Visible Spectrophotometric Estimation of Aripiprazole

Somyadeep Majhi¹, Saroj Kumar Patro¹, P. Sudhir Kumar²

¹Department of Pharmaceutical Analysis and Quality Assurance, Institute of Pharmacy and Technology, Salipur, Cuttack, Orissa - 754202

²School of Phrmceutical Sciences, Siksha “O“ Anusndhan University, Bhubaneswar

*Corresponding Author E-mail: skpatro69@gmail.com

ABSTRACT:

Three new, simple, sensitive and economical methods (Method I, II and III) were developed for the determination of aripiprazole in pure and pharmaceutical formulation. Method-1 is based on the formation of condensed product obtained when Aripiprazole in acidic conditions reacted with 2, 4-dinitrophenylhydrazine in methanol to form yellowish orange chromogen (hydrazone formation) exhibiting λmax at 480nm. Method-II is based on the formation of Yellow colored chromogen (formation of chalcone) by reacting with vanillin in the presence of sulphuric acid medium exhibiting λmax at 560nm. Method-III is based on the formation of dark yellow colored chromogen (cyclic chalcone) by reacting with Cinamaldehyde in the presence of nitric acid exhibiting λmax at 580nm. These methods obeyed Beer’s law in the concentration range of 2.5-40µg/ml, 2-9 µg/ml and 2.5-15 µg/ml respectively. The results of analysis for the three methods were validated statistically and by recovery studies. The colored chromogens formed are stable for more than 8 hours in all the three methods. The results obtained with the proposed methods are in good agreement with labeled amounts when marketed pharmaceutical preparations are analyzed.

KEYWORDS: Visible Spectrophotometry, Aripiprazole, Tablet analysis.

INTRODUCTION:

Aripiprazole, (7-[4-[4-(2, 3-dichlorophenyl)-1 -piperazinyl] butoxy]-3, 4-dihydrocarbostyril, is a psychotropic agent belonging to the chemical class of benzisoxazole derivatives and is indicated for the treatment of schizophrenia^1,2. In the references, gas chromatography-mass spectrometry³, LC–MS/MS⁴, capillary electrophoresis⁶, methods are reported for the analysis of aripiprazole in biological fluids. HPLC is the technique that most commonly used for the determination of aripiprazole in plasma^5-9, UPLC-MS in in-vitro samples ¹⁰, RP-HPLC method in bulk drug and solid dosage forms by internal standard method^11,16, UV spectrophotometric method^17,24, capillary electrophoresis¹⁸, HPTLC method²⁰ also reported.

In this paper we describe a simple, accurate, sensitive and validated visible spectrophotometric method for analysis of aripiprazole in tablet formulation. This method has been successfully used for quality-control analysis of drugs and for other analytical purposes.

EXPERIMENTAL:

A reference standard of aripiprazole was obtained from Orchid Healthcare (Chennai, India). A pharmaceutical product containing the same drug (30 mg per tablet), obtained from the local pharmacy and was used in the experiments. Double distilled water, 2, 4-dinitrophenyl Hydrazine (An accurately weighed quantity of 1.2375 gm of 2, 4-dinitrophenyl Hydrazine was transferred to 50 ml volumetric flask, which was then dissolved and made up to volume with methanol in order to get 0.125 M) and 2 ml of Hydrochloric Acid (50%, v/v), 2ml Vanillin solution (15.2 gm of Vanillin was transferred to 50 ml volumetric flask, which was then dissolved and made up to volume with methanol to get 2 M) and 2 ml of Sulphuric Acid (50%, v/v), 2ml Cinamaldehyde (50%, v/v), and 2 ml of Nitric Acid (50%, v/v) were used. All the reagents were analytical grade and were purchased from SD Fine Chemicals (Bombay, India) Qualigens etc. Whatman no. 41 filter papers (obtained commercially) were used for preparation of sample solutions.

Instrument used: A Shimadzu UV-Visible double beam Spectrophotometer (Pharmaspec 1700) with 1 cm matched quartz cells was used for all spectral measurements, Single pan electronic balance i.e. Contech.

Standard Stock Solution – An accurately weighed ten (10mg) of Aripiprazole was transferred into a 100ml volumetric flask containing 1ml of Dimethyl Formamide (DMF) and the volume was made up to the mark with methanol in order to get 100µg/ml.

Method: 1

Working Standard stock solution: 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 ml of standard stock solution was further diluted up to 10ml with methanol in a 10ml volumetric flasks in order to get 2.5, 5, 10, 15, 20, 25, 30, 35 and 40µg/ml concentration of Aripiprazole respectively.

Construction of Calibration curve of Aripiprazole:

2ml of Aripiprazole working standard stock solution ranging from 2.5 - 40 μg/ml were transferred into a series of 10 ml volumetric flasks. To each 2ml of 2, 4-dinitrophenyl Hydrazine (0.125 M) and 2 ml of Hydrochloric Acid (50%) were added. Then it was further diluted up to the mark with the methanol. It was kept aside for 20 minutes for completion of reaction at the room temperature. The absorbance of the Yellowish Orange chromogen was measured at 480nm against reagent blank. Calibration curve was prepared by plotting concentration versus absorbance and found to be linear in the concentration range of 2.5 - 40 μg/ml. The linear regression equation was found to be Y = 0.0253 x +0.0266, r 2 = 0.9973. The calibration curve is shown in the Fig 1. The linearity data of Aripiprazole is given in the Table 1.

Table: 1 Linearity data of Aripiprazole

Concentration (µg/ml)	Absorbance
0	0
2.5	0.094
5	0.151
10	0.277
15	0.412
20	0.551
25	0.691
30	0.792
35	0.897
40	1.012

Fig: 1 Calibration Curve of Aripiprazole

Method: II

Working Standard Stock Solution

0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9mL of standard stock solution was transferred into eight different 10ml volumetric flasks and diluted up to the mark with methanol in order to get concentration of drug as per given in the Table 2.

Construction of calibration curve:

2ml of Aripiprazole working standard stock solution ranging from 2-9μg/ml were transferred into a series of 10ml volumetric flasks. To each 2ml Vanillin and 2ml of sulphuric acid (50%) or 1N H₂SO₄ were added. Then it was further diluted up to the mark with the methanol. It was kept aside for 20 minutes for completion of reaction at the room temperature. The absorbance of the Yellow chromogen was measured at 560nm against reagent blank. Calibration curve was prepared by plotting concentration versus absorbance and found to be linear in the concentration range of 2-9μg/ml. The linear regression equation was found to be Y = 0.1380x – 0.1341, r² = 0.9972. The linearity data of Aripiprazole is given in the Table 2. The calibration curve of Aripiprazole is shown in the Fig 2.

Table 2: Linearity data of Aripiprazole

Concentration (µg/ml)	Absorbance
0	0
2	0.135
3	0.279
4	0.429
5	0.549
6	0.678
7	0.868
8	0.976
9	1.087

Figure 2: Calibration curve of Aripiprazole

Method: III

Working Standard Stock Solution : 2.5, 5, 7.5, 10, 12.5, 15 ml of standard Stock Solution was further diluted up to 100ml with methanol in a 100ml volumetric flask in order to get 2.5, 5, 7.5, 10, 12.5, 15 µg/ml concentration respectively.

Construction of Calibration curve:

2ml of 2.5, 5, 7.5, 10, 12.5, 15 µg/ml of working standard stock solution were transferred into six different 10ml volumetric flasks. To each 2ml Cinamaldehyde (50%) and 2ml of Nitric Acid (50%) were added. Then it was further diluted up to the mark with the methanol. It was kept aside for 25 minutes for completion of reaction at the room temperature. The absorbance of the dark yellow chromogen was measured at 580nm against reagent blank. Calibration curve was prepared by plotting concentration versus absorbance and found to be linear in the concentration range of 2.5-15μg/ml. The linear regression equation was found to be Y = 0.0757 x – 0.0064, r² = 0.9971. The linearity data is given in the Table 3. The calibration curve of Aripiprazole is shown in the Fig 3.

Table 3: Linearity data of Aripiprazole

Concentration (µg/ml)	Absorbance
0	0
2.5	0.16
5	0.38
7.5	0.55
10	0.78
12.5	0.96
15	1.1

Figure 3: Calibration curve of Aripiprazole

Fig 4: Proposed reaction mechanism of Aripiprazole

i.-Hydrated hydrazine (hydrazones); ii.-Vanillin (chalcone); iii.-Cinnamaldehyde (cyclic chalcone)

Analysis of Commercial formulation (Tablet): Ten tablets of three different brands were weighed and ground to a fine powder separately. An accurately weighed powder sample equivalent to 5, 10 and 20 mg of Aripiprazole as per label claim were transferred to a three different100 ml volumetric flasks. Then the powder was dissolved in 1 ml of dimethyl formamide and the volumes were made up to 100ml with methanol. The solutions were then filtered through Whatmann Filter paper no 41. 0.5 ml of this filtrate (three different strength) were transferred into four different 10 ml volumetric flasks and then follow all the three proposed methods. Amount of Aripiprazole was determined by employing all the three Linear regression equations. The result of tablet analysis is given in the Table 4.

Validation: As per ICH guide lines Q2 B ^13-15

Linearity:

Precision: It was performed to find out intra-day (within a day) variations in the estimation of Aripiprazole of different concentrations with the proposed method. Standard deviation (SD) was found to be less than 1% for within a day variations, which proves that method is precise. The precision data of Aripiprazole is given in the Table 5.

Accuracy: It was found out by recovery study using standard addition method. Known amounts of standard Aripiprazole was added to pre-analyzed samples at a level 80%, 100% and 120% and then subjected to the proposed method. The result of recovery studies is given in the Table 6.

^\

Table 4: Analysis of Pharmaceutical dosage form T1 (Arip MT 10), T2 (Aria), T3 (Arpizol)

Method-1
Brand	Formulation (µg/ml)	Label claim	Found conc.	C. I.	SD	%RSD	SE
		(mg/tablet)
1	5	5	5	100.0±1.743	1.095	1.095	0.547
2	5	10	9.96	99.6±2.162	1.358	1.364	0.679
3	5	20	20.137	100.687±1.66	1.049	1.041	0.524
Method-II
1	5	5	5.01	100.35±2.387	1.5	1.494	0.75
2	5	10	9.99	99.975±3.151	1.98	1.980	0.99
3	5	20	20.155	100.775±1.700	1.068	1.060	0.534
Method-III
1	5	5	5.027	100.55±2.401	1.508	1.500	0.754
2	5	10	10.05	100.50±2.09	1.314	1.307	0.657
3	5	20	20.14	100.712±1.661	1.044	1.036	0.522

Table 5: Precision data of Aripiprazole

Method	Concentration (µg/ml)	Absorbance.	SD
I	10	0.3145	0.033
	15	0.4745	0.053
	20	0.5685	0.047
II	5	0.573	0.019
	6	0.6835	0.11
	7	0.8725	0.024
III	5	0.4025	0.074
	7.5	0.585	0.069
	10	0.845	0.072

Table 6: Recovery data of Aripiprazole

Method	% Level of recovery	Formulation (µg/ml)	Amount Of Pure drug added (µg/ml)	Amount of pure Drug found (µg/ml)	C.I.	% RSD	SE	t
I	80	5	4	3.9575	99.44±1.65	1.046	0.5205	1.066
	100		5	5.02	100.4±1.855	1.161	0.583	0.686
	120		6	6.005	100.083±1.234	0.776	0.387	0.214
II	80	5	4	3.9575	99.87±1.75	1.101	0.55	0.236
	100		5	5.0075	100.150±1.455	0.911	0.457	0.328
	120		6	6.005	100.083±1.234	0.776	0.387	0.214
III	80	5	4	4.0075	100.187±2.438	1.529	0.766	0.244
	100		5	5.02	100.4±1.44	0.905	0.454	0.879
	120		6	5.9975	99.958±2.10	1.322	0.661	0.063

SD: Standard deviation, % SE: Percent standard error, C.I.: Confidence Interval within which true value may be found at 95% confidence level = R ± ts/√n, R: Mean percent result of analysis of Recovery study (n = 4). Theoretical ‘t’ values at 95% confidence level for n - 1 degrees of freedom t (0.05, 3) = 3.182

Sensitivity: The sensitivity of all the three methods was determined with respect to LOD and LOQ. The LOD and LOQ were separately determined based on the standard calibration curve. LOD = (3.3 x S.D /S and LOQ = 10 x S.D/S, where, S.D is the standard deviation of the y- intercepts of regression line and S is the average slope of the calibration curve. The LOD and LOQ values of all the three methods are given in the Table 7.

Table 7: Comparison of recovery study of proposed methods and Reported method:

% Level of recovery	Method	Label claim (mg/tab)	Proposed method (% Recovery ± RSD)	Reported method^22 (% Recovery ± RSD)
80	I	10	1.046	0.86
	II		1.161
	III		0.776
100	I	10	1.101	0.95
	II		0.911
	III		0.776
120	I	10	1.529	0.97
	II		0.905
	III		1.322

Table 8: Optical characteristics and Precision (Aripiprazole)

Optical Characteristics and Precision	Method - I	Method-II	Method-III
λ max (nm)	480	560	580
Beer's law limits (µg/ml)	2.5 - 40	2-9	2.5 - 15
Molar extinction coefficient ( lit. mole^-1. cm^-1)	12728.4165	48051.61445	249441.5576
Sandell's sensitivity (mcg/cm^2/0.001 abs.unit)	0.0357	0.0097	0.0136
Quantitation equation, ( Y = mx + C)*
Slope	0.0253	0.1380	0.0757
Intercept	0.0266	– 0.1341	– 0.0064
Correlation coefficient (r2)	0.9973	0.9972	0.9971
LOD	0.148	0.158	0.149
LOQ	0.571	0.461	0.451

RESULTS AND DISCUSSION:

In method-1, the absorption spectra of the Yellowish Orange chromogen colored product with λmax = 480 nm are shown in measurement. The reagent blank has practically negligible absorption at this wavelength. In method-II, the absorption spectra of the Yellowish chromogen colored product with λmax = 560 nm are shown in measurement. The reagent blank has practically negligible absorption at this wavelength. In method-III, the absorption spectra of the dark yellow chromogen colored product with λmax = 580 nm are shown in measurement. The reagent blank has practically negligible absorption at this wavelength. It was found that 20 to 25 minutes were required to form stable chromogens in all the three methods. The chromogen was stable for 8 hours in method I and for 10 hours in method II and III. The proposed reaction mechanisms for all the three methods are given in the Fig 4. All the three colorimetric methods were validated as per ICH guide lines Q 2B. The reproducibility and accuracy of methods were found to be less than % 1.5 SD. Recovery studies found satisfactory in the range of 98% to 102%. The recovery obtained in each instance was compared with theoretical value of 100 percent by means of student’s ‘t’ test at a 95% confidence level, which revealed, that there was no interference from common adjuvant used in the formulation, indicating accuracy and reliability of the methods. The recovery results of the proposed methods were well agreed with the reported RP-HPLC method for Aripiprazole tablets²². So the proposed method has been found to be new, accurate, simple, sensitive, precise, and convenient and is suitable for routine analysis in laboratory. It can be used in the determination of Aripiprazole in bulk drug and its pharmaceutical preparations in a routine manner.

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Received on 05.12.2015 Modified on 25.12.2015

Accepted on 28.12.2015 © AJRC All right reserved

Asian J. Research Chem. 8(12): December 2015; Page 739-744

DOI: 10.5958/0974-4150.2015.00119.4