Advancement and Validation of new Derivatives Spectrophotometric Method for Individual and Simultaneous Estimation of Diclofenac Sodium and Nicotinamide

Derivative spectrophotometry, which is primarily based on the first and second derivative spectra of absorption, was applied for individual and simultaneous spectrophotometric determination of diclofenac sodium (DS) and nicotinamide (NAM) in the ultraviolet region. The method depends on 1st and 2nd derivative UV spectrophotometry, with the amplitude of peak-to-base line, peak to peak, the area under peak at selected spectrum intervals and zero-crossing at certain wavelengths for each compound measurement. Under optimal conditions, a linear working range of 5-80 μg.ml-1 and 10-140 μg.ml-1 for (DS) and (NAM) with correlation coefficient R2 between 0.9938-0.9998. The mean % recoveries were found to be in the range of 97.95-102.50 % for two drugs. The proposed technique has been effectively applied to the estimation of (DS) and (NAM) in pharmaceutical formulations.


INTRODUCTION
Derivative spectrophotometry is a technique of incredible usefulness for separating both qualitative and quantitative information from spectra made of uncertain bands by utilizing the first or higher derivatives of absorbance concerning wavelength 1 .This technique offers different advantages over the customary absorbance methods, for example: the separation of the sharp spectral features over the huge bands and the improvement of the resolution of overlapping spectra 2 what's more, allow the assay of certain analyses from complex mixtures or matrices via mathematical interpretation of the absorption signal 3 .UV-Vis spectroscopic approach for analysis is broadly utilized within the determination of drug in pharmaceutical preparations and for separation studies, which disposes interference from the formulation of matrix by utilizing zero-crossing techniques 4 .Many reports were found for the analysis in individual form particularly for DS and NAM including spectrophotometric method5-8, chromatographic method 9,10 , flow injection 11 , voltammetry 12 , potentiometric 13 , electrochemical method 14 , capillary electrophoresis 15,16 .

Instruments
UV-Visible double beam spectrophotometer with 10 mm quartz cell Shimadzu 1800, a personal computer.

Chemicals and reagents
Pharmaceutical grade DS and NAM powder received in pure form (99.99 %) was provided as an endowment from the State Company for Drug manufacture and Medical Appliances Samara-Iraq (SDI), methanol (99.7 %) provide by (SCR).All chemical substance utilized were of analytical grade.

preparation of standard stock solution (200 µg. mL -1 ), Diclofenac sodium and nicotinamide
The standard solution of (DS) and (NAM) were prepared by dissolving accurate weighted 20.0 mg of pure drug in 10.0 ml of methanol and further diluted to 100 ml with distilled water.

preparation both Diclofenac sodium and nicotinamide from dosage form
The content of 10 Tablets and capsules was grinded and blended well.Take accurately weighted from a specific quantity of the fine powder to give an equivalent to 50 mg for DS Tablets and 20 mg for NAM capsules and dissolve in ten mL of methanol then diluted to the mark with distilled water in a volumetric flask 100 ml.The solution was filtered by utilizing filter paper (Whatman No.41) to evade any undissolved or suspended components before use; also the first part of the solution filtrate was rejected.

procedures Individual determination of Diclofenac sodium and nicotinamide
In 10 mL calibrated flask transfer aliquots of (DS) standard solutions containing 50-800 μg (or NAM standard solutions containing 100-1400 μg), and dilute with 10% methanol solution to the mark.The spectrum for each solution was recorded against a 10% methanol solution as blank.Zero order spectrums were then manipulated for each to get its first derivative (D1) and second derivative (D2).

Simultaneous determination of Diclofenac sodium and nicotinamide
(I) The content of a series of 10 mL calibrated flasks containing different amounts (50-800) μg of DS standard solutions and 50 μg of (NAM) solution the mixture was then diluted with 10% methanol solution to the mark.Record the spectrum for each solution against a 10% methanol solution as a blank.The recorded spectra were then manipulated to get D1 and D2.
(II) The content of a series of 10 mL calibrated flasks containing different amounts (100-1400) μg of NAM standard solutions and 100 μg of DS solutionthe mixture was then diluted with 10% methanol solution to the mark.The spectrum for each solution was recorded against a 10% methanol solution as a blank.The recorded spectra were then manipulated to get D1 and D2.

Absorption spectra
The absorption spectra were recordof each compounds DS, NAM then their mixtureagainst 10% methanol solution as a blank.Fig. 1 (a) demonstrates the absorption spectrum of DS 10 μg.mL -1 with maximum wavelength at 276 nm, (b) demonstrates the absorption spectrum of (NAM) 50 μg.mL -1 with two maxima wavelength of absorption at 214 nm and 262 nm.The aggregate spectrum to the mixture is demonstrating in (c) with two λmax 212 and 266 nm.

First derivative mode and 2nd derivative mode
The derivative spectra 1st, 2nd order of DS and NAM and for their blend are appeared within Fig. 2, Fig. 3 respectively.Clearly there is a large overlap in spectra of diclofenac sodium and nicotinamide hence, their determination, making use of the zero order absorption measurements, at the point when present in the same solution it is very difficult when utilizing customary two wavelengths of λmax or the tangential base-line approach strategies 17 .Then again, derivative technique is of a specific utility in finding the concentration of single component in a blends, with a large overlapping in spectrum.Consequently, both first, second order derivative spectrophotometric methods have been applied.I n the existing wor k, graphically peak-to-baseline, peak to peak, zero-crossing technique in addition to peak area were utilized to deal with derivatives spectra to complete the data.Truth be told, Theany one of these techniques in the 1st and 2nd derivative modes indicate good proportion to diclofenac sodium and nicotinamide amounts in their blends.
Figure 4 and Fig. 5 illustrate sets of 1st order spectra of medley containing (5-80) μg.ml -1 of diclofenac sodium in the existence of (5 μg.ml -1 ) nicotinamide and (10-140) μg.ml -1 of nicotinamide Diclofenac sodium in the existence of (10 μg.ml -1 ) diclofenac sodium respectively.Fig. 4 indicate that when the amount of nicotinamide is kept constant and varied the concentration of diclofenac sodium, the peak-to-base line, peak to peak, peak areas and zero crossing of nicotinamide were proportional to the concentration of diclofenac sodium.Moreover, the same features were found for the determination of nicotinamidein Fig. 5, i.e. area under peak, peak-to-baseline, peak to peak and zero crossing of diclofenac sodium were in proportion to the concentration of nicotinamide (Table 1 and 2).In the further sets of 2nd derivative of the same above blend, as illustrated in Fig. 6 and Fig. 7.By applying the same mentioned approached in account peak amplitudes (in millimeter) at peak-to-baseline, peak to peak and at zero crossing point of the other compound, and peak areas at selected wavelengths intervals enable the measurement of diclofenac sodium and nicotinamide respectively (Table 1 and 2).

Calibration graph and statistical analysis
The analytical characteristics and most statistical data for each of the proposed methods are given in (Table 1 and 2).Under optimum conditions, linear calibration curve was obtained in the range of (5-80 μg.ml -1 ) for DS and (10-140 μg.ml -1 ) for NAM with correlation coefficient values ranging between (0.9938-0.9998)

Accuracy and precision
Under the optimum conditions, the accuracy and precision of the proposed method (peak to baseline, zero cross and area under the peak for each of first and second order derivative modes) were checked.Table 3 shows the values of relative error percent and relative standard deviation percent for two different level of concentration of diclofenac sodium and nicotinamide with three replicate.

Application in dosage form
The proposed method first and second procedures were successfully applied for direct determination of diclofenac sodium in tablets and nicotinamide in capsules.The results obtained are presented in Table 4, and are in quite agreement with the spiked values.Good recovery 97.95-102.50 % and

CONCLUSION
Derivative Spectrophotometric technique was found to be sensitive, simple, rapid, economical, the results indicates that a good accuracy and precision of the proposed method, good recovery from the results applications in dosage form and it can be used in routine analysis of diclofenac sodium and nicotinamide in their pure forms, dosage form without prior separation or treatment.
In this work, 1st and 2nd derivative modes indicate good proportion to diclofenac sodium and nicotinamide amounts in pure form and in their blends, by graphically peak-to-baseline, peak to peak, zero-crossing technique in addition to peak area were utilized to deal with derivatives spectra to complete the data.