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International Journal of Analytical and Bioanalytical Chemistry

Universal Research Publications. All rights reserved

ISSN-2231-5012
Original Article
Development and validation of a simple and economical spectrofluorimetric
method for estimation of ciprofloxacin in pharmaceutical dosage forms
Serigne Omar SARR1,2,*, Serigne Momar NDIAYE1, Djibril FALL2,3, Adama DIEDHIOU2,3, Amadou DIOP2, Bara
NDIAYE2, Yrim Mbagnick DIOP1,2
1
Laboratoire National de Contrle des Mdicaments, 39, Avenue Pasteur, BP 6303, Dakar-toile, Sngal
2
Laboratoire de Chimie Analytique et Bromatologie, Facult de Mdecine, de Pharmacie et dOdontologie, Universit
Cheikh Anta DIOP de Dakar, BP 5005 Dakar-Fann, Sngal
3
Laboratoire de Chimie Organique et Thrapeutique, Facult de Mdecine, de Pharmacie et dOdontologie, Universit
Cheikh Anta DIOP de Dakar, BP 5005 Dakar-Fann, Sngal
*
Corresponding author : sosarr1@yahoo.fr
Received 23 June 2013; accepted 16 July 2013
Abstract
A new simple, precise, accurate and green spectrofluorimetric method for the determination of ciprofloxacin both as a
bulk drug and in pharmaceutical formulations was developed and validated using water as solvent. At a predetermined
excitation wavelength (280 nm) and emission wavelength (450 nm), it was proved linear in the concentration range of 20250 ng/mL, exhibited good correlation coefficient (R2= 0.9993) and excellent mean recovery (98.40-102.26%). The results
of the recoveries studies showed that the method was not affected by the presence of common excipients. The method was
applied for the analysis of the drug in the pure, tablet and injectable forms. The method was validated for various
parameters according to ICH guidelines. LOD and LOQ for ciprofloxacin were found to be 4.53 ng/mL and 5.67 ng/mL
respectively. The method has been successfully applied for the analysis of marketed formulations available in Senegal.
2013 Universal Research Publications. All rights reserved
KEY WORDS: Spectrofluorometric analysis, validation, ciprofloxacin, green method.
1. INTRODUCTION
Ciprofloxacin is a synthetic antibiotic of the
fluoroquinolone drug class and is chemically cyclopropyl6-fluoro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic
acid. It is a second-generation-fluoroquinolone anti-bacterial.
Literature survey revealed that ciprofloxacin is very
sensitive to ultraviolet light (UV) and will fluoresce, due to
its highly conjugated resonance structure as shown with the
chemical structure in Fig. 1 [1].

Fig.1. Chemical structure of ciprofloxacin

This fluorescence propriety is exploited to detect this
substance in various analytical methods. Then several
researchers have focused on the development of various
analytical methods to determine ciprofloxacin in
pharmaceutical formulations and/or biological fluids [1-4].

72

The compendious methods in the British Pharmacopeia and

the United States Pharmacopeia for the assay of
ciprofloxacin either in pure form or in different
pharmaceutical dosage forms are performed using HPLC
with UV detection [5-6].
Also the International Pharmacopeia published by World
Health Organization described a titrimetric method [7] for
the assay of ciprofloxacin. However, it is well known that
titrimetric methods lack of specificity when compared to
spectrofluorimetry for quantitative analysis.
To date and to our best knowledge, all these methods
described for the analysis of ciprofloxacin in
pharmaceutical formulations use organic solvents of which
difficult removal threatens the environment and sustainable
development especially in underdeveloped countries.
In this present work, a spectroscopic method has been
developed for the estimation of ciprofloxacin in the bulk
and capsule dosage form using only water. The method
developed is precise, accurate, specific, economical and
reproducible. This simple and rapid method was validated
as per ICH guidelines and Pharmacopeias requirements
[6,8].

International Journal of Analytical and Bioanalytical Chemistry 2013; 3(3): 72-77

2. MATERIALS AND METHODS

2.1. Instruments
Spectrophotometric measurements were performed using a
Jasco V-570 UV-visible spectrophotometer with matched
1-cm quartz cells (Jasco instruments, Tokyo, Japan). The
instrument is interfaced to a computer loaded with spectra
manager software and the IBM computer is connected to a
Lexmark Z2420 printer. Working standards were scanned
between 200 - 500 nm to choose the maximum wavelength
absorbance.
Perkin Elmer luminescence spectrometer model LS 45
(Perkin Elmer instruments, Massachusetts, USA) connected
to a Fujitsu Siemens computer loaded with the FLwinlab
application software was used. All the measurements took
place in a standard 10 mm path-length quartz cell, thermo
stated at 25.0 0.5 C, with 10 mm bandwidth for the
emission and excitation monochromators.
Varian ProStar HPLC system connected with PDA detector
and Empower software for data acquisition were used to
run chromatographic analysis.
2.2. Reagents and materials
The ciprofloxacin standard (EDQM, Strasbourg, France)
and the Pharmaceutical grade excipients were provided by
the Senegalese National Medicines Control Laboratory
(LNCM, 39, Avenue Pasteur, Dakar).
Six commercially available formulations codified A, B, C,
D and E were purchased from local drugstores and
analyzed with this method.
Distilled water produced in situ was used to prepare
solutions. No organic solvent was used.
Solutions were filtered through Whatman cellulose filter
grade 42 (GE Healthcare, France).
2.3. Selection of wavelengths
Determination of excitation and emission wavelengths
Excitation and emission spectra were obtained by
Luminescence spectrophotometer. Excitation and emission
wavelengths were respectively determined at 280 nm and
450 nm.
2.4. Preparation of standard stock solution
An amount of 4 mg of USP standard of ciprofloxacin
hydrochloride accurately weighed was transferred to 200
mL volumetric flask and dissolved in distilled water. A
volume of 2 mL of this solution was again transferred to
100 mL volumetric flask and completed to the mark with
distilled water.
2.5. Preparation of calibration curve
From the above stock solution (Cs), an aliquot was further
diluted with distilled water to get working standard
solutions (Cw) of 20-40-50-100-200-250 ng/mL.
2.6. Statistical analysis
All experiments were performed at least in triplicate and
the results are expressed as mean values standard
deviations (SD) and/or relative standard deviations (RSD).
Excel software version 2007 (Windows XP) was used to
analyze obtained data.
Suitable statistical tests were performed on validation data
[9].
3. METHOD VALIDATION
The method was validated for the following parameters
according to the International Conference Harmonization

73

(ICH) guidelines [8] (specificity, linearity, limit of

detection (LOD), limit of quantification (LOQ), accuracy,
precision.
3.1. Specificity in the presence of excipients
Solutions of ciprofloxacin were prepared in both the
selected media along with and without common excipients
(starch, gelatin, gum arabic and talc) separately. Spectra
recorded from 200 nm to 600 nm for blank, sample and
common excipients were compared in order to verify
interference [11].
3.2. Linearity and range
For linearity, five solutions at different concentration (2040-50-100-200-250 ng/mL) were prepared using seven
different aliquots of Cs. The obtained data were used for
the linearity calibration plot. The measured fluorescence
intensity was plotted versus the final concentration to
obtain the calibration graph. Least square regression
analysis is done for the obtained data.
The limit of detection (LOD) and the limit of quantification
(LOQ) for the assay were also determined by using
calibration standards [8, 10, 12].
3.3. Accuracy/recovery studies
Accuracy was determined by the recovery studies in the
formulation of ciprofloxacin. Recovery studies were carried
out by addition of known quantities of standard drug
solution to pre-analyzed sample at five different
concentrations- 60%, 80%, 100%, 120% and 140% of
reference solution.
The percentage recoveries were calculated. Accuracy was
expressed as relative errors which can be calculated by the
equation:
Relative error(%)=(Mean determined value-Theoretical(added amount)/
Theoretical

3.4. Precision
Repeatability results indicate the precision under the same
operating conditions over a short interval of time and interassay precision. Intermediate precision expresses withinlaboratory variations in different days and in different
instruments.
In this work, intra-day precision (repeatability) and interday precision study (intermediate precision) of the method
were assessed at three concentration levels (100, 200 and
250 ng/mL) (n=3) against a qualified reference standard.
The inter-day precision study was performed using the
same protocol on three different days i.e day 1, day 2 and
day 3 at different concentration levels (100, 200 and 250
ng/mL) (n=3). These inter-day studies form part of
robustness. The relative standard deviations (% RSD) of
the obtained assay values at three different concentration
levels were calculated.
3.5. Stability studies
Samples prepared for repeatability study were preserved for
24h at room temperature and were analyzed on the
following day to test for short-term stability [11]. Mean
percentage recovery was determined.
3.6. Assay of content of ciprofloxacin in selected
marketed brands
Five market brands of ciprofloxacin tablets or bolus were
randomly selected and analyzed using the newly developed
and validated method. An exact amount of powder or

International Journal of Analytical and Bioanalytical Chemistry 2013; 3(3): 72-77

suspension of ciprofloxacin was accurately weighed or

measured and transferred to 200 mL volumetric flask and
made up to the mark with distilled water. Then the solution
was shaken for 20 min. The resulting solution was further
diluted with distilled water and filtered through whatman
cellulose filter grade 42. A volume of 1 mL of the above
solution was pipetted out into 200 mL and 100 mL
volumetric flask and made up to the mark with distilled
water. The fluorescence was measured against the blank.
The amount of the drug in a sample was calculated from
the calibration curve using the following equation.
Content of ciprofloxacin (%) per tablet = 200(mL)
200

Or

100

331.34

367.81100

331.34

200(mL) 100 100 367.81 100

presence of excipients (fig. 2b) and excipients mixture (fig.

2c). The excitation and emission spectrum of ciprofloxacin
was not changed in the presence of common excipients in
both the selected media. No fluorescence was noted with
excipients (fig. 2c) at the measuring conditions. The
fluorescence curve at 280 nm (specific to ciprofloxacin)
appears only in fig. 2a and 2b as expected. For the fig. 2a,
2b and 2c, the curve at the excitation wavelength (280 nm)
is known as Raman scattering and due to inelastic
scattering in solvents. This curve is always close to the
curve known as Rayleigh scattering (elastic scattering)
always more intense [13]. This result is thus in accordance
with excipients chemical structures. Therefore the proposed
method is specific for the analysis of drugs without prior
extraction.

Where is the concentration found based on the

fluorescence intensity (ng/mL), Ws is the weight of generic
sample powder (g), Wst is weight of the dosage
ciprofloxacin announced (g), W is the average weight of
tablet (g), P is the potency of ciprofloxacin hydrochloride
standard, 331.34 g/mol
the molecular weight of
ciprofloxacin and 367.81 g/mol the molecular weight of
anhydrous ciprofloxacin hydrochloride .
4. RESULTS AND DISCUSSIONS
4.1. Method development and optimization
Media optimization showed that Ciprofloxacin is freely
soluble in aqueous medium at concentration much higher
than our working and stock concentrations. Then no
organic solvent was used. The final decision of using water
as a media is based on criteria like solubility, sensitivity of
the method, cost, ease of preparation and lack of toxicity.
During the development phase, the maximum absorption
wavelength in water appeared at 280 nm (fig. 1) while the
maximum excitation and emission wavelengths appeared
respectively at 280 nm and 450 nm (fig. 2). The maximum
absorption and excitation wavelength is the same although
different values can be observed.

Fig.2: Spectra of excitation and emission of pure

ciprofloxacin (a), ciprofloxacin with excipients (b) and
excipients mixture (c)

Fig.1: UV spectrum of ciprofloxacin.

The linear regression equation obtained is absorbance at
450 nm Y =1.041 - 4.222 with a regression coefficient of
0.9993.
Otherwise, the analysis time did not exceed five minutes.
4.2. Analytical validation
4.2.1. Specificity in the presence of excipients
Fig. 1 shows the UV absorption spectrum of pure
ciprofloxacin while fig. 2 shows spectra of excitation and
emission of pure ciprofloxacin (fig. 2a), ciprofloxacin in

74

4.2.2. Linearity, Range, LOD and LOQ

The calibration curve was linear over the concentration
range 20-250 ng/mL and the regression equation was found
to be y = 1.041 x - 4.222 with correlation coefficient (R2) of
0.9993.
The correlation coefficient greater than 0,99 means that the
method is linear [2,10, 11, 14]. The two concentration
limits are measured under repeatability conditions (n=6).
The ration of the two variances (Fcalc) is compared with
F0,99 (Snedecor law) [15]. Since Fcalc (4.18) < F0,99 (10,67),
the linearity range is acceptable [15]. A good linear
relationship was observed in the concentration range of 20250 ng/mL.
The LOD and LOQ were calculated as 4.53 ng/mL and
5.67 ng/mL respectively [16].

International Journal of Analytical and Bioanalytical Chemistry 2013; 3(3): 72-77

Table I: Recovery/accuracy for five different concentration of ciprofloxacin by the proposed method (n=8).
Dosage

Label claim

Pre-formulated
granulated

100 mg

Amount added (%)

60
80
100
120
140

Amount foundSD
157.44 0.25
178.90 0.40
201.70 0.30
224.99 0.41
244.58 0.25

RSD (%)
0.16
0.23
0.14
0.18
0.10

Relative error (%)

-1.60
-0.61
0.85
2.27
1.90

% recovered
98.40
99.40
100.80
102.26
101.91

Table II: Repeatability and intermediate precision determined for three different concentrations of ciprofloxacin (n=3).
Repeatability and intermediate precision determined for three different concentrations of ciprofloxacin (n=3)
Repeatability
Intermediate precision
Declared
concentration
(Mean SD
RSD
Average
RSD
Average
(Mean SD ng/mL)
(ng/mL)
ng/mL)
(%)
potency (%)
(%)
potency (%)
100
102.86 0.20
0.25
102.86
102.25 0.30
0.29
102.25
200
192.53 0.20
0.14
96.26
192.77 0.31
0.21
96.38
250
255.21 0.24
0.12
102.08
254.69 0.29
0.15
101.88
The slope of 1.041 and the low limits of detection and
quantization reaching both nanogram per liter indicate
clearly that the developed method is sensitive. These results
are in accordance with the sensitivity of fluorimetric
analytical techniques [17].
No significant difference was observed in the slopes of
calibration plots prepared on different days (ANOVA, P >
0.05). It can be concluded that the developed method is
sensitive.
4.2.3. Accuracy/recovery studies
The excellent % recovery (nearly 100%) and their low
standard (<0,5) represent accuracy. In water, the mean
percentage recoveries for concentration were found to be in
the range of 98.40 to 102.26% (Table 1). The % RSD in
accuracy studies was calculated at each concentration
levels and was found to be less than 0.5% (Table 1). Also
relative error (inaccuracy) values ranged from -1.87% to
2.27% as shown in table 1.
Since Ccalc (0.32) < C0,99 (0.63), the Cochrans test revealed
no high standard deviations. Also, since Gcalc < G0,99 (1.97)
(for all means at each concentration level), the Grubbs test
showed no outlying means.
No significant difference was observed between the mean
values of amounts added and found (p > 0.05).
These results revealed that any change in the drug
concentration in the solution can be accurately determined
by these proposed methods [18].
The high percentage recoveries obtained in Table 1 for
various amounts of ciprofloxacin in formulated mixture
with excipients suggested that there is no interference from
any of the excipients (such as starch, gelatin, gum arabic
and talc) as evidenced by the lack of absorbance and
emission at the specified max for the excipients and blank
solutions [11].
4.2.4. Precision
The RSD in precision studies was found to be 0.12-0.25 %
for repeatability (Intra-day precision) and 0.15-0.29% for
intermediate precision (Inter-day precision) (Table 2).
In all cases, R.S.D. values were within the acceptable range
indicating that this method has excellent repeatability and
intermediate precision.
The RSD in precision studies was found to be 0.12-0.25 %
(Intra-day) and 0.15-0.29 % (Inter-day) (Table 2). The
intra-day and inter-day precision study (Table 2) of the

75

developed method confirmed adequate sample stability and

method reliability where all the RSDs were < 2%. These
results clearly indicate that the method is precise enough
for the analysis of the drug.
4.2.5. Stability studies
Table 3 shows the short-term stability study results which
are in the acceptance range over 24h. Stability studies are
in the acceptance range (Table 3) with average potencies
ranged from 96.3% to 102.85% with RSD < 2% at each
level, after one day storage at room temperature [11].
4.5. Assay of content of ciprofloxacin in selected
marketed brands
The proposed method was applied to the determination of
ciprofloxacin content of five marketed products randomly
selected (five tablets samples coded A to D and one
injectable sample coded E). The results of these assays
yielded 95.19 % to 102.2% for tablets and 101.3% for the
injectable ciprofloxacin sample (table 4).
Ciprofloxacin content of all analysed products was in good
agreement with the label claims and the Pharmacopeias
specifications [6,7]. All calculated RSDs values were less
than 1% (table 4). These values meet official requirements
[8] and many authors considered that these values are very
satisfying [2, 10, 11].
The USP reference method, which involves the use of
reverse-phase C18 column, mobile phase consisting of
0.025 M phosphoric acid and acetonitrile (87:13), flow rate
= 1.5 mL/min and detection wavelength at 278 nm [6], was
compared to our developed method by assessing the
ciprofloxacin content of four other locally marketed
products randomly selected.
For each pharmaceutical preparation, the results of the
proposed methods were statistically compared with those of
the reference method (for accuracy and precision) using the
T-test and the one-way analysis of variance [19-21]. The
calculated T-values and F-values did not exceed the critical
value (critical T-value (p=0,05) = 4,30; critical F-value
(p=0,05) = 161) (table 5). These results indicate that there
were no significant differences between the proposed
methods together with the reference method (Table 5). The
accuracy and precision of the proposed methods are
satisfactory to the label claim amount as indicated from the
% recovery, RSD (%) and relative errors values (Table 5).
To our best knowledge, this analytical fluorimetric method

International Journal of Analytical and Bioanalytical Chemistry 2013; 3(3): 72-77

Table III: Short-term stability determined by the proposed method (n=3)

Short-term stability determined by the proposed method (n=3)
Concentration found
Prepared concentration (ng/mL)
(Mean SD ng/mL)
RSD (%)
20
19.26 0.07
0.32
40
41.17 0.06
0.15
50
48.91 0.04
0.07

Average potency (%)

96.30
102.85
97.82

Table IV: Content of ciprofloxacin in five marketed products determined by the proposed method (n=3)
Concentration found
Drug found (%)
(mg)
Sample N Brand name (code) Label claim (mg)
Mean
SD
Mean
SD
1
A
750
743.62
0.19
99.15
0.03
2
B
500
475.99
0.01
95.19
0.05
3
C
500
495.21
0.05
99.04
0.02
4
D
500
511.03
0.01
102.20
0.05
5
E
100
101.30
0.01
101.30
0.01

RSD (%)
0.50
0.21
0.08
0.19
0.50

Table V: Application of the USP reference method (method A) and the proposed method (method B) for the determination
of ciprofloxacin in four pharmaceutical preparations (n=2).
Method A (HPLC)
Method B (Fluorimetry)
relative
Sample code
T- value F-value
error (%)
MeanStandard deviation (%)
MeanStandard deviation (%)
E
96.81 0.91
97.08 2.77
1.09a
9.31b
0.28
a
F
103.20 1.30
103.48 6,03
0.06
21.51b
0.27
G
103.88 2.22
100.52 1.06
0.46a
4.41b
-3.24
H
108.04 0.57
110.26 2.15
1.70a
13.94b
2.04
a
critical T-value (p=0,05) : 4,30
b
critical F-value (p=0,05) : 161
is the only ciprofloxacin assay method using water as
Stationery Office: London, 2009.
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Such a method can be called green analytical technique.
Formulary NF29, vol. 2, Rockville, 2011.
7. WHO, World Health Organization, The International
5. Conclusion
The proposed method is simple, sensitive, cheap, less time
Pharmacopoeia, Quality Specifications, third ed, vol.5,
consuming and reliable (RSD <1%) with good precision
World Health Organization, Geneva, 2003.
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estimating the commercial formulations without
Validation of Analytical procedure: Methodology, ICH
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guideline, 2005.
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Source of support: Nil; Conflict of interest: None declared

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International Journal of Analytical and Bioanalytical Chemistry 2013; 3(3): 72-77