1. INTRODUCTION:

Ofloxacin, is an antimicrobial drug and chemically it is 9-fluro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperizinyl)-7-oxo-7H-pyrido [1,2,3-de]-1,4-benzoxaine-6-carboxylicacid [1]. Ketorolac Tromethamine, has anti- inflammatory and analgesic activity. Chemically it is 5-benzoly-2,3-dihydro-1H-pyrrolizine-1-carboxylicacid,2-(hydroxymethyl)-1,3 propanediol and official only in USP^[1]. Literature survey reveals that various analytical methods have been reported for the estimation of OFLOX in single and in combination dosage form such as, HPLC ^[2-13], Spectrophotometric^[^14-19], Potentiometry and Conductometry^[20]. Literature survey showed that very few analytical methods have been reported for the estimation of KETO alone such as, Spectrophotometric^[^21], HPLC^[22-26], and HPTLC^[27].

Fixed dose combination containing OFLOX and KETO in ophthalmic dosage form is recently available in the market and a single method using methanolic HCl as a solvent by simultaneous equation and absorbance ratio method has been reported for the simultaneous estimation of both these drugs^[28]. No method has been reported using Distilled water and by First order derivative spectroscopy for the simultaneous estimation of both these drugs.

2. MATERIALS AND METHODS:

2.1 Instrumentation:

A Double beam UV-Visible spectrophotometer (Jasco V 530) with 10 mm matched quartz cells was used. All weighing were done on single pan balance (Shimadzu).

2.2 Reagents and chemicals:

OFLOX and KETO reference standards were kindly provided by Litaka Pharmaceuticals Pvt. Ltd, Pune. All the reagents were of analytical grade. Ophthalmic formulation eye drops (KETOFLOX) containing Ofloxacin (3mg) and Ketorolac Tromithamine (5mg) per ml was purchased from local market. OFLOX and KETO are available in the ratio of 3:5 respectively in the formulation and were used in same ratio for preparation of calibration curves.

2.3 Determination of λmax andpreparation of calibration curves:

Standard stock solutions of OFLOX (300 µg/ml) and KETO (500 µg/ml) were prepared separately in Distilled water. For the selection of analytical wavelength solutions of OFLOX (9 µg/ml) and KETO (15 µg/ml) were prepared separately by appropriate dilution of standard stock solution with Distilled water and scanned in the spectrum mode from 200 to 400 nm. From the overlain spectra of these drugs [Fig. 1], wavelengths 289 nm (λ_max of OFLOX), 322 nm (λ_max of KETO) and 301 nm (isobestic point) were selected for analysis. The calibration curves (Fig. 2 and 3) for OFLOX and KETO were plotted in the concentration range of 1.5-10.5 µg/ml and 2.5-17.5 µg/ml respectively for both drugs at the selected wavelengths. Absorptivity values were calculated from the calibration curves.

Fig. 1: Overlain spectra of OFLOX and KETO

Figure 2: Calibration Curve for OFLOX

2.4 Determination of Linearity:

Standard stock solutions of pure drugs containing 30 mg of OFLOX and 50 mg of KETO/100 ml were prepared in distilled water. The working standard solutions were obtained by appropriate dilutions of the stock solution in distilled water. Series of dilutions with concentrations 1.5-10 µg/ml and 2.5 – 17.5 µg/ml of OFLOX and KETO respectively were prepared. Solutions were scanned and Beers law limits were determined.

igure 3: Calibration Curve for KETO

2.5 Formulation analysis:

For analysis of marketed formulation, sample solution equivalent to OFLOX (3 mg) and KETO (5mg) was pipetted and diluted to 100 ml with Distilled Water. The solution was then filtered though Whatmann filter paper No. 41. Appropriate aliquots were taken for further analysis.

2.5.1 Method A- Simultaneous Equation Method:

Sample stock was appropriately diluted with distilled water to obtain final concentration of 3 µg/ml of OFLOX and 5µg/ml of KETO and absorbance was measured at selected wavelengths. The concentration of drugs was determined by using the Equations 1 and 2.

Using absorptivity values following equations were developed for determining concentration of OFLOX and KETO in ophthalmic formulation.

A₁ = 74.0 C_OFLOX +15.63 C_{KETO----------}(1)

A₂= 28.63_OFLOX +52.23C_{KETO--------------}(2)

Where, A1, A2 are absorbance of the sample solution at 289 and 322 nm, respectively.

C_OFLOXis the concentration of OFLOX in gms/lit

C_KETO is the concentration of the KETO gms/lit

2.5.2 Method B- Absorption Ratio Method (Q Method):

For Q method, 301 nm (isobestic point) and 289 nm (λmax of OFLOX) were selected as wavelengths of measurements.

Concentrations of OFLOX and KETO were determined using following equations,

C_OFLOX = (Qm-Q_KETO). A₁ / (Q_OFLOX-Q_KETO). a_OFLOX1

C_KETO = (Qm-Q_OFLOX). A₁ / (Q_KETO-Q_OFLOX). a_KETO1

Where, Qm = A₂/ A₁

Q_OFLOX = a_OFLOX2/a_OFLOX1and Q_KETO = a_KETO2/a_KETO1

A₂andA₁₌ Absorbance of Mixture at 289 nm and 301 nm respectively

a_OFLOX1= absorptivity of OFLOX at 301 nm (44.4)

a_KETO1= absorptivity of KETO at 301nm (28.75)

a_OFLOX2= absorptivity of OFLOX at 289 nm (74.0)

a_KETO2= absorptivity of KETO at 289 nm (15.63)

2.5.3 Method C- First Order Derivative Spectroscopy:

Standard solutions of 1.5-10 µg/ml and 2.5 – 17.5 µg/ml of OFLOX and KETO respectively were scanned separately in the range of 200-400 nm. These spectrums were converted to first order derivative spectra (Fig. 4) by using derivative mode with 21 data point. For this method, 262 nm and 274 nm were selected as wavelengths of measurements for KETO and OFLOX respectively. There was proportionate increase in amplitude at 262 nm and 274 nm for KETO and OFLOX respectively.

Fig. 4: Overlay of Derivative Spectra of OFLOX and KETO

2.6 Method Validation

The methods were validated for the parameters like Linearity, Precision, Accuracy, Limit of detection and Limit of quantitation as per ICH guidelines.

2.6.1 Determination of Linearity:

2.6.2 Determination of precision:

The precision of method was determined in two ways, intraday precision and inter day precision.

For intraday precision, appropriate dilutions of the formulation equivalent to 100% were prepared and amount of drug content was determined. The same procedure was repeated six times in a day and % RSD was determined. Inter day precision was determined by following the above procedure three times a day and for three consecutive days and % RSD was determined.

2.6.3Accuracy studies:

Accuracy of method was done by % recovery analysis, where standard drug sample equivalent to 50%, 100% and 150% of formulation is spiked and amount recovered is calculated.

3. RESULTS AND DISCUSSION:

The proposed methods for simultaneous estimation of OFLOX and KETO in combined dosage form were found to be simple, accurate and precise which can be well understood from validation data. The % RSD was found to be less than 2, which indicates the validity of methods.

Linearity was observed by linear regression equation method for OFLOX and KETO in different concentration range. Linearity (Table 1) was found to be in a range of 1.5-10.5 μg /ml for OFLOX and 2.5-17.5 μg /ml for KETO by all the methods. The Correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity.

The assay results (Table 2 to 4) obtained by proposed methods lie in a range of 98-102% are in fair agreement; hence it can be used for routine analysis of two drugs in combined dosage forms. There was no interference from Benzylkonium Chloride was observed in these methods. It can be easily and conveniently adopted for routine quality control analysis.

Results of accuracy studies (Table 5) shows amount of % recovery in a range of 97.33-101.33% for Oflox and 98.4-101.6 % for Keto, indicate accuracy of the methods. The intraday and inter day precision data (Table 6) shows RSD values less than 2, explains methods are precise in nature.

Table 1: Linearity data for OFLOX and KETO for all three methods

Sr. No.

Parameters

OFLOX

KETO

Method A

Method B

Method C

Method A

Method B

Method C

Linearity (μg/ ml)

Correlation Coefficient (r²)

1.5-10.5

0.998

1.5-10.5

0.998

1.5-10.5

0.998

2.5-17.5

0.998

2.5-17.5

0.998

2.5-17.5

0.997

N=6

Table 2: Assay results for the determination of OFLOX and KETO in its ophthalmic formulation by method A

Drug

Label Claim (μg/ ml)

Amount Found (μg /ml)

% label Claim

S. D. (±)

OFLOX

KETO

2.94

4.91

98.0

98.20

1.13

1.25

Table 3: Assay results for the determination of OFLOX and KETO in its ophthalmic formulation by method B

Drug

Label Claim (μg/ ml)

Amount Found (μg /ml)

% label Claim

S. D. (±)

OFLOX

KETO

2.96

4.95

98.66

99.00

1.22

1.25

N=6

Table 4: Assay results for the determination of OFLOX and KETO in its ophthalmic formulation by method C

Drug

Label Claim (μg/ ml)

Amount Found (μg /ml)

% label Claim

S. D. (±)

OFLOX

KETO

3.02

5.1

100.66

102

0.93

1.44

N=6

Table 5: Result of Recovery studies by the proposed methods

Drug

Addition

Amount

added

(μg/ ml)

Amount recovered

(μg/ ml)

(Method A)

Recovery

+S.D.

(Method A)

Amount recovered

(μg/ ml)

(Method B)

Recovery

+S.D.

(Method B)

Amount recovered

(μg/ ml)

(Method C)

% Recovery

+S.D.

(Method C)

Oflox

100

150

1.5

3.0

4.5

1.46

2.95

4.43

97.33+ 1.04

98.33+1.02

98.44+1.07

1.48

2.97

4.45

98.66 + 1.24

99.0+1.33

98.88+1.55

1.52

3.03

4.54

101.33+ 1.13

101 + 1.02

100.88 + 0.94

Keto

100

150

2.5

5.0

7.5

2.46

4.92

7.40

98.4+1.22

98.4+1.24

98.66+1.54

2.49

4.96

7.45

99.6+0.93

99.2+0.95

99.33+ 0.86

2.54

5.04

7.53

101.6+ 1.03

100.8+ 0.84

100.4+ 0.75

N=6

Table 6: Intra and Inter-day Precision

Drug	Method A				Method B				Method C
	Intraday Precision		Inter day Precision		Intraday Precision		Inter day Precision		Intraday Precision		Inter day Precision
	SD	%RSD	SD	%RSD	SD	%RSD	SD	%RSD	SD	%RSD	SD	%RSD
Oflox	1.14	1.25	1.15	1.26	1.27	1.30	1.26	1.38	0.94	0.92	0.95	1.01
Keto	1.27	1.38	1.29	1.40	1.28	1.39	1.28	1.35	1.07	1.12	1.09	1.10

N=6

From the above discussion we can claim that developed methods are simple, accurate, precise and economic and are validated as per ICH guidelines.

5. ACKNOWLEDGEMENT:

The authors are thankful to Litaka Pharmaceuticals Pvt. Ltd., Pune, for providing gift samples of Ofloxacin and Ketorolac Tromethamine. The authors are thankful to Management of PES Modern College of Pharmacy, Pune for providing necessary facility for the work.

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Received on 16.04.2013 Accepted on 28.05.2013

Asian J. Pharm. Ana. 3(2): April- June 2013; Page 53-57