Development and Validation of New Reversed Phase High Performance Liquid Chromatography Method for the Estimation of Efavirenz in Bulk and in Pharmaceutical Dosage Forms

 

B. Syama Sundar* and K. Balamuralikrishna

Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar 522 510 Andhra Pradesh, India

*Corresponding Author E-mail: profbsyamsundar@yahoo.co.in

 

ABSTRACT:

A simple, rapid, accurate, precise and reproducible reverse phase high performance liquid chromatographic method has been developed for the estimation of Efavirenz in bulk and in pharmaceutical formulations. The quantification was carried out using Luna column C18 (250 4.6mm i.d., 5m particle size) column in an isocratic mode, with mobile phase comprising a mixture of Methanol, Water and Tetrahydrofuran in a ratio of 30:50:20 (% v/v/v). The flow rate was 1.0 ml/min and the detection was carried out at 259 nm. The retention time of the drug was found to be 3.675 min and the method produced linear response in the concentration range of 20-60 g/ml (r=0.9993). The recovery studies were also carried out and % RSD from reproducibility was found to be 0.86. The proposed method was statistically evaluated and can be applied for routine quality control analysis of Efavirenz in tablets.

 

KEYWORDS: RP-HPLC, Efavirenz, Tablets.

 


 

INTRODUCTION:

Efavirenz [Fig 1] is a non nucleoside reverse transcriptase inhibitor with activity against HIV. It is used with other antiretrovirals for combination therapy of HIV infection and AIDS. It is also used as postoperative prophylaxis [1]. The molecular formula is C14H9ClF3NO2 and its molecular weight is 315.7. Literature survey revealed that a few analytical methods have been reported for the determination of Efavirenz in pure drug, pharmaceutical dosage forms and in biological samples using high performance thin layer chromatography [2] and liquid chromatography [3-17] either in single or in combined forms. The objective of this study is to develop a simple, fast, selective, accurate, precise and sensitive RP-HPLC-UV method for the determination of Efavirenz in bulk and in pharmaceutical dosage forms (Tablets) suitable for routine quality control analysis. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH) [18] for the determination of Efavirenz in bulk and in tablet dosage form.

 

Fig 1: Structure of Efavirenz

 

MATERIALS AND METHODS:

Reagents and chemicals:

Efavirenz working standard was received as gift sample from sun pharmaceuticals Ltd, Mumbai, India. Efferven 600 mg tablets, manufactured by Ranbaxy Laboratories Ltd, Gurgaon, were procured from local pharmacy. HPLC grade Methanol and Tetrahydrofuran- HPLC grade were purchased from Merck, Mumbai.

 

Instruments and chromatographic conditions:

The method development study was carried out isocratically on a high performance liquid chromatography using waters-2487 seperation module equipped with a rheodyne injector, quaternary pump, 20 L fixed sample loop, 25L Hamilton syringe and detection was carried out using Ultraviolet detector. A Mettler Toledo balanced was used for weighing purpose. Chromatographic separation was carried out at room temperature with Luna column C18 (250 4.6mm with 5m particle). Mobile phase containing a mixture of Methanol, Water and Tetrahydrofuran in a ratio of 30:50:20 v/v/v, degassed in a sonicator for 10 min before use. The flow rate of mobile phase was maintained at 1.0 ml/min and detection was done using UV detector at 259 nm. The injection volume of both standards and sample were 20 L.

 

Procedure:

Preparation of standard:

About 100 mg of Efavirenz was weighed accurately and transferred into a 100 mL volumetric flask containing 20 mL of mobile phase. The solution was sonicated for 20 min and then the volume was made up with a further quantity of mobile phase to get a 1mg/mL solution. This solution was suitably diluted with the mobile phase to get a working standard solution of 100 μg/mL of Efavirenz. Peak area was recorded for all the peaks. The plot of peak area vs. the respective drug concentration gives the calibration curve. The retention time of Efavirenz standard was found to be 3.675 min as shown in Fig. 2.

 

Fig 2: A typical chromatogram of Efavirenz standard

 

Analysis of Efavirenz in tablet dosage forms:

Twenty tablets of Efavirenz were weighed and powdered into uniform size in a mortar. From this the average weight of a tablet was calculated. An accurately weighed portion from this powder equivalent to 100 mg of Efavirenz was transferred to a 100mL volumetric flask containing 20 mL of the mobile phase. The contents of the flask were sonicated for about 20 min for complete solubility of the drug and the volume was made up to 100 mL with water. Then the mixture was filtered through 0.45μ membrane filter. From the above solution a 4 mL of aliquot was taken into a separate 100 mL volumetric flask and made up to the volume with mobile phase and mixed well. The above solution (20 μL) was then injected eight times into the column. The mean peak area of the drug was calculated and the drug content in the formulation was calculated by the regression equation of the method.

 

RESULTS AND DISCUSSION:

The present study was carried out to develop a simple, fast, accurate and precise RP-HPLC method for the analysis of Efavirenz in bulk and in tablet dosage forms. For the determination of Efavirenz, different compositions of mobile phases were employed. Initially, a mobile phase consisting of Methanol and water in the ratio of 50:50 (% v/v) was tried where broad peak shape and more retention time were observed. Finally the ratio was changed to 30:50:20 (% v/v/v) (Methanol :Water: Tetrahydrofuran), where Efavirenz was eluted around 3.675 min with symmetric peak shape and shorter retention time. The results of system suitability parameters were given in Table-1

 

Table 1: Results of system suitability parameters of Efavirenz in standard and tablet formulations

S. No.

System suitability parameter

Standard

Sample

1

USP Tailing

1.26

0.5 - 2.0

2.

Retention time

3.675

3.723

3.

Peak area response

220423.61

210968.32

 

Linearity was determined from calibration graph plotted using peak area vs concentration of the standard solutions and it was found to be obeyed in the concentration range of 20-60 g/ml with a good linear relationship (r=0.9993) was shown in Fig.3. The regression curve was constructed by linear regression fitting and its mathematical expression was Y=5510 X -417.60 (where Y is the peak area and X is the concentration of Efavirenz).

 

Fig 3: Linearity of Efavirenz

 

Precision of the developed method was studied by repeatedly injecting Efavirenz standard and sample solutions for six times (n=6). The % RSD was found to be 0.43 and 0.82 respectively.

 

The drug content (Assay) in the tablets was quantified using the proposed RP-HPLC method. The mean amount of Efavirenz in brand of tablet dosage forms is shown in Table-2. The tablets were found to contain 99.66 1.3 % of the drug. It can be concluded that the proposed RP-HPLC method is sufficiently sensitive and reproducible for the analysis of Efavirenz in tablet dosage forms within a short analysis time.


Table 2: Results of assay in Marketed formulation

S. No.

Brand

Standard area peak

Sample area peak

Labeled amount (mg/tab)

Amount found (mg/tab)

1

EFAVIR

220681.36

220813.28

600

598 0.83

 

 

Table 3: Summary of Validation Parameters

S. No.

Parameter

Value

1

Linearity

20-60 mcg/ml

2

System precision

0.11

3

Method precision

0.25

4

Accuracy

50-150 %

5

Assay

99.66

6

specificity

No interference

 

 


The accuracy of the method was evaluated by performing recovery studies by analyzing three different concentration levels ranging from 50-150 % of the standard concentrations. The percentage recovery was calculated.

The developed method was validated according to the standard procedure and the summaries of results obtained are presented in Table-3.

 

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Received on 19.12.2010 Modified on 10.01.2011

Accepted on 28.01.2011 AJRC All right reserved

Asian J. Research Chem. 4(4): April, 2011; Page 555-557