INTRODUCTION:

Ofloxacin (OFL) is chemically (RS)-7-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-4-oxa-azatricyclo [7.3.1.0^5,13] trideca-5(13), 6, 8, 11-tetraene-11-carboxylic acid (Figure 1). Ofloxacin is a second-generation fluoroquinolone and it is measured to be a synthetic antibiotic of the fluoroquinolone drug. Ofloxacin is a consists of 50% of its mirror image and half of levofloxacin (the biologically active component), which is a racemic mixture. Ofloxacin is an antibiotic which cover the broad-spectrum activity. It is inhibits DNA gyrase enzyme activity, a class of topoisomerase II, and topoisomerase IV which is an enzyme inhibiting bacterial cell division therefore essential to discrete (mostly in prokaryotes, as in bacteria) replicated DNA. It is used for the treatment of definite infections instigated by microorganisms, such as pneumonia, bronchitis, urinary tract contaminations, skin, prostate, and venereal disease^1-8.

Figure 1: Structure of Ofloxacin

The chemical name of Ketorolac tromethamine (KET) is (±)-5-benzoyl-2, 3-dihydro-1H-pyrrolizine-1-carboxylic acid, 2-amino-2-(hydroxymethyl)-1, 3-propanediol (Figure 2). It belongs to heterocyclic acetic acid derivatives drug and shown analgesic and non-steroidal anti-inflammatory activity. It was introduced as an Acular eye drops by Allergan and the ophthalmic form was official by Food and Drug Administration (FDA). It is inhibiting cyclooxygenase (COX) enzyme competitively and prevent the prostaglandin synthesis. Ketorolac tromethamine acts as a non-selective inhibitor of COX enzyme^9-16.

Figure 2: Structure of Ketorolac tromethamine

These drugs have encouraged the development of various methods for its evaluation due to therapeutic importance. The literature survey has exposed to spectrophotometric and titrimetric methods. In addition, a number of ways have been reported for quantification in plasma using ultraviolet (UV) or fluorescence detection and high-performance liquid chromatography (HPLC)^17-34. The purpose of this research was to expand an easy and sensitive simultaneous spectrophotometric method for the quantitation of KET and OFL in pharmaceutical formulations and pure drug. This technique uses the recognized simultaneous estimation method.

RESULTS AND DISCUSSION:

The standard plot of OFL and KET were drawn individually at 296nm and 322nm.. The rapport between the concentration and the absorbance of OFL and KET was got to be linear in the concentration value of 2-10 and 3-15µg/mL. The illustrative lined equations were evaluated by the least squares method and the value of correlation coefficients designated very good linearity. The standard addition method was performed and found to have good accuracy of the projected method. It was verified by good percent recovery. It ranged between 99.00 and 100.26%. In the present case the method developed, wavelengths selected were 296nm and 322 nm. Two equations are constructed for a method of simultaneous equation. The concentration of both the components in the mixture can be calculated using these simultaneous equations (1) and (2).

Using these equations, the concentrations of OFL and KET were estimated in commercial formulations. The results of the analysis showed lower values of standard deviation, standard error of mean, coefficient of variation and percentage range of error (within 95% confidence limit) and thus showed the precision of methods. The overlain UV absorption was found to be simple, precise, exact, and economic. Hence the method can be employed for the routine analysis of these two drugs in combined form.

EXPERIMENTAL:

Instrumentation:

A Labindia model 3000+UV-VIS spectrophotometer equipped with 10mm matched quartz cells. A Citizen scale, Japan analytical balance was used.

Materials and Reagent:

Ketorolac Tromethamine and Ofloxacin procured from Sun Pharmaceutical Ltd., Mumbai. All the reagents procured during the experiment were of analytical grade. Marketed formulation Ketoflox (Piramal Healthcare Limited, Dhar, Madhya Pradesh) were taken for analysis.

Simultaneous Estimation of Ketorolac tromethamine and Ofloxacin:

Standard Stock Solution Preparation:

Ketorolac (10mg) was accurately weighed and poured to volumetric flask of capacity 10mL, and the Methanol (diluent) was added to the ring mark to have stock solution of 1000µg/mL, similarly the standard stock solution of Ofloxacin (1000µg/mL) was prepared.

Determination of l_maxof Drugs:

Standard solutions (10mg/mL) of procured ketorolac tromethamine and Ofloxacin were prepared. These standard solutions were run in UV range and scanned. The absorption maxima shown were at 322.0 and 296.0 nm for ketorolac and Ofloxacin respectively. The overlay graph was recorded (figure 3).

Figure 3: Overlay Spectra of Ketorolac and Ofloxacin

Calibration Curves Preparation:

The stock solution of ofloxacin was further diluted with methanol to give standard drug solutions of 2, 4, 6, 8, 10 mg/ mL concentration and solution of Ketorolac diluted to had standard drug solutions of 3, 6, 9, 12, 15, µg/ mL concentration. The calibration curve was plotted for both of the drugs (Figure 4 and 5).

Figure 4: Calibration Curve of Ketorolac tromethamine

Figure 5: Calibration Curve of Ofloxacin

Absorption coefficient of ofloxacin:

Aliquots having ofloxacin concentrations 2, 4, 6, 8, 10 µg/ mL their readings were taken at 296nm and 322nm. The absorption coefficients were calculated for these concentrations give the value of ax₂and ax₁.

Absorption coefficient of ketorolac tromethamine:

Aliquots having ketorolac tromethamine concentrations 3, 6, 9, 12, 15µg/mL their absorbances were measured at 296nm and 322nm. The absorption coefficients were calculated for these concentrations give the value of ay₂and ay₁.

Development of method:

Absorbance of the both drug solutions at 322.0nm and 296.0nm was taken and from the observed absorbance readings, the drug concentration in the solution to analyzed was determined by using Vierordt's formula. The drug concentrations were determined using the following equations,

K_X = (B₂ ay₁ -B₁ ay₂ )/(ax₂ ay₁ -ax₁ ay₂ )

K_Y = (B₁ ax₂ - B₂ ax₁ )/(ax₂ ay₁ -ax₁ ay₂ )

Where,

ax₁ and ax₂ = The absorptivity values of ketorolac at 322.0nm and at 296.0nm respectively,

ay₁ and ay₂ = The absorptivity values of ofloxacin at 322.0nm and at 296.0nm respectively

B₁ = Sample solution absorbance at 322.0 nm

B₂ = Sample solution absorbance at 296.0 nm

Kx = Concentration of ketorolac tromethamine

Ky = Concentration of ofloxacin

The concentration of each component is calculated by substituting the value of absorbances (B₁ and B₂). values.

Analysis of Eye drop Formulation:

Eye Drop equivalent to 5mg Ketorolac tromethamine and 3mg Ofloxacin was weighed and taken to a 10mL volumetric flask and made up to 10mL volume with methanol. Resultant solution was sieved through Whatmann filter paper and its concentration was noted out (Table 1).

Table 1: Estimation of Eye Drop Formulation

Brand Name	Keterolac		Ofloxacin
Brand Name	Label Claim (mg)	% Obtained*	Label Claim (mg)	% Obtained *
Ketoflox	5	99.89	3	99.56

*Denotes average of five determinations

Recovery study:

To validate the accuracy the recovery studies were performed in the newly developed method. During this the a definite concentration of standard drug (80%, 100%, and 120%) was added to the reanalyzed sample solution, and then the analysis of its recovery done and results were updated in Table No.9 and the recovery studies statistical validation data depicted in Table 2 and 3.

Table 2: Recovery Studies

Level of

Recovery (%)

100

120

KET

OFL

KET

OFL

KET

OFL

Amount Present

(mg)

Amount of Std.

Added

(mg)

2.40

3.6

Amount

Recovered

(mg)

3.98

3.95

2.40

2.38

2.41

5.05

4.99

5.00

2.98

2.95

3.02

5.98

5.99

6.01

3.61

3.59

3.60

% Recovery

99.50

98.75

100.00

99.17

100.42

101.00

99.80

100.00

99.33

98.33

100.67

99.67

99.83

100.17

100.28

99.72

100.00

Table 3: Statistical Validation of Recovery Studies

Level of Recovery (%)	Drug	% Recovery	Standard Deviation*	% RSD
80	KET	99.00	0.435	0.437
80	OFL	99.86	0.636	0.637
100	KET	100.26	0.643	0.641
100	OFL	99.44	1.171	1.177
120	KET	99.88	0.255	0.255
120	OFL	100.00	0.278	0.278

*Denotes average of three determinations

CONCLUSION:

The reproducibility, repeatability, and accuracy of this method were found to be good, which is evident by low standard deviation values. The percent recovery experiment values obtained indicates non-interference from the excipients used in the formulations. The percentage recovery was close to 100% for these methods. Thus, it can be concluded that the method established was simple, exact, sensitive and precise. Hence, the above method can be functional effectively in simultaneous estimation of Ketorolac tromethamine and Ofloxacin in sold formulations.

The analysis of eye drop formulation was done and the results obtained were within the limits. The results obtained for validation study were within the limit specified by the ICH guidelines and hence the method was found to be precise and linear. The recovery study data were within ICH limits, thus indicating the accuracy of method.

ACKNOWLEDGMENT:

The authors thank to Sun Pharmaceutical Ltd., Mumbai for providing gift sample of drug.

REFERENCES:

1. O'brien TP, Maguire MG, Fink NE, Alfonso E, McDonnell P. Efficacy of Ofloxacin vs Cefazolin and Tobramycin in the Therapy for Bacterial Keratitis: Report From the Bacterial Keratitis Study Research Group. Archives of Ophthalmology. 1995; 113(10): 1257.

2. Liu JL. K-ratio and Differential Spectrophotometry of Ofloxacin and Metronidazole in compound Ofloxacin Ear Drops. Chinese Journal of Pharmacy. 1997; 28: 514-515.

3. Donnenfeld ED, Perry HD, Snyder RW, Moadel R, Elsky M, Jones H. Intracorneal, aqueous humor, and vitreous humor penetration of topical and oral ofloxacin. Archives of Ophthalmology. 1997; 115(2): 173-176.

4. Choe CH, Bouhaouala SS, Brook I, Elliot TB, Knudson GB. In vitro development of resistance to ofloxacin and doxycycline in Bacillus anthracis Sterne. Antimicrobial agents and chemotherapy. 2000; 44(6): 1766.

5. Emmanuelle C, Bonnafous P, Evelyne P, Sougakoff W, Baohong J, Vincent J. Molecular Detection of Rifampin and Ofloxacin Resistance for Patients Who Experience Relapse of Multibacillary Leprosy. Clinical Infectious Diseases. 2002; 34(1): 39-45.

6. Senthilraja M. Simultaneous UV Spectrophotometric Method for the Estimation of Nitazoxanide and Ofloxacin in Combined Dosage Form. Research Journal of Pharmacy and Technology. 2008; 1(4): 469-471.

7. Pattanayak P, Sahoo S, Mohanty B, Padhi SK. Anthelmintic and Preliminary Phytochemical Screening of Nymphaea nouchali Burm.f. Against Intestinal Helminthiasis. Research Journal of Pharmacy and Technology. 2009; 2(3): 537-539.

8. Patel AN, Patel HU, Patel CN, Jayswal UP. Estimation of Cefixime Trihydrate and Ofloxacin in Combined Dosage Form by UV-Spectrophotometric Methods. Research Journal of Pharmacy and Technology. 2011; 4(6): 976-978.

9. O'Hara DA, Fragen RJ, Kinzer M, Pemberton D. Ketorolac tromethamine as compared with morphine sulphate for the treatment of postoperative pain. Clinical Pharmacology & Therapeutics. 1987; 41(5): 556-561.

10. Brocks DR, Jamali F. Clinical Pharmacokinetics of Ketorolac Tromethamine. Clinical pharmacokinetics. 1992; 23(6): 415-427.

11. Singer AJ, Mynster CJ, McMahon BJ. The effect of IM ketorolac tromethamine on bleeding time: a prospective, interventional, controlled study. The American Journal of Emergency Medicine. 2003; 21(5): 441-443.

12. Chawla A, Ahuja M. In Vitro and In Vivo Evaluation of the Chitosan Microparticulate Ocular Delivery System of Ketorolac Tromethamine. Research Journal of Pharmacy and Technology. 2009; 2(3): 456-462.

13. Begum MY, Shaik MR, Abbulu K, Sudhakar M. Ketorolac Tromethamine Loaded Liposomes: Development, Characterization and In Vitro Evaluation. Research Journal of Pharmacy and Technology. 2011; 4(11): 1766-1771.

14. Annapurna MM, Vellanki Sevyatha SV, Sushmitha M. Simultaneous determination of Ketorolac tromethamine and Fluorometholone in Eye drops by spectrophotometry. Research Journal of Pharmacy and Technology. 2017; 10(4): 1179-1183.

15. Chaithanya SM, Annapurna MM. Method Development and Validation of a new RP-HPLC method for the simultaneous Assay of Ketorolac Tromethamine and Fluorometholone. Research Journal of Pharmacy and Technology. 2018; 11(7): 3119-3122.

16. Khatri U, Rathore KS, Saini S, Bharkatiya M. Formulation and Evaluation of Ketorolac Tromethamine using 32 Factorial Design. Research Journal of Pharmacy and Technology. 2020; 13(6): 2556-2562.

17. Fegade JD, Mehta HP, Chaudhari RY, Patil VR. Simultaneous Spectrophotometric Estimation of Ofloxacin and Ketorolac Tromethamine in ophthalmic dosage form. International Journal of ChemTech Research. 2009; 1(2): 189–194.

18. Razzaq SN, Ashfaq M, Khan IU, Mariam I. Stability indicating HPLC method for the simultaneous determination of ofloxacin and ketorolac tromethamine in pharmaceutical formulations. Analytical Methods. 2012; 4(7): 2121-2126.

19. Vichare V, Tambe V, Joshi SV, Dhole SN. New Simultaneous UV-Visible Spectrophotometric Methods for Estimation of Ofloxacin and Ketorolac Tromethamine in Ophthalmic Dosage Form. Asian Journal of Pharmaceutical Analysis. 2013; 3(2): 53-57.

20. Gandla K, Kumar JM, Bikshapathi DB, Spandana R. A validated RP-HPLC method for simultaneous estimation of febuxostat and ketorolac tromethamine in pharmaceutical formulations. Journal of Drug delivery and Therapeutics. 2012; 2(3): 173-176.

21. Sunil G, Jambulingam M, Thangadurai SA. Kamalakannan D, Sundaraganapathy R, Jothimanivannan C, Development and validation of ketorolac tromethamine in eye drop formulation by RP-HPLC method. Arabian Journal of Chemistry. 2017; 10(1): S928-S935.

22. Bhatt YJ, Sharma SK, Multani PJ. A Validated UV Spectrophotometric Method for the Estimation of Olopatadine and Ketorolac Tromethamine in Ophthalmic Dosage Form. International Journal of Pharmaceutical Sciences Review and Research. 2013; 20(2): 118-120.

23. Patil VP, Devdhe SJ, Angadi SS, Kale SH, Phalke SD, Shelke SD, Patil RH. Validated Spectrophotometric Method for the Estimation of Ketorolac Tromithamine in Bulk and Tablets Using Ninhydrin: A Modified Approach. Asian Journal of Research in Chemistry. 2014; 7(1): 19-24.

24. John P, Azeem W, Ashfaq M, Khan IU, Razzaq SN. Stability indicating RP-HPLC method for simultaneous determination of piroxicam and ofloxacin in binary combination. Pakistan journal of pharmaceutical sciences. 2015; 28(5): 1713-1721.

25. Parmar R, Tandel F, Patel N. Ratio spectra derivative UV Spectrophotometric method for simultaneous estimation of ketorolac tromethamine and phenylephrine hydrochloride in immediate release tablet. International Journal of Pharmacy and Pharmaceutical Sciences. 2015; 7(11): 13-16.

26. Chintawar PP, Pawar PN, Harde MT, Joshi SV, Chaudhari PD. Spectrophotometric Methods for Simultaneous Estimation of Moxifloxacin HCl and Ketorolac Tromethamine. Asian Journal of Research in Chemistry. 2010; 3(3): 767-771.

27. Khairnar DA, Chaudhari CS, Anantwar SP. Method Development and Validation of Ketorolac Tromethamine in Tablet Formulation by RP-HPLC Method. International Journal of Pharmaceutical Sciences and Research. 2014; 5(9): 3696-3703.

28. Bhole RP, Jagadale PD, Chitlange SS, Wankhede SB. A Simple and Sensitive HPTLC Method for Simultaneous Analysis of Phenylephrine hydrochloride and Ketorolac tromethamine in Combined Dose Formulation. Analytical Chemistry Letters. 2015; 5(4): 206-215.

29. Rao J, Sethy K, Yadav S. Validated HPTLC Method for Simultaneous Quantitation of Cefixime and Ofloxacin in Bulk Drug and in Pharmaceutical Formulation. International Journal of Comprehensive Pharmacy. 2011; 2(4): 1-5.

30. Nanjwade BK, Manjappa AS, Murthy RSR, Pol YD. A novel pH-triggered in situ gel for sustained ophthalmic delivery of ketorolac tromethamine. Asian Journal of Pharmaceutical Sciences. 2009; 4(3): 189-199.

31. Devarajan PV, Gore SP, Chavan SV. HPTLC determination of ketorolac tromethamine. Journal of Pharmaceutical and Biomedical Analysis. 2000; 22(4): 679-683.

32. The United States Pharmacopoeia. National Formulary-USP XXIV. Rockville MD. United States Pharmacopeial Convention Inc. 1998; 2149-2152.

33. Kothapalli LP, Mare SG, Thomas AB, Nanda RK, Deshpande AD. Simultaneous Spectrophotometric Methods for Estimation of Metformin and Fenofibrate in synthetic mixture. Research Journal of Pharmacy and Technology. 2008; 1(2): 86-88.

34. Kakde RB, Kotak VH, Barsagade AG, Chaudhary NK, Kale DL. Spectrophotometric Method for Simultaneous Estimation of Amlodipine Besylate and Bisoprolol Fumarate in Pharmaceutical Preparations. Research Journal of Pharmacy and Technology. 2008; 1(4): 513-515.

Received on 14.07.2020 Modified on 12.02.2021

Research J. Pharm. and Tech. 2022; 15(6):2498-2502.

DOI: 10.52711/0974-360X.2022.00417