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AND DROTAVERINE HYDROCHLORIDE IN TABLET FORMULATION.
5.1 OBJECTIVE
To develop a simultaneous assay determination method for
Drotaverine hydrochloride and Mefenamic acid in tablet formulation
5.2 INTRODUCTION
Drotaverine is structurally related to papaverine, an antispasmodic
drug. Drotaverine is a selective phosphodiesterase 4 inhibitor, and has
no antichlolinergic effects. A small study found Drotaverine to be nearly
80% effective in treating renal colic; it has also been used to accelerate
labor [184]. Drotaverine is a benzylisoquinoline derivative, an analogue of
papaverine with smooth muscle relaxant properties. It has antispasmodic
activity due to inhibition of phosphodiesterase IV and is a non
anticolinergic antispasmodic. Mefenamic acid [185-186] is an anthranilic
acid derivative, is a non- steroidal anti-inflammatory drug (NSAID) with
demonstrated anti-inflammatory, analgesic antipyretic activity in
laboratoty animals. The mode of action is not clearly understood. The
anti-inflammatory activity is assumed due to its prostaglandin
synthetase inhibition. The literature survey reveals that there are no
methods reported for simultaneous determination of drotaverine
hydrochloride and mefenamic acid by high performance liquid
chromatography. There are very few methods reported for determination
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of mefenamic acid along with other components in human serum by high
performance liquid chromatography.
5.3 LITERATURE REVIEW
Mohammad-Reza R. et al were reported a simple, rapid and specific
method for analysis of mefenamic acid in serum by a sensitive
HPLC.Diclofenac was used as internal standard. Chromatographic
separation was carried out using C8 column to resolve mefenamic acid
peak from endogenous peaks, and a mobile phase consisting of
acetonitrile and water in the ratio 50:50, v/v, and adjusted pH 3.
Mefenamic acid and Diclofenac were eluted at 7.4 and 5.4 min,
respectively. All validation parameters were complies ICH criteria [187].
Erdal D. et al were reported four new methods for the simultaneous
determination of mefenamic acid and paracetamol in their combination.
In this method, ratio spectra derivative method, analytical signals were
estimated at the wavelengths corresponding to either maximums or
minimums for both drugs in the first derivative spectra of the ratio
spectra obtained by dividing the standard spectrum of two drugs in 0.1
M NaOH:methanol (1:9). The absorbance data was obtained by the
measurements at wavelength range 235–355 nm in the absorption
spectra [188]
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Yogini J. et al were reported HPLC method for the estimation of
ethamsylate and mefenamic acid in tablets. An isocratic RP-HPLC with
PDA was used for detection. Resolution was achieved by using a C8
column with water and acetonitrile in the ratio of 40:60, v/v as mobile
phase adjusted to pH 2.5 with acetic acid. The mobile phase flow rate
was adjusted to 1.0 ml/min and detected at 300 nm [189].
Khuhawar M.Y. et al were reported an acid – 2 - hydroxy
naphthaldehyde (Tr-HN) derivative, with enhancement of
spectrophotometric sensitivity. Tr -HN was eluted from C18, 150×4.6 mm
id and resolved completely from the derivatizing reagent HN. Tr-HN also
was resolved from paracetamol and mefanemic acid. Quantitation was
carried at 317 nm. The method was used for the determination of Tr-HN
from pharmaceutical preparations and blood samples after 2–3 hrs of
administration of the drug [190].
Niopas I. et al were reported a simple, rapid, sensitive and reliable
HPLC method for the determination of indomethacin and mefenamic acid
in human plasma. The chromatographic separation carrried using a C18
column, 250 x 4.6 mm I.D using acetonitrile and 10 mM phosphoric acid
in the ratio of 40:60 v/v as the mobile phase and both drugs were
detected at 280 nm [191].
Alarfaj N.A. et al were reported simple, rapid and accurate
spectrophotometric methods for the determination of mefenamic acid.
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The methods were based on the reaction of mefenamic acid as N -donor
with p-chloranilic acid as acceptor, N-bromosuccinamide oxidation, 3-
methylbenzo-thiazolin-2-one hydrazone oxidation coupling reaction [192]
Fadia H.M. was reported the quantitative predictive abilities of the new
and simple bivariate spectrophotometric method for mixtures of the two
drugs Drotaverine hydrochloride and Nifuroxazide were resolved by
application of the bivariate method. The different approaches were
applied also with previous optimization of the calibration matrix, as they
are useful in simultaneous inclusion of many spectral wavelengths [193]
Vivek S.R. et al were reported three accurate, precise, sensitive and
economical procedures for simultaneous determination of Drotaverine
hydrochloride and Aceclofenac in tablets. The methods used were
Absorbance Ratio Method (I), Simultaneous equation method (II) and
First Order Derivative Spectroscopic Method (III). The first method
employs 230 nm as λ1 and 242 nm as λ2 for formation of equations. The
second method employs determination of a drug concentration by
selecting λmax where the absorbance of these drugs is maximum. So λ
max for Aceclofenac and Drotaverine hydrochloride is 273 nm and 242
nm respectively. The third method was developed based on first order
derivative spectroscopy. Wavelengths 226 nm and 250 nm were selected
for the estimation of the Aceclofenac and Drotaverine hydrochloride
respectively [194].
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Prasad P.D. et al were reported Organic solvent system used for liquid
extraction composed of dichloromethane, and isopropyl alcohol in the
ratio 80:20 v/v. The compounds were separated on a C8, 25.0 cm×4.6
mm, 5 μm particle size columns in isocratic mode by using ammonium
acetate buffer 20 mM, pH 3.5 ± 0.05 adjusted with 85% phosphoric acid
and acetonitrile mixture in a ratio of 55: 45 v/v, as the mobile phase, at
a flow rate of 1.0 mL min-1. The effluent was monitored by UV detection
at 230 nm [195].
Dahivelkar P.P. et.al, were reported two new, simple, accurate and
economical spectrophotometric methods for simultaneous estimation of
drotaverine hydrochloride and mefenamic acid in two-component tablet
formulation. The method developed is first derivative spectrophotometry,
using zero crossing technique and multicomponent analysis [196].
Bolaji O.O. et al were reported a simple and sensitive HPLC method
for the determination of drotaverine in human plasma and Urine.
Chromatographic separation was carried on a C18 -column using a
mobile phase consisting of methanol and 0.02 M sodium dihydrogen
phosphate in the ratio of 70:30, v/v, pH adjusted to 3.2, eluent was
monitored at 254 nm. Drotaverine was resolved from the plasma
constituents and internal standard [197].
Ziyatdinova G.K. et al were reported Isoquinoline derivatives papaverine
and drotaverine are oxidized at a graphite electrode in a 0.1 M sulfuric
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acid solution to give voltammetric waves at 1.1 V for papaverine and at
1.05 and 1.28 V for drotaverine [198].
Fadia H.M. were reported Three new, different, simple, sensitive, and
accurate methods were developed for quantitative determination of
nifuroxazide (I) and drotaverine hydrochloride (II) in a binary mixture.
Spectrophotometry allowed determination of I in the presence of II using
a zero-order spectrum. Determination of second in presence of first was
obtained by 2nd derivative spectrophotometry at 243.6 nm. Both drugs
were evaluated by spectrodensitometry on a silica gel plate using
chloroform, acetone, methanol and glacial acetic acid (6:3:0. 9:0.1) as the
mobile phase and UV detection at 365 nm. The third method was
reversed-phase liquid chromatography using acetonitrile and water in the
ratio 40:60, v/v; adjusted to pH 2.55 with orthophosphoric acid as the
mobile phase and pentoxifylline was used as the internal standard at a
flow rate of 1 mL/min with UV detection at 285 nm at ambient
temperature [199]
5.4 THEORETICAL ANALYSIS
Sample Information:
Drotaverine hydrochloride [200] is a molecule having chemical name
as1,2,3,4-Tetrahydro-6,7-diethoxy-1-((3,4diethoxyphenyl) methylene)-
isoquinoline hydrochloride, and having a molecular formulae of
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171
(C24H31NO4HCl) and molecular weight reported was 433.97, having the
melting point 208-2120C.
Figure 5.01
Drotaverine Structural Formula
Systematic (IUPAC) name is (Z)-1-(3,4-diethoxybenzylidene)-6,7-diethoxy-
1,2,3,4-tetrahydroisoquinoline, formula is C24H31NO4 and mol. mass is
397.507 g/mol. Tablet formulation is easily disintegrated and dispersed
in water with shaking. Mefenamic acid and Drotaverine Hcl are having
UV absorption.
Mefenamic acid [201] is a molecule having chemical name as 2-[(2,3-
dimethyl phenyl)amino]benzoic acid, and having a molecular formulae of
C15H15NO2 and molecular weight reported was 241.29, having the
melting point 230-2310C, and Pka 4.2, C 74.67%, H 6.27%, N 5.80%, O
13.26%. Mefenamic acid is white to almost white, micro-crystalline
powder. Odorless, very little initial taste with bitter after taste darkens on
prolonged exposure to light, non-hygroscopic, decarboxylates at
temperatures above its melting point. Mefenamic acid is practically
insoluble in water, slightly soluble in alcohol and in methylene chloride.
It dissolves in dilute solutions of alkali hydroxides.
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Figure 5.02
Mefenamic Acid Structural Formula
Systematic (IUPAC) name: 2-(2, 3-dimethylphenyl) aminobenzoic acid
Formula : C15H15NO2
Mol. mass : 241.285 g/mol
Solubility Study:
Mefenamic acid is soluble in solutions of alkali hydroxides; sparingly
soluble in chloroform; slightly soluble in alcohol and in methanol;
practically insoluble in water. Drotaverine Hcl is readily soluble in
Chloroform, soluble in 95 % Alcohol, sparingly soluble in water.
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5.5 EXPERIMENTAL INVESTIGATIONS
5.5.1 Experiment No. I
Reagent and Chemicals
Sodium Acetate Trihydrate (GR grade), Acetic Acid, Acetonitrile HPLC
grade, Water (Milli Q), Mefenamic Acid WS and Drotaverine HCl WS
reagents and chemicals were used for experiment.
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (40:60; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Inertsil ODS-3V,
250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The mobile
phase flow rate was maintained at 1.0 ml/min. Standard Mefenamic acid
and drotaverine HCl solution was prepared at concentration 200 μg/mL
of mefenamic acid 32 μg/mL of drotaverine in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 350 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
Both Mefenamic acid and drotaverine hydrochloride peaks were eluted
within 15 minutes. Since peak shape found was not satisfactory, next
experiment carried with changing mobile phase composition.
5.5.2 Experiment No. 2
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The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (80:20; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Inertsil ODS-3V,
250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The mobile
phase flow rate was maintained at 1.5 ml/min. Standard Mefenamic acid
and drotaverine HCl solution was prepared at concentration, 200 μg/mL
of mefenamic acid and 32 μg/mL of drotaverine in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 350 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
Both Mefenamic acid and drotaverine hydrochloride peaks were eluted in
10 minutes. But found was not satisfactory and coelution peaks
observed, therefore next experiment carried with changing mobile phase
composition.
5.5.3 Experiment No. 3
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (65:35; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Kromasil C18,
250 mm x 4.6 mm, 5 µ and maintained tempewrature 25 °C. The mobile
phase flow rate was maintained at 1.0 ml/min. Standard Mefenamic acid
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and drotaverine HCl solution was prepared at concentration, 200 μg/mL
of mefenamic acid and 32 μg/mL of drotaverine in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 350 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
Both Mefenamic acid and drotaverine hydrochloride peaks were
eluted within 10 minutes. Peak shape found was not satisfactory and
tailing found more than 2, next experiment carried with changing mobile
phase composition.
5.5.4 Experiment No. 4
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (60:40; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Inertsil ODS-3V,
250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The mobile
phase flow rate was maintained at 1.5 ml/min. Standard Mefenamic acid
and drotaverine HCl solution was prepared at concentration, 200 μg/mL
of mefenamic acid and 32 μg/mL of drotaverine in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 350 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
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Both Mefenamic acid and drotaverine hydrochloride peaks were
eluted within 12 minutes. Peak shape found was not satisfactory and
tailing found more than 2, next experiment carried with changing mobile
phase composition.
5.5.5 Experiment No. 5
The mobile phase was acetonitrile and a solution of 50 mM potassium
dihydrogen phosphate buffer adjusted pH to 3.0 with 10% solution of
phosphoric acid, (60:40; v/v). Mobile phase was filtered through 0.45 μ
membrane filter. Column equipped with instrument was Zorbax SB
Phenyl, 250 mm x 4.6 mm, 5 µ and maintained temperature 25 °C. The
mobile phase flow rate was maintained at 1.5 ml/min. Standard
Mefenamic acid and drotaverine HCl solution was prepared at
concentration, 200 μg/mL of mefenamic acid and 32 μg/mL of
drotaverine in mobile phase. 20 μL standard solutions were injected two
times and average detector response measured at 350 nm.
Chromatograms evaluated with respect to retention time, resolution and
peak shape.
Both Mefenamic acid and drotaverine hydrochloride peaks were
eluted within 15 minutes. Since peak shape found was not satisfactory,
next experiment carried with changing mobile phase composition.
5.5.6 Experiment No. 6
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The mobile phase was acetonitrile and a solution of 50 mM sodium
acetate buffer adjusted pH to 4.5 with 10% solution of acetic acid,
(55:45; v/v). Mobile phase was filtered through 0.45 μ membrane filter.
Column equipped with instrument was Inertsil ODS-3V, 250 mm x 4.6
mm, 5 µ was maintained temperature 25 °C. The mobile phase flow rate
was maintained at 1.5 ml/min. Standard Mefenamic acid and
drotaverine HCl solution was prepared at concentration, 200 μg/mL of
mefenamic acid and 32 μg/mL of drotaverine in mobile phase. 20 μL
standard solutions were injected two times and average detector
response measured at 350 nm. Chromatograms evaluated with respect to
retention time, resolution and peak shape.
Both Mefenamic acid and drotaverine hydrochloride peaks were
eluted within 15 minutes. Run time and peak shapes were satisfactory.
5.5.7 Experiment No. 7
Standard Preparation:
Weigh accurately about 200 mg of Mefenamic acid working standard
and 32 mg Drotaverine Hcl working standard. Transfer it into a 100 mL
volumetric flask; add 10 mL of water, 80 mL of acetonitrile sonicate to
dissolve the drug completely, make up the volume with acetonitrile and
mix sonicate for few minutes (Conc. of Mefenamic acid is 2000 μg/mL,
Conc. Of Drotaverine Hcl is 320 μg/mL). Pipette out 5 mL of above
solution in 50 mL volumetric flask and make up the volume with
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diluent.Filter it through 0.45µ nylon filter discarding first few milliliters.
(Conc. of Mefenamic acid is 200 μg/mL, Conc. Of Drotaverine Hcl is 32
μg/mL)
Sample Preparation:
Weigh 20 tablets and calculate the average weight. Crush the tablets
to fine powder. Weigh accurately powder equivalent to 80mg of
Drotaverine HCL & 500mg of Mefenamic acid, transfer it into a 250 mL
dry volumetric flask. Add 25 mL of water, sonicate at least for 5 minutes
add 215 ml of acetonitrile, sonicate for 10 - 15 minutes. Allow to cool at
room temperature. Make up the volume with acetonitrile and mix well.
Filter this solution through 0.45µ nylon filter discarding first few drops.
(Conc. of Mefenamic acid is 2000 μg/mL, Conc. of Drotaverine Hcl is 320
μg/mL). Pipette out 5 mL of above filtrate in to 50 mL volumetric flask
and make up the volume with diluent mix well and inject. (Conc. of
Mefenamic acid is 200 μg/ mL, Conc. of Drotaverine Hcl is 32 μg/mL)
Preparation of blank preparation:
Pipette 4.5 ml of acetonitrile and 0.5ml of water in to 50 ml
volumetric flask and make up volume with diluent
5.4.8 Experiment No. 8 (Validation procedure)
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The objective of validation of an analytical procedure is to
demonstrate that the method suitable for its intended purpose. Method
validation for linearity, specificity, robustness precision, accuracy, and
system suitability.
Selectivity:
Blank, standard solution containing Mefenamic acid, drotaverine and
sample solution of Mefenamic acid and drotaverine tablets were prepared
as per methology given in Experimental results by using photodiode
array detector. Two types of specificity experiments were performed. In
the first one, specificity examined by comparing the chromatograms got
from the pharmaceutical preparation and the standard solution with
those got from excipients which used in the tablets and examining the
absence of interferences. In the second type, forced degradation
performed in order to check the suitability of analytical conditions for
stability study of Mefenamic acid and Drotaverine. The accelerated
degradation conditions applied were: light (UVC), acid, basic and oxidant
media. Samples were analysed with a freshly prepared control sample
and under light protection. The peak purity was decided using the tools
of the Waters software. Excipient solutions were submitted to the same
degradation conditions in order to administer no interference. Specific
details of the experiments conditions are described below:
Effect of UV light:
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To a 1 cm closed quartz cell 1 ml of a solution containing 2.0 mg/mL
of Mefenamic acid and 0.32 mg/mL of Drotaverine in acetonitrile was
placed. The cell was exposed to a UV chamber (100 x 18 x 17 cm) with
internal mirrors and UV fluorescent lamp CRS F30W T8 emitting
radiation at 254 nm for 15, 30, 60, 120 and 180 minutes. The same
procedure was realized for preparation for LC analysis. Samples,
protected in aluminum foil (in order to perotect from light) were
submitted simultaneously to identical conditions and used as control.
After the degradation treatment, the samples were diluted to 200 μg/ml
of Mefenamic acid and 32 μg/ml of drotaverine with a mixture of
acetonitrile:water (6:4; v/v) and analysed immediately.
Effect of Oxidation:
Mefenamic acid and Drotaverine standards were dissolved in
acetonitrile (2.0 mg/mL of Mefenamic acid and 0.32 mg/mL of
drotaverine), 5 ml of this solution was transferred to a volumetric flask,
where hydrogen peroxide solution (30%) was added until the final
concentration of 10 % and the volume was completed with acetonitrile.
After 20 hours the solution was diluted until the final concentration of
200 μg/mL of Mefenamic acid and 32 μg/mL of drotaverine, filtered and
analysed. Similar procedure was realized for tablets, when 25 ml of the
initial solution 0.2 mg/mL of Mefenamic acid and 0.032 mg/mL of
Drotaverine, obtained as described in sample preparation for LC
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analysis, were transferred to a volumetric flask and submitted to
degradation. A controlsolution containing the excipients was prepared
under the same circumstances of the tablets.
Effect of Acid Hydrolysis:
5 ml of 2.0 mg/mL of Mefenamic acid and 0.32 mg/mL of Drotaverine
reference standard solution was transferred to a volumetricflask and HCl
(acid degradation) was added until the final concentration of 1M HCl.
After 5 hours and 1 and 6 days, one aliquot of the solution was
neutralized with NaOH 1M and diluted with acetonitrile and water
(55:45, v/v) until the final concentration of 200 μg/ml of Mefenamic acid
and 32 μg/ml of Drotaverine for LC analysis. Similar procedure was
realized with the tablets, when 25 ml of the initial solution 0.2 mg/mL of
Mefenamic acid and 0.032 mg/mL of Drotaverine (obtained as described
in sample preparation for LC analysis) were transferred to a volumetric
flask and submitted to the degradation. A control solution containing the
excipients was prepared under the same conditions of the tablets.
Effect of Alkaline Hydrolysis:
5 ml of 2.0 mg/mL of Mefenamic acid and 0.32 mg/mL of Drotaverine
reference standard solution was transferred to a volumetricflask and
NaOH (alkaline degradation) was added until the final concentration of
1M NaOH. After 5 hours and 1 and 6 days, one aliquot of the solution
was neutralized with HCl 1M and diluted with acetonitrile and water
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(55:45, v/v) until the final concentration of 200 μg/ml of Mefenamic acid
and 32 μg/ml of Drotaverine for LC analysis. Similar procedure was
realized with the tablets, when 25 ml of the initial solution 2.0 mg/mL of
Mefenamic acid and 0.32 mg/mL of Drotaverine (obtained as described
in sample preparation for LC analysis) were transferred to a volumetric
flask and submitted to the degradation. A control solution containing the
excipients was prepared under the same conditions of the tablets.
During the stability assays the peak purity as applied to find the absence
of other substances co-eluting in the same retention time.
Linearity and Range:
To test linearity, standard plots were constructed with six
concentrations in the range of 100-300 μg/mL of Mefenamic acid and
16-48 μg/mL of Drotaverine were prepared in triplicates. The linearity
was evaluated by the least square regression.
Precission:
The repeatability was verified from six independent sample
preparations in the same day, obtained as described in Sample
preparation for LC analysis. The intermediate precision was tested by
assaying freshly prepared sample solutions on different day. Precision
was reported as %RSD.
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Six replicate injections of the standard preparation were made into
the HPLC used the methodology given in experimental result.
Six spiked sample preparations and one control sample preparation of
Mefenamic acid and drotaverine were prepared and injected into the
HPLC using the method as described under experimental result.
Accuracy:
The accuracy was estimated by the recovery of known amounts of
Mefenamic acid and drotaverine standards added to the placebo in the
beginning of the preparation method. The added levels were 80, 100 and
120% of the nominal drug concentrations. The results were expressed as
the percentage of Mefenamic acid and Drotaverine reference standards
recovered from the sample.
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Ruggedness:
Six spiked sample preparations and one control sample preparations
of Mefenamic acid and drotaverine were analysed by a different analyst,
using different column, on different day and injected into a different
HPLC using the method as described in experimental result, along with
standard preparation.
Robustness:
Standard preparation, diluent, placebo preparation and sample
preparation in triplicate of the sample of Mefenamic acid and drotaverine
tablet were prepared as described in experimental result. The samples
along with standard and placebo were injected under different
chromatographic conditions as shown below.
Stability of Analytical Solution:
Standard solution, Sample solution were analysed initially and at
different time intervals at room temperature. The system suitability was
verified through the evaluation of the obtained parameters for the
standard elution, such as theoretical plates, peak asymmetry and
retention factor, verified in different days of the method validation.
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5.6 EXPERIMENTAL RESULTS
On the basis of experiments of Drotaverine HCl and Mefenamic acid
method development, we can conclude that the RP-HPLC method
developed for simultaneous determination of Drotaverine HCl and
Mefenamic acid can be used for routine analysis.
Reagent and Chemicals
Sodium Acetate Tri hydrate (GR grade), Acetic Acid, Acetonitrile
HPLC grade, Water (Milli Q), Mefenamic Acid WS and Drotaverine HCl
WS reagents and chemicals were used for experiment.
Prepared buffer solution by dissolving 6.8 g of Sodium Acetate Tri
hydrate in 1000 mL of water, adjusted pH to 4.5 (±0.05) with Acetic Acid.
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Table 5.01
Chromatographic Conditions for Mefenamic Acid and Drotaverine
Column : Inertsil ODS-3V, 250 mm x 4.6 mm, 5 µ.
Column Oven Temp : 25°C
Wavelength : 350 nm.
Flow Rate : 1.5 mL / min
Injection Volume : 20 µL.
Run Time : 15 min.
Mobile phase : Buffer:Acetonitrile (45:55) v/v
Diluent : Water:Acetonitrile in ratio of 45:55 v/v
Mix Standard Preparation
Weigh accurately about 200 mg of Mefenamic acid working standard
and 32 mg Drotaverine Hcl working standard. Transfer it into a 100 mL
volumetric flask; add 10 mL of water, 80 mL of acetonitrile sonicate to
dissolve the drug completely, make up the volume with acetonitrile and
mix sonicate for few minutes (Conc. of Mefenamic acid is 2000 μg/ mL,
Conc. of Drotaverine Hcl is 320 μg/mL ).Pipette out 5 mL of above
solution in 50 mL volumetric flask and make up the volume with
diluent.Filter it through 0.45µ nylon filter discarding first few
milliliters(Conc. of Mefenamic acid is 200 μg/ mL, Conc. of Drotaverine
HCl is 32 μg/mL).
Sample Preparation:
Weigh 20 tablets and calculate the average weight. Crush the tablets
to fine powder. Weigh accurately powder equivalent to 80 mg of
Drotaverine HCL & 500 mg of Mefenamic acid; transfer it into a 250 mL
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dry volumetric flask. Add 25 mL of water, sonicate at least for 5 minutes
add 215 ml of acetonitrile, sonicate for 10 - 15 minutes. Allow to cool at
room temperature. Make up the volume with acetonitrile and mix well.
Filter this solution through 0.45µ nylon filter discarding first few drops
(Conc. of Mefenamic acid is 2000 μg/ mL, Conc. of Drotaverine Hcl is
320 μg/mL). Pipette out 5 mL of above filtrate in to 50 mL volumetric
flask and make up the volume with diluent ( acetonitrile and water
55:45, v/v ) mix well and inject (Conc. of Mefenamic acid is 200 μg/ mL,
Conc. of Drotaverine Hcl is 32 μg/mL).
Preparation of Blank Preparation:
Pipette 4.5 ml of acetonitrile and 0.5ml of water in to 50 ml
volumetric flask and make up volume with diluent
Procedure for Injection:
Separately inject equal volume of Blank preparation, Standard
Preparation in five replicates and one sample Preparation in duplicate
into the HPLC system. Record the Chromatograms and measure the
response of the peaks due Drotaverine Hcl (1st peak) and Mefenamic acid
(2nd peak). Evaluate the system suitability
System Suitability Parameter
Theoretical Plates: NLT 2000 for both analytes
Tailing Factor: NMT 2.0 for both analytes
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RSD for five injection of Standard solution is NMT 2.0 % for both
analytes.
Note:
Flushing solution used was acetonitrile and water in the ratio 55:45
v/v.
5.7 DISCUSSION OF RESULTS
Linearity and range: the correlation coefficients are less than 0.9995
for Drotaverine and Mefenamic acid.
Precision: system precision RSD is less than 2% and method
precision RSD is less than 2% for Drotaverine and Mefenamic acid.
Accuracy: the mean recoveries for Drotaverine and Mefenamic acid
are within 98 -102 %.
Specificity: Retention time of Drotaverine and Mefenamic acid peaks
in sample preparation is comparable with respect to retention time of
Drotaverine and Mefenamic acid peaks in standard preparation. Peak
purity passes for Drotaverine and Mefenamic acid peaks in standard and
sample preparations. No intereference was observed at the retention time
of Drotaverine and Mefenamic acid peaks. Peak purity passes for all
degradation conditions.
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Ruggesness: the RSD of twelve results obtained from two different
analysts are within 10 %.
Robustness: Drotaverine and Mefenamic acid peaks were resolved
with each other and system suitability complies for all variable
conditions, the test method is robust for all variable conditions.
Stability in analytical solution: Standard and sample solutions are
stable for 12 h at room temperature
System suitability: Theoretical plates are less than 2000, tailing factor
is less than 2.0 and relative standard deviation is less than 5.0 for six
standard replicate injections.
Table 5.02
Peak Purity Data of Drotaverine and Mefenamic acid
Sr. No. Name
Purity Criteria
1 Drotaverine HCl in standard solution
Pass
2 Drotaverine HCl in sample solution Pass
3 Mefenamic acid in standard solution
Pass
4 Mefenamic acid in sample solution Pass
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Table 5.03
Recovery Data of Drotaverine HCl by Placebo Spiked Recovery Method
Level
Amount Added (mg)
Average Area
Amount recovered
Recovery (%)
Mean
Recovery (%)
80 Spl. 1 66.39 429850 67.01 100.93
100.60
80 Spl. 2 66.39 425826 66.38 99.99
80 Spl. 3 66.39 429668 66.98 100.89
100 Spl. 1 82.98 534325 83.29 100.37
100.75
100 Spl. 2 82.98 538087 83.88 101.08
100 Spl. 3 82.98 536670 83.66 100.81
120 Spl. 1 99.58 647581 100.95 101.37
101.50
120 Spl. 2 99.58 648033 101.02 101.44
120 Spl. 3 99.58 649680 101.27 101.70
Mean 100.95
S.D 0.48
%RSD 0.48
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Table 5.04
Recovery Data of Mefenamic Acid by Placebo Spiked Recovery Method
Level
Amount
Added
Average
Area
Amount
recovered
Recovery
(%)
Mean
Recovery
(%)
80 Spl-1 401.10 2930431 403.05 100.49
100.52
80 Spl-2 401.81 2943060 404.79 100.74
80 Spl-3 400.78 2923445 402.09 100.33
100 Spl-1 500.68 3662173 503.69 100.60
100.97
100 Spl-2 502.06 3702201 509.20 101.42
100 Spl-3 501.30 3677136 505.75 100.89
120 Spl-1 602.21 4440365 610.73 101.41
101.31
120 Spl-2 602.09 4426259 608.79 101.11
120 Spl-3 601.82 4436918 610.25 101.40
Mean 100.93
S.D 0.40
%RSD 0.39
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Table 5.05
Linearity Data of Drotaverine HCl
Linearity Level
Conc. in
ppm Experimental
Area (a) Predicted Area (y)
Residuals (b) (x)
y=mx+c b= a-y
Level-50% 16.06 267019 259153 -7866
Level-80% 25.70 428930 429981 1051
Level-90% 28.91 484134 482645 -1489
Level-100% 32.12 536038 551864 15826
Level-110% 38.54 590624 593935 3311
Level-120% 48.18 642146 638221 -3925
Level-150% 3.02 805057 798237 -6818
Correlation 0.99931
Intercept (c) -1112
Slope (m) 61557
Table 5.06
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Linearity Data of Mefenamic acid
Linearity Level
Conc. in ppm
Experimental Area (a)
Predicted Area (y)
Residuals (b) (x)
y=mx+c b= a-y
Level-50% 100 1811359 1819225 -7866
Level-80% 160 2915043 2913982 1061
Level-90% 180 3277421 3278900 -1479
Level-100% 200 3659545 3643819 15726
Level-110% 220 4012059 4008738 3321
Level-120% 240 4369721 4373656 -3935
Level-150% 300 5461585 5468413 -6828
Correlation 0.99997
Intercept (c) -7348
Slope (m) 10465
Table 5.07
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194
Precision Data of Drotaverine HCl.
Sample Test wt (mg) Avg Area mg/Unit % Assay
Spl. 1 891.97 524551 80.319 100.40
Spl. 2 891.91 522971 80.082 100.10
Spl. 3 892.01 516780 79.125 98.91
Spl. 4 891.89 524033 80.246 100.31
Spl. 5 890.99 524551 80.407 100.51
Spl. 6 892.03 517557 79.242 99.05
Average 99.88
SD 0.71
RSD (%) 0.71
Note: Average Weight of Tablets was 891.92 mg.
Table 5.08
Method Precision Data of Mefenamic acid
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195
Sample Test wt(g) Avg Area mg/Unit % Assay
Spl. 1 891.97 3653268 503.60 100.72
Spl. 2 891.91 3605211 497.01 99.40
Spl. 3 892.01 3645108 502.45 100.49
Spl. 4 891.89 3642206 502.12 100.42
Spl. 5 890.99 3654175 504.28 100.86
Spl. 6 892.03 3623346 499.44 99.89
Average 100.30
SD 0.55
RSD (%) 0.55
Table 5.09
Intermediate precision Data of Drotaverine HCl.
Sample Test wt(g) Avg Area mg/Unit % Assay
Spl. 1 891.21 524378 80.288 100.36
Spl. 2 891.36 521759 79.888 99.86
Spl. 3 892.68 516960 79.152 98.94
Spl. 4 891.30 527485 80.768 100.96
Spl. 5 890.72 521274 79.816 99.77
Spl. 6 892.44 517414 79.224 99.03
Average 99.82
SD 0.77
RSD (%) 0.77
Table 5.10
Intermediate Precision Data of Mefenamic Acid
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196
Sample
Test
wt(g) Avg Area mg/Unit
%
Assay
Spl. 1 891.21 3653268 506.40 101.28
Spl. 2 891.36 3605211 495.60 99.12
Spl. 3 892.68 3645108 506.65 101.33
Spl. 4 891.30 3642206 507.60 101.52
Spl. 5 890.72 3654175 502.35 100.47
Spl. 6 892.44 3623346 499.45 99.89
Average 100.60
SD 0.95
RSD (%) 0.95
Figure 5.03
Standard Chromatograph for Mefenamic Acid and Drotaverine
Figure 5.04
Sample Chromatograph for Mefenamic Acid and Drotaverine
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5.8 SUMMARY CONCLUSION AND RECOMMENDATIONS
The proposed simultaneous estimation method method was found to
be simple, precise, accurate and rapid for the determination of
Drotaverine and Mefenamic acid from tablets forms, the mobile phase is
simple to prepare and economical. The sample recoveries in all the
formulations were in good agreement with their respective label claim
and their suggestive not interference of formulation excipients in the
estimation.
Hence this method can be conveniently adopted for routine analysis
of Drotaverine and Mefenamic acid in the pharmaceutical dosage forms
and also for stability analysis.