INTRODUCTION:

Diclofenac sodium, chemically is sodium 2-[(2,6-dichclorophenyl)-amino] phenyl acetate, is a broadly used non-steroidal anti-inflammatory drug for the treatment of inflammatory conditions such as rheumatoid arthritis, osteoarthritis and ankylosing spondilytis.^1-2 Omeprazole chemically is 5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]sulfinyl]-1H-benzimidazole.³ It is a proton pump inhibiter, used in certain conditions where there is too much acid in the stomach. It is used to treat gastric and duodenal ulcers, erosive esophagitis, and gastroesophageal reflux disease (GERD).⁴Literature survey revealed spectrophotometric and chromatographic methods reported for estimation of DFS^5-11and OME^12-19individually or in combination with other drugs.

However, there is no analytical method reported for the simultaneous determination of these drugs in a pharmaceutical formulation. Present work describes simple, rapid, accurate and precise method for simultaneous determination of DFS and OME in Capsules. The proposed method was validated as per ICH guidelines.²⁰

MATERIAL AND METHOD:

Instrument:

A double-beam Shimadzu UV- Visible spectrophotometer, 1700 Pharmaspec, with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of solution.

Material:

Standard gift samples of Diclofenac sodium and Omeprazole were provided by Maxheal Ltd Nashik and Emcure Ltd, Pune respectively. Combined dose capsule formulation containing Diclofenac sodium (100 mg) and Omeprazole (20 mg), manufactured by Themis Labs Ltd, and were purchased from local market.

Solvent used:

Methanol- AR was used as solvent:

Preparation of Standard Stock Solution:

A-Accurately weighed quantity of DFS (25 mg) was transferred to 50.0 ml volumetric flask added 30.0 ml of methanol and ultrasonicated for 5 min and diluted up to the mark with methanol. From this solution 5.0 ml is transferred to 25.0 ml of volumetric flask and diluted up to the mark with methanol. (Concentration: 100μg/ml).

B- Accurately weighed quantity of OME (25 mg) was transferred to 50.0 ml volumetric flask, added 30.0 ml of methanol and ultrasonicated for 5 min and diluted up to the mark with methanol. From this solution 5.0 ml is transferred to 50.0 ml volumetric flask and diluted up to the mark with methanol. (Concentration: 50μg/ml).

Determination of absorption maxima:

By appropriate dilution of two standard drug solutions with methanol containing 25 μg/ml of diclofenac sodium and 5 μg/ml of omeprazole were scanned separately in the range of 200- 400 nm to determine the wavelength of maximum absorption for both the drugs in solvent. Showed absorbance maxima at 248 nm for diclofenac sodium and 301.5nm for omeprazole. Both the drugs were found to have same absorbance at 255.5 nm (iso-absorptive point) (Fig. 1).

Fig 1 Overlain spectra of DFS and OME.

Method I: Simultaneous Equation Method

For the selection of analytical wavelength, standard solution of DFS (25 μg/ml) and OME (5 μg/ml) were prepared separately by appropriate dilution of standard stock solution with methanol and scanned in the entire UV range to determine λ max of both the drugs. The λ max of DFS and OME were found to be 248 nm and 301.5 nm, respectively. A series of standard solutions were prepared having concentration range of 10-50 μg/ml for DFS and 5-25 μg/ml for OME. The absorbance of resulting solutions was measured at 248 nm and 301.5 nm and calibration curves were plotted. Both the drugs obeyed linearity in the concentration range under study. Absorptivity values were then determined for both the drugs at selected wavelengths. Two simultaneous equations (in two variables C1 and C2) were formed using absorbtivity coefficient values obtained and are as follows:

A₁ =24.32C 1 + 27.3C2 (1)

A ₂ =18.25C 1 + 46.0C (2)

Where, A₁ and A₂ are the absorbance of sample solution at 248 nm and 301.5 nm respectively. C1 and C2 are the concentrations of DFS and OME measured in mg/ml, in sample solutions. Absorptivity values 24.32 and 18.25 are of DFS at 248.0 nm and 301.5 nm, respectively. Similarly, 27.3 and 46.0 are absorptivity values of OME at 248 nm and 301.5 nm, respectively. By applying the Cramer's rule to equation 1 and 2, the concentration C_DFS and C_OME, can be obtained as follows,

A₂ (27.3) – A₁ (46.0)

C_DFS = -------------------------------------- (3)

620.495

A₁(18.25) – A₂(24.32)

C_OME= ------------------------------------- (4)

620.495

Method II: Absorption Ratio Method

Q–absorbance ratio method uses the ratio of absorbance measured at two selected wavelengths, one at isoabsorptive point and other being the λ max of one of the two compounds. From the stock solution (100 μg/ml) working standard solution of Diclofenac sodium (25 μg/ ml) and Omeprazole (5 μg/ml) were prepared by appropriate dilution and were scanned in the entire uv range to determine the maximum absorbance (λ max) and (iso-absorptive point), Diclofenac sodium has λ max at 248.0 nm and omeprazole has λ max at 301.5 nm Both the drugs were found to have same absorbance at 255.5 nm (iso-absorptive point) the wavelength selected for analysis were 255.5 nm and 301.5 nm, respectively. A series of standard solution ranging from 10-50 μg/ml for Diclofenac sodium and 5-25 μg/ml for Omeprazole both were prepared and absorbance of solution was recorded at 255.5 nm and 301.5 nm, respectively, to plot a calibration curve of absorbance vs. concentration. The calibration curve was found to linear in the concentration range under study. Absorptivity values of Diclofenac sodium and Omeprazole were determined at selected wavelengths. The concentration of two drugs in the mixture was calculated by using following equation:

C_{DFS =}Qm– Qy/ Qx –Qy X A1 /ax1…… (5)

C_{OME =} Qm– Qx / Qy –Qx X A1 / ay1….. (6)

Qm =A2/A1, Qx =ax2/ax1 and Qy =ay2/ay1

Where A1 and A2 are the absorbaces of mixture at 255.5 nm and 301.5 nm and ax₁(30.42), ax₂ (18.25) and ay₁ (34.48), ay₂(46.0) are absorptivities A(1%,1cm) of Diclofenac sodium and Omeprazole at 255.5 nm and 301.5 nm.

Analysis of marketed capsule formulation:

For the estimation of drugs in the commercial formulation, twenty capsules were weighed; capsule powder was then removed and mixed. Empty capsules were again weighed; difference in the weight of filled capsules and empty capsule shells represents the amount of powder present in twenty capsules. Average weight of capsule powder calculated. A quantity equivalent to about 12.5 mg of OME was transferred to 25.0 ml volumetric flask; 15.0 ml methanol was added and sonicated for 20 min, volume was then made up to the mark with methanol. The resulting solution was mixed and filtered through Whatmann filter paper and filtrate was appropriately diluted to get approximate concentration of 5 μg/ml of OME and 25.0 μg/ ml of DFS. In method I, the concentration of both DFS and OME were determined by measuring absorbance of sample solution at 248.0 nm and 301.5 nm and using equations (3) and (4).

Table No – 1: Result of Analysis of Capsule

Method	Drug	Label Claim (mg/capsule)	Amount *Found (mg/ cap)**	*Label Claim (%)**	*S. D.(±), R.S.D.**
I	DFS	100	99.75	99.95	0.4365, 0.4367
I	OME	20	19.84	99.25	1.256, 1.265
II	DFS	100	99.63	99.63	0.1401, 0.1406
II	OME	20	20.19	100.91	0.6773, 0.6711

* Mean of six determinations. DFS=Diclofenac sodium, OME = Omeprazole S.D = Standard Deviation

Table No – 2: Result of Recovery Studies

Level of recovery	Wt of capsule powder taken (mg)	Amount of pure added (mg)		Method-I				Method-II
		Amount of pure added (mg)		Amt of drug recovered		Percent Recovery		Amt of drug recovered		Percent Recovery
		DFS	OME	DFS	OME	DFS	OME	DFS	OME	DFS	OME
80%	93.88	50.2	10.1	49.97	10.4	99.54	99.40	49.97	9.94	99.54	98.41
	93.78	50.4	10.0	50.38	09.80	99.96	98.00	49.88	10.10	98.96	101.0
	93.76	50.1	10.0	49.55	10.18	98.90	101.80	49.95	9.93	99.70	99.30
100%	93.75	62.7	12.4	62.60	12.30	99.84	99.19	62.45	12.55	99.60	101.2
	93.88	62.3	12.5	62.32	12.44	100.03	99.52	62.37	1254	100.1	100.3
	93.79	62.5	12.2	62.58	12.20	100.12	99.59	62.43	12.55	99.88	102.4
120%	93.87	75.3	15.0	75.32	14.94	100.02	99.60	74.97	14.94	99.56	99.60
	93.73	75.1	15.3	74.97	15.21	99.82	99.41	74.92	15.11	99.76	98.75
	93.85	75.5	15.0	74.99	15.04	99.32	100.26	75.09	14.84	99.45	98.93
Mean % recovery						99.72	99.64			99.61	99.98
S. D.(±)						0.4019	1.004			0.3160	1.331
C.V.						0.4030	1.007			0.3172	1.331

DFS – Diclofenac sodium, OME– Omeprazole, S.D– Standard deviation, C.V-– Coefficient of variance.

In method II, concentration of both DFS and OME were determined by measuring absorbance of sample solution at 255.5 nm and 301.5 nm and determined concentration of both the drugs. Results of capsule analysis are shown in Table No. 1

Validation:

The proposed methods were validated as per ICH guidelines.

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of diclofenac sodium and omeprazole. For all two methods, the beer lambert’s concentration range was found to be 10-50 μg/ ml and 5-25 μg/ ml for diclofenac sodium and omeprazole respectively.

Accuracy:

To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and120%). The results of recovery studies, expressed as percent recovery, were satisfactory and are presented in Table No.2

Precision:

The reproducibility of the proposed methods was determined by analyzing capsule at different time intervals on same day in triplicates (Intra-day assay precision) and on three different days (Inter-day assay precision). Coefficient of variance for intra-day assay precision was found to be 0.3566 (for Diclofenac sodium) and 1.531 (for Omeprazole) in simultaneous equation method and 0.098 (for Diclofenac sodium) and 0.5259 (for Omeprazole) in absorbance ratio method. Inter-day assay precision coefficient of variance was found to be 0.3263 (for Diclofenac sodium) and 1.104 (for Omeprazole) in simultaneous equation method and 0.1446(for Diclofenac sodium) and 0.8127 (for Omeprazole) in absorbance ratio method.

Specificity:

The specificity of the method was confirmed by comparing the λ max of standard with that of diclofenac sodium and omeprazole in the marketed formulation. There is no interference from the excipients commonly present in the capsules. Hence the developed method is specific and selective.

RESULTS AND DISCUSSION:

The methods discussed in the present work provide a convenient and reliable way for quantitative determination of DFS and OME in combined dose capsule formulation. Wavelength of maximum absorbance for DFS (248 nm) and OME (301.5 nm) were selected for analysis by simultaneous equation method (method I). In Q-absorbance method (Method II) quantitative determination was carried out at wavelength range 255.5 nm (for DFS) and 301.5 nm (for OME) Linearity for DFS and OME was observed in the concentration range of 10-50 μg/ml and 5-25 μg/ml, respectively. Percent label claim for DFS and OME in capsule analysis, by both the methods, was found in the range of 99.25 to 100.91%. Percent recovery for DFS and OME, by both methods, was found in the range of 98.0 % to 102.4 % with standard deviation well below 2 indicating accuracy of the methods. Intra-day and Inter-day precision studies were carried out by analyzing tablet formulation, by both methods, three times on the same day and on three different days, respectively. Standard deviation and coefficient of variance for intra-day and inter-day precision studies was satisfactorily low indicating high degree of precision and reproducibility of proposed methods.

CONCLUSION:

Based on the results obtained, it can be concluded that the proposed UV-Spectrophotometric method (simultaneous equation method) for simultaneous determination of Diclofenac sodium and Omeprazole is rapid, economical, accurate and precise. The utility of the developed method has been demonstrated by analysis of combined capsule formulation. Hence, the proposed method can be used for quantitative determination of pharmaceutical formulation containing these ingredients in combination.

ACKNOWLEDGEMENT:

The authors gratefully acknowledge Dr. P. D. Patil, Chairman, Dr. D. Y. Patil Vidya Pratishthan Society and Dr. A. D. Deshpande, Director of Pharmacy for providing excellent infrastructure facility to carry out this research work. Thanks also go to and Maxheal Ltd Nashik and Emcure Ltd, Pune for providing pure drug samples.

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Received on 19.05.2011 Modified on 25.05.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1172-1175