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27 TJPS 2018, 42 (1): 27-36
http://www.tjps.pharm.chula.ac.th
Intended high-performance liquid
chromatography procedure for the
quantification of norfloxacin and
its potential impurities in active
pharmaceutical ingredient and tablet
dosage forms
Bikshal Babu Kasimala1, Useni Reddy Mallu2,
Venkateswara Rao Anna1, L. Maheshwara Reddy3
1Department of Chemistry, K L University, Guntur, Andhra Pradesh, India, 2Celltrion
Pharm Inc., Seoul, South Korea, 3Department of Chemistry, YSR Engineering College
of Yogi Vemana University, Kadapa, Andhra Pradesh, India
INTRODUCTION
Norfloxacin (NFXC) (1-ethyl-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid) is a broad-spectrum antibiotic drug belongs to
a group of medicines called quinolone antibiotics, especially
4-quinolone antibacterial agents.[1] NFXC used in the
prophylaxis of bacterial infections in cirrhotic patients with
gastrointestinal hemorrhage.[2] NFXC is a fluoroquinolone
antimicrobial agent used for the treatment of uncomplicated
and complicated urinary tract infection.[3] NFXC is a derivative
of nalidixic acid, but it possesses greater antibacterial activity
against Gram-positive and Gram-negative bacteria than
nalidixic acid.[4]
DNA gyrase is the essential enzyme for DNA replication.
NFXC works by inhibiting the subunit of DNA gyrase.[5] No
abnormal side effects were observed for NFXC. The side effects
occur with the use of NFXC was common such as nausea,
diarrhea, heartburn, dizziness, stomach cramps, headache,
and weakness.[6]
Literature survey reveals that few methods were high-
performance liquid chromatography (HPLC) analytical methods
were available for the analysis of NFXC in pharmaceutical
formulations[7-9] and biological samples.[10] Rao and Nagaraju[11]
developed a HPLC method for the determination of NFXC
along with synthetic impurities, impurity A, 7-chloro-6-fluoro-
1-methyl-4-oxo-1,4-dihydro-3-qui-nolinecarboxylic acid and
ethyl-7-chloro-6-fluoro-4-oxo-1,4-dihydro-3-quinolinecarbo-
xylate (CAT). CAT and EAT are constitutional isomers. No
methods were detected so far for the analysis of NFXC along
with its synthetic impurities, impurity A [7-chloro-1-ethyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] and
Corresponding Author:
Useni Reddy Mallu, Celltrion
Pharm Inc., Seoul, South Korea.
E-mail: drusenireddymallu@gmail.
com
Received: Dec 20 2016
Accepted: Jan 16 2018
Published: Feb 20 2018
Keywords:
Forced degradation studies,
impurities analysis,
norfloxacin,
reversed-phase
high-performance liquid
chromatography method
Thai Journal of Pharmaceutical Sciences
Original Article
ABSTRACT
Objectives: Norfloxacin (NFXC) is an active pharmaceutical ingredient belongs to 4-quinolone
antibacterial agent used for the prophylaxis of Gram-positive and Gram-negative bacterial infections.
The present work aimed to develop a reliable stability-indicating reversed-phase high-performance
liquid chromatography method for the separation and quantification of NFXC along with its
synthetic impurities A and B. Results: High resolution and precise results were achieved on waters
C18 Column (250 mm × 4.6 mm; 3.5 μm particle size) with a mobile phase of methanol, 0.01M
sodium perchlorate (pH 3.1) in the ratio of 15:85 (v/v) at a flow rate of 0.8 ml/min in isocratic
mode. Eluents were detected using ultraviolet detector at 220 nm. All the validation parameters are
under the acceptance limit. Forced degradation studies were carried under acidic, basic, oxidative,
photolytic, and thermal conditions. NFXC was found to be stable in all the stress conditions and
proposed method can separate the known and unknown impurities. Conclusion: The developed
method was found to be simple, precise, accurate, stable and was found to be suitable for the routine
analysis of NFXC and its impurities A and B in bulk and pharmaceutical formulations.
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
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impurity B [7-[(2-aminoethyl)amino]-1-ethyl-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid]. Hence, we attempted
to develop a reversed-phase-HPLC method for the separation
and analysis of NFXC and its impurities A and B. The molecular
structure of NFXC, impurities A and B were given in Figure 1.
MATERIALS AND METHODS
Instrumentation
The separation and estimation of NFXC with impurities
A and B was done on PEAK HPLC (India) system. Mobile
phase was pumped into column using LC-P7000 isocratic
pump. 20 μl fixed volume sample was injected for the analysis
using rheodyne injector (model 7725) with fixed 20 μl loop.
Variable wavelength programmable ultraviolet (UV)/visible
detector was used for detecting the compounds. The detector
response signals were monitored and integrated using WS-100
Workstation software (Cyberlab, USA). Samples were injected
using Hamilton (USA) manual HPLC syringe. Double beam
UV-visible spectrophotometer (Teccomp UV-2301 - India) was
used for spectral analysis. Denver electronic analytical balance
(SI-234) was used for weighing the standards and samples.
pH of the mobile phase was adjusted using Systronics (India)
digital pH meter (Sr No S 1326).
Chemicals and Reagents
NFXC active pharmaceutical ingredient (API) and its two
impurities A and B were obtained from Dr. Reddy’s Laboratories
Private Limited, India. The marketed formulation of NFXC
(Gramoneg© - 500 mg) was purchased in local pharmacy.
Laboratory reagent grade sodium perchlorate and perchloric
acid were purchased from SD Fine-Chem Limited, Mumbai.
HPLC grade methanol, acetonitrile, and water were purchased
from Merck chemicals, Mumbai. 0.2 μ nylon membrane filter
papers were used for filtration of samples and mobile phase
and were purchased from millipore (India).
Preparation of NFXC, Impurities A and B
Solutions
About 100 mg of standard drug NFXC was weighed accurately
and was dissolved in 50 ml methanol. Mixed the solution till
the drug dissolved completely in methanol. Then, the final
volume was made up to 100 ml with methanol. Filter the final
solution using 0.2 μ nylon membrane filter paper. A standard
stock solution concentration of 1000 μg/ml was obtained.
Necessary dilutions required for the analysis were prepared
from this stock solution.
Preparation of Formulation Solution
The marketed formulation tablets of NFXC (Gramoneg©- 500)
were finely powdered using clean, dry mortar, and pestle. An
amount of the uniform fine powder equivalent to 100 mg of NFXC
standard was weighed accurately and was dissolved in 75 ml
methanol. Keep the solution in an airtight 100 ml volumetric flask
and was kept in a rotatory orbital shaker to 24 h. Then, it was
filtered and make up to the mark with methanol. Sample solution
concentration of 1000 μg/ml was obtained. Then, the solution was
diluted properly to get a final sample concentration of 300 μg/ml.
METHOD DEVELOPMENT
Method development was instantiated by checking the solubility
of standard drug and both impurities. NFXC, impurities A
and B solutions were soluble in methanol. Hence, methanol
was used as diluents for the analysis. Spectrophotometer
analysis was carried for the determination of suitable common
wavelength for NFXC, impurities A and B.
A concentration of 10 μg/ml of NFXC, impurity A and
impurity B were scanned in the region of 800 nm–400 nm
using UV/visible spectrophotometer. The wavelength maxima
were found to be 261 nm for NFXC [Figure 2], 276 nm for
impurity A [Figure 3], and 220 nm for impurity B [Figure 4].
The isoabsorption wavelength of NFXC, impurities A and B
were found to be 220 nm. Hence, 220 nm was found to be
suitable for the analysis of NFXC and its impurities A and B
simultaneously by HPLC-UV detection method.
The optimization of suitable mobile phase was carried
based on the chemical properties of the three molecules in
the study. Based on the properties and available literature,
method has development have been initiated with ionic buffer,
i.e., sodium perchlorate salt as aqueous solvent and methanol
as organic modifier.
Trial and error method was followed for the optimization
of chromatographic conditions. Method development was
initiated with low volume of 0.01 M sodium perchlorate and
higher volume of organic modifier methanol in the ratio of
25:75 (v/v) on C18 column (100 mm × 4.6 mm × 3.5 μ).
In this condition, no clear baseline was observed. Further, the
pH of the mobile phase was adjusted to pH 3.1 gives a clear
baseline, but the resolution was found to be very poor and
similar retention times were observed for NFXC, impurities A
and B. Short length column 100 mm influencing the interaction
time of molecules with stationary phase.
Method optimization was obtained in between the three
compounds NFXC, impurity A, and impurity B [Figure 5]
Figure 1: Molecular structures of norfloxacin and impurities in the study
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
29 TJPS 2018, 42 (1): 27-36
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with mobile phase composition of methanol, 0.01 M sodium
perchlorate (pH 3.1) in the ratio of 15:85 (v/v) at a flow rate of
0.8 ml/min. Waters C18 column (250 mm × 4.6 mm; 3.5 μm)
and 220 nm, 20 μl injection volume was selected for the analysis.
In the optimized condition, peak purity of NFXC was studied
separately using PDA detector. The purity angle of NFXC was
found to be less than purity threshold confirms that the peak
purity for NFXC peak was within acceptance criteria. The peak
purity was found to be 99.3% and the peak purity index was
found 0.993 which was within the acceptance limit of ≥0.990.
METHOD VALIDATION
Method validation was performed as per ICH guidelines for
NFXC and synthetic impurities impurity A and impurity B.[12]
Specificity and System Suitability
The chromatogram of standard and placebo solution obtained
in the optimized method was confirm the specificity. It was
observed that no chromatographic detection was observed
in the retention times of NFXC and both related substances
for placebo analysis confirm that the method developed was
found to be suitable for the separation and analysis of NFXC
and its related impurities A and B.
The standard solution of NFXC and impurities at a
concentration of 10, 20, and 30 μg/ml was prepared separately
and was mixed in equal proportions separately. These
solutions were analyzed triplicates in the developed method.
The number of theoretical plates, tailing factor, and resolution
factor was used for the determination of system suitability. The
results of system suitability [Table 1] confirm that the number
of theoretical plates was found to be more than 2500, <2
tailing factor, and >2 resolution factor was observed for NFXC
and impurities A and B. Hence, the method developed was
found to be suitable and specific.
Relative Retention Time (RRT) and
Relative Response Factor (RRF)
RRT determines the stability of the elution time of the
impurities of NFXC in the developed method. RRF was used
to determine the reproducible response of the pharmaceutical
impurities in the developed method. RRF also helpful for
the determination of actual amount of impurities present
in pharmaceuticals. The ratio between the signals obtained
for impurity to the signal produced by the NFXC API in
the same experimental condition was considered as RRF.
Standard NFXC and impurities at a concentration of 10 μg/
ml, 20 μg/ml, and 30 μg/ml were prepared and were mixed
in equal volume. The solutions were analyzed in triplicates
in the developed method and RRT and RRF were calculated.
The RRT and RRF were found to be within the acceptance
limit of <2 [Table 2] for both impurities A and B in all three
concentration levels. Hence, reproducible retention time and
response were observed in the developed method for the
analysis of NFXC and impurities A and B.
Figure 2: Ultraviolet absorption spectrum of norfloxacin
Figure 3: Ultraviolet absorption spectrum of impurity A
Figure 4: Ultraviolet absorption spectrum of impurity B
Figure 5: Standard chromatogram for norfloxacin (4 μg/ml)
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
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Linearity
A 6 points calibration curve was constructed for NFXC and
both impurities A and B. A combined solutions containing
equal concentration of NFXC and impurities were prepared in
different concentration range and the solutions were analyzed
in triplicates in the optimized method. The response observed at
the retention time (Tr) of compound was used for constructing
the calibration. Calibration curve was constructed by taking
response at Tr of compound on x-axis and concentration
prepared on y-axis. Accurately correlated calibration curve was
observed in the concentration range of 1–6 μg/ml for NFXC,
impurities A and B. Correlation and regression results were
given in Table 3 and calibration curve results were given in
Table 4. Calibration curve was given in Figure 6 for NFXC and
impurities A and B.
Recovery
The accuracy and recovery of the method developed for the
analysis of NFXC was determined by spiked recovery method.
50%, 100%, and 150% concentrations were spiked for a fixed
concentration of 2 μg/ml and the solution was analyzed in
triplicates. The result obtained in spiking experiment was
compared with the standard calibration curve values and %
recovery and % relative standard deviation (RSD) of recovery
was calculated. The % recovery was found to be in the range of
98–100. The % RSD was found to be 0.122, 0.305, and 0.147 in
50% spiked level, 0.106, 0.383, and 0.541 in 100%, and 0.170,
0.212, and 0.153 in 150% spiked level for impurity A, standard
and impurity B, respectively. % RSD was found to be very
less confirmed that the method developed for NFXC was very
accurate. Table 5 summarizes the recovery results for NFXC.
Table 1: System suitability results for NFXC
Concentration (µg/mL) Compound RT (min) Theoretical plates Tailing factor Resolution
10 Standard 6.90 81002 1.38 13.88
Impurity A 5.61 67996 1.37 …
Impurity B 8.82 154807 1.35 21.34
20 Standard 6.93 91722 1.42 14.03
Impurity A 5.61 53276 1.36 …
Impurity B 8.87 159953 1.48 21.49
30 Standard 6.87 91439 1.31 14.74
Impurity A 5.49 47173 1.36 …
Impurity B 8.85 145733 1.54 21.32
Three replicate measurements average values given in the table. NFXC: Norfloxacin
Table 2: RRT and RRF results
Concentration (µg/mL) Compound RT (min) RRT RF RRF
10 Standard 6.90 … 10676 …
Impurity A 5.61 0.81 11764 1.10
Impurity B 8.82 1.28 8629 0.81
20 Standard 6.93 ... 6288 …
Impurity A 5.61 0.81 6644 1.06
Impurity B 8.87 1.28 4616 0.73
30 Standard 6.87 … 4592 …
Impurity A 5.49 0.80 4965 1.08
Impurity B 8.85 1.29 3737 0.81
Three replicate measurements average values given in the table; RRT: Relative retention time, RRF: Relative response and keep
Table 3: Correlation and regression results for NFXC
Compound Slope Intercept r2
Standard 27234.67±222.28 45697.67±62.93 0.998
Impurity A 18458.00±263.50 37557.33±485.97 0.999
Impurity B 22858.67±76.27 44389.00±622.34 0.998
#Values given in table is the average±standard deviation of peak response in three replicate experiments. NFXC: Norfloxacin
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
31 TJPS 2018, 42 (1): 27-36
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Force degradation
Force degradation studies were used to identify and quantify
known and unknown impurities in NFXC. Force degradation
studies were performed and procedure was summarized in
Table 6.
Results confirmed that stress condition does not influence
the elution of standard drug NFXC. Both impurities A and B were
observed in all the stress studies. In addition to these known
impurities, two unknown impurities were detected. NFXC was
very sensitive to basic condition, 7.187% degradation was
observed in 5 h [Figure 7]. High % degradation about 11% was
observed in photolytic degradation in 24 h [Figure 8]. The effect
of other stress conditions was found to be nominal [Figures 9
and 10]. All force degradation studies results were tablets in
Table 7.
Limit of detection (LOD) and limit of
quantification (LOQ)
LOD and LOQ are calculated theoretically based on the response
factor of the NFXC, impurities A and B. S/N ratio approach was
used for LOD and LOQ concentrations determination. LOD
was established with 0.03 μg/ml, 0.01 μg/ml, and 0.01 μg/
ml for NFXC, impurities A and B, respectively [Figure 11]. The
LOD and LOQ results [Table 8] were confirmed the developed
method has high sensitivity.
NFXC Drug Substance Analysis
NFXC drug substance was used to prepare 300 μg/ml solution
and analyzed in the finalized procedure. Results were
satisfactory. Chromatogram was represented in Figure 12.
Method confirming that finalized method has high detectability.
Hence, method can be applied for the identification and
estimation of synthetic impurities in NFXC drug synthesis.
Formulation Analysis
Gramoneg© - 500 tablets, market sample was purchased
for analysis. 300 μg/ml concentration sample was analyzed
and spiked sample was analyzed with two impurities A and B
spiking. No other additional impurities were detected in the
market sample. Spiked sample was represented in Figure 13.Figure 6: Calibration curve
Table 4: Calibration curve results
Concentration in µg/ml Peak area obtained#
Standard Impurity A Impurity B
1 75008.00±168.88 56148.00±549.80 67169.33±723.08
2 99980.33±545.88 74573.00±142.17 89447.67±570.00
3 123727.70±1006.24 92111.00±1500.53 113535.30±1260.81
4 154502.70±2864.39 112166.30±920.52 137669.00±1561.52
5 180580±888.64 1296970±1208.98 156299.30±875.24
6 209883.30±1131.39 148269.70±1087.35 182241.00±1264.01
#Values given in table is the average±standard deviation of peak response in three replicate experiments
Table 5: Recovery results for NFXC in the developed method
Recovery
level
Compound Concentration in µg/ml Amount found
Mean±SD
% recovered
Mean±SD
% RSD of Recovery
Target Spiked Final
50% Standard 2 1 3 2.956±0.009 98.522±0.301 0.305
Impurity A 2 1 3 2.948±0.004 98.267±0.120 0.122
Impurity B 2 1 3 2.962±0.004 98.733±0.145 0.147
100% Standard 2 2 4 3.957±0.015 98.917±0.378 0.383
Impurity A 2 2 4 3.927±0.004 98.183±0.104 0.106
Impurity B 2 2 4 3.951±0.021 98.767±0.535 0.541
150% Standard 2 3 5 4.914±0.0104 98.287±0.208 0.212
Impurity A 2 3 5 4.926±0.008 98.527±0.168 0.170
Impurity B 2 3 5 4.908±0.008 98.167±0.150 0.153
NFXC: Norfloxacin, RSD: Relative standard deviation, SD: Standard deviation
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
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Figure 7: Base degradation chromatogram (BH)
Figure 8: Photolytic degradation chromatogram (PD 2)
Table 6: Forced degradation study procedure for NFXC
Degradation type Experimental conditions Sample code
Acid hydrolysis (AH) 50 mg of drug was mixed with 50 ml of 0.1N HCl
solution. The solution was neutralized and diluted up
to standard concentration and was analyzed in the
developed method condition
5 h (AH 1)
24 h (AH 2)
Base hydrolysis (BH) 50 mg of drug was mixed with 50 ml of 0.1N NaOH
solution. The solution was neutralized and diluted up
to standard concentration and was analyzed in the
developed method condition
5 h (BH)
Oxidative degradation (OD) 50 mg of drug was mixed with 50 ml of 3% peroxide
solution. The solution was neutralized and diluted up
to standard concentration and was analyzed in the
developed method condition
5 h (OD 1)
24 h (OD 2)
Photolytic degradation (PD) 50 mg of drug sample was kept in UV light (254 nm).
After the selected time of light expose, the drug solution
was prepared and was analyzed
5 h (PD 1)
24 h (PD 2)
Thermal degradation (TD) 50 mg of drug sample was kept in oven at 60°C. After
the selected time of light expose, the drug solution was
prepared and was analyzed
5 h (TD 1)
24 h (TD 2)
NFXC: Norfloxacin
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
33 TJPS 2018, 42 (1): 27-36
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Figure 9: Acid hydrolysis chromatogram (PD 2)
Figure 10: Acid hydrolysis chromatogram (AH 2)
Table 7: Forced degradation study results for NFXC
Condition No of addition peaks observed Area obtained % Obtained % degradation
Standard …. 618822 - -
AH 1 0 600497 97.039 2.961
AH 2 2 587762 94.981 5.019
BH 1 2 574346 92.813 7.187
OD 1 0 608235 98.289 1.711
OD 2 1 581180 93.917 6.083
PD 1 1 575315 92.969 7.031
PD 2 2 550696 88.991 11.01
TD 1 0 607299 98.138 1.862
TD 2 2 596906 96.458 3.542
NFXC: Norfloxacin, AH: Acid hydrolysis, BH: Base hydrolysis, OD: Oxidative degradation, PD: Photolytic degradation, TD: Thermal degradation
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
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Figure 12: Norfloxacin drug substance + impurities spiked chromatogram
Figure 11: Limit of detection chromatogram of norfloxacin
In literature, few methods were reported previously
for the estimation of NFXC in pharmaceutical formulations.
The method described by Sharma et al., 2014; Ashok et al.,
2014, and Paulo et al., 2009, were found to be a routine
assay methods and they did not study the stability of NFXC
in stress conditions and the method cannot be applicable
for the separation and estimation of impurities. Lucas et
al., 2013, studied the stability of the drug in different stress
degradation conditions. However, the method fails to analysis
the impurities. The method developed by Mahmood et al.,
2010, and Nageswara et al., 2004, were for the estimation
of drug in plasma and synthetic impurities, respectively, and
these methods were not in our area of study. Hence, the
method developed here was found to be the simple and novel
Mallu, et al.: HPLC analysis of norfloxacin and its potential impurities
35 TJPS 2018, 42 (1): 27-36
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Figure 13: Gramoneg© - 500 tablets+ impurities spiked sample
Table 8: Sensitivity results
Compound Theoretical value Instrumental results
LOQ (µg/ml) LOD (µg/ml) LOQ (µg/ml) LOD (µg/ml)
Standard 0.01 0.003 0.1 0.03
Impurity A 0.005 0.0015 0.04 0.01
Impurity B 0.02 0.004 0.04 0.01
LOD: Limit of detection, LOQ: Limit of quantification
method for the separation, qualitative determination and
quantification of NFXC and its potential related compounds
A and B.
CONCLUSION
Isocratic HPLC procedure was developed for NFXC and its
impurities (A and B). Method was validated as per ICH Q2
guideline. All method validation parameter results found within
the acceptance limit. Method is simple, accurate, and reliable
and can be applied for drug substance synthesis and tablet
dosage forms. Methods for the separation and estimation of
NFXC and its impurities A and B were not reported previously,
and hence, the method found to be novel and suitable for
stability testing, impurity profile analysis of NFXC.
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