INTRODUCTION:

Ciprofloxacin hydrochloride is, chemically, 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1, 4-dihydroquinoline-3-carboxylic acid hydrochloride. The molecular formula is C₁₇H₁₈FN₃O₃.HCl, (fig. 1) representing a molecular weight of 367.8 and belongs to the group of synthetic fluoroquinolone antibiotics with broad antimicrobial activity ^[1], and is structurally related to nalidixic acid. It is believed that the mode of action of this family of drugs is through binding DNA-gyrase enzyme ^[2]. It is also reported that there is a direct correlation of fluoroquinolone bonding with inhibition of DNA gyrase enzyme activity and induction of DNA breakage. Because of this special mechanism of action, fluoroquinolones are considered to be the most effective gram-positive-gram-negative pathogens to combat infections caused by micro organisms that are resistant to other microbial, such as tetracycline. The drug is official in British Pharmacopoeia ^[3]and United States Pharmacopoeia ^[4] which describes a high performance liquid chromatographic (HPLC) method for its assay.

Fig. 1 Structure of Ciprofloxacin hydrochloride

Ciprofloxacin hydrochloride is a pale yellow, crystalline powder, slightly hygroscopic that is freely Soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, practically insoluble in acetone, in ethyl acetate and in methylene chloride ^[3].

Literature survey revealed that various analytical methods such as LC^[5], HPLC^[6-8], HPTLC^[9] and capillary electrophoresis ^[10-12] methods are used for estimation of Ciprofloxacin hydrochloride in single as well as in combination with various drugs. Hence, an attempt has been made to develop new simple, sensitive UV spectrophotometry method for its estimation either in bulk or pharmaceutical tablet dosage form with good accuracy, simplicity, precision and economy.

EXPERIMENTAL:

Instrumentation: Spectral and absorbance measurements were made on UV spectrophotometer. Digital balance (1 mg sensitivity) Sansui was used for weighing the samples. Commercially available tablets of Ciprofloxacin hydrochloride were procured from the local market and estimated.

Chemicals and reagents: -

1. Distill Water In-house

2. Ciprofloxacin hydrochloride (99%) Shreya Life Sciences Pvt. Ltd., Roorkee

Apparatus / Instruments: -

Name	Model	Manufacturer
UV spectrophotometer	UV1800	Shimadzu
Micropipettes	----	Impulse
Tube and micro-pipette tips	----	Eppendorf

OPTIMIZATION

Scanning and determination of maximum wavelength (l_max): In order to ascertain the wavelength of maximum absorption (λ _max) of the drug (10 ppm) in distill water was scanned using UV spectrophotometer within the wavelength region of 220 – 800 nm against distill water as blank. The resulting spectrum is shown in fig. (2) And the absorption curve showed characteristic absorption maxima at 274 nm for Ciprofloxacin hydrochloride.

Fig. 2 Spectrum of Standard Ciprofloxacin hydrochloride (10ppm)

METHODS:

Preparation of standard stock solutions: Standard stock solution was prepared by dissolving 100 mg of Ciprofloxacin hydrochloride in distill water and volume made up to 100 ml with distill water to get concentration of 1mg/ml (1000 ppm) solution.

Preparation of working standard solutions and construction of standard graph: The prepared stock solution was further diluted with distill water to get working standard solutions of 2, 4, 6, 8, and 10 ppm of Ciprofloxacin hydrochloride to construct Beer’s law plot for pure drug, the absorbance was measured at λ _max 274 nm, against distill water as blank. The results are shown in table (1). The standard graph was plotted by taking concentration of drug on X-axis and absorbance on Y-axis and is shown in fig (3). The drug has obeyed Beer’s law in the concentration range of 2-10 ppm. The linearity curve data is shown in table (2).

Table 1: Linearity table of Ciprofloxacin hydrochloride in working standard

S. No.	Concentration (ppm)	Absorbance
1.	2	0.186
2.	4	0.383
3.	6	0.558
4.	8	0.751
5.	10	0.962

Fig. 3 Linearity curve of Ciprofloxacin hydrochloride

Table (2) Linearity curve data

Beer’s Law limit (ppm)	2-10
Correlation coefficient (R²)	0.99907
Regression equation (y*) y= 0.09606x - 0.00859
Slope (m)	0.09606
Y-Intercept (c)	0.00859
Chi square	0.00055
Standard error of estimate	0.01073
RSD	0.00929

y=mx+c where ‘x’ is the concentration of Ciprofloxacin hydrochloride in ppm and y is the absorbance

Preparation of sample stock solutions and working sample solutions:

Ten tablets were accurately weighed and average was calculated. The tablets were then crushed to obtain fine powder. An accurately weighed quantity of tablet powder equivalent to about 100 mg of Ciprofloxacin hydrochloride was transferred to 100 ml volumetric flask, add 50 ml of distill water and shaken for 1 hour. The volume was made up to the mark with distill water and filtered through Whatman filter paper and required dilutions were made from sample stock solution. The spectrum of sample is shown in fig. 4 and recovery study from formulation is shown in table (3).

Table 4: Accuracy Readings

Sample ID	Concentration (ppm)			%Recovery of Pure drug	Statistical Analysis
Sample ID	Pure drug	Formulation	Absorbance	%Recovery of Pure drug	Statistical Analysis
S₁ : 80 %	4	5	0.834	97.46	Mean=97.54 SD=0.133 %RSD=0.136
S₂ : 80 %	4	5	0.834	97.46
S₃ : 80 %	4	5	0.836	97.69
S₄ : 100 %	5	5	0.930	97.71	Mean=97.74 SD=0.058 %RSD=0.059
S₅ : 100 %	5	5	0.930	97.71
S₆ : 100 %	5	5	0.931	97.81
S₇ : 120 %	6	5	1.045	99.71	Mean=99.65 SD=0.11 %RSD=0.11
S₈ : 120 %	6	5	1.045	99.71
S₉ : 120 %	6	5	1.043	99.52

% Recovery= amount recovered / amount introduced X 100

VALIDATION:

Accuracy: To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of bulk samples of Ciprofloxacin hydrochloride within the linearity range were taken and added to the preanalyzed formulation of concentration 5 ppm. From that percentage recovery, values were calculated. The results are shown in table (4).

Fig. 4 Spectrum of Ciprofloxacin hydrochloride sample (10 ppm)

Precision: The precision of the proposed method was ascertained by actual determination of six replicates of fixed concentration of the drug within the Beer’s range and finding out the absorbance by the proposed method. From this absorbance, mean, standard deviation and % RSD was calculated. The system precision and method precision readings are shown in table 5 (a) and 5 (b) respectively.

Table 5 (a): System precision readings

Concentration (ppm)	Absorbance	Statistical analysis
6	0.524	Mean = 0.5245 SD = 0.00055 %RSD = 0.105
6	0.524
6	0.525
6	0.525
6	0.525
6	0.524

Table 5 (b): Method precision readings

Concentration (ppm)	Absorbance	Statistical analysis
6	0.578	Mean = 0.573 SD = 0.0075 %RSD = 1.31
6	0.579
6	0.577
6	0.562
6	0.575
6	0.564

Limit of detection (LOD): LOD calculation is based on the standard deviation of the response (σ) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula:

LOD = 3.3(σ /S)

LOD=0.32 ppm

Limit of Quantitation (LOQ): LOQ calculation is based on the standard deviation of the response (σ) and the slope of the calibration curve (S) at levels approximating the

LOQ according to the formula:

LOQ = 10(σ /S)

LOQ = 0.97 ppm

RESULTS AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that Ciprofloxacin hydrochloride obeys linearity within the concentration range of 2-10 ppm. From the results shown in accuracy table (4), it was found that the percentage recovery values of pure drug from the preanalyzed solution of formulation were in between 97.46– 99.71, which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method. From the results shown in table 5 (a) and 5 (b) it was found that the % RSD is less than 2, which indicates that the system as well as method has good reproducibility. LOD and LOQ were found that 0.32 and 0.97 ppm respectively.

CONCLUSION:

The proposed method was simple, sensitive and reliable with good accuracy and precision. The proposed method is specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the routine determination of Ciprofloxacin hydrochloride in bulk and pharmaceutical tablet dosage form.

ACKNOWLEDGEMENTS:

The authors thank Shreya Life Sciences Pvt. Ltd., Roorkee for providing the gift sample of Ciprofloxacin hydrochloride. Authors are also thankful to Dr. K. N. Modi foundation, Modinagar, Uttar Pradesh for providing necessary facilities for the work.

REFERENCES

1. Goodman LS and Gillman AG (1996) The Pharmacological Basis of Therapeutics, 9th ed. (McGraw-Hill, New York) pp 1065.

2. Wise R, Andrews JM and Edwards LJ (1983) Antimicrob Agents Chemother 23, 559.

3. The British Pharmacopoeia (1999) Vol. 2 (Her Majesty’s Stationery Office, London) pp 1714.

4. The United States Pharmacopoeia 24 (1999) (United States Pharmacopoeial Convention, 12601, Twinbrook Parkway, Rockville, MD 20852) pp 420.

5. Zatou A and Miltiadou N (2002) Sensitive LC-determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection, J Pharm Biomed Anal 28, 559-568.

6. Vega E. Dabbane V, Nassetta M and Sola N (1999) Validation of reversed phase LC method for quantitative analysis of intravenous admixtures of ciprofloxacin and metronidazole, J Pharm Biomed Anal 21, 1003-1009.

7. Lacriox PM, Curran NM and Sears RW (1996) High pressure liquid chromatographic methods for ciprofloxacin hydrochloride and related compounds in raw materials, J Pharam Biomed Anal 14, 641-654.

8. Husain S, Khalid S, Nagaraju V and Nageshwara Rao R (1995) High performance liquid chromatographic separation and determination of small amounts of process impurities of ciprofloxacin in bulk drugs and formulations, J ChromatogrA 705, 380-384.

9. Novakovic J, Nesmerak K, Nova H and Filka K (2001), HPTLC method for the determination and the purity control of ciprofloxacin HCl in coated tablets, J Pharm Biomed Anal 25, 957-964.

10. Wang PL, Feng YL and Chen LA (1997) Validation of capillary electrophoresis method for the determination of quinolone antibiotic and its related impurities, Microchem J 56, 229-235.

11. Altria KD and Chanter YC (1993) Validation of a capillary electrophoresis method for the determination of a quinolone antibiotic and its related impurities, J Chromatogr A 652, 459-463.

12. Yin C and Wu YT (1997) Determination of four quinolones by high performance capillary electrophoresis, Yaowu Fenxi Zazhi 17, 371-373.

Received on 04.02.2012 Modified on 25.02.2012

Asian J. Research Chem. 5(3): March 2012; Page 336-339

Formulation	Labeled amount (mg)	UV spectrophotometry method*
Formulation	Labeled amount (mg)	Mean ± s. d (amount mg recovered)	%Labeled amount	% RSD
Ulticept-250 (tablets)	277.5	263.33±0.001155	94.98±0.4965	0.523