MEDICINAL CHEMISTRY RESEARCH
Med Chem Res (2010) 19:1259–1272 DOI 10.1007/s00044-009-9268-7 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 Ó Birkha¨user Boston 2009
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 kmax values. The increase in absorbance was directly proportional to the concentration of quinolones 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 International Conference on Harmonization. The proposed methods were successfully employed for determination of these quinolones in pharmaceutical formulations. Keywords Ciprofloxacin Gatifloxacin Norfloxacin Levofloxacin Ofloxacin Pefloxacin
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:
[email protected];
[email protected] M. S. Arayne e-mail:
[email protected] N. Sultana Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi, Karachi 75270, Pakistan
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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 LambertBeer 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
Fig. 1 Ciprofloxacin
HN N
N
F
COOH O
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Fig. 2 Gatifloxacin
Fig. 3 Norfloxacin
CH3
HN N
N
F
CO O H O
H3C
CH3
O
N N
N
COOH
F O Fig. 4 Levofloxacin/ofloxacin
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.
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H3 C
CH3
N N
N
COOH
F O Fig. 5 Pefloxacin
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, levofloxacin, 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-1 for ciprofloxacin, 10–250 lg mL-1 for gatifloxacin, 6–300 lg mL-1 for norfloxacin, 10–200 lg mL-1 for 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
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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-1 ciprofloxacin, gatifloxacin, ofloxacin, and pefloxacin, 10–160 lg mL-1 levofloxacin, and 6–300 lg mL-1 norfloxacin, 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 Ciprofloxacin
375
A = 0.0107 Cx ? 0.0667
0.9951
0.1599
0.022
0.066
Gatifloxacin
473
A = 0.0021 Cx – 0.0079
0.9952
0.6632
0.445
1.347
Norfloxacin
442
A = 0.0066 Cx – 0.0041
0.9999
0.1549
0.106
0.321
Levofloxacin
375
A = 0.0139 Cx ? 0.0459
0.9980
0.5142
0.050
0.153
Ofloxacin
375
A = 0.0123 Cx ? 0.091
0.9950
0.5098
0.171
Pefloxacin
434
A = 0.0050 Cx – 0.0487
0.9998
0.2233
4.574
0.517 13.86
With C6H8O7FeNH3 Ciprofloxacin
375
A = 0.0076 Cx ? 0.1145
0.9959
0.1487
2.057
6.234
Gatifloxacin
360
A = 0.0109 Cx – 0.0052
0.9996
0.1515
0.043
0.129
Norfloxacin
432
A = 0.0072 Cx – 0.0257
0.9952
0.2154
0.745
2.258
Levofloxacin
375
A = 0.0072 Cx – 0.0174
0.9958
0.6566
1.329
4.026
Ofloxacin
360
A = 0.0135 Cx ? 0.0364
0.9971
0.3684
0.311
0.942
Pefloxacin
426
A = 0.0031Cx ? 0.0085
0.9963
0.5846
0.527
1.596
With FeNO3 Ciprofloxacin
447
A = 0.0056 Cx ? 0.0031
0.9997
0.5050
0.167
0.505
Gatifloxacin
447
A = 0.0041 Cx ? 0.5307
0.9951
0.2707
0.250
0.757
Norfloxacin
445
A = 0.0061 Cx ? 0.0007
0.9995
0.2333
0.497
1.507
Levofloxacin
375
A = 0.0137 Cx ? 0.051
0.9985
0.3269
0.698
2.116
Ofloxacin
370
A = 0.0135 Cx ? 0.0273
0.9975
0.2987
0.319
0.952
Pefloxacin
434
A = 0.0049 Cx ? 0.0056
0.9998
0.2846
0.143
0.432
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 determination 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
Med Chem Res (2010) 19:1259–1272
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Fig. 6 UV spectra of quinolone/FeCl3 complexes: (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
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).
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Fig. 8 UV spectra of quinolone/FeNO3 complexes: (a) ciprofloxacin; (b) gatifloxacin; (c) levofloxacin; (d) norfloxacin; (e) ofloxacin; (f) pefloxacin
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 ð1=eÞ þ ð1=Kce Þ ð1=Cb Þ where Ca and Cb are the concentrations of the acceptor and donor, respectively, A is the absorbance of the complex, e is the molar absorptivity of the complex, and Kc is the association constant of the complex. The calculated association constants are reported in Table 2. The low values of Kc are 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 Kc values 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-1 deg-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-1 for six quinolones. Absorbances were plotted against concentrations 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.
Med Chem Res (2010) 19:1259–1272 Table 2 Association constants and standard free energy changes
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DG
Kc
log Kc
Ciprofloxacin
–0.349
1.80
0.256
Norfloxacin
–0.348
1.79
0.255
Pefloxacin
–0.349
1.80
0.256
Levofloxacin
–0.350
1.81
0.259
Ofloxacin
–0.350
1.81
0.259
Gatifloxacin
–0.346
1.79
0.254
Ciprofloxacin
–0.586
2.69
0.430
Norfloxacin
–0.584
2.68
0.428
Pefloxacin
–0.586
2.69
0.429
Levofloxacin
–0.653
3.01
0.480
Ofloxacin
–0.590
2.70
0.430
Gatifloxacin
–0.585
2.68
0.429
Ciprofloxacin
–0.776
3.70
0.569
Norfloxacin
–0.776
3.71
0.570
Pefloxacin
–0.777
3.71
0.570
Levofloxacin
–0.778
3.72
0.571
Ofloxacin
–0.781
3.74
0.573
Gatifloxacin
–0.774
3.70
0.568
Drug Ferric chloride
Ferric nitrate
Iron ammonium citrate
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.
Norfloxacin
Mean
Gatifloxacin
Mean
Ciprofloxacin
-1
99.9
101.3
100.6
100.5
100.0
20
40
60
80
100
97.9
200
100.8
99.0
120
6
100.0
100
100.3
200
103.8
100.2
170
101.7
100.7
140
80
99.9
60
99.7
60
100
99.5
104.0
20
40
100.1
100.0
5
Recovered (%)
10
Added (lg mL )
FeCl3
Table 3 Accuracy and precision of proposed method
0.01
0.35
0.39
0.88
0.11
0.55
1.06
1.51
0.69
0.00
2.66
1.16
0.33
0.5
0.20
0.16
0.50
0.09
0.18
2.75
0.00
0.10
% RSD
-1
160
120
80
40
20
99.2
99.1
103.3
97.8
99.4
95.6
104.7
200 10
97.6
97.0
99.9
100.4
101.5
99.8
100.6
99.9
99.8
99.4
102.2
103.0
105.2
Recovered (%)
160
120
80
40
20
10
200
160
120
80
40
20
10
Added (lg mL )
C6H8O7FeNH3
0.55
0.61
2.31
1.61
0.45
3.21
1.25
3.27
1.74
2.17
0.07
0.29
1.04
0.14
1.18
0.40
0.08
0.16
0.41
1.53
2.07
3.61
% RSD
160
120
80
40
20
10
180
140
100
60
40
10
180
140
100
60
40
20
10
Added (lg mL-1)
FeNO3
98.7
100.0
97.3
101.0
98.6
98.0
100.1
100.0
98.4
100.1
102.5
99.9
99.5
99.2
100.6
100.2
103.8
98.5
100.7
Recovered (%)
0.93
0.04
1.95
0.73
0.99
1.43
0.52
0.05
0.01
1.13
0.09
1.77
0.08
0.82
0.35
0.55
0.45
0.11
2.66
1.10
0.49
% RSD
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Mean
100.1
99.6
100.6
100
140
160
0.44
0.28
0.04
0.08
99.4
100.0
160
0.04
0.44
0.04
94.7
101.3
104.0
101.0
40
60
100
140
0.69
2.80
0.89
3.89
100.1
60
120
0.25
Pefloxacin
99.6
40
1.15
0.38
101.6
20
Mean
99.5
10
0.38
99.9
60
1.00
0.33
98.6
40
0.15
1.26
2.27
0.52
Mean
100.2
103.3
300
10
100.7
200
% RSD
Ofloxacin
Levofloxacin
Recovered (%)
-1
Added (lg mL )
FeCl3
Table 3 continued
-1
60
40
20
99.3
101.8
100.0
102.4
100.0
200 10
104.8
97.8
99.2
101.2
100.4
101.3
160
120
80
40
20
10
99.3 100.1
200
98.5
100.0
100.1
99.6
100.0
Recovered (%)
160
120
80
40
20
10
Added (lg mL )
C6H8O7FeNH3
0.52
1.23
0.00
1.67
1.08
0.02
3.34
1.60
0.61
0.87
0.26
0.90
0.28
0.04
0.48
1.06
0.00
0.09
0.29
0.00
1.25
% RSD
80
40
20
10
200
160
120
80
40
20
10
160
120
80
40
20
10
200
Added (lg mL-1)
FeNO3
96.1
102.4
100.0
99.4
97.2
100.0
100.0
101.5
100.0
98.3
100.0
100.0
99.9
97.2
102.5
99.8
102.6
98.8
Recovered (%)
2.79
1.66
0.00
0.41
0.60
2.01
0.00
0.00
1.02
0.02
1.21
0.00
0.95
0.00
0.05
1.98
1.75
0.14
1.80
0.99
0.88
% RSD
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Mean
Recovered (%)
99.8
99.0
160
200
-1
Added (lg mL )
FeCl3
Table 3 continued
1.52
0.69
0.17
% RSD
-1
99.9 100.0
180
100.0
Recovered (%)
140
100
Added (lg mL )
C6H8O7FeNH3
0.50
0.00
0.08
0.00
% RSD
200
160
120
Added (lg mL-1)
FeNO3
101.9
100.0
101.8
Recovered (%)
1.07
1.33
0.03
1.29
% RSD
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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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