Simultaneous Estimation of Amoxycillin Trihdrate and Dicloxacillin Sodium in Formulation by UV-Spectroscopy

M. Manu, W.D. Sam Solomon*and R. Venkatnarayanan

Department of Pharmaceutical Analysis, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore - 641402, Tamilnadu.

*Corresponding Author E-mail: samwd_2000@yahoo.com

ABSTRACT:

A simple UV- Spectrophotometric method has been developed for simultaneous estimation of Amoxycillin Trihydrate and Dicloxacillin Sodium from Pharmaceutical dosage forms. Methanol: water (1:1) was used as solvent. The method involves the measurement of absorbance at two wavelengths 290 nm (λmax for Amoxycillin) and 274nm (λmax for Dicloxacillin). The linearity lies between 2-10µg/ml for Amoxycillin and10-50 µg/ml for Dicloxacillin. The accuracy and precision of the methods were determined and validated statiscally. The method showed good reproducibility and recovered with %RSD less than 2. The method are found to be rapid, specific, precise and accurate and can be successfully applied for the routine analysis of simultaneous estimation of Amoxycilin and Dicloxacillin in bulk and combined dosage form.

KEYWORDS: Simultaneous, UV-Spectrophotometric, Amoxycilin, Dicloxacillin.

INTRODUCTION:

Amoxycillin is a broad spectrum, semisynthetic antibiotic active against many gram positive and gram negative aerobic and anaerobic bacteria. Amoxycillin is chemically[2s-[2α,5,α,6β(s*)]]-6-[[amino(4hydroxyphenyl)acetyl]amino]3,3,dimethyl,-7-oxo-4-thia-lazabicyclo[3.2.0]thopentane-2-carboxylic acid. Dicloxacillin is a narrow spectrum beta-lactum antibiotic of pencillin class. Dicloxacillin is chemically 6R)-6[3-(2, 6,-dichlorophenyl)-5methylisoxazole-4carboxamido] pencillanic acid¹. Literature survey revealed that various Spectrophotometric^2-8 and HPLC^8-13 methods, have been reported for the determination of Amoxycillin and Dicloxacillin, individually and combination with some other drugs. No UV- Spectroscopic method for simultaneous estimation of Amoxycillin and Dicloxacillin in combined dosage forms has so far been reported. The review of literature prompted us to develop a precise simultaneous method for the estimation of Amoxycillin and Dicloxacillin in combined dosage forms.

EXPERIMENTAL METHOD:

An ELICO model SL - 164 double beam UV -Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm, and a pair of 10 mm matched quartz cells, was used to measure absorbance of the resulting solutions. Amoxycillin and Dicloxacillin capsules (suprimox plus, Ranbaxy) were procured from the market. They had labeled content of 250mg Amoxycillin and Dicloxacillin respectively. Methanol: water (1:1) was used as solvent.

Preparation of stock solution:

Accurately weighed quantity of Amoxycillin (100 mg) and Dicloxacillin (100 mg) was transferred to two separate 100 ml volumetric flask, dissolved in Methanol: water (1:1) and diluted to the mark with the same solvent (stock solution-1000 μg/ml).

Linearity and Range:

The aliquot portion of stock standard solutions of Amoxycillin and Dicloxacillin were diluted with water to get the series of concentration 2-10 µg/ml and 10-50 µg/ml respectively. Absorbtivity values (A^1%_1cm)of Amoxycillin and Dicloxacillin were found to be 736 at 290 nm and 249 at 274nm respectively. (Fig-1).

ANALYSIS OF FORMULATION:

Twenty capsules are weighed and powdered. A quantity equivalent to 250 mg of Amoxycillin and 250 mg of Dicloxacillin was taken in 100 ml volumetric flask, dissolved in 30 ml Methanol: water (1:1) and sonicated for 10 min. The volume was then made up to the mark using same solvent. The resulting solution was filtered through Whatmann filter paper grade 1 and from filtrate 1ml was taken and the volume was made up to 100 ml to get the approximate concentration of 250 μg/ml of Amoxycillin and 250 μg/ml of Dicloxacillin. Absorbance of sample solutions were recorded at 290 nm and 274 nm and the concentration of two drugs in the sample were determined by using simultaneous equation. The results are shown in table 3.

Fig 1: Overlain Spectra of Amoxycillin Trihydrate and Dicloxacillin Sodium

METHOD VALIDATION:

Linearity and Range:

The aliquot portion of stock standard solutions of Amoxycillin and Dicloxacillin were diluted with sodium hydroxide to get the series of concentration 2-10 µg/ml and 10-50 µg/ml respectively. The regression coefficient for Amoxycillin and Dicloxacillin was found to be 0.9998 at 290 nm and 0.9997 at 274nm.

Precision:

Precision and accuracy together determine the error of an individual determination. The overlain spectra of both drugs showed that the peaks were well resolved and satisfying the criteria for precision.

Recovery studies:

To study the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels. A known amount of drug was added to preanalyzed capsule powder and percentage recoveries were calculated. The results of recovery studies were satisfactory (Table 2).

RESULTS:

For the reported method linearity was observed in the concentration range of 2-10 μg/ml for Amoxycillin (Fig.-2) and 10-50 μg/ml for Dicloxacillin (Fig.-3). Marketed brand of tablet was analyzed and amount of Amoxycillin and Dicloxacillin determined by proposed methods ranges from 99.56 - 99.94 as shown in Table-3. The proposed methods were validated as per ICH guideline (Table-1). The accuracy of method was determined by calculating mean percentage recovery at 80,100 and 120 % level. The % recovery ranges from 99.97 to 100.71 for Amoxycillin and Dicloxacillin. Precision was calculated as repeatability (% RSD is less than 2) and inter and intraday variations (%RSD is less than 2) for both drugs.

Table 1: Validation Parameter

Parameters	Amox at 290nm	Diclox at 274nm
Linearity Range(μg/ml)	2-10	10-50
Coefficient of Correlation	0.9998	0.9997
Slope	0.0327	0.0419
Intercept	0.0015	0.0002
Repeatability (% RSD) Intraday precision Interday precision	0.71-0.88 0.53-1.12	0.57-0.93 0.43-1.32

Fig 2: Calibration Curve of Amoxycillin Trihydrate

DISCUSSION:

The proposed methods were found to be simple, precise and sensitive for the routine determination of Amoxycillin and Dicloxacillin in capsule formulation. To study the validity and reproducibility of proposed methods, recovery studies were carried out. The methods were validated in terms of linearity, accuracy, precision, specificity and reproducibility. The proposed method can be successfully used for simultaneous estimation of Amoxycillin and Dicloxacillin in combined dosage form.

Table 2: Recovery Studies

Recovery Level	Amt. of drug added (mg)		Amt. of drug found (mg)		% Recovery *
Recovery Level	Amox	Diclox	Amox	Diclox	Amox	Diclox
80%	200	200	201.21	200.64	100.60+0.147	100.32+0.997
100%	250	250	249.93	250.32	99.97+0.134	100.12+0.123
120%	300	300	302.14	301.68	100.71+0.547	100.56+1.203

* RSD for Six Determinations Amox = Amoxycillin, Diclox = Dicloxacillin

Table 3: Analysis of Formulation

Formulation

Label claim

Amount found % RSD*

% Assay RSD*

Suprimox plus capsules

Amoxycillin

250mg

249.87

99.94

Dicloxacillin

250 mg

248.92

99.56

* RSD for Three Determinations

Fig 3: Calibration Curve of Dicloxacillin Sodium

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