Journal of Applied Pharmaceutical Science 01 (08); 2011: 239-243

Sani A. Ali
Department of Pharmaceutical Chemistry, University of Maiduguri , Nigeria.

ISSN: 2231-3354
Received on: 25-09-2011
Revised on: 08:10:2011
Accepted on: 15-10-2011

Sani A. Ali, Chijioke C.

Mmuo Department of
Pharmaceutical Chemistry,
University of Maiduguri ,
Nigeria.

Rafat O. Abdulraheem
Department of Food Science
and Technology, University of
Maiduguri , Nigeria.

Sikirat S.
Abdulkareem
Department of
Chemistry, Federal
Polytechnic, Damaturu,
Yobe State, Nigeria.

Emmanuel T. Alemika
Department of
Pharmaceutical and
Medicinal Chemistry,
University of Jos , Jos,
Nigeria.

Musa A. Sani
Department of Heamatology,
University of Ilorin Teaching
Hospital,Ilorin, Nigeria.

Mohammed Ilyas
Department of
Pharmaceutical and
Medicinal Chemistry, Ahmadu
Bello
University, Zaria, Nigeria.

For Correspondence
 reverse phase high performance liquid chromatography assay has been developed for the
estimation of Ciprofloxacin Hydrochloride in tablet formulation. The separation was achieved by
®
using C-18 column (LichroCART 125x4mm, 5µm) coupled with a guard column of silica in
mobile phase methanol: buffer (0.025M Orthophosphoric acid with the pH adjusted to 3.0±0.1
with triethylamine) (40:60v/v). The flow rate was 2.0ml/min and the drug was detected using UV
detector at the wavelength of 278nm. The retention time was within 1.753 – 1.757 minutes. The
method was validated as per ICH guidelines . The proposed method was found to be accurate,
repeatability and consistent. It was successfully applied for the analysis of the drug in marketed
formulation and could be effectively used for the routine analysis of formulation containing the
High drug without any alteration in the chromatography conditions.

Perform Keywords: Ciprofloxacin, HPLC, Liquid Chromatography.

ance
Liquid
Chromat INTRODUCTION

ography Ciprofloxacin Hydrochloride is a pale yellow, slightly hygroscopic, crystalline powder,

(HPLC) soluble in water, very slightly soluble in dehydrated alcohol, practically insoluble in acetone, in
dichloromethane, and in ethyl acetate and slightly soluble in methyl alcohol. A 2.5 % solution in
Method water has a pH of 3.5 to 4.5. Store in airtight containers. Protect from light (Martindale, 2009).
Develop Drug profile

ment 3-quinoline carboxylic acids, I cyclopropyl -6- fluro -1,4-dilydro-4-oxo-7-(1-

piperazinyl)-monohydrochloride.
and Chemical structure
Validatio
n
Indicatin .HCl
g Assay Molecular weight: 367.82g/mol
for Molecular Formular: C17H18FN3O 3.HCl
Uses
Ciproflox Ciprofloxacin is the most commonly used fluoroquinolones and it is a broad-spectrum antibiotic,
effective against both gram positive and gram negative organisms (Rang, et al 2008). It is particularly active
acin
Hydrochl
oride

Sani A. Ali,
Chijioke C. Mmuo,
Rafat O.
Abdulraheem,
Sikirat S.
Abdulkareem,
Emmanuel T.
Alemika, Musa A.
Sani and
Mohammed Ilyas

ABSTRACT

A new
simple, rapid,
selective, precise and
accurate isocratic
 Journal of Applied Pharmaceutical Science 01 (08); 2011: 239-243

against gram negative bacteria, including Salmonella, Shigella, scanned absorption spectrum for Ciprofloxacin HCl. The spectrum
Campylobacter, Neisseria and Pseudomonas. Ciprofloxacin has was scanned over the range of 200-400nm and was obtained by
only moderate activity against gram positive bacteria such as measuring the absorption of 20µg/ml solution of Ciprofloxacin
Streptococcus, Pneumoniae and Enterococcus faecalis, it should HCl in mobile phase, prepared from a stock solution. The
not be used for pneumococcal pneumonia (BNF, 2010). Also use spectrum was obtained by using a 1cm silica cell and the reference
for Chronic bacterial prostatitis ( Dimitrakov, etal,2009) to Lower cell contained mobile phase; as a result a wavelength of 278nm
respiratory tract infection ( this two are not recommended as a was chosen which correspond with USP standard.
first-line antibiotic choice) (Vardakas, etal,2008) for treatment of
tuberculosis (Thomas, et al, 2008). OPTIMIZATION OF CHROMATOGRAPHIC CONDITION
Spectroscopic analysis of Ciprofloxacin HCl showed that
Availability it has a maximum UV absorbance at 278nm. Therefore, the
Ciprofloxacin is available for oral administration in chromatographic detection was performed at 278nm using a UV-
250mg, 500mg and 750mg tablets, parental administration in Visible detector. It was observed that when Ciprofloxacin HCl was
2mg/ml, ciprofloxacin ophthalmic solution in 0.3% as base. injected at the mobile phase composition ACN: buffer (13:87)
Several papers have been described for the determination (USP) as shown in fig I, it indicates non specificity in activity.
of ciprofloxacin in biological fluids by HPLC with UV (Gladys,
1992) or fluorescence (El-Yazigi A. and Al- Rawithy S., 1990) PREPARATION OF MOBILE PHASE
detector or by microbiological methods (Jehl et al, 1985). Poor
Preparation of Buffer
reproducibility and accuracy for the last method has been reported
1.6ml of 15.7M of Orthophosphoric acid was dissolved in
(Jehl et al, 1990). However, HPLC is the analytical method of
choice for measuring and assaying ciprofloxacin (Teja-Isavadharm 1000ml distilled water and final concentration made up to 0.025M.
et al, 1991). Literature survey has also revealed a few HPLC Triethylamine was used to adjust the pH to 3.0 ± 0.1.
methods for the estimation of Ciprofloxacin HCl and Tinidazole
Mobile Phase
(George et al, 2000; Garcia and Albero, 2001). Also some HPLC
Put 400ml of methanol into a 1000ml beaker, to this add
methods are available for the determination of Ofloxacin and
Ornidazole (Bakshi et al, 2001; Natrajan and Raman, 2005). 600ml of the buffer (Orthophosphoric acid + Triethylamine), it
Similarly some HPLC methods are reported for the estimation of was sonicated for 10minutes using the sonicator and filtered using
Ofloxacin and Tinidazole (Panzade and Mahadik, 2000; Amini et the vacuum pump.
al, 2005; Salomies and Salo, 2005; Behl et al, 2005). HPLC
Mobile Phase Ratio
method for simultaneous estimation of Ciprofloxacin HCl,
Methanol: Buffer (Orthophosphoric acid + Triethylamine)
Tinidazole, Ofloxacin and Ornidazole was also carried out (Ranjit
= 40:60
et al, 2009).

METHODOLOGY Standard Preparation

Method that was carried out using the template and the 25mg of the standard Ciprofloxacin HCl was dissolved in
sketch below as a guiding principle. The rationale behind the use 50ml of the mobile phase. Dilution (1:25) from this solution was
of this method is that Ciprofloxacin HCl has a molecular weight made to obtain a concentration which contains 0.02mg/ml of the
less than 1000, soluble in water, and ion forming, thus the use of standard solution and 20µl was injected to obtain the peak area as
Reversed phase chromatography. Methanol was thought of to shown in fig IV.
replace acetonitrie because of its greater polarity in silica column.
Sample Preparation
MATERIALS 20 intact tablets (Ciprofloxacin) were weighed accurately
The Chromatographic system consisted of HITACHI L- to obtain the average tablet weight; the tablets were then crushed
2130 pump connected to an autosampler L-2200 syringe loading and triturated in a mortar until a fine powder was obtained. An
sample injector valve fitted with a 20µl sample loop of 200vials, a amount of the powder equivalent to one tablet (25mg) was
variable wavelength UV-VIS detector L-2420. A column oven L- weighed accurately and taken into a 50ml volumetric flask. The
2300, packed with silica C18, 5µm particle size, an organizer and powder was dissolved in 50ml of the mobile phase. Dilution (1:25)
diode Arrary Detector L-2455. Standard Ciprofloxacin HCl was from this solution was made and the clear solutions were then
obtained from NAFDAC Drug Laboratory, Maiduguri. ready for injection (20µl was injected).
Ciprofloxacin tablets were obtained from pharmacy outlets and
CONDITIONS OF CHROMATOGRAPHIC METHOD
patent medicine stores within Maiduguri metropolitan of Borno
Chromatographic separation was performed using an
State.
isocratic elution at 40oc on C-18 column (5µm particle size) parked
DETECTION OF WAVELENGTH with silica. The mobile phase was composed of Methanol: Buffer
In setting up the conditions for development of the assay (40:60v/v) and pH of mobile phase was adjusted to 3.0 ± 0.1 with
method, the choice of a detection wavelength was based on the triethylamine. The flow rate is 2.0ml/min and the injection volume
 Journal of Applied Pharmaceutical Science 01 (08); 2011: 239-243

was 20µl. UV measurement was made at wavelength of 278nm. Sample B

The retention time was between 1.750 to 1.753minutes for
Sample Name : B – 20 µg/ml
samples.
System : HPLC
Table I: Uniformity weight of tablets. Detector : UV – VIS
S. No. Sample Average weight 0f Amount
tablets (mg) ± SD weighed (mg)
Type of Analysis : Peak Area
1 A 919.2 ± 0.01 45.96 Peak area : 4628085.7
2 B 681.2 ± 0.03 34.06
3 C 766.9 ± 0.01 38.35 Retention time : 1.750 minutes
4 D 740.4 ± 0.01 37.02
5 E 833.6 ± 0.02 41.68
Fig VB

RESULTS
UV-VIS

METHOD VALIDATION Retention Time

The method was validated in terms of the following 75 75

parameters; linearity, specificity, accuracy, precision and system

50 50
suitability (ICH).

mAU

mAU
Calculations 25 25

Mg/tab = Asa × Mstd × P × AVem

7501.
Astd × Msa × 100
0 0

Asa = Area of sample peak 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Astd = Area of standard peak Minutes

Mstd = Mass of standard weighed (in mg)

Msa = Mass of sample weighed (in mg) Mg/tab = 4628085.7 × 25 × 99.7 × 681.2 = 528.13mg (105.6%)
P = Potency of standard in % 4368440.7 × 34.06 × 100
AVem = Average mass of tablets (in mg)

Sample A Sample C

Sample Name : C – 20 µg/ml

System : HPLC

Sample Name : A – 20 µg/ml Detector

25
System : HPLC Type of Analysis
0.063

Detector : UV – VIS
Peak area
1.757

0
Type of Analysis : Peak Area 0 0

Retention time
Peak area : 4724281.3 1
.
1
. 2. 2.
3
. 3. 4. 4. 5.

Retention time : 1.757 minutes 0.0 0.5 0 5 0 5 0 5 0 5 0

1.
0.0 0.5 0
Fig VC Minute
s

Fig VA

UV-VIS

100 UV-VIS 100 Retention Time

Retention Time

75

50
mAU

mAUmAU

50 50
: UV – VIS 75

: Peak Area
50
: 4646783

mAU
: 1.753 minutes
25

1.753
0

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Minutes

Mg/tab = 4724281.3 × 25 × 99.7 × 919.2 = 539.11mg (107.8%) Mg/tab = 4611585.7 × 25 × 99.7 ×766.9 = 526.18mg (105.2%)

4368440.7 × 45.96 × 100 4368440.7 × 38.35 × 100

 Journal of Applied Pharmaceutical Science 01 (08); 2011: 239-243

Sample D could be as result of less potency of the standard Ciprofloxacin

HCl obtained.
Sample Name : D – 20 µg/ml
System : HPLC Table II: Evaluation of Ciprofloxacin in pharmaceutical formulations.
Detector : UV – VIS S.No Sample Labelled Amount Percentage
Type of Analysis : Peak Area amount (mg) obtained by recovery*
Peak area : 4987905 proposed
method
Retention time : 1.753 minutes 1 A 500 539.11 107.8%
2 B 500 528.13 105.6%
3 C 500 526.18 105.2%
Fig VD 4 D 500 569.19 113.8%
5 E 500 473.84 94.8 %
*Average of three determinations

METHOD VALIDATION FOR

UV-VIS

100 Retention Time 100

CIPROFLOXACIN HYDROCHLORIDE
Linearity
Five different concentrations (10-50µg/ml) of
mAU

mAU
50 50 Ciprofloxacin HCl were prepared for linearity studies. The
responses were measured as peak areas as shown in fig VI-X. The
calibration curve obtained by plotting peak area against
0.040

concentration showed linearity in accordance to Beer’s law over

1.753

0 0
this range and the linearity equation was y=211063x – 6934.9 and
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
the regression coefficient was r=0.9993 (n=5).
Minutes
Table III: Linearity of Ciprofloxacin Hydrochloride.
Mg/tab = 4987905 × 25 × 99.7 × 740.4 = 569.19mg S. No. Concentration (µg/ml) Peak area
1 10 2071855
(113.8%) 4368440.7 × 37.02 × 100
2 20 4361218
3 30 6234483
Sample E 4 40 8303177
5 50 10654017
Sample Name : E – 20 µg/ml
System : HPLC
Detector : UV – VIS
Type of Analysis : Peak Area
Peak area : 4152365.7
Retention time : 1.753 minutes

Fig VE
analysis of the formulations are tabulated in table I. The amounts
UV-VIS obtained by the proposed method are between 94.8% and 113.8%,
Retention Time within the acceptance level of 90% to 110% (USP) except for
75 75 sample D which falls out of the range. The reason for the variation

50 50
mAU

mAU

25 25
0.063

1.753

0 0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Minutes

Mg/tab = 4152365.7 × 25 × 99.7 × 833.6 = 473.84mg (94.8%)

4368440.7 × 41.68 × 100

An RP-HPLC method for the determination of

Ciprofloxacin HCl was developed and validated. Results of
Graph I: Linearity curve of Ciprofloxacin Hydrochloride.

Table IV: Specificity of method (retention time of standard is 1.753).

Sample Retention time
A 1.757
B 1.750
C 1.753
D 1.753
E 1.753

SPECIFICITY
The specificity of the method was ascertained by
analyzing standard drug and sample. The retention time (RT) of
Ciprofloxacin HCl confirmed by comparing the RT with that of
the standard at 20µg/ml.
 Journal of Applied Pharmaceutical Science 01 (08); 2011: 239-243

REPEATABILITY Antibiotics For The Treatment Of Category IIIb Chronic Pelvic Pain
Three 20µl injections from a standard solution were Syndrome In men. Third International Chronic Prostatitis Network. 2009:
injected on to the analytical column and the peak areas data Retrived from: http://www.prostatitis.org/a92000.html.
El-Yazigi A. and Al-Rawithy S. A direct liquid chromatography
obtained were used in assessing system suitability and quality of
quantitation of ciprofloxacin in microsamples of plasma with fluorometric
analysis by calculating the %relative standard deviation detection. Therapeutic Drug Monitoring, 1990; 12:378-81.
(%RSD=0.12 n=3) at 20µg/ml. The lower %RSD indicates that Evans W C: General Methods associated with the photochemical
there are less variation and there are high precision in the values. Investigation of herbal products In: Trease and Evans Pharmacognosy,
th
15 edition, Elsevier, New Delhi, (2006) 139-145
Table V: Repeatability of method. Garcia M S. and Albero M I. Analysis of ciprofloxacin by high
No of Values of Mean of SD %RSD %CV liquid performance chromatography. Indian Journal Pharm. Biopharm.
injection peak areas values 2001; 61:87
3 4361218, 4368441 5108.665 0.12 0.12 George J K, Anson J N and Dieter B. Liquid Chromatography
4372202, analysis of Ciprofloxacin and Ciprofloxacin metabolites in body fluids.
4371902 Journal liquid chromatography relat. Tech 2000; 9: 2897.
Gladys M., : Improved high-performance liquid
ACCURACY AND PRECISION chromatographic determination of ciprofloxacin and its metabolites in
The method was established by carrying out analysis of human specimens. Journal chromatography B., 1992; 582: 263-7.
the analyte (standard Ciprofloxacin HCl) using proposed method. Jehl F., Gallion C., Debs J., Borgard JM., Monteil H and Minck
The low value of % CV showed that the method is precise within R.: A direct liquid chromatography quantitation of ciprofloxacin in
microsamples of plasma with fluorometric detection. Journal
the acceptance limit of 2%. The results are shown in Table V.
Chromatography 1985; 339: 347-57.
Jehl F. Gallion, C., and Montreal H.: High performance liquid
CONCLUSION chromatography of antibiotics. Journal of Chromatography 1990; 531:
An RP-HPLC method for the determination of 509-518.
Ciprofloxacin HCl was developed and validated. Results of th
Martindale: The complete drug reference, 36 edition, (2009)
analysis of the formulations are tabulated in table I. The amounts 243.
obtained by the proposed method are between 94.8% and 113.8%, Natrajan S and Raman B: Development and Validation of
within the acceptance level of 90% to 110% (USP) except for stability indicating method for simultaneous estimation of ofloxacin and
sample D which falls out of the range. Ornidazole. Indian Pharm. 2005; 4(33):79
Panzade P D and Mahadik K R: Simultaneous estimation of
The results obtained indicate that the proposed method is
ofloxacin and Tinidazole in tablet dosage form. Indian drugs 2000;
simple, rapid, accurate, and specific. Linearity was observed over
38(7):368.
a concentration range of 10 to 50µg/ml for the standard Rang H.P., Dale M.M., J.M. Ritter and R.I. Flower:
Ciprofloxacin Hydrochloride. This project, therefore describes an th
Antibacterial drug: In Rang and Dale Pharmacology 6 edition, Churchill
isocratic HPLC method using UV detection, which provides Livingstone Elsevier publication, 2008; pp 672 – 674.
adequate sensitivity for routine use and diminishing the time of Ranjit Singh, Mukesh M, Shailendra K. Saraf, Shubhini Saraf
sampling and chromatographic analysis. and Ram C. Gupta: Simultaneous estimation of Ciprofloxacin
Hydrochloride, Ofloxacin, Tinidazole and Ornidazole by Reverse phase-
RECOMMENDATION High Performance Liquid Chromatography. Eurasian Journal. Anal.
It can be used for routine analysis of formulations Chem., 2009; 4(2):161-167.
containing Ciprofloxacin Hydrochloride without any alteration in Salomies H and Salo J P: An HPLC study of tinidazole.
the assay. The main advantage of the method is the simple Chromagraphia 2005; 36(1):79.
Sani A. A., Celestine C. E., Ojuolape R. A., Taiwo E A.,
chromatographic conditions adopted. Therefore, the proposed
Ibrahim M. S., Mohammed I., Abdul K. H. and Abdulkareem S. S.:
method reduces the retention time and as such saves time and
Quality Assessment of Artemisinin-Based Drugs Marketed and Used In
quantity of mobile phase used. Maiduguri Metropolitan Council Borno State, Nigeria : Research Journal
REFERENCES of Pharmaceutical, Biological and Chemical Sciences 2010; 2 (2) Pp. 615-
Amini M, Abdi K, Darabi M and Shafiee A J Determination of 623
ofloxacin in plasma by HPLC with UV detection. Journal Applied Sci. Teja – Isavadharm P., Keeratatithakul D., Watt G., Webster
2005; 5(9):1655 HK., and Edstein MD.: Measurement of ciprofloxacin in human plasma,
Behl A, Ahuja M and Dhake A S Reverse phase high whole blood and erythrocytes by high performances liquid
performance liquid chromatography method for quantification of chromatography. Therapeutic Drug Monitoring 1991; 13: 263-7.
ofloxacin tablets. Indian Journal Pharm. Sci., 2005; 7: 479 Thomas Lemke, David Williams, Victoria Roche and William
th
Bakshi M, Singh B, Singh A and Singh S The ICH guidance in Zito : In Foye’s Principles of Medicinal Chemistry, 6 editions, LWW,
practice: Stress degradation studies on Ornidazole and development of a New Delhi, 2008; pp 1139.
validated stability- indicating assay. Journal Pharm. Biomed. Anal. 2001; Vardakas KZ, Siempos II, Grammatikos A, Athanassa Z,
26: 891 Korbila I, and Falagas, M. x:“Respiratory fluoroquinolones for the
British National Formulary 59, March 2010 treatment of community-acquired pneumonia: a meta-analysis of
Dimitrakov J., Tchitalov J., Zlatanov T., Dikov D. “ A randomized controlled trials”. CMAJ 2008; 179 (12): 1269–77.
Prospective,Randomized, Double-Blind, Placebo-Controlled Study of