Spectrophotometric Determination of Zn(II) in Pharmaceuticals Using (Di Amino di Hydroxy Pyrimidine) as Complexing Agent

INTRODUCTION

Zinc is the second most copious transition metal particle in the human body. It is a vital component for all creatures including individuals. Zinc is found in a few sustenances, for example, rice, oats, meat, liver, shellfish, cheeses nuts, in a few catalysts and DNA-restricting proteins. It assumes a noteworthy physiological job in individuals like mammalian proliferation, quality interpretation, insusceptible capacity, mind capacity and pathology. Zinc is a fundamental follow component to vegetation and fauna1,2, that can cause side effects of deficiency 3,4and can be dangerous when exposures surpass physiological needs5,6. It is a basic constituent of roughly 33% of all proteins7. Zinc will assume essential jobs in all replications, Gene articulation, energy transduction, cell flagging, arrangement of endo and exo skeletons and data transfer8,9. Checking of zinc in pharmaceuticals and other genuine examples is vital, normally the investigative strategies for the assurance of zinc are neutron initiation analysis (NAA), Atomic retention and outflow spectrophotometry, Inductively coupled plasma spectrophotometry (ICPMS) are the most broadly employed10. Be that as it may, in spite of the fact that these techniques are solid and touchy they experience the ill effects of the impediment of being fairly costly, tedious and not in every case promptly11,12. So as to accomplish precise, solid and touchy outcomes, spectrophotometric techniques which will in general be more affordable and work escalated are accessible choices, to those strategies requiring progressively advanced instruments. Further, the advancement of subsidiary spectrophotometry is exceptionally valuable methodology for deciding the centralization of parts in the blends with the covering spectra as it takes out a significant part of the interference13-16. Further, it was generally utilized in the analysis of pharmaceutical examples protein analysis, ecological example analysis17. The determination of trace amounts of Zinc(II) by ultra violet and visible spectrophotometry typically relies on four distinct characteristics9 apart from the divalent cations. First when coordinated by ligand in any Geometry it has stereo chemical flexibility, second in terms of the hard-soft acid-base theory it has amphoteric property, third divalent zinc(II) has no redox activity18. Finally Zn2+ has chemical stability and it undergoes the complexation with selected sensitive chromophoric chelator19. Among the many reagents reported so far with heterocyclic rings20-24 the present heterocyclic pyramidine (Scheme 1) has been shown to serve as an

Scheme1. Formation of 2,5-diamino-4,6-dihydroxypyrimidine

2,5-Diamino-4,6-dihydroxypyrimidine was set up in three stages by the strategies detailed in the literature25-26. Diethylmalonote (47.4 mL) was blended with the blend of frosty acidic corrosive and water (1:1.5) and moved in to 500 mL round base jar fitted with mechanical stirrer and thermometer. The jar was cooled in an ice shower 0.65 g of NaNO2 was included parts by keeping up the temperature around 5 0C with constant blending. After the expansion of all out NaNO2 the ice shower was evacuated blending was proceeded for four hours. The response blend was separated with ether and dissipated to get diethyl-isonitrosomalonote. Diethyl isonitrosomalonote blended with1:3 blend of acidic anhydride and frosty acidic corrosive in a three necked round base carafe fitted with mechanical stirrer, thermometer and dropping channel. 78.5 g of Zn dust was included little parts by keeping up the temperature at 40-50 0C pursued by discontinuous cooling. The response blend was sifted with suction, the filtrate and washings were vanished on the steam shower and after that cooled in an ice shower by fast mixing, diethyl acetamidomalonote isolates out as a white crystalline strong. The yield was 35-40 g, M.P was (95-97 0C). Diethyl acetamidomalonote was blended with guanidinium carbonate and EtOH refluxed for 36 h, at that point the response blend was sifted by suction. The filtrate and the washings were dissipated and cooled in an ice shower to isolate out the 5-acetamido-2-amino-4,6-hydroxypyrimidine. This was refluxed with 100 mL of 1 M HCl for 1 h at that point cooled in an ice shower sifted pursued by washings with HCl and CH3)2CO then air dried at 40 0C to secure the 2,5-diamino-4,6-dihydroxypyrimidine (25 g). The structure was affirmed from Mass, IR, H1 NMR Spectral examinations. AnalaR grades from Fisher Scientific Qualigens India. Zn(II)- arrangement Stock standard Zn(II) arrangement was set up by dissolving 0.4397 g of Zn(II) sulfate hepta hydrate in twofold refined water containing 1000 µg/mL. The arrangement was institutionalized by complexometric titration utilizing EDTA27. The working standard solutions were set up by appropriate weakening of the stock arrangement. Support solutions. Cushion solutions were set up by utilizing 0.1 M Acetic corrosive, 0.1 M sodium acetic acid derivation in the pH extend 2-8. Solutions of assorted particles. Solutions of differing particles containing 1000 µg/mL were set up by dissolving required measures of salts of the comparing particles in twofold refined water. Ligand arrangement. The reagent stock arrangement (0.1 M) was set up by dissolving 1.421 g of DADHP in ethylene glycol. This was weakened to the required focus utilizing ethylene glycol. Instruments. Elico smaller scale processor based twofold bar UV-obvious spectrophotometer SL 210 furnished with 1 cm quartz cells were utilized for spectrophotometric estimations. The pH estimations are made with Elico computerized pH meter L.1 127 model. General procedure to evaluate different parameters

To evaluate the optimum conditions for the determination of Zn(II) an aliquots of solution containing microgram quantities of Zinc(II) were taken in a series of comparison tubes followed by the addition of 2 mL of pyridine and 1 mL of 2 M HCl, then the pH was adjusted to 6 using acetic acid and sodium acetate buffer. The solutions are equilibrated with 5 mL of DADHP (3 x 10-3 M) reagent solution and made up to 20 mL with double distilled water. The absorption of the ripen-mango-colour complex was measured at 480 nm against a similarly prepared reagent blank. The composition of the complex was ensured by mole ratio, slope ratio and Job‘s continuous variation methods. The calibration plot was obtained in the range 1-6 µg. obeying the Beer‘s law.

For the above solutions the derivative spectras (1st, 2nd & 3rd order) were recorded with group size 9 and

peak heights were plotted against the amount of zinc to obtain the calibration. The amount of zinc present in the pharmaceutical sample solutions were computed from the calibration plots, both in direct and derivative spectrophotometry. The calibration plots follow the straight line equation ChemSci Trans., 2013, Y=A+Bx where x is the concentration of solution, Y is the measured absorbance or peak height, A is the intercept and B is the slope. By substituting the experimental data in least square analysis, the equations were calculated as Y=-0.01143+0.0309x for zero order spectrophotometry ∂A/∂λ=0.00396+0.00469x for first order derivative, ∂2A/∂λ2 = 0.00329+ 0.00247x for second order derivative and ∂3A/∂λ3 = 0.00362+0.00200x for the third order derivative spectrophotometry respctively.

The yellow-mango-colour complex of [Zn(II) -DADHP] absorption spectra was examined in the wave length range 400-600 nm against the reagent blank. The complex shows the maximum absorption at 480 nm in acetic acid and sodium acetate buffer (Figure 1).

Figure 2. Influence of pH on the absorbance of Zn(II)-DADHP complex (a) Direct spectrophotometry (b) First derivative (c) Second derivative and (d)

The complexation selectivity was investigated in different volumes of pyridine in the Figure 3. Effect of salting out agent on the absorbance of Zn(II)-DADHP complex (a) Effect of pyridine; (b) Effect of HClconcentrartionZn(II) =7.645x10-6 M (10 μg); DADHP = 7.645x10-4 M

To achieve the complete complexation of Zn(II) and for the maximum colouration hundred folds excess of the reagent was necessary. The ripen-mango- colour formation between the Zn(II) and the reagent was instantaneous and the colour was stable for more than 48 h, (Figure 4). Figure 4: Effect of reagent on the absorbance of Zn(II)-DADHP complex Zn(II) =7.645x10-6 M (10 μg); DADHP = 7.645x 10-4 M ChemSci Trans., 2013, Composition of the complex Elating of experimental observations in mole ratio, Job‘s continuous variation method and slope ratio method confirms Zn(II) forms the 1:6 complex with the reagent and the

Figure 5. Evaluation of the composition of Zn(II)-DADHP complex; (a) Mole ratio method; Job‘s continuous variation method; (c) Slope ratio method; Zn(II) =3x10-3 M = DADHP

Calibration plots constructed in the range 1-6 µg obeying the Beer‘s law for both direct and derivative spectrophotometry, the molar absorptivity of the complex is 0.1484x104 L.mole-1.cm-1 and the Sandell‘s sensitivity of the method was 0.04545 µg cm-2.In the derivative spectrophotometry it was clearly reflects the peak height is proportional to the amount of Zn(II) present in the solution (Figure 6). The standard deviation correlation co- efficient and other statistical parameters of the method were evaluated to ten replicate determinations (Table 1). Figure 6. Derivative spectra of [Zn (II)-DADHP] system (a) First order; (b) Second order; Third order Zn(II) µg/mL (1) 0.2; (2) 0.4; (3) 0.6

The pharmaceutical samples are prepared by an established procedure to destroy the organic matters present29-31 based on the use of 0.01 M hydrochloric acid for repeated evaporation. Finally, the residue left over was shaken well with double distilled water, sonicated and filtered. The filtrate was diluted to 100 mL, 2 mL of aliquots of the solution was used for the quantification of zinc. The measured absorbance and amplitude values are compiled with calibration plots (Figure 7) and the results are summarized in Table 2. Figure 7. Calibration curve of Zn(II) obeying the Beer‘s law (0.5-6.0 µg/mL) (I) Direct spectrophotometry; (II) A- First; b- Second; c-Third derivative spectrophotometry

The effect of various anions and cations on the determinations of Zn(II) (4 µg/mL) in the developed optimal conditions was examined and the tolerance limits were defined as the concentration of added solution causing less than ±2% relative error on the absorbance. The results are presented in the Table 3. The interference due to Cu2+, Ni2+, Cd2+, Mn2+, Fe2+ and Ag+ was eliminated using thiourea, citrate, ascorbic acid, fluoride as appropriate masking agent. But it was noticed that there was strong interference of EDTA in the determination of Zn(II).

In most of the part of the cases the recuperations are under 100%, in first, second and third subsidiary spectrophotometric estimations. Just in few it is more prominent than 100%. Further, the third subordinate spectrophotometric technique was observed to be more delicate and precise than immediate, first and second subsidiary strategies, in which the recuperations are equivalent to 100% yet more prominent than half. The loss of the objective compounds could be brought about by a few variables. The most critical one is, the examples were estimated after absorption process with acids, in the interim the estimations of standard solutions of Zn(II) for setting up the alignment bends were done with no assimilation procedure. The principle favorable position of the proposed technique is adaptability of the framework, completely robotized and easy to collect and work. Tasteful recuperations were seen in the analysis of pharmaceutical arrangements, only in third subordinate technique the outcomes are great concurrence with the affirmed qualities. Table.4. Comparison of Direct and Derivative Spectrophotometry

The results shown in the Table (4) indicates the recovery of Zn(II) from the pharmaceutical samples.

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Research Scholar of Rayalaseema University, Kurnool, Andhra pradesh