an average coefficient of determination (R²) is 0.9995(Table 4).

3.3.3 Accuracy:

Accuracy was calculated as the % recovery of known spiked amounts of Aripiprazole in the Placebo Preparations using 5 concentration levels covering the specified range (25, 50, 100, 125, 150 µg mL^-1). The accuracy of the method is determined by recovery experiments. The recovery is performed by Aripiprazole sample at 80%, 100% and 120% levels against Aripiprazole working standard. At each level, three sample preparations were used. The average % recovery on three preparations at each level, as 100.00%, 100.00% and 99.95% respectively (Table 6).

Fig. 3: A Typical Linearity Plot for Aripiprazole.

3.3.4 LOD and LOQ:

The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be readily detected but not necessarily quantified. It is usually regarded as the amount for which the signal-to-noise ratio (SNR) is 3:1. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte that can be quantified with acceptable precision and accuracy. It is usually regarded as the amount for which the SNR is 10:1. Two types of solution, blank solution and solutions containing known, progressively decreasing concentrations of the analyte were prepared and analyzed. The signal-to-noise ratios 3.2 for LOD and 11.4 for LOQ. LOD and LOQ were 0.5 μg mL^-1 and 2.8 μg mL^-1, respectively.

3.3.5 Robustness:

To determine the robustness of developed method, experimental conditions were purposely altered. The flow rate of the mobile phase was 1.0 mL min-1. To study the effect of flow rate on the retention, flow was changed by 0.2 units from 0.8 to 1.2 mL min^-1. The effect of the percent organic strength on the retention and peak area was studied by varying Acetonitrile by -3 to +3 %. The pH of the mobile phase was altered by +0.2 units. The % RSD was Less than 1.0% (Table 7).

3.3.6. Stability of analytical solution and mobile phase

The % RSD for retention times of different intervals of 0hr, 24hrs, 30hrs and 48hrs was found to be 0.13% and 0.46% for mobile phase and analyte solution respectively, (Table 8).

Table 3: Results for Degradation behaviour of Aripiprazole.

Degradation mechanism / condition	% Assay	% Degradation	Purity Angle	Purity Threshold	Remarks
Un-degraded sample	99.6	-	0.322	0.953	Passed
Thermal at - 125°C for 6 Hr	97.0	2.6	0.068	0.258	Passed
photolytic at 254 nm for 168 Hrs	99.8	-	0.046	0.261	Passed
Acid /1 N HCl Heat on water bath for 2 Hrs	92.0	8.0	0.060	0.273	Passed
Base /1 N NaOH Heat on water bath for 2 Hrs	93.0	6.6	0.178	0.278	Passed
Peroxide /10.0% H₂O₂ Heat on water bath for 2 Hrs	96.7	2.9	0.080	0.390	Passed

Table 4: Results for Linearity Plot for Aripiprazole.

Linearity Level	Concentration (µg/mL)	Response	Statistical Analysis
L1-25%	25.0	1422111	Slope	55537.79
L2-50%	50.0	2845226	y-Intercept	63573.671
L3-100%	100.0	5588341	% of y- Intercept	1.14
L4-150%	150.0	8542654	Correlation Coefficient	0.99974
L5-200%	200.0	11076877	Regression Coefficient-r²	0.99947

Table 5a: Results of System Precision.

Concentration 100%	Injection	Peak Area	Theoretical Plates	Tailing factor
	1	5418465	4127	1.22
	2	5428133	4125	1.23
	3	5448332	4110	1.23
	4	5418455	4091	1.22
	5	5438415	4089	1.22
Statistical Analysis	Mean	5430044	4108	1.22
	SD	11653.85
	% RSD	0.21%

Table 5b: Results for Inter and Intraday of Aripiprazole.

Preparation No.	Inter Day	Intraday
Preparation No.	% Assay	% Assay
01	98.7	99.2
02	100.3	100.1
03	100.0	100
04	99.7	99.9
05	99.5	99.1
06	99.4	99.6
Average	99.6	99.7
Std.Dev.	0.55	0.42
%RSD	0.55	0.42

Table 6: Results for Accuracy (% recovery) of Aripiprazole.

% Spiked to the label amount	Amount added (mg)	Amount found (mg)	% Recovery	Mean % Recovery N=3,+SEM
80% - 1	24.01	24.01	100.00	100.00+0.01
80% - 2	24.04	24.03	100.04
80% - 3	24.02	24.03	99.96
100% - 1	30.03	30.02	100.03	100.00+0.02
100% - 2	30.01	30.04	99.90
100% - 3	30.03	30.01	100.07
120% - 1	36.01	36.02	99.97	99.95+0.01
120% - 2	36.02	36.03	99.97
120% - 3	36.01	36.04	99.92

Table 7: Results for Robustness of Aripiprazole.

Robustness Parameter	Retention time	Tailing Factor	RSD (%)
Mobile Phase Composition (%v/v)
32:68	5.4	1.03	0.11
40:60 (Optimized)	5.6	1.11	0.05
38:62	5.7	1.21	0.12
Flow rate variation (mL min-¹)
0.8	6.2	1.23	0.28
1.0 (Optimized)	5.6	1.14	0.25
1.2	5.1	1.11	0.35
Buffer pH
3.0	5.3	1.07	0.41
3.2 (Optimized)	5.6	1.12	0.35
3.4	6.2	1.15	0.38

Table 8: Results for Stability of analytical solution and mobile phase.

Time Interval	Retention time ( in minutes)	Mobile phase stability	Analyte solution stability
0 Hour (Initial)	5.63	99.89	99. 92
12 Hours	5.64	99.72	99.88
24 Hours	5.65	99.72	99.71
30 Hours	5.65	99.77	99.81
48 Hours	5.64	99.85	99.87
Mean ± SEM (n= 4)	5.64 ± 0.01	99.79 ± 0.08	99.82 ± 0.08
%RSD	0.15	0.08	0.08

4.0 CONCLUSION:

Forced degradation study on Aripiprazole in tablet formulation was carried out under the conditions of Photolysis, hydrolysis, Oxidation and thermal. Based on the information generated by forced degradation, stability indicating LC method was developed and validated. The Assay method adopted for Aripiprazole is specific, precise, linear and accurate. The Analyte solution was found to be stable up to 48 hours under ambient conditions and mobile phase was found to be stable up to 48 hours. Hence this method can be used for routine analysis and stability study.

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Received on 12.01.2011 Accepted on 22.02.2011

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