Extractive Spectrophotometric Determination of Ofloxacin and Tinidazole with Bromophenol Blue.

 

Dr. Y. Haribabu, Mrs. Sheeja Velayudhan Kutty, Vidhya P.M.*,Greeshma S.

Grace College of Pharmacy, Palakkad, Kerala

*Corresponding Author E-mail:- vidhyapm24@yahoo.com

 

The study aims to develop a simple, sensitive,rapid,accurate and precise extractive spectrophotometric method for the determination of ofloxacin and tinidazole in bulk drug and tablet dosage form. This method is based on the formation of yellow-ion pair complex between the drug and the anionic dye bromophenol blue in phthalate buffer at ph 3.6. The formed complexes were extracted with chloroform and measured at 426nm for ofloxacin – bromophenol blue and 312nm for tinidazole - bromophenol blue. Beer’s law was obeyed in the concentration range of 2-12µg/ml for ofloxacin and tinidazole with a linear regression value 0.994 and 0.996 respectively. Inter day and Intraday studies showed high degree of repeatability. Recoveries obtained do not differ significantly from 100% showed that there was no interference from the common excipients used in the tablet formulation indicating accuracy and reliability of the method.The proposed method was successfully applied for the determination of ofloxacin and tinidazole in commercial tablet containing 200 mg of ofloxacin and 300 mg of tinidazole ( oflox TZ ). As per ICH guidelines the results of the analysis were validated statistically and were found to be satisfactory.

 

 

 

INTRODUCTION:

Ofloxacin , (+) 9-fliuoro-2,3, dihydro -3- methyl -10 –(4-methyl-1-piperaznyl)-7-oxo-7H –pyrido [1,2,3-d,e]-1,4-benzoxazine -6-carboxylic acid, is one of the new generation of fluorinated quinolone structurally related to nalidixic acid ^[1][Fig 1]. This agent is a new broad spectrum antibacterial drug active against most Gram negative, Gram positive bacteria and some anaerobes. This broad spectrum of antibacterial activity and wide spread distribution to most tissues and body fluids at relatively high concentration after oral administration have made this drug useful for the treatment of systemic infections including urinary tract, respiratory  and gastro intestinal infections ^[2]. Tinidazole [1-(2-ethylsulfonyethyl)-2-methyl-5-nitroinidazole] is a 5 –nitroimidazole derivative, an antiparasitic drug used against protozoan infections. It is also used in the treatment of a variety of amebic and parasitic infections [Fig 2].

 

Literature survey reveals that OFL was determined by several methods including spectrophtotmetric, HPLC, extractive spectrophotometric method^[4] and chemiluminescene method.

 

Capillary electrophoresis, HPLC,spectrophotometric and capillary electrophoresis methods have been reported for the estimation of OFL in combination with other drugs ^[5]. TNZ was determined by spectrophotometric, HPLC, and electrochemical study ^[6]. Some methods have also been reported for the determination of TNZ in combination with other drugs including spectrophotometric, capillary electrophoresis and differential pulse polarography ^[7].Literature survey revealed that spectrophotometric and HPLC methods have been reported for the estimation of OFL and TNZ in pharmaceutical formulations ^[8-11]. The aim of this paper was to explore the possibility of using extractive spectrophotometric technique for the estimation of ofloxacin and tinidazole in bulk and in their combination. The propose methods are simple, precise, accurate and economical than reported methods.

 

Figure 1: structure of ofloxacin

 

Figure 2: structure of tinidazole

 

MATERIALS AND METHOD:

Instrumental:

A SHIMADZU model PHARMASPEC-1800 UV-Vis spectrophotometer with 1.0 cm matched cells was used for the electronic spectral measurements. Ofloxacin, tinidazole and all other chemicals used were analytical reagent grade. Ofloxacin and tinidazole pure drug was generously provided by Torrent Pharmaceuticals Pvt ltd (Ahmadabad, India), as a gift sample. The commercially available tablet “oflox –TZ” (Cipla pharmaceuticals) containing 200 mg ofloxacin and 600 mg tinidazole was procured from the local market. Freshly prepared phthalate buffer of ph 3.6, bromophenol blue, chloroform and ethanol were used in the present analysis.

 

Solvents:

Preparation of standard stock solution

The standard ofloxacin and tinidazole 10 mg was weighed and made up to 100 ml with distilled water to get a concentration of 100µg/ml.

 

Preparation of bromophenol blue solution:

140 mg of bromophenol blue was dissolved in20 ml ethanol and the volume was made upto 00 ml with ethanol.

 Preparation of phthalate buffer

1.020g of potassium hydrogen phthalate was dissolved in little amount of water and the ph of the solution was adjusted to ph3.6by the addition of 0.1M Hcl.

 

Procedure:

Extractive spectrophotometric determination

Aliquots of 0.2 – 1.2 ml of standard drug solutions were transferred to stoppered flask and added 1ml of buffer solution of ph 3.6, 5 ml of bromophenol blue solution. The mixture was extracted with 5ml of chloroform by shaking for 1 minute.1 ml of organic extract was taken and completed to 10 ml with ethanol in volumetric flask. The absorbance of yellow coloured extracts was measured at 424 and 312 nm for ofloxacin and tinidazole against the corresponding reagent blank [Fig 3] [Fig 4].

 

Tablet assay procedure:

Twenty tablets were weighed and powdered .An amount equivalent to one tablet was weighed and extracted with 25 ml 0.5 N acetic acid for four times and filtered . Necessary amount of filtrate was diluted to 100ml and the same procedure was applied as described under the procedure for bulk samples [Table 1].

 

 

Table 1: Analysis of marketed formulation

Formulation	Label claim  (mg)		Amount estimated		% Amount estimated		%RSD
	OFL	TNZ	OFL	TNZ	OFL	TNZ	OFL	TNZ
Tab oflox- TZ	200	600	199.65	599.83	99.82	99.97	0.435	0.507
Tab oflomac	200	600	198.87	600.93	99.43	100.15	0.426	0.483

 

Figure 3: Overlay spectra of ofloxacin in bromophenol blue at 426nm

 

Figure 4: Overlay spectra of tinidazole in bromophenol blue at 312 nm

 

Method validation:

Linearity:

Working standard solution was prepared by series of dilutions of 0.2 – 1.2 ml of standard stock solution and added 1ml of buffer solution of ph 3.6, 5 ml of bromophenol blue solution. The mixture was extracted with 5ml of chloroform by shaking for 1 minute.1 ml of organic extract was taken and completed to 10 ml with ethanol in volumetric flask. The absorbance of yellow coloured extracts was measured at 424 and 312 nm for ofloxacin and tinidazole against the corresponding reagent blank. Calibration curve was prepared by plotting concentration versus absorbance and found to be linear in the concentration range of 2-12µg/ml [Fig 5] [Fig 6] [Table 2] [Table3].

 

Figure 5: Linearity of ofloxacin

 

Figure 6: Linearity of tinidazole

 

Table 2 : Linearity of Ofloxacin

Sl no:	Concentration  (μg/ml )	Absorbance at 426 nm
1	2	0.198
2	4	0.392
3	6	0.537
4	8	0.699
5	10	0.878
6	12	0.982

 

Table 3 : Linearity of Tinidazole

Sl no	Concentration  (μg/ml)	Absorbance at 312 nm
1	2	0.120
2	4	0.228
3	6	0.321
4	8	0.424
5	10	0.502
6	12	0.592

 

Precision:

Precision of the method was determined by performing Interday variation, intraday variation and repeatability studies and expressed in the forms of %RSD. In Interday variation, the absorbances of working standard solutions of ofloxacin and tinidazole  (2-12 µg/ml) were measured on three consecutive days. In intraday variation the absorbance were measured three times a day. In repeatability study, six determinations of the fixed concentration of the drug were analyzed [Table 4].

 

LOD and LOQ:

In this study, LOD and LOQ were based on the standard deviation of the response (σ) and the slope of the corresponding curve (S) using the following equation.

LOD = 3.3σ/S

LOQ = 10σ/S

Where

σ is the standard deviation of the response of blank,

S is the slope of calibration curve.

 

Accuracy (Recovery studies):

The accuracy of the proposed method was determined by calculating the recoveries of ofloxacin and tinidazole by the standard addition method. It was determined by preparing solutions of different concentrations at 80%, 100% and 120% in which the amount of marketed formulation was kept constant and the amount of pure drug was varied [Table 5].

 

Robustness:

The robustness of the proposed method was carried out by changing the pH of the system to 3.1 [Table 6] .

 

RESULTS:

The presence of piperazine ring in ofloxacin and imidazole ring in the tinidazole offers a basic character to the drugs. So when treated with an acid dye such as bromophenol blue at pH 3.6of potassium hydrogen phthalate buffer, a yellow ion pair complex which is extracted with chloroform is formed. The absorbance of the formed ion-pair complexes were measured at 426 and 312 nm for ofloxacin and tinidazole respectively. Beer’s law was obeyed in the concentration range of 2-12µg/ml with a linear regression value 0.997. Interday and Intraday studies showed high degree of repeatability of an analytical method under normal operating conditions. The %RSD for precision, which was less than 2% Indicates that the method is precise. Recoveries obtained do not differ significantly from 100% showed that there was no interference from the common excipients used in the tablet formulation indicating accuracy and reliability of the method [Table 7].

 

DISCUSSION:

The method was found to be simple, economical, selective and sensitive. The statistical parameters clearly indicate the reproducibility and accuracy of the method. Analysis of ofloxacin and tinidazole in its dosage forms showed no interference from the common excipients and additives. Extractive spectrophotometry by indicating pH of the medium may be recommended for routine and quality control analysis of the investigated drug in tablets.

 

 

Table 4: Precision data

	Fortified amount (µg/ml		Amount found (µg/ml		%RSD
	OFL	TNZ	OFL	TNZ	OFL	TNZ
Intraday (n=3)	8	8	7.98	7.95	0.68	0.79
	10	10	10.12	9.98	0.83	0.62
	12	12	11.96	12.01	0.47	0.76
Inter day (n=3)	8	8	8.05	7.98	0.35	0.56
	10	10	9.97	9.96	0.68	0.94
	12	12	12.14	12.15	0.52	0.83
Repeatability (n=6)	8	8	7.96	8.24	0.79	0.61
	10	10	10.22	10.08	0.98	1.05
	12	12	11.97	12.06	0.87	0.53

 

 

Table 5: Recovery study

Level of addition (%)	Formulation (µg/ml)		Addition of pure drug (µg/ml)		% Recovery of pure drug		%RSD
	OFL	TNZ	OFL	TNZ	OFL	TNZ	OFL	TNZ
80 n=3	10	30	8	24	99.86	99.53	0.068	0.066
100 n=3	10	30	10	30	99.44	100.20	0.061	0.124
120 n=3	10	30	12	36	100.36	100.15	0.285	0.117

 

 

Table 6 : Robustness data

	Fortified amount (µg/ml		Amount found (µg/ml			%RSD
Robustness n=3	OFL	TNZ	OFL	TNZ	OFL	TNZ
	6	6	5.99	5.96	0.63	0.85
	8	8	7.89	8.05	0.66	0.48
	10	10	9.96	10.03	0.95	0.75

 

 

Table 7: Validation parameters

Parameters	values
	OFL	TNZ
λ max	426 nm	312 nm
Linearity range	2-12µg/ml	2-12µg/ml
Regression equation	Y= 0.0824x + 0.0323	Y= 0.0489x + 0.0193
slope	0.0824	0.0489
intercept	0.0323	0.0193
Correlation coefficient	0.9946	0.9961
LOD	0.4007	0.567
LOQ	1.21	1.72

CONCLUSION:

The proposed method was found to be simple, sensitive, and precise for the determination of Ofloxacin and Tinidazole from pure and its tablet dosage forms. The sample recoveries in all formulation were in good agreement with their respective label claims without the interference of excipients and the other additives. Thus the proposed method can be used for the routine analysis of the samples in pure and its dosage forms.

 

ACKNOWLEDGMENT:

The authors are thankful to the Torrent pharmaceuticals Ltd, Ahmadabad for providing the drug samples and also to the Principal and to the Staffs of Grace College of Pharmacy for providing the facilities to carry out the research work.

 

REFERENCES:

1      Martindale the complete drug reference, thirty fifth edition volume 1, page no.277; 764

2      K D Tripathi Essentials of Medical Pharmacology Sixth edition page no. 647-651; 750- 752

3.     The Merck Index, thirteenth edition, page no 1213; 1685

4.     Ncilay suslu and Ayla tamer.application of bromophenol blue and bromocresol purple for the   extractive spectrophotometric determination of ofloxacin., Analysis Letters 36:6, 1163- 1181; 2003.

5      Dhandapani B, thirumoorthy N, Rasheed SH, Kotaiah MR, Anjaneyalu N.Method development and validation for the simultaneous estimation of ofloxacin and Ornidazole in tablet dosage form by RP-HPLC . Int J Pharmaceut Biomed Res 2010; 1:42-8.

6.     Umadevi Kothapalli, Kothakota Vandana, Arun Kumar Dash, Siva Kishore, Loya Harika, Kishanta kumar Pradhan. A validated UV –Spectrophotometric method for the estimation of tinidazole in bulk and pharmaceutical dosage form. IJPBA 2011;2(4): 1152-1156

7.     Bombale M V, Kadam S.S and Dhaneshwar S R. Simultaneous spectrophotometric estimation of Ciprofloxacin and Tinidazole from a combined dosage form, Indian Journal of Pharmaceutical Sciences, 1997, 59,265-268

8.     Kareti Srinivasa Rao, Arijit Banerjee, Nargesh Kumar Keshar. Spectrophotometric methods for the simultaneous estimation of ofloxacin and tinidazole in bulk and in pharmaceutical dosage form. Chron Young Sci 2011;2:98-102.

9.     Rama Kotaiah, Shaik Harun Rasheed, Narasimha Rao, Venkateswarlu, Konda Ravi Kumar. Simultaneous estimation of ofloxacin and tinidazole in tablet dosage form by RP-HPLC.RJPBCS, 2010: 1(4) : 460-466

10.   Dharuman , M.Vasudevan, K.N.Somasekaran, B.Dhandapani, Prashant D. Ghode, M. Thiagarajan. RP-HPLC method development and validation for the simultaneous estimation of ofloxacin and tinidazole in tablets. IJPRIF, Vol 1, No.2 2009:121-124.

11    Santosh Kumar Vobbilireddi , K. Sujana and Dr. A. Prameela Rani. New Validated UV- Spectrophotometric Method for the Determination of Telmisartan in Bulk and Dosage Form. Research J. Pharm. and Tech. 5(9): September 2012.1209-1212.

 

 

Received on 11.03.2013          Modified on 01.04.2013

Accepted on 12.04.2013         © RJPT All right reserved