165

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

166

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]

167

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.

168

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].

169

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

170

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

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.

172

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.

173

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

174

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

175

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.

176

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

177

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

178

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)

179

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:

180

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

181

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

182

(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.

183

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.

184

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.

185

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.

186

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

187

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

188

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.

189

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

190

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

191

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

192

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

193

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

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

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

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

197

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.