Determination of Camylofin Dihydrochloride and Nimesulide in Pharmaceutical Preparation by Gas Chromatography

doi:10.4236/ajac.2011.28110

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American Journal of Anal yt ical Chemistry, 2011, 2, 944-952

doi:10.4236/ajac.2011.28110 Published Online December 2011 (http://www.SciRP.org/journal/ajac)

Determination of Camylofin Dihydrochloride and

Nimesulide in Pharmaceutical Preparation by Gas

Chromatography

Rajeev Kumar R. Singh1,2, Manapragada V. Rathnam1, Sangeeta J. Singh2, Raju V. K. Vegesna2

1Department of Chemistry, B. N. Bandodkar C oll ege of Science, Thane, India

2Versapharm Inco rp orated, Warminister, USA

E-mail: [email protected]

Received February 27, 20 1 1; revised May 22, 2011; accepted June 1, 2011

Abstract

This research paper describes si mple analytical method for determination of Camylofin dihydrochloride and

Nimesulide in tablet formulation by Gas chromatography method. Benzoic acid was used as internal standard.

Validation was carried out in compliance with the International Conference on Harmonization guidelines.

The method utilized GC (Agilent Technologies 6890 N Network GC system with FID detector), and RTX-5

capillary column (5% diphenyl-95% dimethyl polysiloxane), 30 m × 0.53 mm, 1.5 µm as stationary phase.

Helium was used as the carrier gas at a flow rate of 1.5 mL·min–1. The proposed method was validated for

linearity, LOD, LOQ, accuracy, precision, ruggedness and solution stability. It can be conveniently adopted

for routine quality control analysis.

Keywords: Capillary Column, Gas Chromatography, Pharmaceutical Preparations, Camylofin

Dihydrochloride, Nimesulide

1. Introduction

Camylofin dihydrochloride is 3-methylbutyl 2-(2-diethy-

laminoethylamino)-2-phenyl-acetate hydrochloride is a

drug used an antispasmodic [1]. Nimesulide N-(4-Nitro-

2-phenoxyphenyl) methanesulfonamide.Nimesulide is a

relatively COX-2 selective, non-steroidal anti-inflam-

matory drug (NSAID) with analgesic and antipyretic

properties. Its approved indications are the treatment of

acute pain, the symptomatic treatment of osteoarthritis

and primary dysmenorrhoea in adolescents and adults

above 12 years old [2].Th e structur e of the drug is shown

in Figure 1. One such combination contains 50 mg of

Camylofin dihydroch loride and 100 mg of Nimesulide.

The literature survey revealed that there is no method

for the simultaneous determination of these compounds.

There are other publications for determination of these

compounds but in combination with other components

by other analytical techniques like HPLC, spectropho-

tometry and colorimetric [4-16].There is a GC method

reported for the analysis of Camylofin dihydrochloride

[17]. There are, however, no publications for simultane-

ous determination of these drugs in such pharmaceutical

preparation. Therefore a GC method was developed for

2HCl

Camylofin dihydrochloride ( C19H 32N 2O 2,2HCl)

NO 2

Nimesulide (C13H12N2O5S)

Figure 1. Structure of camylofin dihydrochloride and ni-

mesulide.

R. K. R. SINGH ET AL.945

determination of camylofin dihydrochloride and Nime-

sulide from their dosage form. The method described is

simple, fast, precise and accurate for simultaneous de-

termination of Camylofin dihydrochloride and Nimesu-

lide from pharmaceutical preparation. The method is

very cost and time effective since it does not require any

mobile phase preparation and can be easily adapted to

Quality control testing laboratory.

2. Materials and Methods

2.1. Chemicals and Reagents

Anafortan N tablets manufactured by Khandelwal lab,

India were procured from the market. Anafortan N tab-

lets is a combination of Camylofin dihydrochloride 50

mg and Nimesulide 100 mg. Methanol was from Quali-

gens. All dilutions were performed in standard volumet-

ric flasks.

2.2. Apparatus

The analysis was performed by using the analytical bal-

ance Mettler Toledo, the GC used is of Agilent Tech-

nologies 6890 N Network GC system with FID detector.

Column used in GC is a capillary column RTX-5, 30 m ×

0.53 mm, 1.5 µm. Photo stability studies were carried out

in a photo stability chamber (Sanyo, Leicestershire, UK).

Thermal stability studies were carried out in a dry air

oven (Lin dberg-Blue, USA).

2.3. Experimental

Method development and optimization of chroma-

tographic condition s:

To develop a suitable GC method for the analysis of

camylofin dihydrochloride and Nimesulide in their dos-

age form, different capillary columns were tried [18].

The criteria employed for selecting the columns for the

analyses of the drugs were cost involve, time required for

the analysis, better separation of the components. Chro-

matographic separation was preformed with Agilent

Technologies 6890 N Ne twork Gas chromatography sys-

tem, equipped with auto sampler and a flame ionization

detector. Chromatograms and data were recorded by

means of Empower software. RTX-5 capillary column

(Crossbond 50% diphenyl-95% dimethyl polysiloxane)

was used for analysis. The column dimension was 30 m

× 0.53 mm, 1.5 µm. The system was run at a flow rate of

1.5 mL·min–1, 1 µL of sample was injected in the chro-

matographic system and flame ionization detector was

used for simultaneous determination of Camylofin dihy-

drochloride and Nimesulide. Helium was used as a car-

rier gas. Oven temperature was kept 180˚C and increased

at a rate of 10˚C·min–1 to 280˚C and held at 280˚C for

15.0 minutes. Injector temperature and detector tem-

perature were kept at 250˚C and 280˚C respectively. The

split ratio was kept at 50:1. A summary of method de-

velopment and optimization is described in Table 1.

2.3.1. Preparation of Standard Stock Solutions

The stock solution of Camylofin dihydrochloride (1250

µg·mL–1) was prepared by dissolving 126.1 mg of Ca-

mylofin dihydrochloride (99.9%) in methanol in a stan-

dard 100 mL volumetric flask (stock solution A). The

stock solution of Nimesulide (2500 µg·mL–1) was pre-

pared by dissolving 250.8 mg of Nimesulide (99.8%) in

methanol in a standard 100 mL volumetric flask (stock

solution B). Internal standard (benzoic acid) stock solu-

tion (5000 µg·mL–1) was prepared by dissolving 500.9

mg of benzoic acid (99.6%) in methanol in a 100 mL

standard volumetric flask (stock solution C).

Transferred 10.0 mL of each stock solution A, B & C

to a 50 mL volumetric flask and diluted up to the mark

with methanol. This is working standard solution.

2.3.2. Sample Preparation

For analysis of the tablet dosage form, twenty tablets

were weighed individually and their average weight was

determined. The tablets were crushed to fine homoge-

nous powder and quantity equivalent to ten tablets were

transferred in a 200 mL volumetric flask. Add ed about 100

mL of Methanol to the volumetric flask, shaken for 10

minutes and then sonicated for 15 minutes. The solu tion

Table 1. Summary of optimization of chromatographic conditions.

Column used Carrier gas Flow rate Observation Result

DBWax, 30 m × 0.53 mm, 1.0 µm

capillary column Helium 1.2 mL·min–1 No peaks observed Method rejected

DB624, 30 m × 0.32 mm, 1.8 µm

capillary column Helium 1.2 mL·min–1 Peak shape for both components not good Method rejected

RTX1, 30 m × 0.53 mm, 1 .0 µm

capillary column Helium 1.5 mL·min–1 Poor resolution and low response Method rejected

RTX5, 30 m × 0.53 mm, 1 .5 µm

capillary column Helium 1.5 mL·min–1 Good resolution and good peak shape Method accepted

R. K. R. SINGH ET AL.

946

was allowed to stand at room temperature for 20 - 30

minutes and filtered throug h Whatman no. 41 filter paper.

The residue was washed with Methanol and the com-

bined filtrate was made up to the mark with the same

solvent.

5.0 mL of filtrate was quantitatively transferred to a 50

mL volumetric flask, 10.0 mL of internal standard solu-

tion was added to it, and solution was diluted up to the

mark with methanol. The identities of both the com-

pounds were established by comparing retention time of

the sample solution with those of standard solution.

2.4. Validation Parameters

The method validation was carried out as per ICH guide-

lines [19]. Various method validation parameters were

performed.

2.4.1. System Suitability Test

System suitability tests are used to verify that the repro-

ducibility of the equipment is adequate for the an alysis to

be carried out. System suitability tests were performed as

per the USP 31 to confirm the suitability and reproduci-

bility of the system. Various parameters such as tailing

factor and resol ut i on be t w een the peaks were obtained.

2.4.2. Specificity

Specificity of the method was evaluated by injecting

diluents, placebo, individual Camylofin dihydrochloride

and Nimesulide and sample solution in to the GC system

to check any interference to the peaks.

2.4.3. Linearity

Linearity was evaluated by analysis of working standard

solutions of Camylofin dihydrochloride and Nimesulide

of seven different concentrations. The range of linearity

was from 250 - 750 µg·mL–1 for Nimesulide and 125 -

375 µg·mL–1 for Camylofin dihydrochloride. The peak

area ratio and concentration of each drug was subjected

to regression analysis to calculate the calibration equa-

tions and correlation coefficients.

2.4.4. LOD and LOQ/Sensitivity

Sensitivity was determined by establishing the limit of

detection (LOD) and limit of quantification (LOQ). The

limit of detectio n (LOD) and limit of quantitation (LOQ)

were established at signal-to-noise ratio of 3:1 and 10:1

respectively.

2.4.5. Accu racy

Accuracy was determined over the range 50% to 150%

of the sample concentration. Calculated amount of Ca-

mylofin dihydrochloride and Nimesulide from standard

stock solution was added in placebo to attain 50%, 100%

and 150% of sample concentration. Each sample was

prepared in triplicate at each level. Blank and standard

preparations were injected and chromatograms were re-

corded.

2.4.6. Precision

Repeatability was studied by carrying out system preci-

sion. System precision was determined from results for

six replicate injections of the mixed standard solutions.

Method precision was determined from results from five

independent determinations at 100% of the test concen-

trations of Camylofin dihydrochloride and Nimesulide in

the product.

2.4.7. Ruggedness (Intermediate Precision)

Ruggedness study was demonstrated by injecting six

individual sample preparations at 100% of the test con-

centrations of Camylofin dihydrochloride and Nimesu-

lide on different day using another column and system.

2.4.8. Robustness

By deliberate change in experimental condition the reso-

lution between Methylparaben, Camylofin dihydrochlo-

ride and Nimesulide were evaluated. To study the effect

of flow rate on system suitability parameters, 0.2 units

changed i.e. 1.3 and 1.7 mL·min–1 . T he effect of column

temperature was studied at 170˚C and 190˚C. The injec-

tor temperature and detector temperature were kept con-

stant.

2.4.9. Stability of Solution

The solution stability of Camylofin dihydrochloride and

Nimesulide was carried out by leaving the test solutions

of sample in a tightly capped volumetric flask at room

temperature for 72 hours. The same sample solutions

were assayed for 24 hours interval up to the study period

against freshly prepared standard solution.

2.4.10. Stress Testing (Forced Degradation Study)

To further confirm the stability indicating nature of the

method, the drug was subjected to stress conditions as

per the ICH recommended test conditions [20,21].

To study the effect of acid, 5 mL of 2 M HCl was

added to the sample and the mixture was kept for 48

hours at room temperature. To study the effect of base, 5

mL of 1 N NaOH solution was added to the sample and

the mixture kept for 3 hours at room temperature. To

study the effect of oxidizing conditions, 5 mL of 3% v/v

H2O2 was added to the sample and the mixture was kept

for 48 hours at room temperature.

To study the effect of temperature sample was kept in

an oven at 8 0˚C for 5 days.

R. K. R. SINGH ET AL.947

To study the effect of light sample was and kept in a

photostability chamber for 5 days.

3. Results and Discussion

3.1. System Suitability

System suitability tests are used to verify that the repro-

ducibility of the equipment is adequate for the an alysis to

be carried out. System suitability tests were performed as

per the USP 31 to confirm the suitability and reproduci-

bility of the system. The % RSD values were found to be

satisfactory and meeting the requirements of USP 31

(RSD less than 2.0%). A typical GC chromatogram for

simultaneous determination of camylofin dihydrochlo-

ride and Nimesulide from pharmaceutical formulation is

shown in Figure 2 and Figure 5. System suitability pa-

rameters are mentioned in Table 2.

3.2. Specificity

No peak was observed at the retention ti me of Camylofin

dihydrochloride, Nimesulide and Benzoic acid in dilu-

ents and Placebo chromatogram. Hence the method was

specific.

3.3. Linearity

Linearity was evaluated by analysis of working standard

solutions of Camylofin dihydrochloride and Nimesulide

of seven different concentrations. The range of linearity

was from 125 µg·mL–1 to 375 µg·mL–1 (250 µg·mL–1 is

100% level) for Camylofin dihydrochloride and 250

µg·mL–1 to 750 µg·mL–1 (500 µg·mL–1 is 100% level) for

Nimesulide. The peak area ratio and concentration of each

drug was subjected to regression analysis to calculate the

calibration equations and correlation coefficients. Figure 6

represents the linearity plots of Camylofin dihydrochloride

and Nimesulide. The regression data obtained for the Ca-

mylofin dihydrochloride and Nimesulide is represented in

Table 3. The result shows that within the concentration

range mentioned above, there was an excellent correlation

between peak area ratio and concentration.

3.4. LOD and LOQ/Sensitivity

The LOD and LOQ of Camylofin dihydrochloride and

Nimesulide was experimentally determined by six injec-

tions of each drug. The LOD of Camylofin dihydrochlo-

ride and Nimesulide was found to be 1.2 µg·mL–1 & 1.6

µg·mL–1 respectively. The LOQ of Camylofin dihydro-

chloride and Nimesulide was found to be 2.1 µg·mL–1 &

2.7 µg·mL–1 respectively.

3.5. Accuracy

Accuracy was expressed as the percentage of analytes

recovered by the assay. Table 4 lists the recoveries of the

drug from a series of spiked concentrations. The results

indicate the method is highly accurate for simultaneous

determination of Camylofin dihydrochloride and Nime-

sulide.

Figure 2. Chromatogram of diluent.

R. K. R. SINGH ET AL.

948

Figure 3. Chromatogram of placebo.

Figure 4. Chromatogram of Camylofin dihydrochloride and Nimesulide with benzoic acid (internal standard) in standard

preparation. Nimesulide-25.744.

3.6. Precision

The values of the relative standard deviation of five rep-

licate injections of the standard solution containing both

the analytes of interest were within the limits of not more

than 2.0%. Ref e r Table 5.

3.7. Ruggedness (Intermediate Precision) and

Robustness

Ruggedness study was done by injecting six individual

sample preparations at 100% of the test concentrations of

amylofin dihydrochloride and Nimesulide on different C

R. K. R. SINGH ET AL.

949

Figure 5. Chromatogram of Camylofin dihydrochloride and Nimesulide with Benzoic acid (internal standard) in sample

preparation.

Table 2. Results of system suitability.

Parameters Benzoic

acid Camylofin

dihydrochloride Nimesulide

Resolution NA 78.2 42.8

Tailing factor 1.0 1.2 1.0

Theoretical plates 62551 375888 211127

% RSD NA 0.45 0.51

day and different GC system. The mean % Assay ob-

tained was compared with mean % Assay of precision

study. The relative standard deviation (RSD) was less

than 2%. Refer Table 6.

The method was not affected by deliberate variations

such as flow rate and column temperature.

3.8. Solution Stability

The % assay of Camylofin dihydrochloride and Nimesu-

lide were checked in the test solutions. The % RSD of

assay of Camylofin dihydrochloride and Nimesulide

during solution stability experiment was within 1.0. No

significant changes were observed in the content of Ca-

mylofin dihydrochloride and Nimesulide during solution

stability experiment. Sample solutions used during the

experiment were stable up to the study period of 72 hours.

The results are reported in Table 7.

Figure 6. Linearity plot of camylofin dihydrochloride and

imesulide. n

R. K. R. SINGH ET AL.

950

Table 3. Results of linearity study.

Analyte Slope Intercept Correlation coefficient (r2) (n = 7)

Camylofin dihydrochloride 0.0244 –0.0713 0.9998

Nimesulide 0.0068 –0.0049 0.9999

Table 4. Accuracy of method.

Analyte Recovery

Level (%) Amount added

(µg·mL–1) Amount recovered

( µg·mL–1 ) RSD (%)

N = 3 (%) Recovery

Camylofin dihydrochloride 50

100

150

125.12

250.24

375.36

124.88

251.91

375.94

0.32

0.22

0.21

99.81

100.67

100.15

Nimesulide 50

100

150

250.74

501.48

752.22

249.15

500.58

751.45

0.25

0.24

0.19

99.37

99.82

99.90

Table 5. Results of precision experiment.

Results Camylofin dihydrochloride Nimesulide

Drug found in m g/tab (mean) 49.81 100.22

% Mean Assay 99.62 100.22

% RSD 0.47 0.29

Table 6. Ruggedness of assay experiment.

Results Camylofin dihydrochloride Nimesulide

Drug found in m g/tab (mean) 50.23 100.64

% Mean Assay 100.46 100.64

% RSD 0.55 0.42

% Difference wr.t. Precision 0.84 0.42

Table 7. Results of solution stability.

Condition % Assay of Camylofin

dihydrochloride % Difference w.r.t.

initial assay % Assay of Nimesulide % Difference w.r.t.

initial assay

Initial 99.6 NA 100.2 NA

24 hours 99.4 0.2 100.0 0.2

48 hours 99.0 0.6 99.5 0.7

72 hours 98.4 1.2 99.1 1.1

3.9. Stress Testing (Forced Degradation Study)

The % degradation of Camylofin dihydrochloride in acid

hydrolysis, base hydrolysis, oxidation, thermal and

photolytic was 10.43, 18.20, 4.87, 1.53 and 2.20 respec-

tively with respect to the control sample. The % degrada-

tion of Nimesulide in acid hydrolysis, base hydrolysis,

oxidation, thermal and photolytic was 4.34, 21.99, 3.97,

1.97 and 7.34 respectively with respect to the control

sample. The mass balance was found to be more than

97.0%. The peaks of the degradation products were well

resolved from the principle peaks. The results of stress

studies are tabulated in Tables 8(a)-(b).

4. Conclusions

The method after being completely validated showed

atisfactory data for all the method validation parameters. s

R. K. R. SINGH ET AL.

951

Table 8. (a): Summary of forced degradation results for Camylofin dihydrochloride; (b): summary of forced degradation

results for Nimesulide.

(a)

Stress condition Time % Assay of Camylofin 2HCl % Degradation

w.r.t control Mass balance

(% assay+ % degradation products)

Control NA 99.74 NA 100.14

Acid hydrolysis (2 M HCl) 48 h 89.31 10.43 98.01

Base hydrolysis (1 N NaOH) 3 h 81.54 18.20 97.52

Oxidation (3% H2O2) 48 h 94.87 4.87 99.19

Thermal (80˚C) 5 day 98.24 1.53 98.21

Light (photolytic degradation) 5 day 97.54 2.20 98.54

(b)

Stress condition Time % Assay of Nimesulide% Degradation

w.r.t control Mass balance (% assay+ %

degradation products)

Control NA 100.21 NA 100.14

Acid hydrolysis (2 M HCl) 48 h 95.87 4.34 98.01

Base hydrolysis (1 N NaOH) 3 h 78.22 21.99 97.52

Oxidation (3% H2O2) 48 h 96.24 3.97 99.19

Thermal (80˚C) 5 day 98.24 1.97 98.21

Light (photolytic degradation) 5 day 92.87 7.34 98.54

Method validation study showed that the method is spe-

cific, linear, accurate, easily reproducible and can be

used for simultaneous determination of camylofin dihy-

drochloride and Nimesulide from pharmaceutical prepa-

rations. Stress testing showed that all degradation prod-

ucts were well separated from Camylofin dihydrochlo-

ride and Nimesulide, confirming its stability indicating

capability. The method seems to be suitable for quality

control in the pharmaceutical industry because of its sen-

sitivity, simplicity and selectivity.

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