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
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
NH
O
O
N
2HCl
Camylofin dihydrochloride ( C19H 32N 2O 2,2HCl)
O
NH
NO 2
S
O
O
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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