1. INTRODUCTION:

Aripiprazole is chemically a quinolinone derivative, used as an anti-psychotic agent. Chemically, Aripiprazole is known as 7-[4-[4-(dichlorophenyl) piperizin-1-yl]-3,4-dihydroquinolinone. The empirical formula is C₂₃H₂₇Cl₂N₃O₂ and its molecular weight is 448.38. It is a white crystalline powder and practically insoluble in water and its solubility is pH dependent. The chemical structure is given below ^1-6

It is not official in any pharmacopoeia and few liquid chromatography procedures have been reported for the determination of Aripiprazole in Bulk and Pharmaceutical dosage forms. However there are no publications concerning the analysis of Aripiprazole in presence of its intermediates in bulk and Pharmaceutical dosage forms ^7-9. So it is felt necessary to develop a new Rapid Resolution liquid chromatographic (RRLC) procedure which would serve as a rapid and reliable method for the determination of Aripiprazole in respective related impurities [Fig 1] in bulk and pharmaceutical dosage forms. In the proposed method, related impurities were well separated and eluted before 10min .Finally the method was thoroughly validated for the assay and it is related impurities.

2. EXPERIMENTAL^10-13

2.1 Instrumentation : Agilent RRLC 1200 series equipped with binary pump SL and DAD SL detector was used .The output signal was monitored and integrated using waters Empower 2 software

Solutions:

2.2 Preparation of pH 3.0 Buffer solution:

Weigh and dissolve 2.72 g of potassium dihydrogen orthophosphate in 1000 mL of milli-Q water in a suitable container, add 10 mL of triethylamine and mix, and adjust the pH of the solution to 3.0 ± 0.05 with dilute ortho phosphoric acid.

Impurity-A Impurity-B

Impurity –C Impurity –D

N-Oxide

2.3 Preparation of Mobile phase:

A mixture of pH 3.0 buffer, Acetonitrile and Methanol in the ratio 60:20:20 (v/v/v) was prepared and filtered through 0.45µm nylon membrane filter prior to use.

Diluent : Mix pH 3.0 buffer and Acetonitrile in the Ratio 62:38(v/v)

2.4 Preparation of Standard solution (120µg/ml): About 50mg of Aripiprazole (ARPI) working standard was accurately weighed and transferred in to a 50mL volumetric flask and dissolved in 70ml of diluent and diluted to volume with diluent and mixed well. Further 3mL of the Resulting solution was taken into 25mL volumetric flask and made up to volume with diluent and mixed well. Solution was filtered through 0.45µm nylon membrane filter prior to use.

2.5 Preparation of Test Solution: Twenty tablets were taken weighed and powdered .Sample equivalent to about 50mg of Aripiprazole (ARPI) was accurately weighed and transferred in to a 50mL volumetric flask 70ml of diluent was added and sonicated for 30min and diluted to volume with diluent and mixed well. 3ml of the Clear Centrifuge is taken in to 25ml volumetric flask and made up to volume with diluent. Filtered through 0.45µm nylon membrane filter prior to use.

2.6 Preparation of Degradation samples for Specificity Study:

For Acid degradation Aripiprazole sample was refluxed with 1N HCl at 80ºC for 4hrs and then neutralized by adjusting pH to 7.0 with 1N NaOH .The Solution was further diluted to required concentration with diluent.

For basic degradation Aripiprazole sample was refluxed with 2N NaOH for 1hr and then neutralized by adjusting pH to 7.0 with 2N HCl .The Solution was further diluted to required concentration with diluent.

For Oxidative degradation Aripiprazole sample was refluxed 1%H₂O₂by heating on water bath at

40ºC for 1hour .The Solution was further diluted to required concentration with diluent.

For Photolightic Stress the samples were exposed to UV at 254nm for 63hrs and visible light for 288hrs meeting the specification of ICH i.e UV(200watt/m²) and Visible(1.2million Lux hours).

For Thermal Degradation Samples were Exposed to Temperature at 105ºC for 22hrs.

The above stressed samples i.e Photolightic and Thermal stress samples solutions were prepared to required concentration with diluent.

2.7 Chromatographic Conditions:

A Hypersil Gold C18 (50 x 4.6mm;5µm packing) column was used for analysis at column temperature 40ºC .The mobile phase was pumped through the column at a flow rate of 1.0mL/min. The Sample injection Volume was 5µL.The photodiode array detector was set to a wavelength of 252nm for the detection

3.0 RESULTS AND DISCUSSION:

3.1 Method development^11-14

3.1.1 Separation of Known degradant impurities:

To develop a suitable and robust RRLC method for the determination of Aripiprazole different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a Peerless basic C18 250 x 4.6mm, 5µ column using a mobile phase of pH 2.0 buffer: Acetonitrile in the ratio 55:45.In the above condition elution was very broad for Aripiprazole. Early elution with little separation was observed with mobile phase consisting of pH 2.0 buffers: Acetonitrile in the ratio 55:45 using column Purosphere STAR, RP-18e, 250 x 4.6 mm, 5 µm. Finally the mobile phase consisting of pH 3.0 buffer: Acetonitrile: Methanol in the ratio 60:20:20 was found to be appropriate ,allowing good separation and symmetrical peak at a flow rate of 1.0mL/min using Hypersil Gold, C18, 50 x 4.6 mm, 5 µm.. The Chromatogram of Aripiprazole sample spiked with the related compounds using the proposed method is shown in Fig.2.In the proposed method the resolution is more than 2 between the Aripiprazole and N-Oxide impurity .System suitability results of the method are presented in Table 1.Aripiprazole and its related compounds show significant UV absorbance at Wavelength 252 nm .Hence this wavelength has been chosen for detection in the analysis of Aripiprazole.

TABLE-1 SYSTEM SUITABILITY REPORT

Compound	Tailing Factor ^a	Resolution^a	%RSD^a	Theoretical Plates
Aripiprazole	1.1	----	0.5	3116
N-Oxide	1.0	2.4	0.8	3291

^a Number of samples analyzed are six

Fig -2 TYPICAL CHROMATOGRAM OF ARIPIPRAZOLE AND ITS RELATED IMPURITIES

3.1.2 Column Selection:

Based on the retention time and separation of the compounds Hypersil Gold , (50 x 4.6 mm, 5 µm) column was selected as suitable column for the analysis of Aripiprazole.

3.2 Method Validation:^15-16

The developed RRLC method of Aripiprazole is extensively validated for assay and its Related impurities 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 Aripiprazole peak. This indicates that the excipients used in the formulation do not interfere in estimation of Aripiprazole in Aripiprazole tablets.

Interference from degradation products:

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

3.2.2) limit of detection and limit of QUANTITATION:

A study to establish the Limit of detection and limit of quantitation of Aripiprazole related impurities were conducted.

TABE -2 TABLE RESULTS FOR SPECIFICITY

[INTERFERENCE FROM DEGRADATION PRODUCTS]

Stress Condition	% Degradation	Purity Angle	Purity Threshold	Purity Flag
Acid Stress	2.39	0.067	0.303	No
Base Stress	1.47	0.062	0.302	No
Oxidation Stress	12.87	0.066	0.290	No
Photolightic Stress	Nil	0.074	0.320	No
Thermal Stress	1.94	0.075	0.320	No

Limit of detection and limit of quantitation were established based on signal to noise ratio. A series of solutions having Aripiprazole related impurities were injected. Limit of detection for related impurities were established by identifying the concentration which gives signal to noise ratio about 3. Limit of quantitation was established by identifying the concentration which gives signal to noise ratio about 10.

Precision of Aripiprazole related impurities at about Limit of Quantitation were conducted. Six test preparations of Aripiprazole Tablets having related impurities at about Limit of quantitation was prepared and injected into the RRLC system. The %RSD at LOQ level was calculated for all known impurities and found to be less than 5.0%.

Accuracy of Aripiprazole related impurities at about Limit of Quantitation was conducted. Test solutions spiked with related impurities at about Limit of Quantitation was prepared in triplicate and injected into RRLC system and calculated the % recovery. The mean recovery of Aripiprazole related impurities at about Limit of Quantitation was ranged from 95.2 to 104.8% .The results are given under Table-3

FIG-3 LINEARITY OF DETECTOR RESPONSE GRAPH OF ARIPIPRAZOLE

FIG-4 LINEARITY OF DETECTOR RESPONSE GRAPH OF ARIPIPRAZOLE RELATED IMPURITIES

TABLE-3 TABLE RESULTS FOR LOD AND LOQ OF ARIPIPRAZOLE IMPURITIES

IMPURITY	Limit of detection (LOD)	Limit of Quantification (LOQ)	% RSD*	% Recovery#
IMPURITY	Conc. µg/mL	Conc. µg/mL	% RSD*	% Recovery#
Imp-A	0.015	0.04	1.7	103.8
Imp-B	0.034	0.101	4.6	104.8
Imp-C	0.030	0.090	4.3	95.2
Imp-D	0.033	0.101	4.2	97.8
N-oxide	0.030	0.090	2.8	102.1

^a Number of samples analyzed are six

# Number of samples analyzed are three

TABLE-4 RESULTS FOR PRECISION OF TEST METHOD

Test No	% Assay
Test No	2mg	5mg	10mg	15mg	20mg	30mg
01	99.1	102.6	100.9	97.2	99.4	98.1
02	98.5	100.5	101.0	97.4	98.9	97.9
03	98.6	99.1	98.4	97.7	98.6	98.3
04	98.9	99.8	99.0	97.4	98.7	97.9
05	98.9	99.8	98.2	97.6	98.8	98.3
06	99.0	99.2	99.5	97.3	98.9	97.9
Average	98.8	100.2	99.5	97.4	98.9	98.1
% RSD	0.2	1.3	1.2	0.2	0.3	0.2

S. No	% Impurity
S. No	Imp-A	Imp-B	Imp-C	Imp-D	N-oxide
1	0.5321	0.5557	0.5180	0.5204	0.4930
2	0.5322	0.5569	0.5176	0.5194	0.4929
3	0.5338	0.5581	0.5197	0.5215	0.4934
4	0.5324	0.5571	0.5177	0.5190	0.4949
5	0.5320	0.5587	0.5169	0.5182	0.4939
6	0.5400	0.5648	0.5254	0.5265	0.5021
Average	0.5338	0.5586	0.5192	0.5208	0.4950
% RSD	0.6	0.6	0.6	0.6	0.7

TABLE-5 ACCURACY IN THE ASSAY DETERMINATION OF ARIPIPRAZOLE

Spike level	Average ‘mg’ of Aripiprazole added*	Average ‘mg’ of Aripiprazole Recovered*	Average %recovery*
25%	37.58	36.95	98.3
50%	74.97	74.93	99.9
75%	112.57	111.51	99.1
100%	149.87	147.74	98.6
150%	224.66	222.75	99.1

^a Number of samples analyzed at each spike level are three

TABLE-6 ACCURACY IN THE DETERMINATION OF ARIPIPRAZOLE RELATED IMPURITIES

Spike level	IMP A			IMP B			IMP C
Spike level	µg/ml added	µg/ml found	Avg % Recovery	µg/ml added	µg/ml found	Avg % Recovery	µg/ml added	µg/ml found	Avg % Recovery
50 %	1.2500	1.3333	106.7	1.3050	1.4422	110.5	1.2550	1.2688	101.1
75 %	1.8750	2.0003	106.7	1.9575	2.1470	109.7	1.8825	1.9548	103.8
100 %	2.500	2.6325	105.3	2.6100	2.7977	107.2	2.5100	2.3892	95.2
125%	3.1250	3.3272	106.5	3.2625	3.5520	108.9	3.1375	3.1815	101.4

Spike level	IMP D			N-OXIDE
Spike level	µg/ml added	µg/ml found	Avg % Recovery	µg/ml added	µg/ml found	Avg % Recovery
50 %	1.2007	1.1178	93.1	1.3457	1.5108	112.3
75 %	1.8010	1.7755	98.6	2.0186	2.1483	106.4
100 %	2.4013	2.3390	97.4	2.6914	2.5813	95.9
125%	3.0017	3.0593	101.9	3.3643	3.4082	101.3

^a Number of samples analyzed at each spike level are three

TABLE-7 STABILITY DATA OF ARIPIPRAZOLE 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	99.5*	NA		99.1		98.5	NA	NA
1	100.6	1.1		99.8		99.7	0.7	1.2
2	100.8	1.3		100.0		100.0	0.9	1.5
REFRIGERATOR 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	99.5*		NA	99.1	98.5		NA	NA
1	100.6		1.1	100.2	99.7		1.1	1.2
2	100.1		0.6	99.7	99.5		0.6	1.0

* Potency of Aripiprazole on as is basis

3.2.3) Linearity of Detector Response:

a) Active Ingredient:

Linearity of detector response for Aripiprazole was established by plotting a graph to concentration versus average area and determining the correlation coefficient. A series of solutions of Aripiprazole standard were prepared in the concentration range of about 30.507µg/mL to 183.040µg/mL A graph was plotted to concentration in µg/mL on X- axis versus response on Y-axis. The detector response was found to be linear with a correlation coefficient of 0.999. Linearity graph is shown in Fig-3.

b) Related impurities:

Linearity of detector response of all known Arpiprazole Related impurities is established by plotting a graph to concentration versus area of Aripiprazole related impurities and determining the correlation coefficient. A series of solutions of Aripiprazole related impurities in the concentration ranging from Limit of Quantitation level to about 150% of

target concentration level of Aripiprazole known impurities were prepared and injected into the RRLC system.

The detector response was found to be linear from Limit of quantitation to 150% of target concentration level of Aripiprazole known Impurities. Linearity graph is Shown in Fig-4

3.2.4) Precision of test Method:

a) Active Ingredient:

The precision of test method for Active Substance was conducted by assaying six samples of Aripiprazole tablets. The Average % assay of Aripiprazole in Aripiprazole tablets was found to be 98.8%, 100.2%, 99.5%, 97.4%, 98.9% and 98.1% respectively for 2mg, 5mg, 10mg, 15mg, 20mg and 30mg tablets and RSD was found to be 0.2%, 1.3%, 1.2%, 0.2%,0.3% and 0.2% respectively. The results were given in Table-4.

b) Related impurities:

The precision of test method of all known impurities of Arpiprazole was evaluated by spiking all known impurities at target concentration level on Tablets. The Relative standard deviations of all known impurities were calculated and found to be less than 1.0%. The results were given in Table-5 .

3.2.5) Accuracy:

a) Active Ingredient:

A study of recovery of Aripiprazole from spiked placebo was conducted at five different Spike levels i.e. 25%, 50%, 75%, 100%, and 150% .Samples were prepared by mixing placebo with Aripiprazole raw material equivalent to about of the target initial Concentration of Aripiprazole. Sample solutions were prepared in triplicate for each spike level and assayed as per proposed method. The Slope, intercept, % recovery and Correlation Coefficient were calculated and given in Table-6.The mean Recoveries of Aripiprazole from spiked were found to be in the range of 98.3-99.9%.

b) Related impurities:

A study of recovery of Aripiprazole related impurities in spiked samples of Aripiprazole test preparation was conducted. Samples were prepared in triplicate by spiking of all known impurities in test preparation at 50%, 75%, 100% and 125% of the target concentration level of Aripiprazole known Impurities. The average %recovery for aripiprazole Related Impurities was Calculated and given in Table-6. Aripiprazole related impurities from spiked were found to be in the range of 93.1-112.3%

3.2.6) Ruggedness:

A study to establish the stability of Aripiprazole in standard was conducted on bench top and Refrigerator at Initial, 1 day and 2 days. The assay of Aripiprazole in standard solution was estimated against freshly prepared standard each time. The difference in % assay of Standard from initial to 1 day and 2 days was calculated and given in Table-6. From the above study, it was established that the standard preparation was stable for a period of 2days on bench top and Refrigerator.

A study to establish the stability of Aripiprazole in Test Solution was conducted on bench top and Refrigerator at Initial, 1 day and 2 days. The assay, of Aripiprazole in test solutions was estimated against freshly prepared standard each time. The difference in % assay of test from initial to 1 day and 2 days was calculated and given in Table-7. From the above study, it was established that the Test Solution was stable for a period of 2days on bench top and Refrigerator.

3.2.7) Robustness:

A study to establish the effect of variation in mobile phase composition, Flow, Temperature and pH of buffer in mobile phase was conducted. Standard and test solutions spiked with known related impurities of Arpiprazole prepared as per proposed method were injected into RRLC system. The System suitability parameters, % Assay and RRT of all individual known impurities were evaluated. 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 Aripiprazole .

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Received on 28.12.2009 Modified on 20.02.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 380-385

TABLE-4 RESULTS FOR PRECISION OF TEST METHOD

% Assay

Average

99.5*

99.5*

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