Development and Validation of Analytical Method for Aripiprazole and
Escitalopram Oxalate by Simultaneous Equation Spectroscopic
Method
Divya Solanki1,2*, Hasumati
Raj2, Neelam Prajapati2
1Research Scholar 2014, Gujarat Technological
University, Gujarat
2Quality Assurance Department, Shree Dhanvantary Pharmacy College, Kim, Surat
*Corresponding Author E-mail: divyasolanki844@gmail.com
ABSTRACT:
A simple, accurate and precise spectroscopic method
was developed for simultaneous estimation of Aripiprazole
and Escitalopram Oxalate in synthetic mixture using
simultaneous equation Method. In this spectroscopic method, 255.00 nm and
238.00 nm wavelengths were selected for measurement of absorptivity.
Both the drugs show linearity in a concentration range of 5-30 μg/ml and 15-75 μg/ml
at their respective λmax. Accuracy, precision
and recovery studies were done by QC samples covering lower, medium and high
concentrations of the linearity range. The relative standard deviation for
accuracy, precision studies were found to be within the acceptance range
(<2%). The limit of determination was 0.129 and 0.223μg/ml Aripiprazole and Escitalopram
Oxalate, respectively. The limit of quantification was 0.392 and 0.677 for Aripiprazole and Escitalopram
Oxalate, respectively. Recovery of Aripiprazole and Escitalopram Oxalate in were found to be 100.66 % and
100.70 % respectively confirming the accuracy of the proposed method. The proposed
method is recommended for routine
analysis since they are rapid, simple, accurate and also sensitive and specific
by no heating and no organic solvent extraction.
KEYWORDS:
Aripiprazole, Escitalopram
Oxalate, simultaneous estimation, Simultaneous equation method, analysis method.
1. INTRODUCTION:
Aripiprazole is a psychotropic 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's antipsychotic activity is likely due to a
combination of antagonism at D2 receptors in the mesolimbic
pathway and 5HT2A receptors in the frontal cortex.
Antagonism
at D2 receptors relieves positive symptoms while antagonism at 5HT2A receptors
relieves negative symptoms of schizophrenia. IUPAN name of Aripiprazole is7-[4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]butoxy]-3,4-dihydro-2(1H)-quinolinone(1).
Figure:1 Structure of aripiprazole (2)
Aripiprazole is white, crystalline
solid powder. Solubility is given in practically
soluble in chloroform and ethanol
(3-4).
Escitalopram Oxalate is selective serotonin reuptake
inhibitors (SSRIs) are a group of chemically diverse antidepressant drugs that
specifically inhibit serotonin reuptake. The SSRIs block the reuptake of
serotonin, leading to increased concentrations of the neurotransmitter in the
synaptic cleft and, ultimately, to greater postsynaptic neuronal activity. The combination of both have decrease dose
and improve Depression. IUPAN name of Escitalopram
Oxalate 1-[3-(Dimethylamino)
propyl]-1-(4fluorophenyl)-1,3-dihydro-5-isobenzofuran-carbonitrile oxalate(5).
Figure 2: Structure of
Escitalopram Oxalate
(6)
Escitalopram Oxalate is white, crystalline solid powder. Solubility is given in practically
soluble in methanol(7-8).
The review of literature
regarding quantitative analysis of Aripiprazole and Escitalopram Oxalate revealed that no Simultaneous Equation
method attempt was made to develop analytical methods for Aripiprazole
and Escitalopram Oxalate. Some spectrometric methods and
chromatographic methods have been reported for the estimation of the individual
and combination of drugs(9-14).The focus of the present study was to
develop and validate a rapid, stable, specific, and economic Spectroscopic
method for the estimation of Aripiprazole and Escitalopram Oxalate in Synthetic Mixture.
1.1. Theory:
We can find out
concentration of both the drug from combination mixture using the simultaneous
equation method. In this method using the absorbance of both the drug and
mixture at their wavelength and put this value in following equation and we can
find out the concentration of drugs present in combination.
(A2 × Ay1) – (A1
× Ay2)
Cx =
--------------------------------- ------ (1)
(Ay1
× Ax2) – (Ay2 × Ax1)
(A1 × Ax2) – (A2 × Ax1)
Cy
= --------------------------------- ------ (2)
(Ax2 × Ay1) – (Ax1 × Ay2)
Where,
Cx= Concentration
of drug ESC
Cy = Concentration
of drug ARI
A1
= Absorbanceof mixture at wavelength 238 nm
A2 = Absorbance
of mixture at wavelength 255nm
Ax1 = Absorptivity of drug A at wavelength 238 nm
Ax2 = Absorptivity of drug A at wavelength 255nm
Ay1 = Absorptivity of drug B at wavelength 238
nm
Ay2 = Absorptivity of drug B at wavelength
255nm
2. MATERIALS AND
METHODOLOGY:
2.1.
Apparatus
A double beam UV/Visible spectrophotometer
(Shimadzu
model 2450 , Japan) with spectral
width of 2nm, 1 cm quartz cells
was used to measure absorbance
of all the solutions. Spectra
were automatically
obtained by UV-Probe system software.
2.2.
Reference samples
Aripiprazole and Escitalopram
Oxalate received as gift sample from Cadila Healthcare Limited,
GIDC Estate ankleshwar.
2.3. Materials and reagents
Methanol AR grade (RANKEM)
2.4. Preparation
of Standard Solution and Synthetic Mixture
2.4.1 Preparation
of stock Solution
of Aripiprazole:
·
An accurately weighed quantity
equivalent to 10mg of Aripiprazole was transferred to
100 ml volumetric flask made up to the mark with the methanol to obtain standard solution having
concentration of ARI
(100μg/ml).
·
2.4.2 Preparation
of standard stock
solution of Escitalopram Oxalate:
·
An
accurately weighed quantity of ESC
(10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and
diluted to the mark with methanol to obtain standard solution having
concentration of ESC (100μg/ml).
·
2.4.3. Preparation
of Standard Mixture Solution (ARI+ ESC):
· 1 ml of working standard stock solution of
ARI (100μg/ml) and 3ml of standard Stock solution of ESC (100μg/ml)
were pipetted out into 10ml volumetric flask and
volume was adjusted to the mark with methanol to get 10μg/ml of ARI and
30μg/ml of ESC.
·
2.4.4 Preparation of Test Solution
Weigh Synthetic mixture equivalent to 10 mg of Aripiprazole taken in 100ml Volumetric Flask, dissolved in
25 ml of Methanol, Sonicated for 15min, diluted with 100 ml Methanol. Take 0.5, 1.0,
1.5, 2.0, and 2.5 ml and diluted with
10ml Methanol to make concentration 5, 10, 15, 20 and 25µg/ml of ARI and 15, 30, 45, 60 and 75µg/ml of ESC.
Figure 3 Overlainzero
order
spectra
of ARI and ESC (1:3) ratio,
respectively
2.5 Procedure
Calibration curves for
Escitalopram Oxalate:
This
series consisted of five concentrations of standard ARI solution ranging from
5-25μg/ml. The solutions were prepared by pipette out Standard ARI stock
solution (0.5ml, 1.0ml, 1.5ml, 2.0ml
and 2.5ml) was transferred into a series of 10 ml volumetric flask and
volume was adjusted up to mark with Methanol.
A zero order spectrum measured the absorbance at 255.00nm against a reagent blank solution (methanol).
Calibration
curve for
Aripiprazole:
This
series consisted of five concentrations of standard ESC solution ranging from
15-75μg/ml. The solutions were prepared by pipette out Standard ESC stock
solution (1.5ml, 3.0ml, 4.5ml, 6.0ml
and 7.5 ml) was transferred into a series of 10 ml volumetric flask and
volume was adjusted up to mark with Methanol. A zero order spectrum, measured
the absorbance at 238.00nm
against a reagent blank solution (methanol).
3.
RESULTS AND DISCUSSION:
Validation Parameters
3.1 Linearity
Six point calibration curves were obtained in the
concentration range of 5-25μg/ml for Aripiprazole
and 15-75μg/ml for Escitalopram Oxalate. The
response of drug was found to be linear in investigation range and the
regression equations was found to be y = 0.033x - 0.035 for ARI (n=6), y =
0.038x + 0.095 for ESC (n=6), with the correlation coefficient 0.999 and
0.999 (n=6) respectively, is listed in Table 1.
Table.1
Calibration data for ari and esc at 255.00nm and
238.00nm, respectively. *(n=6)
|
Sr.No |
Escitalopram Oxalate μg/ml |
Absorbance ± SD (n=6) (238nm) |
%RSD |
Aripiprazole μg/ml |
Absorbance±S.D. (n=6) (255
nm) |
%RSD |
|
1 |
15 |
0.639±0.001 |
0.307 |
5 |
0.14±0.0009 |
0.665 |
|
2 |
30 |
1.270±0.001 |
0.160 |
10 |
0.292±0.002 |
0.762 |
|
3 |
45 |
1.807±0.010 |
0.571 |
15 |
0.46±0.001 |
0.253 |
|
4 |
60 |
2.407±0.016 |
0.665 |
20 |
0.627±0.002 |
0.334 |
|
5 |
75 |
2.933±0.023 |
0.793 |
25 |
0.807±0.005 |
0.723 |
FIG. 5 Calibration curve for ESC
at 238.00nm and Aripiprazole AT 255nm
3.2 Precision:
I. Intraday
precision:
The precision of the developed method was assessed by
analyzing combined standard solution containing three different concentrations
30, 45, 60μg/ml for ESC and 10, 15, 20μg/ml ARI. Three replicate
(n=3) each on same day. Intraday precision data presented in Table (A). These% RSD value
was found to be less than 1.0 indicated that the method is precise.
Table (A) Intraday precision data
for
estimation of ESC and ARI (n=3)
|
Conc. (μg/ml) |
Escitalopram
Oxalate + Aripiprazole |
|||
|
ESC
+ ARI |
Absorbance (238nm) |
%RSD |
Absorbance (255nm) |
%RSD |
|
30:10 |
1.58 |
0.630 |
0.466 |
0.643 |
|
45:15 |
2.30 |
0.860 |
0.793 |
0.126 |
|
60:20 |
2.91 |
0.343 |
1.04 |
0.960 |
II. Interday precision
The precision of the developed method was assessed by
analyzing combined standard solution
containing three different concentrations 30, 45, 60μg/ml for ESC
and 10, 15, 20μg/ml ARI triplicate (n=3) per day for consecutive 3 days
for inter-day precision. Interday precision data presented in Table B. These %RSD value
was found to be less than 1.0 indicated that the method is precise.
3.3 Accuracy:
Composition of synthetic mixture
The preparation of synthetic mixture was as per
patent:
·
Aripiprazole: 50 mg
·
Escitalopram Oxalate: 150 mg
·
Starch: 1310 mg
·
Magnesium Stearate: 40 mg
·
Lactose : 950 q.s
· From the Synthetic Mixture weigh accurately
equivalent about 10mg of ARI. Take Four 100ml Volumetric Flask and in each
flask add synthetic mixture equivalent to 10mg of ARI. Flask 1 form as a
Placebo and remaining flask 2, 3,4 spike with 80, 100, 120%. Same way spike ESC
in respectively flask. dissolved in 25 ml Methanol and Sonicated
for 15min. make up the volume with methanol . The solution was filtered through
Whatman filter paper No. 42.
· Finally the solution had concentration
100μg/ml for ARI and 300μg/ml for ESC. From that pipette out 0.5 ml
in 10 ml volumetric flask and volume was made up to mark with Methanol to make
final concentration ARI (5 µg/ml) and ESC (15 µg/ml).
Data from nine determinations over three concentration
levels covering the specified range was determined and % recovery was
calculated.
Table (B) Interday precision data
for
estimation of ESC and ARI (n=3)
|
Conc. (μg/ml) |
Escitalopram
Oxalate + Aripiprazole |
||||
|
ESC |
ARI |
Absorbance (238nm) |
% RSD |
Absorbance (255 nm) |
% RSD |
|
30:10 |
1.57 |
0.732 |
0.471 |
0.760 |
|
|
45:15 |
2.28 |
0.910 |
0.793 |
0.262 |
|
|
60:20 |
2.91 |
0.524 |
1.043 |
0.961 |
|
|
Amount of Formulation (mg) |
Amount of API Spiking (mg) |
Total Amount (mg) |
|||
|
ESC |
ARI |
ESC |
ARI |
ESC |
ARI |
|
30 |
10 |
- |
- |
30 |
10 |
|
30 |
10 |
24 |
8 |
54 |
18 |
|
30 |
10 |
30 |
10 |
60 |
20 |
|
30 |
10 |
36 |
12 |
66 |
22 |
Recovery data
of ESC and ARI *(n=3)
|
%
Recovery |
Total
Conc. |
Conc.
found (µg/ml) |
%
Recovery ± SD |
%
RSD |
||||
|
|
ESC |
ARI |
ESC |
ARI |
ESC
|
ARI |
ESC |
ARI |
|
Control |
15 |
5 |
15.07
|
5.07 |
100.29 ±0.321 |
101.40±0.871 |
0.320 |
0.859 |
|
80% |
27 |
9 |
27.11 |
9.07 |
100.96±0.756 |
101.83±0.803 |
0.749 |
0.789 |
|
100% |
30 |
10 |
30.26 |
10.03 |
101.73±0.070 |
100.62±0.970 |
0.068 |
0.964 |
|
120% |
33 |
11 |
32.89 |
10.97 |
99.42±0.393 |
99.55±0.975 |
0.393 |
0.979 |
3.4 Limit of
Detection and Limit of Quantification
The
Limit of detection and quantitation of the developed
method was assessed by analyzing 10 replicates of standard solutions containing
concentrations 15μg/ml for ESC and 5μg/ml for ARI.
The
LOD and LOQ were calculated as LOD =
3.3*σ/S, and LOQ = 10*σ/S, where σ is the standard deviation
of the lowest standard concentration and S is the slope of the standard curve.
LOD and
LOQ value of ESC and ARI (n=3)
|
Drugs |
LOD (µg/ml) |
LOQ (µg/ml) |
|
|
Escitalopram Oxalate + Aripiprazole |
238 nm |
0.223 |
0.677 |
|
255 nm |
0.129 |
0.392 |
|
Robustness of ESC and
ARI (n=3)
|
Parameters |
|
238nm (esc) |
RSD |
ARI (255nm) |
RSD |
|
Different
instrument |
Inst. 1 |
1.580±0.010 |
0.632 |
0.469±0.003 |
0.639 |
|
Inst. 2 |
1.570±0.010 |
0.636 |
0.472±0.002 |
0.423 |
|
|
Different analyst |
Analyst 1 |
1.580±0.010 |
0.632 |
0.465±0.001 |
0.215 |
|
Analyst 2 |
1.570±0.010 |
0.636 |
0.463±0.001 |
0.249 |
3.5
Robustness:
·
Robustness of the method was determined by subjecting the method to
slight change in the method condition, individually, the :
·
Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450).
·
Change Analyst
% RSD was calculated.
3.6 Ruggedness
·
Ruggedness of the method was determined by subjecting the method to
slight change in the method condition, individually, the :
·
Change in Wavelength from 238.00 and 255.00 to 238±2 and 255±02.
·
Change Ratio
·
Change Solvent
· Three
replicates were made for the same concentration
· % RSD was
calculated.
Ruggedness of ESC and
ARI (n=3)
|
Parameter |
|
238nm (ESC) |
%RSD |
255nm(ARI) |
%RSD |
|
Change wavelength |
240 and 257 nm |
1.580±0.010 |
0.632 |
0.463±0.001 |
0.329 |
|
236 and 253 nm |
1.553±0.015 |
0.743 |
0.475±0.001 |
0.210 |
|
|
Change Ratio |
1:3 |
1.580±0.010 |
0.632 |
0.468±0.001 |
0.213 |
|
3:1 |
0.952±0.001 |
0.105 |
0.921±0.001 |
0.108 |
|
|
1:2 |
1.042±0.001 |
0.146 |
0.399±0.001 |
0.382 |
|
|
2:1 |
0.755±0.001 |
0.132 |
0.625±0.001 |
0.244 |
|
|
Solvent change |
2 % Water |
1.465±0.001 |
0.104 |
0.467±0.001 |
0.326 |
|
5 %
Water |
1.407±0.001 |
0.108 |
0.444±0.003 |
0.851 |
4. Application of the
proposed
method for analysis of ari
and esc in synthetic mixture
The preparation of
synthetic mixture as per patent:
·
Aripiprazole: 50 mg
·
Escitalopram Oxalate: 150 mg
·
Starch: 1310 mg
·
Magnesium Stearate: 40 mg
·
Lactose : 950 q.s
From the Synthesis Mixture weigh accurately equivalent about 10mg of ARI in 100ml Volumetric Flask dissolve in 25ml of Methanol. Sonicate for 15 min. Dilute up to the 100 ml with Solvent. Shake vigorously; filter the solution and further Dilution.
Finally the solution had the
concentration 100PPM and 300PPM
respectively for ARI and ESC. After that from this solution 0.5ml was pipette out and diluted up to 10 ml with Methanol. So the concentration
was 15 PPM and 5 PPM for ESC and ARI respectively.
Table 8
Analysis data
of Synthetic Mixture*(n=3)
|
Drugs |
% Assay ± SD |
% RSD(n=3) |
|
|
Escitalopram Oxalate + Aripiprazole |
(238nm) |
100.29 ± 0.321 |
0.320 |
|
(255nm) |
101.40
± 0.87 |
0.859 |
|
Summary Table:
TABLE.9 Summary of Validation Parameters
|
SR. NO. |
PARAMETER |
Escitalopram Oxalate |
Aripiprazole |
|
1 |
Wave length Max. |
238.00nm |
255.00nm |
|
2 |
Linearity (µg/ml) (n=6) |
15-75 µg/ml |
5-25 µg/ml |
|
3 |
Regression
equation |
y = 0.038x + 0.095 |
y = 0.033x - 0.035 |
|
4 |
Correlation
coefficient (r2) |
0.999 |
0.999 |
|
5 |
Accuracy(%Recovery)
(n=3) |
100.70 |
100.66 |
|
6 |
Precision Intra-day
(%RSD)(n=3) Inter-day
(%RSD)(n=3) |
0.343 - 0.860 0.262 – 0.961 |
0.126 – 0.960 0.524 – 0.910 |
|
7 |
LOD (µg/ml)
(n=10) |
0.223 |
0.129 |
|
8 |
LOQ (µg/ml) (n=10) |
0.677 |
0.392 |
|
9 |
Robustness and
Ruggedness (%RSD) |
0.104-0.743 |
0.108 -0.851 |
|
10 |
Assay |
100.29% |
101.40% |
5. CONCLUSION:
A new, Simultaneous Equation
method has been developed for estimation of Aripiprazole
and Escitalopram Oxalate. The method was validated by
employment of ICH (15)guidelines. The validation data is indicative
of good precision and accuracy, and prove the reliability of the method. The
method involves the generation of absorbance spectra followed by measurement of
the absorbance. The proposed method does not require any sophisticated
mathematical treatment for the absorption data, and it exhibits several
advantages over other Spectrophotometric methods for resolution of binary
mixtures. Therefore, the presented methodology is adequate for the routine
quality control analysis of these fixed-dose combinations.
6. CONFLICT OF INTEREST:
The authors confirm that this article content has no
conflict of interest.
7. ACKNOWLEDGEMENT:
We are sincerely thankful to Shree Dhanvantary
Pharmacy College, Kim, Surat, for providing us
Infrastructure facilities and moral support to carry out this research work. We
are also thankful to SDPARC for giving us their special time and guidance for
this research work. We also thank our colleagues for their helping hand.
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Aripiprazole mechanism :information available from drug bank:
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Escitalopram Oxalate Drug info (database available on internet)
http://www.chemicalbook.com/ChemicalProductProperty_EN_CB5712919. html
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Escitalopram Oxalate Drug info (database available on
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http://www.scbt.com/datasheet-208365-s-citalopramoxalate.html
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Escitalopram Oxalate Drug info (database available on internet):
Wikipedia. Available from: https://en.wikipedia.org/wiki/Escitalopram.
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Received on 21.02.2016 Accepted on 14.03.2016
© Asian Pharma
Press All Right Reserved
Asian J. Pharm.
Ana. 6(1): January-
March, 2016; Page 41-46
DOI: 10.5958/2231-5675.2016.00007.7