Method Development and Validation of a new RP-HPLC method for the simultaneous Assay of Ketorolac Tromethamine and Fluorometholone

 

Sunkara Mrunal Chaithanya*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy,

GITAM (Deemed to be University), Visakhapatnam-530045, India

 

ABSTRACT:

A new RP-HPLC method has been developed and validated for the simultaneous determination of Ketorolac tromethamine and Fluorometholone pharmaceutical formulations. Shimadzu Model CBM-20A/20 Alite with C₈ Phenomenex column (250 mm × 4.6 mm i.d., 5 µm particle size) was used for the proposed work with a mixture of 0.1M ammonium formate and methanol as mobile phase and flow rate 0.8 ml/min (UV detection at 241 nm).  The linear regression equation was found to be y = 35207x + 29529 R² = 0.9995 for Fluorometholone and y = 68207x + 57590 R² = 0.9997 for Ketorolac tromethamine and the method was validated.

 

 

 

INTRODUCTION:

Fluorometholone (FRML) (Figure 1) is used for reducing inflammation during the eye disease after the surgery ¹. Ketorolac tromethamine (KTR) (Figure 2) is known for its anti-inflammatory and analgesic activities. The combination of Fluorometholone and Ketorolac tromethamine is available as eye drops with brand names Eyetrust with label claim: 0.1% and 0.5% (5 ml). Spectrophototmetric^2-3 and liquid chromatographic^4-5 techniques were developed in the literature and in the present study the authors have proposed a new RP-HPLC method for the simultaneous estimation of Fluorometholone and Ketorolac tromethamine in ophthalmic preparations and the method was validated⁶.

 

 

 

Figure 1: Structure of Fluorometholone (FRML)

 

 

Figure 2: Structure of Ketorolac tromethamine (KTR)

 

MATERIALS AND METHODS:

Chemicals and reagents: 

Methanol, sodium hydroxide, hydrochloric acid, ammonium formate and hydrogen peroxide (H₂O₂) were purchased from Merck (India). All chemicals are of HPLC grade. All chemicals were of analytical grade and used as received. 

Chromatographic conditions:

Chromatographic separation was achieved by using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector with C8 Phenomenex column (250 mm × 4.6 mm i.d., 5 µm particle size) maintained at 25 ºC. Isocratic elution was performed using 0.1M ammonium formate and methanol (20:80%, v/v) and the flow rate was 0.8 ml/min (Detection at 241nm).

 

Method validation:

25 mg of FRML and KTR were accurately weighed and dissolved in in methanol in 25 ml volumetric flask (1000 μg/ml) and dilutions were carried out with mobile phase. 1-150 μg/ml solutions were prepared from the stock, diluted with mobile phase and injected in to the HPLC system. The peak area of FRML and KTR were calculated from the respective chromatograms and calibration curve was drawn. The intra-day and inter-day precision studies were performed on the same day and on three different days and the % RSD was calculated from the peak area reported. The accuracy of the method was determined using standard addition and recovery experiments. The robustness of the method was studied at different chromatographic conditions such as wavelength (239 and 243 nm), percentage of methanol in the mobile phase (78 and 82%) and flow rate (± 0.05 ml/min).

 

Assay of marketed formulations:

The marketed brand was procured from the local pharmacy store and extracted with methanol, sonicated, filtered and diluted with mobile phase. The resulting solutions were injected in to the HPLC system and the peak area of both the drugs was observed from the resulting chromatogram.

 

RESULTS AND DISCUSSION:                                    

Method development and optimization:

Initially the stressed samples were analyzed using a mixture of 0.1M ammonium formate: methanol (40: 60% v/v) with a flow rate of 0.8 ml/min in which the peak was obtained at retention time was above 8.85 min for both the drugs but the resolution as well as the peak symmetry were not good. The mobile phase ratio was slightly altered to 30:70 % v/v and a sharp peak was eluted at 7.96 min for Ketorolac tromethamine with slight tailing. Finally, the mobile phase composition consisting of ammonium formate and methanol 20:80 % v/v was chosen for the study where sharp and symmetrical peaks were eluted (UV detection at 241 nm) with retention times 5.247 ± 0.04 min (Fluorometholone) and 4.055 ± 0.03 min (Ketorolac tromethamine) (Figure 3).

 

 

Figure 3: Typical chromatograms of A) Ketorolac tromethamine (Rt 4.055 ± 0.03 min) and Fluorometholone (Rt 5.247 ± 0.04 min) B) Ear drops (FRML: KTR = 1: 5) C) Placebo

Method validation:

 

Fluorometholone and Ketorolac tromethamine have shown linearity 1-150 μg/ml (Table 1) with linear regression equations y = 35207x+29529 (R² = 0.9995) and y = 68207x + 57590 (R² = 0.9997) for FRML and KTR respectively (Figure 4 and Figure 5). The % RSD in intra-day precision, inter-day precision (Table 2), accuracy (Table 3) and robustness (Table 4) studies was found to be less than 2.0 % with recovery of 98.83-99.38 % in accuracy study.

 

Table. 1. Linearity of FRML and KTR

*Mean of three replicates

 

 

Figure 4: Calibration curve of Fluorometholone

 

Table .2. Precision studies of FRML and KTR

Drugs	Conc. (μg/mL)	Intra-day precision		Inter-day precision
		*Conc. (μg/mL)  ± SD	%RSD	* Conc. (μg/mL)  ± SD	%RSD
FRML	10	9.99 ±0.009	0.06	9.98 ±0.005	0.05
	50	49.8 ± 0.12	0.20	49.9 ± 0.11	0.23
	100	99.8 ±0.059	0.05	99.9 ± 0.069	0.06
KTR	10	9.98±0.052	0.05	9.99±0.007	0.06
	50	49.7±0.094	0.09	49.8±0.14	0.20
	100	99.9±0.069	0.06	99.7±0.008	0.04

*Mean of three replicates

 

Table. 3. Accuracy studies of FRML and KTR

*Mean of three replicates

Table. 4. Robustness study of FRML and KTR

*Mean of three replicates

 

 

Figure 5: Calibration curve of Ketorolac tromethamine

 

Assay of marketed formulations:

The proposed method was applied for the determination of FRML and KTR in marketed formulations i.e. Eyetrust eye drops and the respective chromatogram was shown in Figure 3. The % recovery was found to be 98.00 and 99.40 (Table 5). The 3D chromatograms of Fluorometholone and Ketorolac tromethamine obtained for the API (Active Pharmaceutical Ingredient) as well as the marketed formulation (Eye drops) were shown in Figure 6a and 6b.

 

 

 

6a. Eye drops formulation

 

6b. Fluorometholone and Ketorolac tromethamine (API)

Figure 6a, 6b : 3D chromatograms of Fluorometholone and Ketorolac tromethamine

 

_{Table. 5.} Assay of FRML and KTR eye drops

* Mean of three replicates

 

CONCLUSION:

The proposed RP-HPLC method is simple, precise, robust and accurate and can be used for the routine analysis of Fluorometholone and Ketorolac tromethamine in pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

The authors are grateful to GITAM (Deemed to be University), Visakhapatnam, India for providing the research facilities and the authors have no conflict of interest.

 

REFERENCES:

1.     The Merck Index, An Encyclopedia of chemicals, drugs    and    biologicals, (14^th edition). Whitehouse Station, NJ:  Merck Research Laboratories Division of Merck and Co., Inc.  2006

2.     Mathrusri Annapurna M, Sevyatha VSV and 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.

3.     Shah JA and Maheshwari DG. Development and validation of first order derivative UV spectrophotometric method for simultaneous estimation of Fluorometholone acetate and Ketorolac tromethamine in ophthalmic dosage form. International Journal of Pharmaceutical Research. 2014; 2(2): 56- 64.

4.     Priti SC, Rajesh RP  and Dushyant  AS. Development and validation of RP-HPLC method for simultaneous estimation of Ketorolac tromethamine and Fluorometholone in ophthalmic dosage form. Inventi Rapid-Pharm Analysis & Quality Assurance 2014; Inventi:ppaqa/1424/14.

5.     Mathrusri Annapurna M, Narendra A and Sevyatha VSV. A new liquid chromatographic method for the simultaneous determination of Ketorolac tromethamine and Fluorometholone in presence of Hydrochlorothiazide.  International Journal of Green Pharmacy. 2018; 12(1) (Suppl): S220-S224.

6.     ICH validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization, 2005.

 

 

 

 

 

Accepted on 24.07.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 3119-3122.