Simultaneous Spectrophotometric Estimation of Metronidazole and Norfloxacin in Combined Tablet Formulations using hydrotropy
Deepika Maliwal*1, Anurekha Jain1, RK Maheshwari2, Vidyasagar Patidar1
1Department of Medicinal and Pharmaceutical Chemistry, B.R.Nahata College of Pharmacy, Mandsaur (MP) 2Department of Pharmaceutics, G.I.T.S., Indore (MP)
*Corresponding Author E-mail: diyampharm@rediff.com
ABSTRACT:
Quantitative estimation of poorly water-soluble drugs involves use of organic solvents. Major drawbacks of organic solvents include high cost, volatiliy and toxicity. In the present investigation, hydrotropic solubilization is employed to enhance the aqueous solubilities of poorly water-soluble drugs metronidazole and norfloxacin in two-component tablet formulation for simultaneous spectrophotometric determination. Three simple, accurate and economical procedures employed are simultaneous equation method, absorbance ratio method, and dual wavelength method. All methods utilize 8.0M-urea solution as, hydrotropic solubilizing agent. In the urea solution, metronidazole and norfloxacin show maximum absorbance at a wavelength of about 320 and 275 nm respectively and isobestic point is observed at 294nm. The hydrotropic agent and additives used in the manufacture of tablets did not interfere in the analysis. The results of analysis have been validated statistically and by recovery studies.
KEY WORDS: Metronidazole, Norfloxacin, Urea, Hydrotropy, Spectrophotometry.
INTRODUCTION:
The metronidazole and norfloxacin combination has an extended range of antimicrobial spectrum. Chemically, norfloxacin1 is 1-ethyl-6-fluoro-1, 4-dihydro-4-oxo-7- (1-piperazinyl)-3-quinolincarboxylic acid and metronidazole1 is 1-(2-hydroxy ethyl)-2 methyl-5-nitroimidazole. The term hydrotropy has been used to designate the increase in solubility of various substances in water, due to the presence of large amounts of additives. A large number of poorly water-soluble drugs have been solubilized using various hydrotropic solutions- Sodium benzoate, niacinamide, sodium salicylate, sodium acetate, sodium citrate, and urea, have been employed to enhance the aqueous solubility of many poorly water-soluble drug. Maheshwari et al. have developed new analytical methods based on hydrotropic solubilization phenomenon for poorly water soluble cefixime2, frusemide3, ketoprofen4,5, salicylic acid4, tinidazole6, ofloxacin7, metronidazole8, norfloxacin8, nalidixic acid8, aceclofenac9 and hydrochlorothiazide10.
Extensive literature survey revealed HPLC and spectrophotometric methods11-17 for simultaneous and separate estimation of metronidazole and norfloxacin in binary tablet formulation, which require use of costlier and toxic organic solvents, but no method has been reported using hydrotropic solubilizationThe aim of the present work was to develop a simple, rapid, precise, reproducible and economical method for the simultaneous estimation of the binary drug formulation using simultaneous equation method, graphical absorbance ratio method and dual wavelength method.
Materials:
A Thermospectronic Model of Elico India SL-159 UV/VIS Spectrophotometer with 1cm matched quartz cells was used. Commercial two-component tablets of metronidazole and norfloxacin (formulation-Nor-metroyl of Lekar Pharma.ltd) were procured from local market. Metronidazole and norfloxacin were gift sample by Ipca Labs Ltd, Ratlam.
Preliminary solubility studies of drugs:
Solubilities of metronidazole and norfloxacin were determined at 28±1°. An excess amount of drug was added to screw capped 30 ml glass vials containing different aqueous systems viz. distilled water, buffer of pH 8, buffer of pH 8.2, 4.0 M sodium benzoate solution, 2.0M sodium acetate and 8.0M urea solution. The vials were shaken mechanically for 12 h at
Table 1- Results of analysis of marketed tablet formulation with statistical evaluation
Method |
Label claim
|
%Label claim estimated
(Mean ± S.D.) (N-3)
|
IP %
|
%Coefficient of variation
|
Standard error
|
|
Drug
|
mg/tablet
|
|||||
|
Simultaneous equations method |
MZ
|
500 |
99.63 ±0.4726 |
95-105 |
0.4743 |
0.2728 |
NOR |
400 |
99.97±0.2082 |
90-105 |
0.1202 |
0.2082 |
|
|
Absorbance ratio method
|
MZ
|
500 |
99.67±0.4163 |
95-105 |
0.4176 |
0.2404 |
|
NOR |
400 |
100.8±0.8718 |
90-105 |
0.8648 |
0.5033 |
|
|
Dual wavelength method |
MZ
|
500 |
100.1±1.007 |
95-105 |
1.0059 |
0.5812 |
|
NOR |
400 |
100.7±0.6603 |
90-105 |
0.6557 |
0.3812 |
|
MZ-Metronidazole NOR-Norfloxacin
|
Parameters |
SIM |
ARM |
DWM |
|||
|
MZ |
NOR |
MZ |
NOR |
MZ |
NOR |
|
|
Beer’s law limit (µg/ml) |
0-30 |
0-16 |
0-30 |
0-16 |
0-35 |
0-35 |
|
Sandell’s sensitivity (µg/cm2/0.001AU) |
0.013157 |
0.014992 |
0.013157 |
0.014992 |
0.015576 |
0.022631 |
|
Molar extinction coefficient(L / mol-1 / cm-1) |
3.2073´104 |
2.8245´104 |
3.2073´104 |
2.8245´104 |
3.338´104 |
2.8035´104 |
|
Regression equation |
Y = 0.0184 X + 0.0266 |
Y=0.0422X + 0.0955 |
Y = 0.0184 X+ 0.0266 |
Y=0.0422X + 0.0955 |
Y =0.0297 X – 0.0514 |
Y =0.0355 X + 0.1283 |
|
Slope |
0.0184 |
0.0422 |
0.0184 |
0.0422 |
0.0297 |
0.0355 |
|
Intercept |
0.0266 |
0.0955 |
0.0266 |
0.0955 |
0.0514 |
0.1283 |
|
Correlation coefficient |
0.9942 |
0.9885 |
0.9942 |
0.9885 |
0.9964 |
0.9842 |
28±1°, in a mechanical shaker. These solutions were allowed to equilibrate for the next 24 hr, and then centrifuged for 5 min at 2000 rpm. The supernatant of each vial was filtered through Whatmann filter paper No. 41. The filtrates were diluted suitably, and analyzed spectrophotometrically against corresponding solvent blank..
Preparation of standard metronidazole and norfloxacin solutions:
Pure 50 mg of metronidazole 40 mg norfloxacin were dissolved in 40 ml of 8.0M urea solutions separately and stirred for 15 min and the final volume of both solutions was made up to 100ml with distilled water. The solutions were filtered through Whatmann filter paper No. 41 and first few ml were rejected. 20ml of stock solutions were further diluted to 100ml with distilled water to get working concentrations of 100mg/ml and 80mg/ml of metronidazole and norfloxacin respectively.
Method 1: Employing simultaneous equation method:
20 tablets were weighed and ground to a fine powder. Tablet powder equivalent to 50 mg metronidazole (40 mg norfloxacin) was weighed and transferred to a 100 ml volumetric flask 40 ml of 8.0M urea solution was added to the flask and stirred for 15 min to dissolve the drug and the final volume was made up to 100 ml with distilled water.
The solution was filtered through Whatmann fitter paper No. 41 and the first few ml were rejected. The filtrate was diluted suitably with distilled water to get 10mg/ml metronidazole and 8mg/ml norfloxacin. The absorbances at 320 and 275 nm on the spectra of sample solutions were measured and the amount of drug present in the sample solutions was obtained from the simultaneous equation described in literature. From these concentrations, the composition of the tablet was obtained. The results of analysis and statistical validation obtained from two different brands of tablet formulations were recorded in Table –1. After 48 hr, the solutions were reanalyzed to determine chemical stability and precipitation, if any.
Method 2:Employing absorbance ratio method (Q- analysis):
This method depends upon the property that, for the substance that obeys Beer’s law at all wavelengths, the ratio of absorbances at any two wavelengths are constant values, independent of concentration or path length. In the USP, this ratio is referred to as Q ratio.
One of the two selected wavelengths is an isoabsorptive point, if possible, and the other is the wavelength of maximum absorption of one of two components. A simple straight-line graph can be drawn to show the relation between absorbance ratio and fraction of relative concentration of the two components. From the overlain spectra of the two drugs it is evident that metronidazole and norfloxacin show isoabsorptive point at 294nm. Norfloxacin shows absorbance maxima at 275 nm. Therefore the two wavelengths selected are 275 and 294nm.
Table 3-Results of the Application of the Standard Addition Technique to the Metronidazole in Tablet by the Developed Methods
|
Method
|
Amount of drug taken (mg) |
Drug added (spiked) (mg) |
%Recovery estimated (mean ± S.D.) (n-3)
|
%Coeff Icientof variation |
Standard error
|
|
SIM |
100 |
10 |
99.6±0.71 |
0.71 |
0.41 |
|
ARM |
100 |
10 |
100.4±0.58 |
0.58 |
0.34 |
|
DWM |
100 |
10 |
99.1±0.81 |
0.82 |
0.47 |
|
SIM |
100 |
15 |
99.1±0.63 |
0.64 |
0.36 |
|
ARM |
100 |
15 |
99.7±0.80 |
0.80 |
0.46 |
|
DWM |
100 |
15 |
99.3±0.77 |
0.77 |
0.44 |
Table 4 Results of the Application of the Standard Addition Technique to the Norfloxacin in Tablet by the Developed Methods
|
Method
|
Amount of drug taken (mg) |
Drug added (spiked) (mg) |
%Recovery estimated (mean ± S.D.) (n-3)
|
%Coeff Icient of variation
|
Standard error
|
|
SIM |
100 |
10 |
99.9±0.69 |
0.69 |
0.39 |
|
ARM |
100 |
10 |
99.9±0.76 |
0.76 |
0.43 |
|
DWM |
100 |
10 |
99.9±0.91 |
0.92 |
0.53 |
|
SIM |
100 |
15 |
99.7±1.38 |
1.38 |
0.80 |
|
ARM |
100 |
15 |
99.3±0.77 |
0.77 |
0.44 |
|
DWM |
100 |
15 |
99.1±0.68 |
0.70 |
0.39 |
SIM-Simultaneous equation method, ARM-Absorbance ratio method, DWM- Dual wavelength method, CM-Calibration method.
Table 5 Result of Robustness (Metronidazole plus
Norfloxacin) (n=3)
|
|
SIM |
ARM |
DWM |
|
METRONIDAZOLE |
|||
|
Amount found (Mean% ± S.D.)
|
99.45± 0.68 |
99.03± 0.58 |
99.12± 0.62 |
|
Standard error |
0.3926 |
0.3348 |
0.3579 |
|
Precision, (%C.V.) |
0.68 |
0.58 |
0.62 |
|
NORFLOXACIN |
|||
|
Amount found (mean% ± S.D) |
100.04± 0.45 |
100.30± 0.59 |
99.89± 0.17 |
|
Standard error |
0.2598 |
0.3406 |
0.0981 |
|
Precision, (%C.V.) |
0.44 |
0.58 |
0.17 |
Fig .1-Overlain spectra of pure drugs metronidazole and norfloxacin
Method 3: Dual wavelength method:
The absorbance difference between two points on the mixture spectra is directly proportional to the concentration of the component of interest; independent of the interfering component is the basic principle underlying dual wavelength method of analysis. Standard stock solution was prepared as described earlier of strength 100mg/ml each of metronidazole and norfloxacin separately. The six mixed standards of metronidazole and norfloxacin have been prepared were prepared by diluting different aliquots of the stock solutions of metronidazole and norfloxacin with water. . From the overlain spectra of the two drugs (Figure 1) it is evident that Metronidazole and Norfloxacin show absorption maxima at 320 and 275 nm. The wavelength at which one of the peaks of Metronidazole appears i.e., 320nm (where Norfloxacin has considerable absorbance) was selected as the first wavelength (l1) for the estimation of Metronidazole. The wavelength at which Norfloxacin exhibits absorbance equal to that at l1 was selected as the second wavelength (l2), which was 300nm. Similarly, the two wavelengths selected for the estimation of Norfloxacin were 275nm and 245nm.
Procedure for the analysis of tablet formulation: Tablet solution was prepared as described in method I and suitably diluted to get 10mg/ml metronidazole and 8mg/ml norfloxacin. The concentration of metronidazole and norfloxacin was determined by analyzing these solutions. The results of analysis and the statistical parameters are given in Table-1.
Results of solubility studies indicated that enhancement in aqueous solubilities of metronidazole and norfloxacin in 8.0M urea solution were more than 6 and 9 folds, respectively as compared to their solubilities in distilled water. Therefore, this solution was employed to extract metronidazole and norfloxacin from the fine powder of tablet formulation. In order to see the effect of pH on the solubility of metronidazole and norfloxacin, buffer solutions of pH8 and 8.2 were also made and solubilities of drug were determined. In case of metronidazole and norfloxacin, there was negligible effect on solubilities in buffer solutions, as compared to their solubilities in water. This indicates that the enhancement in solubilities of drug in hydrotropic solutions was due to hydrotropy. It is evident from Table-1 that the values of standard deviation, percent coefficient of variation and standard error for all four methods are satisfactorily low showing accuracy of the proposed methods.
Table 6. Result of Repeatability (Metronidazole plus
Norfloxacin) (n=3)
|
METRONIDAZOLE |
|||
|
|
SIM |
ARM |
DWM |
|
Intra Day |
|
|
|
|
Amount found (mean% ± S.D.)
|
99.48± 0.29 |
99.51± 0.24 |
98.30± 0.26 |
|
Accuracy, Bias(%) |
-0.6 |
-0.5 |
-1.7 |
|
Standard Error |
0.1674 |
0.1385 |
0.1501 |
|
Precision, (%C.V.) |
0.29 |
0.25 |
0.27 |
|
Inter Day |
|
|
|
|
Amount found (mean% ± S.D) |
99.55± 1.20 |
99.48± 0.36 |
99.26± 0.89 |
|
Accuracy, Bias (%) |
-0.5 |
-0.6 |
-0.8 |
|
Standard Error |
0.6939 |
0.2078 |
0.5138 |
|
Precision, (%C.V.) |
1.20 |
0.36 |
0.90 |
|
NORFLOXACIN |
|||
|
Intra Day |
|
|
|
|
Amount found (mean% ± S.D.)
|
99.37± 0.23 |
100.43±0.30 |
99.56± 0.29 |
|
Accuracy, Bias (%) |
-0.7 |
-0.6 |
-0.5 |
|
Standard Error |
0.1327 |
0.1732 |
0.1674 |
|
Precision, (%C.V.) |
0.23 |
0.30 |
0.29 |
|
Inter Day |
|
|
|
|
Amount found (mean% ± S.D) |
99.34± 0.39 |
100.95±0.28 |
99.47± 0.35 |
|
Accuracy, Bias (%) |
-0.7 |
-0.1 |
-0.6 |
|
Standard Error |
0.2251 |
0.1679 |
0.2020 |
|
Precision, (%C.V.) |
0.39 |
0.28 |
0.35 |
The optical characteristics such as absorption maxima, Beer’s law limits, correlation coefficient(r), slope (b), Intercept (a)
,
molar absorbtivity, sandell’s sensitivity of all methods are summarized in Table- 2 that shows the precision and sensitivity of the proposed methods.
Percentage recovery values in range of 98-100% with low values of standard deviation, percent coefficient of variation and standard error. The hydrotropic agent (urea) and excipients used in the manufacture of tablet did not interfere in the analysis. Drug content in the extract of 8.0M urea solution was same within 48 hr and also there was no precipitation of drug. This indicates that the extract can be analyzed within 48 hr at least with sufficient accuracy. All the analytical validation parameters were observed and they indicate the validity of method. The results are shown in (Table 3-7).
Table 7 Result of Intermediate Precision (Metronidazole plus Norfloxacin) (n=3)
|
METRONIDAZOLE |
|||
|
|
SIM |
ARM |
DWM |
|
Analyst 1 |
|
|
|
|
Amount found (mean% ± S.D.)
|
99.69± 0.72 |
99.46± 0.45 |
99.39± 0.79 |
|
Standard error |
0.4157 |
0.2598 |
0.4561 |
|
Precision, (%C.V.) |
0.72 |
0.45 |
0.79 |
|
Analyst 2 |
|
|
|
|
Amount found (mean% ± S.D) |
99.22± 0.66 |
99.95± 0.28 |
99.92± 0.48 |
|
Standard error |
0.3810 |
0.1679 |
0.2771 |
|
Precision, (%C.V.) |
0.66 |
0.28 |
0.48 |
|
NORFLOXACIN |
|||
|
Analyst 1 |
|
|
|
|
Amount found (mean% ± S.D.)
|
99.07± 0.32 |
100.08± 0.33 |
100.61± 0.49 |
|
Standard error |
0.1847 |
0.1905 |
0.2829 |
|
Precision, (%C.V.) |
0.32 |
0.33 |
0.49 |
|
Analyst 2 |
|
|
|
|
Amount found (mean% ± S.D) |
99.39± 0.72 |
100.12± 0.77 |
100.68± 0.18 |
|
Standard error |
0.4157 |
0.4457 |
0.1039 |
|
Precision, (%C.V.) |
0.72 |
0.77 |
0.18 |
Various organic solvents like methanol, chloroform, alcohol, dimethyl formamide, and benzene have been employed for the solubilization of poorly water-soluble drugs for spectrophotometric estimations. Drawbacks of organic solvents include higher cost, toxicity, pollution, and error, in analysis due to volatility. Using metronidazole and norfloxacin as a model poorly water soluble drug combination, the author want to emphasize on the use of hydrotropic solutions as solubilizing agents for simultaneous spectrophotometric estimation of drugs having lmax above 250nm.
It is thus concluded, that the proposed method is new, simple, cost effective, accurate, safe, free from pollution and precise, and can be successfully employed in the routine simultaneous estimation of metronidazole and norfloxacin in two-component tablet formulation.
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Received on 26.06.2008 Modified on 20.07.2008
Accepted on 12.10.2008 © RJPT All right reserved
Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 357-361