You are hereUV - Spectrophotometric and RP - HPLC Method Developement for Simultaneous Determination of Paracetamol and Etodolac in Pharmaceutical Dosage Form

UV - Spectrophotometric and RP - HPLC Method Developement for Simultaneous Determination of Paracetamol and Etodolac in Pharmaceutical Dosage Form


2.2. UV- spectrophotometry:
A Thermospectronic model is Systronics-108 (Double beam) Spectrophotometer with 1cm. matched quartz cells was used. Standard stock solutions of Paracetamol of 1000 μg.mL-1 were prepared by dissolving 100 mg of paracetamol in 100 mL of methanol,  From these stock solutions, working standard solutions having concentration 20,30,40,50,60 μg.mL-1 and Standard stock solutions of Etodolac of 1000 μg.mL-1 were prepared by dissolving 100 mg of Etodolac in 100 mL of methanol, From these stock solutions, working standard solutions having concentration 30,40,50,60,70 μg.mL-1each were prepared by appropriate dilutions. They were scanned in the wavelength range of 400−200 nm and the overlain spectrum was obtained (Fig 3). Two wavelengths 256.0 nm (λmax of Paracetamol) and 226.0 nm (λmax of Etodolac) were selected for the formation of simultaneous equation. The calibration curves were found to be linear in the concentration range of 20-60 μg.mL-1 for Paracetamol and 30-70 μg.mL-1 for Etodolac. The absorptivity coefficients of each drug at both wavelengths were determined. The concentration of two drugs in the mixture were calculated using equations [12],

CPCM = A2 ay1 – A1 ay2/ ax2 ay1 – ax1ay2 ……………… … (1)
CETO = A1 ax2 – A2 ax2/ ax2 ay1 – ax1ay2…………………... (2)

Where, A1 and A2 are absorbance of mixture at 256.0 nm and 226.0 nm; ax1 and ax2,
absorptivities of Paracetamol at 256.0 nm and 226.0 nm, respectively; ay1 and ay2
absorptivities of Etodolac at 256.5 nm and 226.0 nm, respectively. CPCM and CETO are
concentration of Paracetamol and Etodolac in mixture. The absorptivities reported are the
mean of six independent determinations (Table 1).

Fig 3. Overlain Spectrum of Paracetamol And Etodolac in Methanol taken on Systronics-108 (Double beam) Spectrophotometer
2.3. HPLC method:

The HPLC system consisted of a YL-9100 pump, a loop injector of injection capacity 20 μL, Detector consists of Photodiode array detector, a Promosil C18 (250 X 4.60 mm) 5μm  column, a HPLC guard cartridge system and a YL Clarity software at ambient temperature.
Different mobile phases were tested in order to find the best conditions, for separating both the drugs simultaneously. The optimal composition of mobile phase was determined to be Acetonitrile: Methanol (23:77, v/v). The flow rate was set to 1.5 mL.min-1 and UV detection was carried out at 239 nm.
Stock solution was prepared by dissolving 100 mg of Paracetamol and Etodolac in 100 mL was taken in separate two100 mL volumetric flask for each drug by using Acetonitrile: Methanol (23:77, v/v) as solvent.

Table 1. Absorptivity Values at 256.0 nm (λmax of Paracetamol) and 226.0 nm (λmax of Etodolac)


Absorptivity at 226.0 nm

Absorptivity at 256.0 nm


Paracetamol

Etodolac

Paracetamol

Etodolac

*Mean

362.25

514.23

472.45

408.28

± S.D.

1.17

0.51

0.54

0.48

* Absorptivity values are the mean of six determinations. S.D. is standard deviation. ax1 and ax2 absorptivities of  at 256.0 nm and 226.0 nm, respectively; ay1 and ay2 absorptivities of Etodolac at 226.0 nm and 256.0 nm, respectively.

All solutions were stored at + 50C in the dark; these solutions were shown to be stable during the period of study.
From the above stock solutions, dilutions were made in the concentration range of 20–60 μg.mL–1 of Paracetamol and 30-70 μg.mL–1 Etodolac. A volume of 20 μL of each sample was injected into column. All measurements were repeated three times for each concentration and calibration curve was constructed by plotting the peak area ratios of analyte to the corresponding drug concentration.

2.4. Analysis of Pharmaceutical Dosage Forms
To determine the content of Paracetamol and Etodolac simultaneously in tablets (label claim: 100 mg Paracetamol and 100 mg Etodolac, film coated); twenty tablets were weighed; their average weight determined and were finely powdered. The correct amount of powder was dissolved in Acetonitrile: Methanol (23:77, v/v).  by stirring for 30 min. The excipients were separated by filtration. After filtration, an appropriate amount of internal standard was added and diluted up to mark with Acetonitrile: Methanol (23:77, v/v). Appropriate aliquots were subjected to above methods and the amount of Paracetamol and Etodolac were determined. The results are reported in Table 2.

Table 2. Analysis data of tablet formulations


Parameters

UV – spectrophotometry

HPLC

Paracetamol

Etodolac

Paracetamol

Etodolac

Label Claim

500

400

500

400

*Drug content

99.96

99.37

100.3

100.0

± S. D.

0.450

0.625

0.624

1.000

% R.S.D.

0.451

0.628

0.622

1.000

* Value for Drug content (%) are the mean of five estimations; S.D. is standard deviation and R.S.D. is relative standard deviation

2.5. Recovery studies
To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiments were carried out by standard addition method, at 80, 100 and 120 % level. From the total amount of drug found, the percentage recovery was calculated. The results are reported in Table 3.

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