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Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1335
SIMULTANEOUS SPECTROPHOTOMETRIC DETERMINATION OF
NORFLOXACIN AND METRONIDAZOLE FROM
PHARMACEUTICAL DOSAGE FORM BY ABSORBANCE RATIO
METHOD
1*Pralhad Rege, 2Kyle Meyers and 3Neha Kapadia
1Assistant Professor and Research guide, Dept of Chemistry, St. Xavier’s College, Mumbai,
5- Mahapalika Marg, Mumbai-400001, Maharashtra, India.
2Lecturer, Dept of Chemistry, St. Xavier’s College, Mumbai, 5- Mahapalika Marg, Mumbai-
400001, Maharashtra, India.
3Lecturer,, Dept of Chemistry, St. Xavier’s College, Mumbai, 5- Mahapalika Marg,
Mumbai-400001, Maharashtra, India.
ABSTRACT
In present study, a successful attempt has been made to develop a
simple, sensitive, accurate and economical spectrophotometric method
for simultaneous estimation of Norfloxacin and Metronidazole in tablet
dosage form. The method is based on Q-analysis (absorbance ratio
method). Norfloxacin and Metronidazole shows absorbance maxima at
275 nm and 325 nm in 0.1N sodium hydroxide, respectively. The
linearity was obtained in the concentration range 2 μg / mL to 20 μg /
mL for Norfloxacin at 275 nm and 2.5 μg / mL to 25 μg / mL for
Metronidazole at 325 nm. In given method the concentration and
amount of drugs was determined by using ratio of absorbance at iso-
absorptive point (290 nm) and at λmax of one of the drugs (275 nm).
The results of the analysis have been validated statistically and by
assay, recovery studies. Therefore, the proposed validated method can be successfully applied
for routine quality control analysis and simultaneous determination of norfloxacin and
metronidazole in combined drug formulations.
KEYWORDS: Norfloxacin, metronidazole, absorbance ratio method, spectrophotometric
method and validation.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 12, Issue 1, 1335-1345 Research Article ISSN 2278 – 4357
*Corresponding Author
Dr. Pralhad Rege
Assistant Professor and
Research guide, Dept of
Chemistry, St. Xavier's
College, Mumbai, 5-
Mahapalika Marg, Mumbai-
400001, Maharashtra, India.
Article Received on
02 Nov. 2022,
Revised on 23 Nov. 2022,
Accepted on 13 Dec. 2022
DOI: 10.20959/wjpr20231-23849
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1336
Some abbreviations used in the paper
NF- Norfloxacin
MNZ- Metronidazole.
INTRODUCTION
In the topical countries like India, the major problems of health arise due to improper
lifestyle, unhealthy environmental conditions, unhygienic and substandard food. Infections
caused by the microorganisms like, fungi, protozoa, are most common. Drugs with antifungal
and antiprotozoal activity have been used in the treatment of the same.
Both the drugs in combined dosage formulations are available in the market and has gained
great acceptance in diarrhea, bacterial and protozoal infections as this combination is broad
spectrum antibiotic. In many cases, drugs with two active ingredients are prescribed to the
patients to have an added advantage. Many of these antibacterial drugs are found in
combination with antifungal and antiprotozoal drugs which are highly effective against
fungal and protozoal infections. It is highly effective for bacterial and protozoan infections
and is available in the tablet form. A literature surveys reveals few Chromatographic methods
i.e., HPLC HPTLC, and very few spectrophotometric methods (simultaneous equation
methods) for the simultaneous determination of Norfloxacin and Metronidazole. Very little
attention has been paid to the use of electroanalytical method.
The present study a successful attempt has been made to quantify both these drugs i.e.,
Norfloxacin and Metronidazole simultaneously in pharmaceutical dosage formulation by a
spectrophotometric method namely, Q-analysis (absorbance ratio method). The proposed
method has been validated as per ICH guidelines.
Norfloxacin
(IUPAC) name:1-ethyl- 6- fluoro- 4-oxo- 7-piperazin- 1-yl- 1 H- quinoline-3-carboxylic acid
Formula: C16H18FN3O3
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1337
Molecular Weight: 319.33 g
Metronidazole
(IUPAC) name2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol
Formula: C6H9N3O3
Molecular Weight: 171.15 g
MATERIALS AND METHODS (EXPERIMENTAL)
Instrument
Systronics UV – VIS spectrophotometer type 118, single beam spectrophotometer with
spectral width of 2.0 nm. Wavelength accuracy is + 1.0 nm and a pair of 10 mm matched
quartz cells was used to measure the absorbance. The wavelength ranges from 200 – 999.9
nm obtained by a 1200 grooves/mm grating with Czerny – Turner Mount. Fused Silica
deuterium lamp and tungsten iodide sources are used to cover the wavelength range. High
sensitivity, wide range, reliable silicon photocell is used as the detector. The photometric
readings of %T (Percent Transmittance), ABS (Absorbance) and CONC (Concentration) are
displayed on an electronic display.
Materials
Pure standard of norfloxacin and metronidazole was obtained from Cipla pharmaceutical Pvt.
Ltd. The tablet formulations of the said combination were purchased from a local pharmacy
(The label claim contained 400mg of norfloxacin and 500mg of metronidazole.) All the
solutions were prepared in double distilled water. All the reagents were of AR grade.
Preparation of standard and sample solutions
Standard drug solution
A Primary Stock solution of Standard NF was prepared by dissolving 20 mg in 50 mL of
0.1N NaOH to obtain a 400ppm solution and Standard MNZ was prepared by dissolving 25
mg in 50 mL of 0.1N NaOH to obtain a 500ppm solution. 5 mL of the primary stock solution
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1338
was diluted to 100 mL with 0.1N NaOH. 20 μg / mL and 25μg / mL of standard NF and
Standard MNZ, respectively, was used for the preparation of the calibration curve.
Sample Solution
Commercial sample (Nor-metrogyl (Lekar Pharma Limited) containing of norfloxacin and
metronidazole in combination was procured. Sample contained a label claim of 400mg of
norfloxacin and 500mg of metronidazole per tablet. Ten tablets of given sample were
weighed and powdered for the analysis. The powder (57 mg) equivalent to 20 mg of
norfloxacin and 25 mg of metronidazole was accurately weighed and transferred to a 50 mL
standard flask and the sample was dissolved in 0.1N NaOH. The mixture was then mixed and
allowed to settle to room temperature. The sample mixture was then filtered with Whatman
filter paper no. 41 to removed additives which did not dissolve in the solvent (0.1N NaOH) use. 5
mL of this solution was then diluted with 0.1N NaOH to 100mL. Absorbances of these solutions were
measured at appropriate wavelengths, i.e. at 275nm and 290 nm.
Absorbance Ratio Method (Q-Analysis)
Absorbance ratio method uses the ratio of absorbance at two selected wavelengths one at iso-
absorptive point and other being the λmax of one of the components. From the overlay
spectra of two drugs, it is evident that Norfloxacin and Metronidazole show iso-absorptive
point at 290nm and the second which is the λmax of Norfloxacin (275 nm). The
quantification of both drugs was carried out at the selected wavelengths. i.e. 275 nm and 290
nm.
ANALYTICAL METHOD VALIDATION
System Suitability
System suitability tests are used to ensure reproducibility of the equipment. The System
suitability was determined with a solution containing 10 μg / mL of NF and 12.5 μg / mL of
MNZ i.e. at the working concentrations of NF and MNZ. For this 5 mL of Standard solution
was diluted to 10mL with 0.1N NaOH. These solutions were analyzed with five replicates
and the mean was used for the whole calculation.
Specificity
The specificity of method was confirmed by recording the spectra of both the standard
solution and the drug sample solutions. The spectra obtained from the drugs sample solution
were found to be identical to those obtained for standard solution.
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1339
The addition of the standard solution to the drug sample solution for recovery analysis
did not change the characteristics of spectra . This gives the validity of method for
the determination of both drugs from combined pharmaceutical formulation.
Linearity and Range
A good linearity was achieved for NF and MNZ in the concentration ranges of 1-10 μg/ml
and 2-20 μg/ml respectively. The calibration curves were constructed with concentration (C)
against absorbance of both drugs. Linearity graphs are indicated in (Fig 1 and Fig 2).
Limit of detection (LOD) and Limit of Quantification (LOQ)
The signal-to-noise ratio of 3:1 and 10:1is usually used to establish LOD and LOQ,
respectively. For LOD and LOQ analysis twenty readings for blank recorded then their
standard deviation calculated i.e. for LOD= (SD×3+ Mean absorbance of Blank) and for
LOQ= (SD×10 + Mean absorbance of Blank.).
Intra-day and Inter-day precision
The intra-day and inter-day precision was used to study the variability of the method.
According to USP, ruggedness is the degree of reproducibility of the results obtained under
variety of test conditions. It is expressed as percent RSD. It is also called as reproducibility or
intermediate precision. It is the analysis of same sample under variety of normal test
condition such as different laboratories, different analysts, different instruments, different lots
of reagents, different days etc.
It was checked by recording the absorbance as well as spectra of standard solutions of NF and
MNZ i.e. working concentrations for NF (8 μg/ml, 10 μg/ml, 12 μg/ml) and for MNZ (10
μg/ml, 12.5 μg/ml, and 15 μg/ml) with five replicates (both at intra-day (five times within 24
hour) and inter-day (two times each. during 3 days intervals) to check the precision.
Sensitivity
Sensitivity refers to the smallest quantity that can be accurately measured. It also indicates
the capacity of the method to record or measure small variations in concentrations. In the case
of spectrophotometric methods, a parameter known as “Sandell’s Sensitivity” is used to
evaluate, the sensitivity of the method. It is the amount required to give an absorbance of
0.001 unit in one square centimeter path.
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1340
Assay
For estimation of drugs from commercial formulations, twenty tablets were weighed
accurately and finely powdered. 57 mg of powder which is equivalent to (20 mg of
norfloxacin and 25 mg of metronidazole) was accurately weighed and transferred to 50ml
volumetric flask and dissolved in 25ml of 0.1N NaOH and sonicated for 10 mins. The
solution was filtered through whatman filter paper No.41 and residue was washed thoroughly
with given solvent. The filtrate and washings were combined in 50ml volumetric flask and
diluted with 0.1N NaOH. The absorbance was measured at 275 nm and 290 nm and finally
concentration of both the drugs was calculated using equations 1 and 2. The amount of NF
and MNZ present in the given sample found out. Assay studies were carried out at three
different levels i.e. 80%, 100%, 120% level.
The validated spectrophotometric method was used for the simultaneous quantitative
determination of NF and MNZ from the formulation. Quantification has been done by
absorbance ratio method (ARM). Two samples of different brands were used for the
determination. The absorptivity coefficients of these two drugs were determined by using
calibration curve equations 1 and 2 mentioned earlier.
Further two more equations were derived with the equations (1) and (2) which are given
below:
CN= Qm - QM X A ……………… (1)
QN - QM AN1
CM= Qm – QN X A ……………… (2)
QM – QN AM1
Where,
CN = Concentration of Standard NF
CM = Concentration of Standard MNZ
Qm= Ratio of absorbance of mixture at max (275 nm) and iso(290 nm)
QN = Ratio of absorptivity of N at max (275 nm) and iso(290 nm)
QM = Ratio of absorptivity of M atmax (275 nm) and iso(290 nm)
A = Absorbance of mixture at iso-absorptive point (290 nm)
AN1 = E (1%, 1 cm) of N at iso(absorptivity of N drug at iso-absorptive point)
AM1 = E (1%, 1 cm) of M atiso(absorptivity of M drug at iso-absorptive point)
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1341
Accuracy (Recovery)
The recovery was used to evaluate the accuracy of the method. Accuracy of the method was
determined using the method of standard addition. A fixed volume of standard NF and MNZ
solution was mixed with different concentrations of preanalyzed sample solutions and
mixtures were analyzed by proposed method. The percent recovery was determined at
different levels i.e. from 80% to 120% level.
Table 1: Method validation parameters for the determination of Norfloxacin and
Metronidazole.
Parameters
ARM
Norfloxacin
Metronidazole
At 275 nm
At 290 nm
At 275 nm
At 290 nm
Correlation coefficient (R2)
0.9991
0.9994
0.9993
0.9990
Linearity range (µg/mL)
1to 10 µg/ml
2 to 20 µg/ml
System suitability (n=5) %RSD
Less than 1 %
Less than 1 %
LOD (μg mL-1)
0.0625 µg/ml
0.0625 µg/ml
LOQ (μg mL-1)
0.125 µg/ml
0.125 µg/ml
Intraday precision (n=5) %RSD
Less than 1 %
Less than 1 %
Interday precision (n=5) %RSD
Less than 1 %
Less than 1 %
Assay
98% to 102%
98% to 102%
Recovery
98% to 102%
98% to 102%
Sandell’s Sensitivity (μg / cm2)
At 290 nm = 0.012
At 290 nm = 0.039
Molar Absorptivity(mole/lit/cm)
At 290 nm =8500
At 290 nm= 2560
ARM: - Absorbance Ratio Method
Sample Used
Brand Name
Nor-metrogyl (Lekar Pharma Limited)
Batch No.
3ANK2037
A.P.I.
Metronidazole- 500mg
Norfloxacin - 400mg
Excipients
q.s.
Colour
Light Melon Orange
Average weight of tablet
1.143 g
Table 2: Result of Assay studies of Norfloxacin and Metronidazole.
Method (SEM)
Norfloxacin
Metronidazole
Labeled claim (mg)
400mg
500mg
Drug found in mg
398.8 mg
492.1 mg
% Assay
99.7%
98.42 %
% RSD (n=5)
Less than 1 %
Less than 1 %
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1342
Table 3: Results of Recovery Experiment.
Method
Name
Level of %
Recovery
% Recovery
Found
Standard Deviation
(SD)
% (RSD)
(N=5)
NF
MNZ
NF
MNZ
NF
MNZ
ARM
0
100.6%
100.2%
0.0196
0.0682
0.582
0.443
80 %
99.8%
99.4%
0.0337
0.1021
0.687
0.972
100 %
101.01%
101%
0.0083
0.0682
0.139
0.538
120%
100.2%
99.7%
0.0151
0.0859
0.223
0.584
RESULTS AND DISCUSSION
1. The proposed method was found to be simple, accurate, sensitive, precise and
economical.
2. The given method has been validated as per as ICH guidelines. Method validation
parameters for the determination of Norfloxacin and Metronidazole is given in (Table.1)
i.e. system suitability, the mean % RSD was found to be less than 1.for both NF and
MNZ, which was found to be well within the acceptable limit. Regression analysis
(Linearity and Range) for both the drugs was > 0.999 indicate the precision of the
validated method. Lower value of LOD and LOQ confirm the sensitivity of the specified
method.
3. Lower values of Sandell’s Sensitivity and higher values for Molar Absorptivity indicates
effectiveness of the method for routine use.
4. Repeatability and also inter and intra-day precision was studied where % RSD was found
to be less than 1) for both drugs. (Table.1)
5. Accuracy was determined for the given method by calculating the percent recovery at
different levels i.e. from 80% to 120% level. The percentage recovery at three different
was found to be from 98.00 % to 102.00 % for both the drugs. (Table 3).
6. Quantification of drugs from formulation was done out by assay analysis for the given
method. Assay studies were carried out at three different levels i.e. 80%, 100%, 120%
level. The percentage assay at three different levels for NF and MNZ was found to be
from 98.00 % to 102.00 %. (Table 2).
CONCLUSION
Application to Analysis of Pharmaceutical Formulation
All these factors mentioned in the above section lead to the conclusion that, above validated
method mentioned in this paper for simultaneous estimation of norfloxacin and
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1343
metronidazole by spectrophotometric technique can be recommended for routine quality
control analysis in their combined pharmaceutical dosage forms.
ACKNOWLEDGEMENT
We thank to our Department of Chemistry St. Xavier’s college for providing us all the
Necessary instrumentation facilities and their technical assistance.
Conflicts of Interest
Authors have declared that no competing interests exist.
Data Availability
All the necessary / required data have been provided in the tabular form.
Funding Source
There is no funding agency is involved in the same. All the necessary expenditure was bear
by all the authors and the students involved
Figure 1: Linearity Graphs of NF and MNZ at 275 nm (λmax of one of the drugs).
Figure 2: Linearity Graphs at Iso-absorptive point (290 nm).
Rege et al. World Journal of Pharmacy and Pharmaceutical Sciences
www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1344
Figure 3: Overlay spectra of Norfloxacin (275nm), Metronidazole (325nm) and Iso-
absorptive point (290nm).
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www.wjpps.com │ Vol 12, Issue 1, 2023. │ ISO 9001:2015 Certified Journal │
1345
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