Q
Absorbance Ratio Method of Levosulpiride and
2-Methoxy 5-Sulfamoylbenzoyl Benzoic Acid Methyl Ester in Their Synthetic
Mixture
Monika A. Rana*, Dr. Hasumati A. Raj
Department of
Quality Assurance, Shree Dhanvantary Pharmacy
College, Kim Surat, Gujarat, India
*Corresponding Author E-mail: monika92rana@gmail.com
ABSTRACT:
A simple, accurate and precise spectroscopic method was developed
for Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl
benzoic acid methyl ester in synthetic mixture using Q absorbance Ratio Method.
In this spectroscopic method, 231.50 nm (as an iso-absorptive
point) and 240.60 nm wavelengths (λmax of any of
the two drugs) were selected for measurement of absorptivity.
Both the drugs show linearity in a concentration range of 10-30 μg/ml at their respective λmax
and at the isoabsorptive point. The relative standard
deviation for accuracy, precision studies were found to be within the acceptance
range (<2%). The limit of determination was 0.084μg/ml and
0.110μg/ml for Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester, respectively. The limit of
quantification was 0.257μg/ml and 0.330 μg/ml for Levosulpiride
and2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester, respectively.
Recovery of Levosulpiride and
2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester were found to be
101.12 % and 101.89 % 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: Levosulpiride, 2-methoxy 5-sulfamoylbenzoyl benzoic acid
methyl ester, Q absorbance ratio method.
1. INTRODUCTION:
Levosulpiride is the levo enantiomer of sulpiride. It is a
substituted benzamide which is meant to be used for
several indications: depression, psychosis, somatoform disorders, emesis and
dyspepsia.[1] It is physically present
as a white crystalline powder. The levoenantiomer
shows better/similar pharmacological actions and lower incidence of toxic
effects than both dextro as well as the racemic forms of the drug. Levosulpride
is an atypical antipsychotic agent that blocks the presynaptic
dopaminergic D2 receptors.1 Like its parent compound,
Levosulpiride shows antagonism at D3 and D2 receptors
present presynaptically as well as postsynaptically in the rat striatum or nucleus accumbens2.
The preferential binding of the presynaptic dopamine
receptors decreases the synthesis and release of dopamine at low doses whereas
it causes postsynaptic D2 receptor antagonism at higher dose.
This receptor profile of the drug along with its limbic
selectivity explains its effectiveness in the management of both positive and
negative symptoms of schizophrenia. The parent drug is given in a dose of
400-1800 mg orally daily although a much lower dose is effective for producing
antidepressant effect (about 50-300 mg).The plasma t1/2 of the drug is about
6-8hours. The drug is chiefly excreted through the renal route.[2] IUPAC name of Levosulpiride
is (S)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide.[3]
Structure of Levosulpiridein
(Figure 1)
Figure
1: Structure of Levosulpiride
Levosulpiride is white or almost white, crystalline
powder.[4] Solubility is given
in soluble in water and freely soluble in Methanol, 0.1 N NaOH
and 0.1 N HCl.
2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester is a white crystalline powder. Its
melting point is 175°C -177°C. [5]Solubility is given in soluble in
water and methanol and freely soluble in 0.1 N NaOH and 0.1 N HCl.
The review of literature regarding quantitative analysis of
Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester revealed that no attempt was made to develop analytical
methods for Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester.Derivative spectrometric methods and chromatographic methods have
been reported for the estimation of the Levosulpiride
drugs. But no Analytical Method have been reported for 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester.The focus of the present study was to develop and validate
a rapid, stable, specific, and economic spectroscopic method for the estimation
of Levosulpirideand 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester in Synthetic mixture.[6]
Structure of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester
in (Figure 2)
Figure
2: Structure of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester
2. MATERIALS AND
METHODOLOGY:
Levosulpiride and its intermidiate
2-Methoxy 5-Sulfamoylbenzoyl Benzoic acid Methyl ester was received as gift
sample from Prayosha Health Care Pvt
Ltd.
A double beam UV/Visible spectrophotometer (Shimadzu model
2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure
absorbance of all the solutions.
Spectra were automatically obtained by UV-Probe system
software.
An analytical balance (Sartorius CD2250, Gottingen,
Germany) was used for weighing the samples.
Sonicator (D120/2H, TRANS-O-SONIC)
Class ‘A’ volumetric glassware
were used (Borosillicte).
2.1. Materials and reagents
2.1.1 Preparation of stock
solution of Levo
Accurately weighed quantity of Levosulpiride
10 mg was transferred to 100 ml volumetric flask, Add 5 ml of Methanol and 20
ml of water, sonicate it for
15min and dilute it up to the mark with Water to give a stock solution having
strength of 100μg/ml.
2.1.2 Preparation of stock
solution of MSB
Accurately weighed quantity of 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester 10mg was transferred to 100 ml volumetric flask, Add 5 ml of
Methanol and 20 ml of water sonicate it for 15min and
dilute it up to the mark with Water to give a stock solution having strength of
100μg/ml.
2.1.3 Preparation of
standard mixture solution
From the stock solution of LEVO take 1 ml and from stock
solution of MSB take 1 ml and transferred in to 10ml volumetric flask and
diluted up to mark with Distilled water to give a solution having strength of
LEVO was 10 μg/ml and MSB was 10μg/ml.
2.1.4 Preparation of test
solution
From the stock solution of LEVO take 1 ml and from stock
solution of MSB take 1 ml and transferred in to 10ml volumetric flask and
diluted up to mark with Distilled water to give a solution having strength of
LEVO was 10 μg/ml and MSB was 10 μg/ml.
2.2. Calibration curves for Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester
Pipette out 1.0, 1.5, 2.0, 2.5 and 3.0 ml of the stock
solution of Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester (100μg/ml) into a series of 10ml volumetric flasks and
the volume was adjusted to mark with Distilled water and measured absorbance at
231.50 nm and 240.60 nm. Plot the graph of absorbance versus respective
concentration of Levosulpiride and 2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester. Linearity range of LEVO and MSB was found with correlation
co-efficient.
3. Q Absorbance ratio method:
Development of Method
Different solutions were prepared in the different solvents
according to the solubility of the drugs. It was found that Distilled water
showing good overlay and distinct λmax of the
both drugs. Therefore, it can be easy to measure the response of the both drugs
in the combined mixture. The λmax of the Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl
benzoic acid methyl ester was
found to be 231.50 nm and 240.60 nm respectively in Distilled water. The
overlain derivative spectra (zero order) of LEVO and MSB at different
concentrations revealed that different concentration of LEVO and MSB possess iso-absorptive point at 231.50 nm. Considering above facts,
wavelength 231.50 nm (λ1) and 240.60 nm (λ2) were selected for the
estimation of both the drugs by absorbance ratio method (Figure 3).
Figure 3:
Overlain zero order spectra of LEVO and MSB in Distilled water (1:1)
Figure 4:
Linearity zero order spectra of LEVO and MSB in Distilled water (1:1)
4. RESULT AND
DISCUSSION
Validation Parameters [7]
4.1. Linearity and Range
Different concentrations of Levosulpiride
(10- 30μg/ml) and 2-methoxy 5-sulfamoylbenzoyl benzoic acid
methyl ester (10-
30μg/ml) were prepared from respective stock solutions. The absorbance was
noted at 231.50 and 240.60nm. It was noted that at the wavelengths 231.50 and
240.60 nm good linearity was observed and hence these wavelengths were fixed
for their Q absorbance ratio method. (figure 4, 5).
Measure the absorbance at 231.50nm (λ1) and 240.60 nm (λ2) for both
drugs. The absorbance was calculated for Levosulpiride
and2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester at the selected wavelengths (Table 1).
Figure 5: Iso absorptive point at 231.50nm in zero order spectra
(1:1)
Table 1: Linearity Results obtained by applying Q Absorbance ratio
methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Conc. (μg/ml) |
LEVO Mean Abs. ± SD (231.50nm) |
LEVO Mean Abs. ±SD (240.60nm) |
MSB Mean Abs. ± SD (231.50nm) |
MSB Mean Abs. ± SD (240.60nm) |
|
10 |
0.577±0.0012 |
0.563±0.0004 |
0.581±0.0007 |
0.640±0.0008 |
|
15 |
0.897±0.0008 |
0.874±0.0005 |
0.895±0.0029 |
0.965±0.0005 |
|
20 |
1.184±0.0010 |
1.152±0.0023 |
1.187±0.0010 |
1.281±0.0007 |
|
25 |
1.397±0.0009 |
1.340±0.0010 |
1.399±0.0005 |
1.637±0.0007 |
|
30 |
1.701±0.0004 |
1.561±0.0018 |
1.710±0.0008 |
1.881±0.0008 |
Correlation coefficient (r2) for calibration curve of LEVO
and MSB at 231.50 nm and 240.60 nm was found to be 0.9961and 0.9973,
respectively.
The regression line equation for LEVO and MSB are as
following,
y = 0.055x - 0.0527for LEV at
231.50nm__(1) (figure 6)
y = 0.0631x - 0.02 for MSB at
240.60nm__ (2) (figure 7)
y = 0.0492x - 0.1139 for LEV at
240.60nm_(3) (figure 8)
y = 0.0552x – 0.0506 for MSB at
231.50nm__(4) (figure 9)
Figure 6:
Calibration graph of LEVOSULPIRIDE at 231.50nm
Figure 7:
Calibration graph ofMSB at 240.60nm
Figure 8:
Calibration graph of LEVOSULPIRIDE at 240.60nm
Figure 9:
Calibration graph of MSB at 231.50nm
Absorbance ratio equation
Cx = {(QM-Qy)/ (Qx-Qy)}*
(A1/ax1)
Cy = {(QM-Qx)/ (Qy-Qx)}* (A1/ay1)
Where, Cx= Concentration of LEVO
Cy= Concentration of MSB
A1 = Absorbance of test at λ1 (iso
absorptive point)
A2 = Absorbance of test at λ2 (λmax
of MSB)
QM = A2/A1
Qx = ax2/ ax1
Qy = ay2 / ay1
ax1 = Absorptivity of x drug at
λ1
ax2 = Absorptivity of x drug at
λ2
ay1 = Absorptivity of y drug at
λ1
ay2 = Absorptivity
of y drug at λ2
4.2. Precision
4.2.1. Intraday precision
Mixed solutions of LEVO and MSB containing 26, 28 and 30 μg/ml and 26, 28 and 30 μg/ml
respectively series were analyzed three times on the same day using developed
spectroscopic method and %RSD was calculated. The % RSD was found to be
0.062-0.120 % for LEVO and 0.038-0.082 % for MSB. These %RSD value was found to
be less than ±2.0 indicated that the method is precise. (Table 2)
4.2.2. Interday precision
Mixed solutions of LEVO and MSB containing 26, 28 and 30 μg/ml and 26, 28 and 30 μg/ml
respectively series were analyzed three times on the different day using
developed spectroscopic method and %RSD was calculated. The % RSD was found to
be 0.084-0.140 % for LEVO and 0.070-0.079 % for MSB. These %RSD value was found
to be less than ±2.0 indicated that the method is precise. (Table 3)
Table 2: Intraday precision results obtained by applying Q
Absorbance ratio methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Conc. (μg/ml) |
Abs.*
(LEVO) Avg. ±
SD(231.50 nm) |
% RSD |
Abs.*
(MSB) Avg.±
SD(240.60nm) |
% RSD |
|
|
LEVO |
MSB |
||||
|
26 |
26 |
2.453 ±0.001 |
0.062 |
2.628 ±0.001 |
0.038 |
|
28 |
28 |
2.642 ±0.002 |
0.078 |
2.833 ±0.001 |
0.061 |
|
30 |
30 |
2.831 ±0.003 |
0.120 |
3.032 ±0.002 |
0.082 |
Table 3: Interday precision results obtained by applying Q
Absorbance ratio methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Conc. (μg/ml) |
Abs.*
(LEVO) Avg. ±
SD(231.50 nm) |
% RSD |
Abs.*
(MSB) Avg.±
SD(240.60nm) |
% RSD |
|
|
LEVO |
MSB |
||||
|
26 |
26 |
2.456 ±0.002 |
0.084 |
2.633 ±0.002 |
0.079 |
|
28 |
28 |
2.645 ±0.002 |
0.095 |
2.836 ±0.002 |
0.070 |
|
30 |
30 |
2.833 ±0.004 |
0.140 |
3.037 ±0.002 |
0.076 |
Table 4: Recovery data results obtained by applying Q Absorbance
ratio methods of Levosulpiride in Distilled water.
|
Conc. of LEVO from
formulation
(µg/ml) |
Amount
of Std. LEVO added
(µg/ml) |
Total amount of LEVO (µg/ml) |
Total amount of LEVO found (µg/ml)* Mean± SD |
% Recovery (n=3) |
% RSD LEVO |
|
10 |
8 |
18 |
18.32±0.001 |
101.80 |
0.098 |
|
10 |
10 |
20 |
20.42±0.002 |
102.10 |
0.195 |
|
10 |
12 |
22 |
21.88±0.0003 |
99.45 |
0.030 |
Table 5: Recovery data results obtained by applying Q Absorbance
ratio methods of 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Conc. of MSB from
formulation
(µg/ml) |
Amount
of Std.MSB added (µg/ml) |
Total amount of MSB (µg/ml) |
Total amount of MSB
found (µg/ml)* Mean± SD |
% Recovery (n=3) |
% RSD MSB |
|
00 |
8 |
8 |
7.95±0.002 |
99.38 |
0.020 0.252 |
|
00 |
10 |
10 |
10.48±0.002 |
104.80 |
0.252 |
|
00 |
12 |
12 |
12.18±0.003 |
101.50 |
0.189 |
4.3. Accuracy
The developed UV spectroscopic method was checked for the
accuracy. It was determined by calculating the recovery of LEVO and MSB from
formulation solution by standard addition method in the combined mixture
solution. The spiking was done at three levels 80 %, 100 % and 120 %. %
recovery for LEVO and MSB by this method was found in the range of 99.45-102.10
% and 99.38-104.80 %, respectively (Table 4 and 5)
The value of %RSD within the limit indicated that the
method is accurate and percentage recovery shows that there is no interference
from the excipients.
4.4. Limit of detection and quantitation
The LOD for LEVO and MSB was conformed to be
0.084μg/ml and 0.110μg/ml, respectively. The LOQ for LEVO and MSB was
conformed to be 0.257 μg/ml and 0.330μg/ml,
respectively. The obtained LOD and LOQ results are presented in Table 6.
Table 6: Limit of detection and limit of quantitation
data results obtained by applying Q Absorbance ratio methods to laboratory prepared mixtures of Levosulpiride and 2-methoxy 5-sulfamoylbenzoyl benzoic acid
methyl ester in Distilled
water.
|
Parameter |
LEVO*(231.50 nm) |
MSB*(240.60 nm) |
|
LOD (µg/ml) |
0.084 |
0.110 |
|
LOQ (µg/ml) |
0.257 |
0.330 |
Table 7: Robustness data results obtained by applying Q Absorbance
ratio methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Condition |
Concentration(µg/ml) |
Different Analyst |
Different
Instrument |
||
|
Analyst 1 Abs. ± RSD |
Analyst 2 Abs. ± RSD |
UV 2450 Abs. ± RSD |
UV 1800 Abs. ± RSD |
||
|
LEV (231.50 nm) |
26 |
2.451±0.081 |
2.454±0.100 |
2.452±0.040 |
2.454±0.062 |
|
28 |
2.640±0.057 |
2.643±0.078 |
2.643±0.075 |
2.645±0.095 |
|
|
30 |
2.831±0.100 |
2.834±0.073 |
2.832±0.081 |
2.835±0.093 |
|
|
MSB (240.60 nm) |
26 |
2.630±0.058 |
2.633±0.075 |
2.628±0.021 |
2.631±0.038 |
|
28 |
2.833±0.073 |
2.834±0.053 |
2.831±0.040 |
2.834±0.070 |
|
|
30 |
3.033±0.068 |
3.035±0.032 |
3.031±0.068 |
3.035±0.019 |
|
Table 8: Assay of formulation results obtained by applying Q
Absorbance ratio methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Drug |
Conc taken(μg/ml) |
Conc found(μg/ml) |
% found |
Limit |
|
LEV |
25 |
25.025 |
100.10% |
98.5 -101 % |
|
MSB |
25 |
24.7 |
98.80% |
- |
Table 9: Assay of formulation results obtained by applying Q
Absorbance ratio methods to available ofLevosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Drug |
Conc taken(μg/ml) |
Conc found(μg/ml) |
% found |
Limit |
|
LEV |
25 |
25.12 |
100.48% |
98.5 -101 % |
Table 10: Summary results obtained by applying Q Absorbance ratio
methods to laboratory prepared mixtures of Levosulpiride
and 2-methoxy 5-sulfamoylbenzoyl benzoic acid methyl ester in Distilled water.
|
Sr no. |
Parameters |
Q absorbance
Ratio Method |
|
|
LEV |
MSB |
||
|
1 |
λmax (nm) |
231.50 nm |
240.60nm |
|
2 |
Linearity range
(µg/mL) |
10 - 30 |
10 - 30 |
|
3 |
Regression
equation |
Y =0.055x +0.0527 |
Y =0.0631x+0.02 |
|
4 |
Correlation
coefficient (r2) |
0.9961 |
0.9973 |
|
5 |
Precision |
||
|
Intraday % RSD (n
= 3) |
0.062 – 0.120 |
0.038 – 0.082 |
|
|
Inter day % RSD
(n = 3) |
0.084 – 0.140 |
0.070 – 0.079 |
|
|
6 |
Accuracy %
Recovery (n = 3) |
101.12 % |
101.89% |
|
7 |
Limit of
detection (µg/mL) (n = 10) |
0.084 |
0.110 |
|
8 |
Limit of
quantification(µg/mL) (n = 10) |
0.257 |
0.330 |
|
9 |
Assay % |
100.48% |
– |
4.5. Robustness and Ruggedness
The obtained Ruggedness and Robustness results are
presented in table 6.24. The % RSD was found to be for 0.040 – 0.1% LEVO and
0.019 - 0.075% for MSB. These %RSD value was found to be less than ± 2.0
indicated that the method is robust and rugged. No significant changes in the
spectrums were observed, proving that the developed method is rugged and robust.(Table 7)
4.6. Application of the proposed method
for analysis of IRB and ATR in formulation
A zero order spectrum of the test solution was recorded and
Measure the absorbance at 231.50nm (λ1) and 240.60nm (λ2) for
estimation of LEVO and MSB. The concentrations of LEVO and MSB in formulation
were determined using the absorbance ratio equation. (Table
8, 9)
5. CONCLUSION:
A new, Q absorbance ratio method has been developed for
estimation of Levosulpiride and2-methoxy
5-sulfamoylbenzoyl benzoic acid methyl ester in synthetic mixture. The method was validated by employment of
ICH (18) guidelines. The validation data is indicative of good precision and
accuracy, and prove the reliability of the method.
6.
REFERANCE:
1.
Depomed,
Inc. Gastric retentive pharmaceutical compositions for immediate and extended
release of levosulpiride. United State patents US
20110052700 A1, 2011.
2.
Gupta
S, Rai Gobind, Halder S and Sharma K, "Levosulpiride:
A Review." Drug Rev.2007,
10, 14-146.
3.
Drug
Profile available from, www.prahoshahealthcare.com
4.
British
Pharmacopeia; The Indian Pharmacopeia Commission,
Ghaziabad, Govt. of India; Ministry of Health and Family Welfare, 1998, VolumeӀ, pp 1252-1253.
5.
www.chemicalbook.com
6.
Monika
A. Rana, Hasumati A. Raj.”A
Review On Analytical Methods For Determination Of Levosulpiride In Pharmaceutical Dosage Forms And Biological
Sample.”Pharma Tutor, 2014.
7.
International
Conference on Harmonization, Harmonized Tripartite Guideline, Validation of
Analytical Procedures Text and Methodology, ICH Q2 (R1), 2005.
Received on 03.03.2015 Accepted on 21.03.2015
© Asian Pharma
Press All Right Reserved
Asian J. Pharm. Ana.
5(2): April-June 2015;
Page 67-73
DOI: 10.5958/2231-5675.2015.00011.3