Diclofenac Sodium Determination in Pharmaceutical Preparations After Complexation With Calcium and Magnesium and Molecular Modelling Study


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Introduction
Calcium is 5 th and Magnesium is 9 th most abundant elements found in the Earth's crust [1] [2]. Both are the key element of a strong nutrition and a mineral essential for life. These are vital for all livings; calcium is essential for the bones and teeth. The chief component of bones is calcium phosphate [3]. Magnesium is an important element for animals as well as plants. In plants photosynthesis process is taking place by chlorophyll that contain magnesium. [4]. Calcium and Magnesium are also essential generally for all cells of human body, the number of cellular processes functioning by calcium ion [5]. Signi cant collaboration between magnesium ions and phosphate is essential for humans. In humans, several enzymes are functioning through magnesium ion which are related compounds like RNA, ATP, and DNA. Magnesium ions form complexes with these compounds [4]. The average human body has approximately 1 kg of calcium and 20g 0f magnesium in intra and extra cellular, especially in bones [6] [7]. Maintenance of the magnesium level in serum is by renal secretion and gastric absorption [8]. Calcium is also essential for pregnant females and children for their growth. De ciency of calcium rises to poor blood clotting and rickets a condition in which the bone weakens and cracks. Calcium complements are used to inhibit calcium de cits. Vitamin D content in things is main source of calcium [9]. Hypomagnesaemia is the cause of magnesium de ciency. Due to increasing loss of magnesium by gastrointestinal and renal also cause of de ciency [10]. [11] [12].
Diclofenac, (2,(2,6-diclorophenylamino) phenylacetate) is a phenylacetic acid derivative [13], the most usually used as pain killer and is chemically used as sodium and potassiumsalts. It is a nonsteroidal anti-in ammatory drug (NSAID) that exposes anti-in ammatory and pain-relieving actions in both animals and human beings [14]. Diclofenac reacts with numerous metals to form complexes. The diclofenac ligand has been found to act as bidentate chelating agent. The diclofenac coordinate through the oxygens of carboxyl group. The molar ratio chelation is 1:2 (M:Diclofenac) with general formula (M(diclofenac)2(H2O)2]nH2O. The complex formation of diclofenac with several metal ions have been reported such as copper, iron, nickel, cadmium, calcium, magnesium and others in the presence of excess of metal ion [15]. Formation of Diclofenac-metal complex depends on nature of metal, pH of the solution and speed for the precipitation. According to survey of literature some diclofenac-metal complexes are soluble, and others are insoluble precipitates in aqueous medium. Structurally calcium ion is comparatively big ion that inclines to accept a great coordination number in the complexes. Preparation and characterization of stable monomeric Calcium and magnesium complexes with anti-in ammatory drug diclofenac is reported with formula [Ca (diclofenac) 2 [15].
Several procedures have been reported for the determination of diclofenac sodium in pharmaceutical preparations including indirect ow injection spectrophotometric method [16], indirect spectrophotometric method [17], square wave voltammatricmethod [18]. Spectrophotometry [19], Spectro uorometry [20,21], Indirect uorometric determination [22], Indirect digital image based (webcam) ame emision spectrometric [23] and indirect atomic spectrometric methods [24] [25]. The reported procedures are sensitive, but the determinations using atomic absorption spectrometry are considered as easy and equipment is also frequently available in most of analytical laboratories. Issa et al determined diclofenac sodium by oxidation of the drug by iron (III). The excess of iron (III) was extracted in diethyl ether and iron (II) remaining in aqueous solution was determined by air-acetylene ame AAS. Currentlydiclofenac sodium has been determined indirectly after complexation with copper or iron [25].
This study focuses on the formation of diclofenac-metal complexes with calcium or magnesium which are in the form of insoluble precipitates. These precipitates were extracted in chloroform or centrifuged from centrifuge machine. For the preparation of diclofenacmetal complexes, rst pure diclofenac drug was used for the formation of complex with metal ion, then used pharmaceutical preparations of diclofenac sodium. The research is indirect determination of diclofenac, therefore after complexation with calcium or magnesium in aqueous solution, the content for metal ion was analysed instead of diclofenac-metal complex.

Materials And Methods
Apparatus and chemicals: The 10 mL of glass test tubes with stand and holder, pipit ller, volumetric asks, funnel,Whatman lter papers, paper tape, iron stand,buret, conical asketc.were used. Diclofenac sodium (Novartis Pharms Jamshoro), Chloroform (Merck,Germany), Copper sulphate, ferrous sulphate, EDTA, murioxide, (Fisher, Scienti c USA) were also used. Granted reagents grade ammonium chloride, potassium chloride, boric acid, acetic acid, sodium tetraborate, ammonium acetate, sodium acetate, ammonia solution and hydrochloric acid (37%) were from Merck, Dramstadt, Germany. Stock solutions of diclofenac drug contain 1 mg/ml was prepared in distilled water. Calcium (II)and magnesium (II) solutions containing 1 mg/mL metal ions were prepared in distilled water from calcium carbonate and magnesium chloride. A few drops of acid were added before the adjusting the volume. Buffer solution (0.1 M) between pH 1 to 10 at unit interval were prepared from the following, potassium chloride adjusted with hydrochloric acid (pH 1-2), acetic acid with sodium acetate (pH 3-6), ammonium acetate (pH 7), boric acid-sodium tetraborate (pH 8-9) ammonium chloride, ammonia (pH 10). ground to ne powder and the powder containing 25 mg of diclofenac sodium was weighed and dissolved in distilled water, ltered and volume adjusted to 25 mL. Each of solution was diluted 10 times. Solution (1 mL) of Ca (II) or Mg (II) containing 100 µg each was transferred to test tube followed by 1.0 mL (100 µg) of drug solution of each sample separately. The remaining procedure was followed as above and analysed through AAS as well as EDTA titration techniques. The concentration of diclofenac sodium drug in sample was calculated from the regression equation of calibration curve: Instruments for samples analysis: The pH of solutions was measured with an Orion 420A pH meter (Orion Research Inc., Boston, USA) combined with glass electrode and reference internal electrode, Air-acetylene ame Atomic Absorption Spectrophotometer (AAS) (Perkin-Elmer AA 800 model Singapore), with standard burner head was operated at the conditions recommended by the manufacturer. The equipment was controlled by the computer with Winlabsoftware. The analysis was carried out at least in triplicate (n=3), with integration time 3 sec and delay time 3 sec. Centrifuged machine (Allegra 64R centrifuge Backman, USA), was used throughout the study.

Computational Study
In this study the computational models showing interactions of calcium with diclofenac and magnesium with diclofenac were also studied. Molecular modelling calculations were conducted using Gaussian 09W software package. The optimizations of molecular models were performed using semi empirical calculation procedure with PM6 level.

Results And Discussion
The diclofenac sodium reacts with Calcium (II) and Magnesium (II) to form metal complexes. The complexes are slightly soluble in water and turbidity or precipitate formation generally takes place in aqueous phase. Therefore, excess of Ca (II) or Mg (II) was added to diclofenac sodium solution and turbidity or precipitate formed was separated by solvent extraction or centrifugation. The decrease in the concentration of Ca (II) or Mg (II) in aqueous phase was proportional to the concentration of diclofenac sodium. The concentration of Ca (II) or Mg (II) in aqueous phase was monitored by ame atomic absorption spectrophotometeror by EDTA titration. The effect of pH on the complexation, solvent extraction or precipitation by centrifugation of Ca (II) or Mg (II) complexes with diclofenac sodium was examined within pH 2-10 at unit interval following analytical procedure. Both the metal ions indicated the formation of whitish precipitates at acidicpH. The pH for Ca (II) and Mg (II) were optimised at 3, , based on the maximum decrease in the concentration of Ca (II) and Mg (II) in aqueous solution, after the addition of same amount of standard of diclofenac solution to Ca (II) or Mg (II) solution. Chloroform, carbon tetrachloride, benzene, n-butanol and amyl alcohol were examined for the extraction of Ca (II) or Mg (II) complex of diclofenac sodium, but chloroform gave better results and was selected. The effect of concentration of diclofenac sodium on the decrease in the concentration of Ca (II) and Mg (II) was examined using both solvent extraction and centrifugation methods. A linear calibration curves were obtained with 40-200 µg/ml diclofenac sodium using either Ca (II) or Mg (II) as monitoring metal ion using either solvent extraction or centrifugation technique. The coe cient of determination (r2) for the calibration curves were obtained with0.9945-0.9938 (Fig. 1, 2) and 0.9897-0.9853 (Fig. 3, 4).
Limit of detection (LOD) measured as 3 times the standard deviation of the slope was calculated 15µg/ml using solvent extraction procedure and centrifugation technique separately using both Ca (II) or Mg (II). In order to test the validity of the calibration curves 4 test solutions of diclofenac sodium within the calibration range for each determination were analyzed and relative errors were obtained within ±4.5%. The effect of drug additives glucose, fructose, gum acacia and methylparaben for their possible interfering effects on the determination of diclofenac sodium were examined using Ca (II) or Mg (II) as indicating ions. The concentration of additives added was at least twice the concentration of the analyte. The results obtained were compared with the analyte without addition of the additives.
The relative error was obtained within ±4.9%. The repeatability of the analytical procedures was examined inter (n=3) and intraday (n=3) by the same operator at the nal concentration of diclofenac sodium at 50 µg/ml. The relative standard deviation obtained were within ±5% and ±4.5% using Ca (II) and Mg (II) as monitoring ion respectively. The analytical methods developed were applied for the analysis of pharmaceutical preparations voltral, voren, Qufen, Dicloplus and Dicloran tablets, each containing 100 mg/tablet diclofenac sodium. The results of analyses are recorded in tablets and agreed with labeled values with RSDs within 1.4-5.3% (Table 1) for all the procedures. Now comparing the all four procedures examined, all indicated acceptable limits of quantitation, but the procedure with solvent extraction indicated better linear calibration curves than centrifugation methods. Again, comparing the results of calcium (II) and magnesium (II), a better linearity of calibration curve was obtained using calcium (II). The method was compared with recent indirect spectrophotometric determination of diclofenac, based on oxidation with bromosuccimimide with linear calibration range 1-18 µg/ml [26] and HPLC analytical method for the determination of diclofenac sodium in tablets with linear calibaration range 10-200 µg/ml and lower limit of detection 12.5 µg/ml [27]. The results indicate comparable similarity, but the use of simple chemicals, together with commonly used ame atomic absorption spectrophotometer are the added advantage of the present methods.

Computational Study Results
During molecular modelling study, the models optimized were Ca interaction with diclofenac and Mg interaction with diclofenac. The Ca and Mg were interacted with carboxyl and hydroxyl oxygen atoms of diclofenac molecule. Carboxyl and hydroxyl oxygen interactions of diclofenac were selected because of negative charge which can easily interact with positive metals (Ca and Mg).

Physical Characteristics of Optimized Molecular Models
Physical properties computed during computational work included number of atoms, spin of structure, total energy, dipole moment and bond lengths. The results of all physical properties of optimized molecular models were shown in table 2.
Number of atoms measured were 67 atoms for both models. Both optimized molecular models exhibited singlet spin. Total energy measured for Ca-Diclo model was -15.83 eV and for Mg-Diclo was -11.44 eV. The total energy of Mg-Diclo model was higher than Ca-Diclo model which indicated least stability in Mg-Diclo structure than Ca-Diclo structure. Similarly, the dipole moment of Ca-Diclo was observed as 7.3956 debye which was higher than dipole moment of Mg-Diclo structure that was 5.8478 debye. Hence the optimized molecular model of Ca-Diclo is more polar than Mg-Diclomolecular model due to high dipole moment. The calculated bond length values of Ca interaction with carboxyl oxygen (Ca-Diclo carboxyl oxygen) was 2.376 A and for Ca-Diclo hydroxyl oxygen was 2.358, which indicated slight shorter and stronger bond than Ca-Diclo carboxyl oxygen. Hence the bond formation between Ca and hyxdroxyl oxygen is slightly stronger than Ca with carboxyl oxygen. On other hand, the bond lengths values of Mg-Diclo carboxyl and Mg-Diclo hydroxyl were 3.474 and 2.050, respectively. For both the metals (Ca and Mg), the hydroxyl interaction is more favourable than carboxyl interaction due to shorter bond lengths.
Consequently, from the computational molecular modelling studies it can be concluded that the complex formation between Ca diclofenac was likely to be more favourable than Mg diclofenac due to less energy and higher polarity.

Conclusion
The determination of diclofenac sodium was carried out indirectly via atomic absorption spectrophotometry from pharmaceutical preparations. Diclofenac sodium reacted with calcium (II) or magnesium (II) and the complexes were formed as precipitates. A decrease in the concentration of metal ion was correlated with the concentration of diclofenac sodium. The complex precipitates were separate out either by solvent extraction or by the centrifugation methods. The excess of Ca (II) or Mg (II), as a probe ion was determined by Atomic Consequently, from the computational molecular modelling studies have been concluded that the complex formation between Ca diclofenac was more favourable than Mg diclofenac due to less energy and higher polarity.   Figure 1 Linear calibration curve between Ca (II) and diclofenac sodium by solvent extraction Figure 2 Page 12/13 Linear calibration curve between Mg (II) and diclofenac sodium by solvent Linear calibration curve between Ca (II) and diclofenac sodium by centrifuge method Figure 4 Linear calibration curve between Mg (II) and diclofenac sodium by centrifuge method Page 13/13 PM6 level optimized molecular model of Mg complex with two Diclofenac molecules