Simple Spectrophotometric Methods for the Determination of      

Zn-Sparfloxacin 1, 10-Phenanthroline Metal complex.

 

Prafulla M Sabale¹*, Jignesh Chavda¹, Vidya Sabale²

¹Dept. of Pharmaceutical Chemistry, Parul Institute of Pharmacy, Limda-391 760, Vadodara, Gujarat, India.

²Dept. of Pharmaceutics, Parul Institute of Pharmacy and Research, Limda-391 760, Vadodara, Gujarat, India.

A simple, rapid, precise and accurate UV Spectrophotometric method was developed and validated for the estimation of Zn-Sparfloxacin1,10-phenanthroline using Distilled methanol. Absorption maxima was determined and found 293nm.from these solvent followed by linearity range determination which was found within the range of 5-60 μg/ml. From the linearity range, calibration curve is prepared in the range of 5-60 μg/ml and validated the various parameters in metal complex. All the experiments were carried out in triplicate, for each parameter relative standard deviation and % recoveries was calculated and the data were statistically interpreted and were found to be significant and within the limit. The proposed methods are further evaluated for precision, robustness studies. The results indicate the methods were precise and robust.

 

 

INTRODUCTION:

Metal complexes are known as coordination compounds, consisting of a central atom or ion (metal) bonded with anions (ligands). The environment around the metal center, coordination geometry, number of coordinated ligands and their donor group is the key factor for metalloprotein to carry out a specific physiological function. [1, 2]. Metal ions can interact with many different kinds of biomolecules including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. The lipophilicity of the drug is increased through the formation of chelates and drug action is significantly increased due to effective permeability of the drug into the site of action. Interaction of various metal ions with antibiotics may enhance their antimicrobial activity as compared to that of free ligands. [3] Metal ions play a key role in the action of synthetic and natural metalloantibiotics and improve specific interactions of these antibiotics with enzyme and bimolecules. The presence of metal ions results in a higher uptake of quinolones by bacterial cells compared to that of the drug alone. Bidentate Nitrogen donors like 2,2'- Bipyridyl and 1,10-phenanthroline form strong complexes with most metal ions. In the presence of ligand such as 1,10-phenanthroline, 2, 2’-Bipyridyl, chelation was through the 3-carboxylate and the 4-oxo group. [4]

 

Fluoroquinolone, such as sparfloxacin, ofloxacin, levofloxacin, pefloxacin, norfloxacin and gatifloxacin possesses broad spectrum antibiotics and capable of forming a well-defined metal-fluoroquinolone complex due to the presence of vicinal oxo and carboxyl groups. Metal ions play a key role in the actions of synthetic and natural metalloantibiotics, and are involved in specific interactions of these antibiotics with proteins, membranes, nucleic acids, DNA and other biomolecules. The presence of metal ion results in a higher uptake of quinolones by bacterial cells compared to that of the drug alone. [5,6] The complex has been prepared between a nitrogen donor atom, quinolone moiety and metal salt. Quinoline moiety is used for preparing complex are known drugs and extension of the work can be done by changing metal having its physiological importance in body may be any inorganic or organic acid salt of zinc, manganese, magnesium, copper, ferrous and bismuth etc.

 

Our research group is working on synthesis, characterizations, analytical method development and validation of metalloantibiotics, we have synthesized different fluoroquinolone complex with N-N donars and metal salts, and are characterized on the basis of spectral interpretations. Metal complexes were also evaluated for antimicrobial activity which exhibited moderate activity against E.coli and S. aureus as compared to parent fluroquinolones. [7-10].

 

Figure 1.Chemical structure of Zn-Sparfoxacin 1, 10-Phenanthroline Metal complex

 

MATERIAL AND METHODS:

Sparfloxacin was a received as gift sample from Alembic Research Center, Vadodara. Zinc –Sparfloxacin 1,10-phenanthroline metal complex was synthesized and its structure was established with spectral studies at Dept of Pharmaceutical Chemistry, Parul Institute of Pharmacy, Vadodara. [6] All other chemicals were purchased from s. d. Fine Chemicals Ltd. Dried and distilled analytical grade methanol was used throughout UV spectrophotometric method development and validation. UV spectrophotometric method was performed on Shimadzu UV/Visible spectrophotometer, (model: 1800 Japan) having two matched quartz cells with 1 cm light path with spectral bandwidth of 2 nm and wavelength accuracy of ±0.5 nm, with automatic wavelength correction was employed. A Mettler toledo electronic balance (ML204/A01) was used for weighing the sample. An ultrasonic cleaner (Jayant Pvt. Ltd) was used for sonicating the sample solution.

 

Preparation of standard stock solution:

Standard stock solution containing 1000 μg/ml of Zinc –Sparfloxacin 1,10-phenanthroline was prepared in distilled methanol solvent by weighing accurately about 10 mg of Zinc –Sparfloxacin 1,10-phenanthroline standard and transferred to a 100 ml volumetric flask and adding 90 ml of respective solvent distilled methanol and shake for 5 minutes to dissolve and dilute to volume with respective solvent distilled methanol. The standard sample solution was prepared by transferred aliquots of standard stock solution into a series of 10 ml volumetric flask and dilute with solvent distilled methanol to get desired concentrations. The method was extended for determination of Zinc–Sparfloxacin 1,10-phenanthroline. The solutions were scanned on spectrophotometer in the UV range 200-400 nm and an absorption maximum was calculated.

 

Sample preparation for analysis

From the standard solution of Zinc –Sparfloxacin 1,10-phenanthroline (1000 μg/ml) to take the 1 ml of solution and dilute up to 10 ml with distilled methanol in volumetric flask so final 100 μg/ml concentration of Zinc –Sparfloxacin 1,10-phenanthroline was prepared.

 

Analytical procedure for method

For   method   an aliquots of Zinc –Sparfloxacin 1,10- phenanthroline ranging from 0.5-6.0 ml of standard solution were transferred into a series of 10ml volumetric flasks. The absorbances were measured at 293 nm against   blank.

 

Fig 2: Absorption maxima of Zinc–Sparfloxacin 1,10-phenanthroline in Distilled Methanol

 

Fig 3: Overlay Absorption maxima of Sparfloxacin and Zinc–Sparfloxacin 1,10-     phenanthroline  in Methanol

 

Fig 4: Linearity curve of Zinc–Sparfloxacin 1,10- phenanthroline in 293 nm

Table1: Calibration curve of Bi- ciprofloxacin 2, 2-bipyridyl

Conc. (µg/ml)	5	10	20	30	40	50	60
Absorbance	0.062	0.214	0.531	0.793	1.076	1.386	1.656

 

 

Table -2: Statistical data of Zinc–Sparfloxacin 1,10-phenanthroline Calibration curve in     293 nm

λmax(nm)	293
Beer’s law limits (μg / ml)	5-60
Molar Absorptivity (mol-1cm-1)	280
Slope	0.028
Intercept	-0.071
Regression coefficient(r2)	0.9998

 

Table 3: Precision

Intraday

Concentration	0 hr	2 hr	4 hr	Mean	S.D	%R.S.D
20	0.552	0.545	0.542	0.5463	0.005132	0.9392
30	0.797	0.800	0.802	0.7996	0.002517	0.3147
40	1.088	1.082	1.085	1.085	0.003	0.2764

 

Interday

Concentration	Day-1	Day-2	Day-3	Mean	S.D	%R.S.D
20	0.545	0.561	0.563	0.5563	0.009866	1.773
30	0.800	0.820	0.810	0.81	0.09	1.234
40	1.082	1.097	1.096	1.0916	0.0083	0.7682

 

 Repeatability

Concentration	Set1	Set2	Set3	Set4	Set5	Set6	Mean	S.D	%R.S.D
30	0.802	0.800	0.809	0.797	0.799	0.800	0.8011	0.004167	0.5201

 

Table 4: Robustness

By different wavelength

Concentration	At 292nm	At 293nm	At 294nm	Mean	S.D	%R.S.D
20	0.545	0.552	0.549	0.5486	0.003512	0.64007
30	0.777	0.791	0.792	0.7866	0.008386	1.066
40	1.051	1,072	1.075	1.066	0.01307	1.2267

 

By different instruments

Concentration	Instrument1	Insrument2	Mean	S.D	%R.S.D
20	0.563	0.561	0.562	0.001414	0.2516
30	0.814	0.820	0.817	0.004243	0.5192
40	1.090	1.097	1.0935	0.00495	0.4526

 

Fig 5: Calibration curve of Zinc–Sparfloxacin 1,10-phenanthroline in 293 nm

 

Table 5: Determination of Accuracy by Percentage Recovery Method

Concentration	Absorbance	Amount Recovered	% Recovery
Set1 20 30 40   Set2 20 30 40   Set3 20 30 40	0.496 0.784 1.062     0.491 0.767 1.069     0.493 0.780 1.073	20.14 30.42 40.35     19.96 29.82 40.60     20.03 30.28 40.75	100.71 101.42 100.89     99.82 99.04 101.51     100.17 100.95 100.87

 

Table 6: Validation parameters

Parameters	Result
Absorption maxima(nm) Linearity Range(µg/ml) Standard regression equation Absorptivity coefficient Corelation coefficient(r2) Accuracy(%recovery) Limit of quantification(µg/ml) Limit of detection(µg/ml)	293 5-60 Y=0.029X-0.071 280 0.9998 98-102 2.2041 0.7273

 

RESULTS AND DISCUSSION:

The absorption spectral analysis shows the λmax of Zinc–Sparfloxacin 1,10- phenanthroline was found to be 293 nm for which shown in Fig. 2 and Fig. 3.The calibration curve was obtained for a series of concentration in the range of 5-60 mcg/ml for these methods (Fig. 4). They were found to be linear and hence, suitable for the estimation of the Zinc–Sparfloxacin 1,10-  phenanthroline. The slope, intercept, correlation coefficient and optical characteristics are summarized in Table 2. Regression analysis of Beer’s law plot revealed a good correlation. The proposed methods were validated as per the ICH guidelines [10-12]. The recovery technique was performed to study the accuracy and reproducibility of the proposed methods. For this, known quantities of the Zinc–Sparfloxacin 1,10- phenanthroline solution were prepared and were analyzed. The recoveries studies of Zinc–Sparfloxacin 1,10- phenanthroline by the proposed methods are satisfactory and the results are shown in Table 5. The precision was measured in terms of inter day and intraday, which was determined by sufficient number of aliquots of a homogenous sample. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the proposed methods.

 

CONCLUSION:

The analytical UV Spectrophotometric methods for the estimation of Zinc–Sparfloxacin 1,10- phenanthroline was developed  and validated thoroughly.  The developed method was found to be simple with high accuracy, precision. Sample recoveries were in good agreement, suggesting validity of the method. The developed methods were stability specific and validated as per ICH guidelines. The method seems to be economical and give an acceptable recovery of the analytes, which can be directly and easily applied to the analysis of Zinc–Sparfloxacin 1,10-phenanthroline metal complex.

 

ACKNOWLEDGMENTS:

We would like to thanks Dr. Devanshu J Patel, Managing Trustee Parul Trust for providing necessary infrastructure and Dr. Rajesh K. S. Principal, Parul Institute of Pharmacy, Limda, Vadodara for offering precious suggestions.

 

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Received on 25.04.2013          Modified on 02.05.2013

Accepted on 10.05.2013         © RJPT All right reserved