Facile and manifest spectrophotometric methods for the determination of six quinolone antibiotics in pharmaceutical formulations using iron salts

Abstract

Simple spectrophotometric methods have been developed for the determination of six quinolone antibiotics, namely, ciprofloxacin,
gatifloxacin, norfloxacin, levofloxacin, ofloxacin, and pefloxacin, in pharmaceutical formulations using three different salts
of iron. These methods are based on the formation of complexes with ferric nitrate, ferric chloride, or iron ammonium citrate
in which the carboxylic group of quinolones undergoes complexation with iron. The complexes formed in these reactions, having
a brown color, were measured at their respective λmax values. The increase in absorbance was directly proportional to the concentration of quinolones and obeys Beer’s law in the
range of 6–300μgmL−1 (r≥0.9999). The proposed methods were optimized and validated per the guidelines of the International Conference on Harmonization.
The proposed methods were successfully employed for determination of these quinolones in pharmaceutical formulations.

KeywordsCiprofloxacin-Gatifloxacin-Norfloxacin-Levofloxacin-Ofloxacin-Pefloxacin

Full text

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ORIGINAL RESEARCH
Facile and manifest spectrophotometric methods
for the determination of six quinolone antibiotics
in pharmaceutical formulations using iron salts
Farhan Ahmed Siddiqui•M. Saeed Arayne•
Najma Sultana•Agha Zeeshan Mirza•
Faiza Qureshi•M. Hashim Zuberi
Received: 3 May 2009/Accepted: 15 September 2009/Published online: 11 November 2009
? Birkha ¨user Boston 2009
Abstract
mination of six quinolone antibiotics, namely, ciprofloxacin, gatifloxacin, norflox-
acin, levofloxacin, ofloxacin, and pefloxacin, in pharmaceutical formulations using
three different salts of iron. These methods are based on the formation of complexes
with ferric nitrate, ferric chloride, or iron ammonium citrate in which the carboxylic
group of quinolones undergoes complexation with iron. The complexes formed in
these reactions, having a brown color, were measured at their respective kmaxvalues.
The increase in absorbance was directly proportional to the concentration of quin-
olones and obeys Beer’s law in the range of 6–300 lg mL-1(r C 0.9999). The
proposed methods were optimized and validated per the guidelines of the Interna-
tional Conference on Harmonization. The proposed methods were successfully
employed for determination of these quinolones in pharmaceutical formulations.
Simple spectrophotometric methods have been developed for the deter-
Keywords
Ofloxacin ? Pefloxacin
Ciprofloxacin ? Gatifloxacin ? Norfloxacin ? Levofloxacin ?
Introduction
Quinolones or fluoroquinolones constitute a large class of synthetic antimicrobial
agents that are highly effective in the treatment of many types of infectious diseases,
F. A. Siddiqui (&) ? M. S. Arayne ? A. Z. Mirza ? F. Qureshi ? M. H. Zuberi
Department of Chemistry, University of Karachi, Karachi 75270, Pakistan
e-mail: farhanchemist@hotmail.com; farhanchemist@gmail.com
M. S. Arayne
e-mail: msarayne@gmail.com
N. Sultana
Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi, Karachi
75270, Pakistan
Med Chem Res (2010) 19:1259–1272
DOI 10.1007/s00044-009-9268-7
MEDICINAL
CHEMISTRY
RESEARCH

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particularly those caused by bacteria. They are widely used to treat human and
veterinary diseases (Currie et al., 1998; Ihrke et al., 1999; Chen et al., 1999). Due to
the fluorine atom at position C-6 and the piperazine or methyl piperazine at position
C-7, these antibiotics exhibit a broad spectrum of activity against Gram-positive and
Gram-negative bacteria. Over time, bacteria become resistant to medicines that are
used to combat them; because of this, the medical world is always in search of new
and improved ways to battle these disease-causing bacteria. New antibiotics are
continually being developed and quinolones are at the forefront of this research
(Wolfson and Hooper, 1989; Carlucci, 1998).
Several analytical methods for quantitative determination of quinolones in
pharmaceutical formulations are described in the literature, including capillary
electrophoresis (Flurer, 1997; Sun and Chen, 1997; Bhowal and Das, 1991) and UV
spectrophotometry (Venugopal and Saha, 2005). Spectrophotometric analysis of
gatifloxacin was done using the latter method, but different buffers were used;
however, in our case no buffer was used, so the method has an advantage over the
previous one. In a method reported by Fratini and Schapoval (1996), the Lambert-
Beer law was obeyed in the concentration range of 20–100 lg mL-1, and also nitric
was used, but in the newly developed method the linearity range is much broader
and, also, the limit of quantification is much less than in this previous work,
titrimetry (British Pharmacopoeia, 1999; Belal et al., 1999), and high-performance
liquid chromatography (HPLC) (Samanidou et al., 2003; Joshi, 2002; Sanzgiri et al.,
1994). Some authors prepared derivatives of different quinolones and compared
their properties against those of quinolone (Shaharyar et al., 2007; Gopalakrishnan
et al., 2007; Jayashree et al., 2009); and the in vitro availability of atorvastatin, in
the presence of ciprofloxacin, gatifloxacin, and ofloxacin has been reported (Arayne
et al., 2009).
The objective of this research was to develop and validate rapid, economical, and
sensitive methods for quantitative determination of six quinolones—ciprofloxacin
(Fig. 1), gatifloxacin (Fig. 2), norfloxacin (Fig. 3), levofloxacin/ofloxacin (Fig. 4),
and pefloxacin (Fig. 5)—in bulk and tablet formulations using different salts of iron.
The major advantage of the proposed methods is that these six flourquinolones can
be determined on a single system with minor modifications in detection wavelength.
The proposed methods were applied successfully to determination of the six
HN
NN
F
O
COOH
Fig. 1 Ciprofloxacin
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quinolones in both reference material and dosage forms with high values of
accuracy and precision. No interference was observed in the assay from common
excipients at levels found in pharmaceutical formulations. These methods were
validated by the statistical data. In addition, the association constant, stochiometric
ratio of reactants, and standard free energy changes (DG?) were determined.
Fig. 2 Gatifloxacin
NN
HN
O
HOOCF
CH3
Fig. 3 Norfloxacin
N
N
F
N
O
H3C
O
CH3
COOH
Fig. 4 Levofloxacin/ofloxacin
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Experimental
Instrumentation
A double-beam UV–vis spectrophotometer (Shimadzu model 1601) equipped with
10-mm quartz cells was used to make absorbance measurements and Shimadzu
UVPC version 3.9 software was used to control the instrument, data acquisition, and
data analysis. Spectra of quinolone–iron complexes were recorded over the
wavelength range 360–800 nm.
Chemicals
All chemicals used were of analytical grade; demineralized double-distilled water
was used throughout the study. Ciprofloxacin, gatifloxacin, norfloxacin, levoflox-
acin, ofloxacin, and pefloxacin were kind gifts from local pharmaceutical
companies. Pharmaceutical formulations were purchased from the market and
ferric chloride, ferric nitrate, and iron ammonium citrate were from Merck,
Germany.
Preparation of standard solutions
Reference stock solutions of ciprofloxacin, gatifloxacin, norfloxacin, levofloxacin,
and pefloxacin were prepared in water at a concentration of 500 lg mL-1, whereas
ofloxacin at the same concentration was prepared in methanol. These stock solutions
were diluted to obtain the desired concentration ranges for different quinolones (5–
200 lg mL-1for ciprofloxacin, 10–250 lg mL-1for gatifloxacin, 6–300 lg mL-1
for norfloxacin, 10–200 lg mL-1for levofloxacin, ofloxacin, and pefloxacin).
Preparation from pharmaceutical formulations
Twenty tablets of each formulation were weighed and powdered. A powdered tablet
equivalent to 50 mg of active substance was transferred to a 100-ml volumetric flask
NN
N
O
COOH
H3C
F
CH3
Fig. 5 Pefloxacin
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and diluted up to the mark with the same solvent as mentioned for the standard
preparation. These solutions were stirred on a magnetic stirrer for 60 min, filtered,
and further diluted to obtain the desired concentration ranges. All solutions were
stored at 4?C. One percent solutions of ferric chloride, ferric nitrate, and iron
ammonium citrate were prepared in double-distilled water.
Quinolone complexes with ferric chloride
To prepare 10–200 lg mL-1ciprofloxacin, gatifloxacin, ofloxacin, and pefloxacin,
10–160 lg mL-1levofloxacin, and 6–300 lg mL-1norfloxacin, different aliquot
portions of reference standard solutions of each drug were transferred into separate
series of 25-ml volumetric flasks. In each flask, 3 ml of 1% ferric chloride solution
was successively added. The volume was made up to the mark with water and set
aside at room temperature for 10 min. The absorbance of quinolone–iron complexes
was measured against a reagent blank at 375, 473, 442, 375, 375, and 434 nm for
ciprofloxacin, gatifloxacin, norfloxacin, levofloxacin, ofloxacin, and pefloxacin,
respectively. The calibration graph was prepared by plotting absorbance versus
concentration of drugs (Table 1).
Table 1 Linear regression functions and their statistical parameters
Drug
k
Regression equation
r
SE LOD
(lg mL-1)
LOQ
(lg mL-1)
With FeCl3
Ciprofloxacin375 A = 0.0107 Cx? 0.06670.9951 0.15990.0220.066
Gatifloxacin 473A = 0.0021 Cx– 0.0079
A = 0.0066 Cx– 0.0041
0.9952 0.66320.4451.347
Norfloxacin 442
0.99990.15490.106 0.321
Levofloxacin375A = 0.0139 Cx? 0.04590.9980 0.5142 0.0500.153
Ofloxacin375A = 0.0123 Cx? 0.0910.99500.50980.1710.517
Pefloxacin434 A = 0.0050 Cx– 0.04870.99980.22334.57413.86
With C6H8O7FeNH3
Ciprofloxacin 375 A = 0.0076 Cx? 0.1145
A = 0.0109 Cx– 0.0052
A = 0.0072 Cx– 0.0257
0.9959 0.14872.0576.234
Gatifloxacin 360
0.9996 0.15150.0430.129
Norfloxacin432
0.9952 0.2154 0.745 2.258
Levofloxacin 375A = 0.0072 Cx– 0.01740.9958 0.65661.3294.026
Ofloxacin360A = 0.0135 Cx? 0.0364 0.99710.3684 0.3110.942
Pefloxacin426A = 0.0031Cx? 0.00850.99630.58460.5271.596
With FeNO3
Ciprofloxacin 447A = 0.0056 Cx? 0.0031
A = 0.0041 Cx? 0.5307
A = 0.0061 Cx? 0.0007
0.9997 0.50500.1670.505
Gatifloxacin 447
0.9951 0.27070.2500.757
Norfloxacin 445
0.99950.2333 0.4971.507
Levofloxacin375 A = 0.0137 Cx? 0.051 0.9985 0.32690.6982.116
Ofloxacin370A = 0.0135 Cx? 0.0273 0.99750.2987 0.3190.952
Pefloxacin434A = 0.0049 Cx? 0.00560.99980.28460.1430.432
SE standard error
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Quinolone complexes with ferric nitrate and iron ammonium citrate
Assays were completed for the formation of complexes of quinolones with ferric
nitrate (using 3 ml of a 1% solution) and iron ammonium citrate (using 1 ml of a
1% solution) as mentioned for ferric chloride.
Results and discussion
Iron(III) salts have been shown to be ideal for the derivatization of carboxylic
groups (Franch et al., 2004; Arayne et al., 2008), which makes them a suitable
reagent for detection and quantification of quinolones via their carboxylic group.
Fe3?holds three molecules of quinolone, resulting in the complex having a brown
color which absorbs radiation in the visible range. In this article, the development
and validation of sensitive and precise spectrophotometric methods for determina-
tion of six quinolones, using this derivatization technique, have been described.
Optimization of derivatization conditions
The optimum reaction conditions for quantitative determination of all quinolones
were established via a number of preliminary experiments. The concentration of
iron(III) was optimized by using 1–5 ml of a 1% iron solution. Steady and
maximum color development of the complex was achieved with a volume of 3 ml
of ferric chloride and ferric nitrate, but in the case of iron ammonium citrate the best
results were observed when 1 ml of salt solution was used. Hence 3 ml of 1% ferric
chloride and ferric nitrate solutions and 1 ml of 1% iron ammonium citrate solution
were used as the optimal concentrations for validation of the method.
Calibration curves
Calibration curves were prepared by linear least squares regression analysis plotting
of the absorbance of quinolone–iron complexes versus the concentration of
quinolone (6–300 lg mL-1) (Table 1).
Reaction of quinolone with ferric chloride
An intense brown color developed in the visible region, showing minor bands at 360
and 440 nm for all quinolones except gatifloxacin, which exhibited absorption at
445 nm (Fig. 6) when the solutions of quinolones were mixed with ferric chloride
solution individually. These bands were attributed to the formation of a quinolone–
iron complex, in which three quinolone rings bind to iron(III).
Reaction of quinolone with ferric nitrate and iron ammonium citrate
An intense brown color developed in the visible region at 360 and 440 nm (Figs. 7
and 8) when solutions of different quinolones were mixed with ferric nitrate and
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iron ammonium citrate. These bands have been attributed to the formation of a
quinolone–iron complex, which is formed by three quinolone rings and iron.
These visible spectrophotometric methods, using aqueous solutions of iron(III)
ions as reagents, have an elegant simplicity; a brownish-green complex was formed
in the proportion 3:1 [quinolone:iron(III)]. The optimized methods were validated
for quinolone–iron complexes in pharmaceutical formulations. Results were of
adequate precision and accuracy. The absorption spectra obtained revealed that all
the quinolones showed almost the same behavior, except for gatifloxacin. In
reaction with ferric chloride, all quinolones show the same curve except for
gatifloxacin (Fig. 6); two bands were found in all quinolones except gatifloxacin,
one at 360 nm, whereas gatifloxacin showed maxiumum absorbance at 440 nm. A
similar trend was observed in the case of iron ammonium citrate and ferric nitrate
(Figs. 7 and 8).
Fig. 6 UV spectra of quinolone/FeCl3complexes: (a) ciprofloxacin; (b) gatifloxacin; (c) levofloxacin;
(d) norfloxacin; (e) ofloxacin; (f) pefloxacin
Fig. 7 UV spectra of quinolone/ferric ammonium citrate complexes: (a) ciprofloxacin; (b) gatifloxacin;
(c) levofloxacin; (d) norfloxacin; (e) ofloxacin; (f) pefloxacin
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Association constants and standard free energy changes
The absorbance of the complex was used to calculate the association constant using
the Benesi–Hildebrand (Benesi and Hildebrand, 1949) equation:
Ca=A ¼ 1=e
ðÞ þ 1=Kc?e
ð Þ ? 1=Cb
ðÞ
where Caand Cbare the concentrations of the acceptor and donor, respectively, A is
the absorbance of the complex, e is the molar absorptivity of the complex, and Kcis
the association constant of the complex. The calculated association constants are
reported in Table 2. The low values of Kcare common in these complexes due to
dissociation of the complex to the radical anion.
The standard free energy changes of complexation (DG?) were calculated from
the Kcvalues by the following equation (Martin et al., 1969):
DG?¼ ?2:303RT logKc
where DG? is the free energy change of the complex (kJ mol-1), R the gas constant
(1.987 cal mol-1deg-1), T the temperature in Kelvin (273 ? ?C), and Kc the
association constant of quinolone–iron complexes (l mol-1).
Validation of methods
Linearity, limits of detection and quantification, and stability
Linearity of the assay was demonstrated by at least six concentrations over the range
6–300 lg mL-1for six quinolones. Absorbances were plotted against concentra-
tions and analyzed using least squares linear regression (Table 1). According to ICH
recommendation the detection and quantification limits of the methods were
calculated using the standard deviation of the response and the slope of calibration
curve as reported in Table 1.
Fig. 8 UV spectra of quinolone/FeNO3complexes: (a) ciprofloxacin; (b) gatifloxacin; (c) levofloxacin;
(d) norfloxacin; (e) ofloxacin; (f) pefloxacin
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Precision and accuracy were assessed in conjunction with the linearity
studies using three spiked samples of three concentrations of each quinolone.
Measured concentrations were determined by application of the appropriate
standard curve obtained on each occasion. Precision was assessed in terms of
percentage RSD values. Percentage recovery values were used to express
accuracy (Table 3).
Sensitivity and interference study
The percentage recovery values of quinolones confirm the high sensitivity of the
proposed methods. The excellent recoveries indicate the absence of interference
from frequently encountered excipients. Percentage RSD values were B3.89
(Table 3). Under the same experimental conditions different excipients at different
concentrations were added and analyzed. Potential interference problems from the
commonly used excipients and other additives such as microcrystalline cellulose,
lactose, povidone, starch, and magnesium stearate were examined, and it was
confirmed that the excipients did not interfere with the assay. Low percentage RSD
values signify good precision of the method.
Table 2 Association constants
and standard free energy
changes
Drug
DGKc
log Kc
Ferric chloride
Ciprofloxacin –0.3491.800.256
Norfloxacin–0.348 1.79 0.255
Pefloxacin–0.3491.80 0.256
Levofloxacin –0.350 1.810.259
Ofloxacin–0.350 1.81 0.259
Gatifloxacin–0.3461.79 0.254
Ferric nitrate
Ciprofloxacin –0.5862.69 0.430
Norfloxacin –0.5842.68 0.428
Pefloxacin –0.586 2.69 0.429
Levofloxacin –0.653 3.01 0.480
Ofloxacin–0.590 2.700.430
Gatifloxacin –0.585 2.680.429
Iron ammonium citrate
Ciprofloxacin–0.7763.70 0.569
Norfloxacin–0.776 3.710.570
Pefloxacin–0.7773.710.570
Levofloxacin–0.778 3.720.571
Ofloxacin –0.7813.740.573
Gatifloxacin –0.7743.700.568
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Table 3 Accuracy and precision of proposed method
FeCl3
C6H8O7FeNH3
FeNO3
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
Ciprofloxacin
5
100.1
0.10
10
105.2
3.61
10
100.7
0.49
10
100.0
0.00
20
103.0
2.07
20
98.5
1.10
20
104.0
2.75
40
102.2
1.53
40
103.8
2.66
60
99.7
0.18
80
99.4
0.41
60
100.2
0.11
100
99.9
0.09
120
99.8
0.16
100
100.6
0.45
140
100.7
0.50
160
99.9
0.08
140
99.2
0.55
170
100.2
0.16
200
100.6
0.40
180
99.5
0.35
200
100.3
0.20
Mean
0.5
1.18
0.82
Gatifloxacin
40
99.5
0.33
10
99.8
0.14
10
99.9
0.08
60
101.7
1.16
20
101.5
1.04
40
102.5
1.77
80
103.8
2.66
40
100.4
0.29
60
100.1
0.09
100
100.0
0.00
80
99.9
0.07
100
98.4
1.13
120
99.0
0.69
120
97.0
2.17
140
100.0
0.01
200
97.9
1.51
160
97.6
1.74
180
100.1
0.05
200
104.7
3.27
Mean
1.06
1.25
0.52
Norfloxacin
6
100.8
0.55
10
95.6
3.21
10
98.0
1.43
20
99.9
0.11
20
99.4
0.45
20
98.6
0.99
40
101.3
0.88
40
97.8
1.61
40
101.0
0.73
60
100.6
0.39
80
103.3
2.31
80
97.3
1.95
80
100.5
0.35
120
99.1
0.61
120
100.0
0.04
100
100.0
0.01
160
99.2
0.55
160
98.7
0.93
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Table 3 continued
FeCl3
C6H8O7FeNH3
FeNO3
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
200
100.7
0.52
200
98.8
0.88
300
103.3
2.27
Mean
1.26
1.25
0.99
Levofloxacin
10
100.2
0.15
10
100.0
0.00
10
102.6
1.80
40
98.6
1.00
20
99.6
0.29
20
99.8
0.14
60
99.9
0.08
40
100.1
0.09
40
102.5
1.75
100
100.1
0.04
80
100.0
0.00
80
97.2
1.98
140
99.6
0.28
120
98.5
1.06
120
99.9
0.05
160
100.6
0.44
160
99.3
0.48
160
100.0
0.00
200
100.1
0.04
Mean
0.33
0.28
0.95
Ofloxacin
10
99.5
0.38
10
101.3
0.90
10
100.0
0.00
20
101.6
1.15
20
100.4
0.26
20
98.3
1.21
40
99.6
0.25
40
101.2
0.87
40
100.0
0.02
60
100.1
0.04
80
99.2
0.61
80
101.5
1.02
120
99.4
0.44
120
97.8
1.60
120
100.0
0.00
160
100.0
0.04
160
104.8
3.34
160
100.0
0.00
200
100.0
0.02
200
97.2
2.01
Mean
0.38
1.08
0.60
Pefloxacin
40
94.7
3.89
10
102.4
1.67
10
99.4
0.41
60
101.3
0.89
20
100.0
0.00
20
100.0
0.00
100
104.0
2.80
40
101.8
1.23
40
102.4
1.66
140
101.0
0.69
60
99.3
0.52
80
96.1
2.79
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Table 3 continued
FeCl3
C6H8O7FeNH3
FeNO3
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
Added (lg mL-1)
Recovered (%)
% RSD
160
99.8
0.17
100
100.0
0.00
120
101.8
1.29
200
99.0
0.69
140
99.9
0.08
160
100.0
0.03
180
100.0
0.00
200
101.9
1.33
Mean
1.52
0.50
1.07
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Application in pharmaceutical formulations
The proposed methods were successfully applied to the analysis of quinolones in
commercial formulations. The results were in good agreement with the declared
contents, and no interference was observed in the assay of all quinolones from
common excipients at levels found in pharmaceutical formulations. These methods
rely on the use of simple and inexpensive chemicals and techniques but have a
sensitivity analogous to that obtained by sophisticated and expensive techniques
such as HPLC and are validated by statistical data. The reaction conditions and
application of the methods for determination of quinolones in pharmaceutical
formulations have been established.
Conclusion
It is rare that ferric salts are used as chromogenic reagents for spectrophotometric
determination of these quinolones. The proposed methods, which are simple and
rapid, offer the advantages of sensitivity over a wide range of concentrations
without the need for extraction or heating. The methods do not entail any stringent
reaction conditions and have been successfully applied to the determination of
quinolones in pharmaceutical formulations.
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