First order derivative spectrophotometric methods for the determination of Fluorometholone in ophthalmic preparations

 

Bulusu Ravi Teja, Bhupathi Raju Kishan Varma, Chandaka Prasanna Kumar, Mukthinuthalapati Mathrusri Annapurna*

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy,GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

Three simple spectrophotometric techniques have been developed for the quantification of Fluorometholone in ophthalmic preparations using sodium acetate buffer (pH 4), phosphate buffer (pH 4) and phosphate buffer (pH 7) solutions. Linearity was observed over the concentration range 2-25, 2-30 and 1-50 µg/mL in sodium acetate buffer, phosphate buffer (pH 4) and phosphate buffer (pH 7) respectively. The three methods were validated and can be applied for the assay of Fluorometholone in pharmaceutical formulations.

 

 

 

 

INTRODUCTION:

Fluorometholone¹, chemically 6α-methyl-9α-fluoro-11β,17α-dihydroxypregna-1,4-diene-3,20-dione (C₂₂H₂₉FO₄) with molecular weight 376.462 g/mol (Fig 1) is used to treat inflammatory eye diseases. (The Merck Index, 2006). Fluorometholone is a corticosteroid, most often used after laser-based refractive surgery. Fluorometholone acetate ophthalmic suspension is indicated for use in the treatment of steroid responsive inflammatory conditions of the palpebral and bulbar conjunctiva, cornea, and anterior segment of the eye. Fluorometholone is a glucocorticoid employed in the treatment of allergic and inflammatory conditions of the eye and in the treatment of various skin disorders topically. It is available in pharma market with brand names Biflace eye drops (Alembic Pharma) Flurisone (Label claim: 0.1% and 0.25%) (Micro Vision), F.M.L. and FML Forte (Allergan India Ltd).

 

Only one spectrophotometric method² is available in the literature and in the present study the authors have proposed three different derivative spectrophotometric techniques for the quantification of FLM in eye preparations and the methods are validated³.

 

Fig 1: Structure of Fluorometholone (FLM)

 

MATERIALS AND METHODS:

Chemicals and reagents:

Fluorometholone stock solution was prepared in methanol and dilutions were made with sodium acetate (Method A), phosphate buffer (pH 4) (Method B) and phosphate buffer (pH 7) (Method C). Fluorometholone was obtained from Alembic Pharma (India) as gift sample and was used as received.

 

 

(A)

 

(B)

 

(C)

 

Fig 2. Overlay first order derivative spectrum of Fluoromethalone in A) Sodium acetate (pH 4) B) Phosphate buffer (pH 4) and C) Phosphate buffer (pH 7)

Instrumentation:

UV-1800 double beam UV-VIS spectrophotometer (Shimadzu) with a pair of 10mm path length matched quartz cells is used for the study. All the solutions were scanned 200-400 nm with medium scanning speed.

 

General procedure:

Three spectrophotometric methods Method A, B and C were developed for the determination of Fluorometholone in sodium acetate buffer (pH 4), phosphate buffer (pH 4) and phosphate buffer (pH 7) solutions. 25 mg of Fluorometholone was weighed and transferred to 25 ml volumetric flask and dissolved in methanol. From this stock solution (1000 µg/mL) dilutions were made with sodium acetate buffer (pH 4), phosphate buffer (pH 4) and phosphate buffer (pH 7) solutions separately for Method A, B and C respectively and scanned against their reagent blank in the UV region.

 

Validation

Linearity:

A series of solutions 2-25, 2-30 and 1-50 µg/mL solutions were prepared for Method A, B and C respectively and scanned (200-400 nm). The zero order spectra so obtained were transformed in to first order derivative spectra with the help of inbuilt software and a maxima was observed in all the three methods. The derivative absorbance observed from the maxima were taken against concentration of the drug solution and plotted.

 

 

Precision and Accuracy studies:

The intra-day and inter-day precision studies were performed at different concentration levels (10, 15 and 20 µg/mL) and accuracy studies were performed by standard addition method (80%, 100%, and 120%) and the % recovery was calculated.

 

Assay of Fluorometholone in ear drops:

Available marketed formulations i.e. eye drops preparation containing Fluorometholone was procured from the local pharmacy store, extracted with methanol and assayed after dilution with sodium acetate buffer (pH 4), phosphate buffer (pH 4) and phosphate buffer (pH 7) solutions for Method A, B and C respectively by following the above procedure.

 

 

RESULTS AND DISCUSSION:

Fluorometholone was quantified spectrophotometrically using first order derivative method with three different buffer solutions i.e. sodium acetate (Method A), phosphate buffer (pH 4) (Method B) and phosphate buffer (pH 7) (Method C). The derivative spectra show maxima at 224.12, 227.36 and 227.42 nm for Method A, B and C respectively (Fig 2).

 

 

Linearity was observed over the concentration range 2-25, 2-30 and 1-50 µg/mL (Table 1) with linear regression equations were found to be y = 0.0019x - 0.0027 (R² = 0.9973), y = 0.0014x - 0.0008 (R² = 0.9993) and y = 0.0014x - 0.0006 (R² = 0.9996) for Method A, B and C respectively (Fig 3). The % RSD in intra-day (0.82-0.91) and inter-day (0.92-1.03) precision was found to be less than 2 in all the three methods A, B and C indicating that all the are precise and in accuracy studies the % recovery was found to be 98.78-99.84 with % RSD less than 2 (0.83-1.12) confirming that the methods are accurate (Table 2).

 

 

Table. 1. Linearity of Fluorometholone

*Mean of three replicates

Table. 2. Accuracy study of Fluorometholone (FLM)

Spiked Conc (μg/ml)	Formulation Conc. (μg/ml)	Method A		Method B		Method C
		%RSD	%*Recovery	%RSD	%*Recovery	%RSD	%*Recovery
4 (80%)	5	1.03	99.82	0.87	99.84	0.93	99.24
5 (100%)	5	0.94	99.19	0.98	98.98	1.12	99.13
6 120%)	5	0.83	99.32	0.93	99.24	0.86	98.78

*Mean of three replicates

 

 

Assay of Fluorometholone:

The % recovery obtained in the assay studies was found to be 99.5-99.9(Table 3) and the three methods can be used for the determination of Fluorometholone in ophthalmic preparations.

 

 

Table. 3. Assay of Fluorometholone

*Mean of three replicates

 

(A)

 

(B)

(C)

 

Fig 3. Calibration curves of Fluoromethalone in A) Sodium acetate B) Phosphate buffer (pH 4) and C) Phosphate buffer (pH 7)

 

CONCLUSION:

The three first derivative spectrophotometric methods are simple, precise and accurate for the routine analysis of Fluorometholone in pharmaceutical formulations successfully.

 

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM University, Visakhapatnam for providing the research facilities and the authors have no conflict of interest.

 

REFERENCES:

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

2.        Narendra A, Deepika D and Mathrusri Annapurna M, New spectrophotometric methods for the quantitative analysis of Fluorometholone in ophthalmic suspensions. Chemical Science Transactions, 3(1), 2014, 445-449.

3.         ICH Validation of analytical procedures: Text and methodology, Q2 (R1), International Conference on Harmonization, 2005.

 

 

 

Accepted on 30.05.2017           © RJPT All right reserved