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Development of an Ecological-friendly Method for Ciprofloxacin

Determination and Cloud Point Extraction in Pharmaceuticals using
Fe(II) (FeSO4.7H2O)
To cite this article: Abbas S. Fahad et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 871 012028

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The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

Development of an Ecological-friendly Method for Ciprofloxacin

Determination and Cloud Point Extraction in Pharmaceuticals using Fe(II)
(FeSO4.7H2O)

Abbas S. Fahad1, Mohammed Z. Thani2,Asmaa M. Abdullah3,SaadiyahA Dhahir4

1,2,3
Department of Chemistry, College of Science, Al-Mustansiriyah University,
Baghdad-Iraq.
4
Department of chemistry, College of science for women, University of Baghdad-
Iraq.
2
Corresponding Author: [email protected].

Abstract. In this study, new spectrophotometric methods development for evaluation of ciprofloxacin are
described. The first method including conversion of ciprofloxacin to colored complex with Fe (II) in the
alkaline medium. The colored product has a yellow color with absorbance at λmax 450 nm. Between the
concentration range (2-90 µg.mL-1) , the Beer,s law is obeyed with correlation coefficient ( R2) as 0.9996 ,
limit of detection as 0.365 µg.mL-1, limit of quantification as 1.189 µg.mL-1. Secondly, cloud point
extraction was used to determine of a trace amount of the colored product in the first method followed by
measuring with a UV-Vis spectrophotometer. The linearity of calibration curve was above the range of (1-
50µg.mL-1) , the correlation coefficient ( R2) was 0.9995. The Limit of detection (LOD) and Limit of
quantification (LOQ) were found to be 0.129 and 0.387 µg.mL-1respectively. This method was successfully
employed for ciprofloxacin detection in the several pharmaceutical samples by Rec. Was rang between
(98-102.8).

Key word: Ciprofloxacin, Metal Fe(II), Cloud point Extraction, Ecological-friendly

1. Introduction
Ciprofloxacin [1- cyclopropyl-6-fluoro- 1,4 – dihydro – 4- oxo -7- ( 1- piperazinyl ) -3- quinolone
carboxylic acid ] (Figureure 1) , is used to for treat the of bacterial infections. It is believed that the mode of
action of the quinolone family is through binding DNA gyrase enzyme [1,2]. It proves to be effective in the
treatment of infections caused by microorganisms such as , Escherichia coli , Haemophilusducreyi,
Neisseriagonorrhoeae, Mycobacterium intracellar, Brucella campylobacter, Pseudomonas aeroginosa ,
Enterobacteriaceae , Vibrio , Haemophilus influenza , Neisseria meningitides , Moraxella catarrhalis and
Bacillus anthracis [3]. Ciprofloxacin can be estimated using different analytical methods like HPLC [4 -9 ],
capillary electrophoresis [10,11], fluorimetry [12], chemiluminometry [13], voltammetry [14 , 15], high
performance capillary electrophoresis [16], spectrofluorimetry [17] and ISE-based potentiometry [18]. The
reproducible, sensitive, accurate and precise analytical methods are required to an evaluation of
ciprofloxacin in the pharmaceutical samples. Therefore, we existed in this work new methods for
determination of ciprofloxacin using complication reaction with iron metal and pre-concentration,
extraction it by cloud point extraction (CPE). CPE technique has become increasing popular compared
with classical extraction methods due to the advantage of low consumption of organic solvents , rapid

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The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

phase separation, high enrichment factor, low cost and high recovery. This technique is attractive that
decreases the consumption and exposures to the solvent is CPE and also, it decreases extraction time and
disposal costs that have been used for pre-concentration of ciprofloxacin after the formation of a complex
which poorly water-soluble [19- 29]. The aim of the present work, is to combine and develop of cloud point
extraction ( CPE) with a spectrophotometric method to determine the ciprofloxacin, as high sensitive
method.

Figurer 1.Ciprofloxacin structure

2. Experimental
The spectra and absorbance measurement were achieved in single beam UV-Vis spectrophotometer 160
equip with (1cm ) quartz cell. The pH values were measured by metlar pH meter. All chemicals and
reagents used without further purification and freshly prepared. However, ciprofloxacin was purchased
from the general company for the manufacture of medicines and medical supplies the state company for
drugs industry and medical appliances samarra –Iraq and (FeSO4.7H2O) from Merck company (MSD). A
stock iron solution (250 µg/ mL) was prepared by dissolving (0.025 g )of FeSO4. 7H2O in D.W and diluting
to (100 mL). Stock ciprofloxacin solution ( 250 µg/ mL) was prepared by dissolving ( 0.025 g ) in D.W and
dilution to the mark in ( 100 mL ) volumetric flask . 0.01M of hydrochloric acid, 0.01 M sodium hydroxide,
10 % of TritonX-100 .

2.1. General procedure of ciprofloxacin complication

An aliquot of (10 mL) solution containing (1 mL) of ciprofloxacin (250 µg/mL), was mixed with (0.2mL)
of pH 8.3(Ammonia, ammonium chloride), (0.6mL) of FeSO4.7H2O ( 250 µg / mL), and then mixture was
completed to 10 mL with D.W. The yellow solution formed that gave absorbance at λmax 450 nm.

2.2. General procedure of CPE for Ciprofloxacin

Various concentrations (1-50µg / mL) of ciprofloxacin complex put in the (10 mL) centrifuge tubes, then
1.8 mL of Triton-X-100 and D.W was added to make total volume of solution to 10 mL. The mixture
solution kept in the thermostatic bath (800C) for 40 min. Separation of two phases was carried out by

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 The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

centrifugation (Hermle Labor Technik GmbH-Z200 A Universal Compact Centrifuge) for 1 min at 1000
rpm. The mixture was cooled to increase the viscosity of the surfactant- rich phase and aqueous phase was
easily disposal by decantation. The rich-surfactant phase from this procedure was diluted with 1 mL of
methanol and transferred into quartz cell to measure its absorbance at 450 nm .

2.3. Pharmaceutical preparations procedure

Ciprofloxacin tablets provided from Dar Al-Dawa or the state company for drugs industry and medical
appliances samarra –Iraq and Pharma International company containing (500 mg) were carefully weight ,
then average tablets weight was extracted. The valet weight (0.737gm for one table) was dissolved in D.W
to ensure the complete solubility, then made up to volumetric flask (100 mL) and the solution was filtrated.

3. Results and Discussion

Absorption spectra of ciprofloxacin and its Fe(II) complex in pH 8.3 were measured against reagent blank
as the reference (Figure.2). The complex formation was accompanied with red shift of λmax of
ciprofloxacin( 450 nm) by125nm. Investigations were achieved to establish the most favorable conditions.
The effect of each of the following variable on the reaction was studied.

3.1 Study of optimization reaction of complication

Different parameter affected absorbance of complication solution, such as pH, type of buffer , volume of
buffer, concentration of metal , temperature and time of reaction . The influence of pH on the absorbance at
a fixed concentration of complex was investigated in the range pH (1-13.5) using NaOH (0.1M) and
HCl(0.IM) .Various types of buffer solutions such as (Ammonia/ammonium chlorid, Sn( OH)2/SnCl2 and
Li(OH).H2O/LiI ) were examined . The best one is ammonium buffer and optimum value was pH8.3(
Figure.3). Thus, (0.2 mL) of buffer solution (Ammonia/ammonium) pH 8.3 was chosen in the subsequent
experiments.

Figure 2. Absorption spectra of ciprofloxacin and its complex Figure 3. Effect of PH on complication of 25 µg.mL-1
Drug, Metal Fe+2 against Water and Complex CIP-Fe+2 Ciprofloxacin with Fe(II)
against a blank prepared under the same conditions without
drug.

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 The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

The influence of the amount of ion metal (Fe+2) was studied by varying concentrations of (Fe+2) used from
5-62.5 μg /mL in the complication process and it was found that 15 μg /mL gave the optimum absorption
intensity as shown in (Figure.4) . The temperature and reaction time was investigated. It was observed that
the temperature 25 0C and reaction time 10 min were the optimum conditions to obtain the heights
absorbance for ciprofloxacin, as shown in (Figure.5) and (Figure.6). The continuous variation and mole
ratio methods were used to estimate the stoichiometry of ciprofloxacin : Fe(II) ratio. The results showed
that the ratio of 1:1 (drug: Fe+2) (Figure.10 and Figure.11) .

Figure 4.Effect of Concentration Fe(II) on complication Figure 5. Effect of Temperature on complication of 25 µg.mL-
process with 25 µg.mL-1 Ciprofloxacin 1
Ciprofloxacin with Fe(II)

Figure 6. Effect of Time on complexation of 25 µg.mL -1 Figure 7. Effect of surfactant volume on complexation of 25
Ciprofloxacin with Fe(II) µg.mL-1 Ciprofloxacin with Fe(II)

Figure 8. Effect of Temperature on CPE of 25 µg.mL-1 Figure 9. Effect of Time on CPE of 25 µg.mL-1 Ciprofloxacin
Ciprofloxacin with Fe(II) with Fe(II)

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The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

Figure10. Mole ratio method of complex(Fe(II):CIPRO) Figure 11. Continuous variation method of complex
(Fe(II):CIPRO)

3.2 Analytical Data: Under the enhanced conditions, the absorption intensity of ciprofloxacin drug
increases linearly as the concentration of ciprofloxacin drug increases.

3.3 Precision and Accuracy: The accuracy was evaluated by determination of the percentage, relative error
and recovery and the precision was estimated by the percentage relative error (RSD%) (Table. 2).
Table. 1 shows the comparison between the spectrophotometric method and cloud point extraction and we
observed that the second method gives the best results.

3.4 Optimization Study of cloud point extraction for ciprofloxacin

A series of different types of surfactant (Triton-X100, TritonX-114, Tween-80, Tween-20, SDS and
CTAB) and volumes in the range ( 0.2-2.6 mL) to improve CPE was investigated. The results illustrate that
the absorption intensity will increasing with surfactant conc., consequently, 1.8 mL of (10%, TX-100) was
chosen as optimum ( Figure.7). The influence of temperature and extraction time has also been investigated
by various temperature ( 60-90 0C ) and extraction time ( 20-70 min). The results showed that 80 0C and 40
min were the best and selected as optimum (Figure.8) and (Figure.9).The centrifugation speed and time
were studied to gain excellent extraction, the centrifugation speed and time were investigated in the range (
1000-6000 rpm) and (1-6 min) respectively. It found that 1000 rpm and 1min were chosen as optimum.
Methanol was added to the surfactant- rich phase after cloud point extraction as the surfactant- rich phase
was highly viscous to ease its transfer into sample cell. The amount of (1 mL) methanol was chosen to have
amount of sample for transferring and measuring the absorption intensity of the sample.

Analytical data of CPE for ciprofloxacin drug: Under the optimum conditions founded by CPE method for
the determination of ciprofloxacin and linear calibration curve was established by plotting concentration of
ciprofloxacin versus absorbance ( 1-50 µg.mL-1) .

Table 1. Comparison the analytical parameter of determination CIP drug between two method

Parameters Before CPE After CPE

λmax nm 450 450

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The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

Color yellow yellow

Regression equation y = 0.0029x + 0.016 y = 0.015x + 0.075
Linearity range( µg/mL-1) 2-90 1-50
Correlation Coefficient (R2) 0.9996 0.9995
Ɛ(L.mol-1.cm-1) 960.915 4970
Sandell,ssensivity (µg . cm-2) 0.344 0.066
Slope (b) 0.0029 0.015
Intercept(a) 0.016 0.075
Limit of detection( µg/mL-1) 0.356 0.129
Limit quantification( µg/mL-1) 1.189 0.387
EF 517.2
PF 13.4
-4
C.L.for the slope(b±tsb) at 95% 0.0029 ± 6.44x10 0.015±1.141 x 10-3
C.L.for the intercept(a±tsa) at 95% 0.016± 8.44x10-7 0.075±2.37 x 10-2
Standard error for regression line (Sy/x) 0.0299 0.01875
C.L for Conc. 10 μg ml-1at 95% 9.83±1.2502 10.06±1.6540
C.L for Conc. 20 μg ml-1at 95% 20.3±0.993 20.3±0.6566
C.L for Conc. 30μg ml-1at 95% 29.9±1.382 29.76±2.069

Table 2. Application of the proposed CPE for the evaluation of ciprofloxine

Before cloud point extraction

drug Conc. of drug Relative Revco Average RSD%
µg/mL-1 Error% % Recov% (n=5)
Taken Found
Ciprodar 10 9.79 -2.1 96.4 98 5.0
20 20.25 1.25 101.2 2.8
30 30.3 1.00 101.0 3.2
Cipopharm 10 9.44 -5.6 98.0 98.9 5.1
20 19.4 -3 99.5 4.4
30 29.6 -1.33 99.3 2.5
After cloud point extraction
10 10.1 1.0 101 102.2 4.2
Ciprodar 20 21.4 7.0 107 1.9
30 29.6 -1.3 98.6 3.9
10 10 0 100 102.8 4.0
Cipopharm 20 22.2 11 111 1.1
30 29.3 -2.3 97.6 1.7

Table 3. Comparison the values of LOD and LOQ of the CPE method with various methods reported in
literature

Method LOD µg/mL LOQ Ref.

µg/mL
Titrimetric 0.05 0.09 [30]
A first-derivative spectrophotometric 0.18 0.60 [31]
Cloud point extraction 0.77 2.57 [32]
HPLC 0.11 0.35 [33]
HPLC 0.0587 0.1779 [34]
Liquid-Liquid 0.19 - [35]
Self-Assembled Gold Nanofilm
HPLC 0.25µM - [36]
HPLC 0.16 0.28 [37]
matrix is potential synchronous spectrometry 0.0135 - [38]
Reverse flow injection spectrophotometric 0.20 0.69 [39]
Cloud point extraction 0.075 0.129 Present work

4. Conclusion

A simple, precise, accurate, ecological-friendly and sensitive spectrophotometric method for the estimation
of trace amount of ciprofloxacin. The first method containing convention ciprofloxacin to the colored
product was measured using UV-Vis spectrophotometry the second method is cloud point extraction that

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The First International Conference of Pure and Engineering Sciences (ICPES2020) IOP Publishing
IOP Conf. Series: Materials Science and Engineering 871 (2020) 012028 doi:10.1088/1757-899X/871/1/012028

was to determination and pre-concentration of ciprofloxacin complex. The method is cheap and less time
consulting compared with the other methods like, SPE , liquid-liquid extraction and HPLC .

5.Acknowledgments

The authors are grateful to the department of Chemistry, college of science, Mustansiriyah College because
of offering laboratory for services in conformity with the provider laboratory for presenting spectra and
analytical enmeshments.

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