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

This research paper describes simple 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. 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.


Introduction
Camylofin dihydrochloride is 3-methylbutyl 2-(2-diethylaminoethylamino)-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-inflammatory 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].The structure of the drug is shown in Figure 1.One such combination contains 50 mg of Camylofin dihydrochloride 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, spectrophotometry and colorimetric [4][5][6][7][8][9][10][11][12][13][14][15][16].There is a GC method reported for the analysis of Camylofin dihydrochloride [17].There are, however, no publications for simultaneous determination of these drugs in such pharmaceutical preparation.Therefore a GC method was developed for determination of camylofin dihydrochloride and Nimesulide from their dosage form.The method described is simple, fast, precise and accurate for simultaneous determination of Camylofin dihydrochloride and Nimesulide 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.

Chemicals and Reagents
Anafortan N tablets manufactured by Khandelwal lab, India were procured from the market.Anafortan N tablets is a combination of Camylofin dihydrochloride 50 mg and Nimesulide 100 mg.Methanol was from Qualigens.All dilutions were performed in standard volumetric flasks.

Apparatus
The analysis was performed by using the analytical balance Mettler Toledo, the GC used is of Agilent Technologies 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 (Lindberg-Blue, USA).

Method development and optimization of chromatographic conditions:
To develop a suitable GC method for the analysis of camylofin dihydrochloride and Nimesulide in their dosage 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.Chromatographic separation was preformed with Agilent Technologies 6890 N Network Gas chromatography system, 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 chromatographic system and flame ionization detector was used for simultaneous determination of Camylofin dihydrochloride and Nimesulide.Helium was used as a carrier 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 temperature were kept at 250˚C and 280˚C respectively.The split ratio was kept at 50:1.A summary of method development and optimization is described in Table 1.

Preparation of Standard Stock Solutions
The stock solution of Camylofin dihydrochloride (1250 µg•mL -1 ) was prepared by dissolving 126.1 mg of Camylofin dihydrochloride (99.9%) in methanol in a standard 100 mL volumetric flask (stock solution A).The stock solution of Nimesulide (2500 µg•mL -1 ) was prepared 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 solution (5000 µg•mL -1 ) was prepared by dissolving 500.9mg 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.

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 homogenous powder and quantity equivalent to ten tablets were transferred in a 200 mL volumetric flask.Added about 100 mL of Methanol to the volumetric flask, shaken for 10 minutes and then sonicated for 15 minutes.The solu tion was allowed to stand at room temperature for 20 -30 minutes and filtered through Whatman no.41 filter paper.
The residue was washed with Methanol and the combined 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 solution was added to it, and solution was diluted up to the mark with methanol.The identities of both the compounds were established by comparing retention time of the sample solution with those of standard solution.

Validation Parameters
The method validation was carried out as per ICH guidelines [19].Various method validation parameters were performed.

System Suitability Test
System suitability tests are used to verify that the reproducibility of the equipment is adequate for the analysis to be carried out.System suitability tests were performed as per the USP 31 to confirm the suitability and reproducibility of the system.Various parameters such as tailing factor and resolution between the peaks were obtained.

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.

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 equations and correlation coefficients.

LOD and LOQ/Sensitivity
Sensitivity was determined by establishing the limit of detection (LOD) and limit of quantification (LOQ).The limit of detection (LOD) and limit of quantitation (LOQ) were established at signal-to-noise ratio of 3:1 and 10:1 respectively.

Accuracy
Accuracy was determined over the range 50% to 150% of the sample concentration.Calculated amount of Camylofin 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 recorded.

Precision
Repeatability was studied by carrying out system precision.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 concentrations of Camylofin dihydrochloride and Nimesulide in the product.

Ruggedness (Intermediate Precision)
Ruggedness study was demonstrated by injecting six individual sample preparations at 100% of the test concentrations of Camylofin dihydrochloride and Nimesulide on different day using another column and system.

Robustness
By deliberate change in experimental condition the resolution between Methylparaben, Camylofin dihydrochloride 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 .The effect of column temperature was studied at 170˚C and 190˚C.The injector temperature and detector temperature were kept constant.

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.

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 H 2 O 2 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 80˚C for 5 days.
To study the effect of light sample was and kept in a photostability chamber for 5 days.

System Suitability
System suitability tests are used to verify that the reproducibility of the equipment is adequate for the analysis to be carried out.System suitability tests were performed as per the USP 31 to confirm the suitability and reproducibility 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 dihydrochloride and Nimesulide from pharmaceutical formulation is shown in Figure 2 and Figure 5. System suitability parameters are mentioned in Table 2.

Specificity
No peak was observed at the retention time of Camylofin dihydrochloride, Nimesulide and Benzoic acid in diluents and Placebo chromatogram.Hence the method was specific.

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 Camylofin 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.

LOD and LOQ/Sensitivity
The LOD and LOQ of Camylofin dihydrochloride and Nimesulide was experimentally determined by six injections of each drug.The LOD of Camylofin dihydrochloride and Nimesulide was found to be 1.2 µg•mL -1 & 1.6 µg•mL -1 respectively.The LOQ of Camylofin dihydrochloride and Nimesulide was found to be 2.1 µg•mL -1 & 2.7 µg•mL -1 respectively.

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 Nimesulide.

Precision
The values of the relative standard deviation of five replicate injections of the standard solution containing both the analytes of interest were within the limits of not more than 2.0%.Refer Table 5.

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  day and different GC system.The mean % Assay obtained 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.

Solution Stability
The % assay of Camylofin dihydrochloride and Nimesulide 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 Camylofin dihydrochloride and Nimesulide during solution stability experiment.Sample solutions used during the experiment were stable upto the study period of 72 hours.
The results are reported in Table 7.

Conclusions
The method after being completely validated showed atisfactory data for all the method validation parameters.s Method validation study showed that the method is specific, linear, accurate, easily reproducible and can be used for simultaneous determination of camylofin dihydrochloride and Nimesulide from pharmaceutical preparations.Stress testing showed that all degradation products were well separated from Camylofin dihydrochloride and Nimesulide, confirming its stability indicating capability.The method seems to be suitable for quality control in the pharmaceutical industry because of its sensitivity, simplicity and selectivity.

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