INTRODUCTION:

Ulcerative colitis (UC) is a chronic inflammation of the large intestine. Patients with ulcerative colitis, ulcers and inflammation of the inner lining of the colon lead to symptoms of abdominal pain, diarrhoea, and rectal bleeding. Ulcerative colitis and Crohn's disease (CD) are caused by abnormal activation of the immune system in the intestines. Long-standing ulcerative colitis is a risk factor for colon cancer.¹ A currently popular antibiotic for gastrointestinal infection is ciprofloxacin, which is employed successfully in UC and CD. Ciprofloxacin is a fluoroquinolone that is well absorbed and is active against Gram-negative facultative anaerobes and microaerophiles, such as salmonella, shigella and campylobacter, and it also has modest activity against Gram-positive organisms such as Enterococcus faecalis.² Ciprofloxacin Hcl functions by inhibiting DNA gyrase, a type II topoisomerase, which is an enzyme necessary to separate replicated DNA, thereby inhibiting cell division. It is rapidly absorbed orally and shows 60-70% oral bioavailability and 3-4 h elimination half-life. Due to its elimination half- life, ciprofloxacin is administered twice to thrice daily.³

The molecular formula is C₁₇H₁₈FN₃O₃ and the molecular weight is 331.3415. CFH is rapidly absorbed from the gastrointestinal tract after oral administration. CFH has a t_1/2of 4.0 h with an oral bioavailability of 70%. CFH chemical structure is given in figure-1 and its IUPAC name is 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid. ⁴

Figure 1: Chemical structure of Ciprofloxacin hydrochloride.

The reported analytical methods for the detection of CFH have used procedures involving LC determination with fluorescence detection, visible Spectrophotometry, HPLC analysis.^5,6 Further a sensitive solid-phase spectrofluorimetry for the analysis of CFH in human urine and serum samples was developed by Navalon A and his co workers.⁷ Several techniques such as Stability indicating reversed-phase liquid chromatography, HPLC, Flow Injection Analysis Based on Chemiluminescence, excitation-emission fluorescence for CFH alone and simultaneous estimation methods were reported.^8,9 Review of past literature showed that there is no reported UV analytical method for the analysis of CFH. Hence the present investigation was undertaken to develop a sensitive, simple UV Spectrophotometric method and its validation for the quantitation of CFH in bulk fluids and characterization studies of the drug candidate.

MATERIALS AND METHODS:

Shimadzu UV 1700 double beam spectrophotometer (Japan) was used for all the spectrophotometric measurements. The absorption spectra of the reference and test solutions were carried out in a 1 cm quartz cells over the range of 200 - 400 nm. All laboratory glassware like volumetric flasks and pipettes were calibrated and used for experimentation. Rotary shaker (konark instruments, Mumbai), Theil’s melting point apparatus was used for drug melting point determination. Dhona 200 D electronic balance (Mumbai) was used for weighing of all samples.

Active pharmaceutical ingredient and Chemicals:

Ciprofloxacin hydrochloride was generously gifted by Granules India Ltd, Hyderabad, India; the drug was used as received. All the laboratory reagent grade solvents like Methanol (Pampasara distillaries) and chemicals n-octanol (INR Chemicals, Mumbai). Potassium dihydrogen ortho phosphate and Sodium hydroxide pellets from S.d. Fine chemicals limited, Mumbai were used in study.

Experimental methodology: The analytical method used for the estimation ciprofloxacin hydrochloride in bulk sample:

Preparation of Stock Solution:

The stock solution of ciprofloxacin hydrochloride is prepared by dissolving 100 mg of drug in 100 ml distilled water in volumetric flask with continuous shaking. 1 ml of sample was withdrawn and diluted to 100 ml to get 10µg / ml of solution. The solution was than scanned in UV range between 200–400 nm UV-VIS Spectrophotometer, (double beam) Shimadzu, Japan to determine the (λ_max) of the drug against blank as distilled water.

Calibration curve of Ciprofloxacin hydrochloride:

The prepared stock solution was subsequently diluted to get 2µg/ml, 4µg/ml, 6µg/ml 8µg/ml , 10µg/ml. The resulting solutions absorbance was measured at wavelength of 271.0 nm using (double beam) UV spectrophotomer against blank of distilled water. The results obtained were tabulated and plotted a calibration curve of absorbance versus concentration. The slope of the calibration curve is determined by regression equation (Table-1) (Fig.-2).

Table 1. Spectrophotometric data for ciprofloxacin hydrochloride at λ_max 271 nm

Sl. no	Concentration (µg/ml)	Absorbance
1	0	0.00
2	2	0.201
3	4	0.401
4	6	0.578
5	8	0.787
6	10	0.993

Y = 0.0985*X+0.0004

Fig 2. Calibration curve of ciprofloxacin hydrochloride at λ_max271 nm

Linearity studies: ¹⁰ The analytical method so developed was validated for linearity. To establish linearity of the proposed method six different concentrations of drug solutions (0 - 10 μg/ml) was prepared from CFH stock solution and analysed for their absorbance at 271 nm. The obtained data was subjected for linear regression analysis by using method of least squares. (Table-2)

Table 2. Linearity studies of ciprofloxacin hydrochloride:

Sl. no	Concentration (µg/ml)	Absorbance (nm)	Regression Data
1	0.00	0.00	*m = 0.0985	C = 0.0004	r = 0. 9997
2	2	0.201
3	4	0.401
4	6	0.578
5	8	0.787
6	10	0.993

*m = slope; c = intercept; r = regression.

Validation of analytical method developed in the laboratory by accuracy and precision study: ¹⁰ Further the analytical method will be validated for other parameters like accuracy, precision. Different concentrations of drug solutions were prepared and analyzed for repeatability of the method. Inter-day and intra-day variation was studied to determine precision. Drug concentrations of 0–10μg/ml were prepared at three different times in a day and studied for intra-day variation. Same method was followed for three different days to study inter-day variation. The relative standard deviation from the regression equation was taken as precision (Table-3).

Methods used for characterization studies of ciprofloxacin hydrochloride:

Melting point determination: Melting point of the drug was determined by taking a small amount of drug in a capillary tube closed at one end and was placed in theil’s melting point apparatus and the temperature at which the drug melts was noted. Average of triplicate readings was noted.

Table 3. Inter day and Intraday Validation studies of ciprofloxacin Hcl (n = 3 )

MORNING	DAY 1	DAY 2	DAY 3	AVG±SD
Concentration (μg /ml)	Absorbance
0	0.000	0.000	0.000	0.000
2	0.197	0.195	0.204	0.199 ± 0.004
4	0.388	0.390	0.402	0.393 ± 0.008
6	0.590	0.594	0.607	0.597 ± 0.01
8	0.788	0.803	0.806	0.799 ± 0.01
10	0.983	0.992	0.993	0.989 ± 0.06
AFTERNOON	Day 1	Day 2	Day 3	AVG
0	0.00	0.00	0.000	0.00
2	0.203	0.200	0.200	0.201 ± 0.002
4	0.392	0.395	0.398	0.395 ± 0.003
6	0.583	0.599	0.591	0.591 ± 0.008
8	0.791	0.796	0.785	0.791 ± 0.006
10	0.993	0.979	0.996	0.989 ± 0.009
EVENING	Day 1	Day 2	Day 3	AVG
0	0.000	0.000	0.00	0.000
2	0.194	0.196	0.200	0.197 ± 0.007
4	0.385	0.388	0.398	0.39 ± 0.003
6	0.580	0.586	0.596	0.587 ± 0.008
8	0.785	0.789	0.789	0.788 ± 0.002
10	0.987	0.966	0.991	0.981 ± 0.013
	Absorbance
Concentration (μg /ml)	Day 1 M* Avg	*Day 2 A Avg**	Day 3 E* Avg	3 Days Avg±SD
0	0.000	0.00	0.000	0.00
2	0.199	0.201	0.197	0.199 ± 0.002
4	0.393	0.395	0.39	0.393 ± 0.003
6	0.597	0.591	0.587	0.592 ± 0.005
8	0.799	0.791	0.788	0.793 ± 0.006
10	0.989	0.989	0.981	0.986 ± 0.005

*M – morning, A- afternoon and E- evening.

Solubility studies: The solubility of CFH was determined according to method proposed by Diez et. al. ¹¹ The excess of amount of drug was taken and dissolved in measured quantity of distilled water in volumetric flask and intermittent shaking for 24 hours to attain the equilibrium with undissolved drug. The solution is filtered after 24 hours and successively diluted with distilled water suitably and the concentration was measured in a UV spectrophotometer at their respective absorbance maxima. Similarly the solubility of drug is determined in different buffers, viz., pH 1.2, pH 7.0, and pH 8.0. respectively.

Partition coefficient determination of Ciprofloxacin hydrochloride:¹²

The partition coefficient of the drug was determined by taking equal volumes of n-octanol and aqueous phases in a separating funnel. A drug solution of 1mg/mL was prepared and 1mL of the solution was added to octanol: water (50:50) was taken in a separating funnel and shaken for 10 minutes and allowed to stand for 1 h and is continued for 24 h. Then aqueous phase and octanol phase was separated, centrifuged for 10 min at 2000 rpm. The aqueous phase and octanol phase were assayed before and after partitioning using UV Spectrophotometer at their respective λ max to get partition coefficient. Triplicate readings (n=3) were taken and average was calculated.

RESULTS AND DISCUSSION:

The U.V. absorption maxima of Ciprofloxacin hydrochloride in distilled water were found to be 271.0nm which is same as literature value 271.4 nm.¹³ The slope (m) of calibration curve of was 0.0985. Linearity studies indicated that estimation of CFH between 2.00 μg / ml to 10.00 μg /ml was found to be linear with slope (m) 0.0985, intercept (c) is 0.0004 (r2 = 0.9997). The estimation of Ciprofloxacin hydrochloride was found to obey beer’s limit in between 2.00 μg / ml to 10.00 μg /ml and the curve was found to be linear (Table-4). The SD values of Inter day and Intra day variation studies indicated that the variation is minimum in UV spectrophotometer (UV 1700, Shimadzu, Japan). Hence the developed analytical method by using UV spectrophotometer was found to be accurate and precise to analyze the drug sample in bulk and dosage forms. Using the proposed analytical technique, further quantitation work of prospective in vitro studies of CFH could be carried out. During characterization studies the melting point of the drug was found to be 324⁰ C that is near to the literature value 318 – 320⁰ C.¹⁴ Later, solubility of ciprofloxacin hydrochloride in distilled water, acidic and alkaline pH buffers was studied. It was found to be 45.58 mg/ml in distilled water, 2.18 mg/ml in pH 1.2, and 0.095 mg/ml in buffer of pH 8.0. The log p value of Ciprofloxacin hydrochloride was observed to be -0.438 which corroborates to the reported literature value -0.56.¹⁵ Results of studies on melting point and log p of drug suggested the values corroborating with previously reported literature values. The characterization studies showed that the bulk sample obtained was pure.

Table 4. Analytical parameters of CFH by UV Spectrophotometry.

Sl. no	Analytical Parameter	Value
1	UV λ maxima	271nm
2	Relative standard deviation	0.0011
3	Beer’s law limits	1-10 (μg / ml)
4	Regression equation	(y) = 0.0985*X 0.0004; ( r = 0.9997)
5	Slope (m)	0.09851
6	Intercept (c)	-0.00040
7	Correlation coefficient(r)	0.99978
8	Linearity range	2-10 μg / ml

CONCLUSION:

Preliminary investigation of past research methods on CFH revealed various literature reports on analytical methods of the drug estimation that are useful in the study. The analytical method developed using UV spectrophotometer is linear and the inter-day and intra-day variation is minimum. Although there are reports and publications of methods like spectrofluorimetry, HPLC and colorimetry for the estimation of CFH, but there is no publication or report corresponding to the development and validation of UV Spectrophotometric method of ciprofloxacin hydrochloride alone. Therefore it could conclude that the proposed investigation is a novel work and the investigation would help in quantification of drug candidate spectrophotometrically in the bulk fluids and dosage forms. The solubility studies indicated that the Solubility of ciprofloxacin HCl in distilled water is higher than in acidic or alkaline pH buffers. The experimental values reproducibility near to the reported ones suggest the bulk drug sample obtained was pure.

ACKNOWLEDGEMENTS:

The authors would like to thank Academy of medical education management and Principal V.L.College of Pharmacy, Raichur, Karnataka, India for providing the research facilities.

REFERENCES:

1) http://www.medicinenet.com/ulcerative_colitis/article.htm accessed on 8/10/2008

2) Cummings JH, Macfarlane GT, Macfarlane S. Intestinal bacteria and ulcerative colitis. Curr. Issues Intest. Microbiol. 2003; 4: 09-20.

3) Moffat AC, Osselton MD, Widdop B. Clarke’s analysis of drug and poison. 3rd ed. London: Pharmaceutical Press; 2004; 3: 111.

4) http://www.drugbank.ca/drugs/DB00537 accessed on 14/12/2009

5) Anastasia Zotou and Niki Miltiadou. Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection. Journal of pharmaceutical and biomedical analysis. 2002; 28 (3): 559-568.

6) Vybiralova Z, Nobilis M, Zoulova J, et al. High-performance liquid chromatographic determination of ciprofloxacin in plasma samples. Journal of pharmaceutical and biomedical analysis. 2005; 37 (5): 851-858.

7) Navalon A, Ballesteros O, Blanc R, et al. Determination of ciprofloxacin in human urine and serum samples by solid-phase spectrofluorimetry. Talanta. 2000; 52 (5): 845-852.

8) Manuela T. Maya, Nuno J. Gonçalves, Nuno B. Silva, et al. Simple high-performance liquid chromatographic assay for the determination of ciprofloxacin in human plasma with ultraviolet detection. Journal of Chromatography B: Biomedical sciences and applications. 2001; 755 (1-2): 305-309.

9) Zhuoyong Zhang, Xia Li, Xiaoli Wang, et al. Determination of Ciprofloxacin by flow injection analysis based on chemiluminescence. System journal of rare earths. 2006; 24 (3): 285-288.

10) Nikhil A Karani, Prashant Pingale. Analytical method development and validation of venlafaxine hydrochloride in solid dosage forms using UV spectrophotometer. Journal of Pharmacy Research. 2009; 2 (7): 1246-1249.

11) Diez Colom H, Moreno Obach R.Comparative in vitro study of transdermal absorption of series of calcium channel antagonists. J Pharm Sci. 1991; 80(10): 932-934.

12) Jona JA, Dittert LW, Crooks PA. Design of novel prodrugs for the enhancement of the transdermal penetration of indomethacin. Int J Pharm 1995; 123: 127-36.

13) Patel SA, Patel NM, Patel MM. Simultaneous spectrophotometric estimation of ciprofloxacin and ornidazole in tablets. Indian journal of pharmaceutical sciences. 2006; 68(5): 665 – 667.

14) http://www.chemblink.com/products/86393-32-0.htm accessed on 18/9/2009

15) http://www.drugbank.ca/drugs/DB00537 accessed on 1/12/2009