VALIDATED STABILITY INDICATING ANALYTICAL METHOD AND IN-VITRO DISSOLUTION STUDIES OF EFAVIRENZ FORMULATION BY RP-HPLC
Venkata Raju Y *, Sunitha G, Ashish Kumar Pal, Haripriya A, Sirisha N, Pani kumar AD.
Gokaraju Rangaraju College of Pharmacy, (affiliated to Osmania University), Bachupally, Nizampet road, Hyderabad, (Andhra Pradesh)-50009
Keywords: RP-HPLC, stability indicating, forced degradation.
Abstract

A reverse phase stability indicating HPLC method was developed for the analysis of efavirenz bulk and pharmaceutical formulations. The developed method was also utilized for in-vitro dissolution studies of efavirenz formulations. Acetonitrile and acetate buffer pH 3.4 was the mobile phase (75:25% v/v), with retention time of 4.007 min at a flow rate of 1.5 mL/min detected at 292 nm wavelength. Linear regression analysis calibration plot showed an excellent linearity between response and concentration in the range of 50-300 µgmL-1. The regression co-efficient was 0.999 and the linear regression equation was y = 7780x+11159. Limits of detection (LOD) and quantification (LOQ) were 0.238 and 0.793 µgmL-1 respectively. The method was validated for accuracy, precision, specificity, robustness, detection and quantification limits, in accordance with ICH guidelines. The specificity of the method was ascertained by forced degradation studies by acid, alkali hydrolysis, oxidation and thermal degradation methods. The degraded products were well resolved from the analysis peak with significant differences at their retention time values. Wide linearity range, sensitivity, accuracy, short retention time and simple mobile phase indicate the method is suitable for routine quantification of efavirenz with high precision and accuracy.

Article Information

Identifiers and Pagination:
Year:2012
Volume:4
First Page:264
Last Page:272
Publisher Id:JAppPharm (2012 ). 4. 264-272
Article History:
Received:July 3, 2012
Accepted:September 22, 2012
Collection year:2012
First Published:October 1, 2012

  1. INTRODUCTION

Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and is used as a part of highly active anti-retroviral therapy (HAART) for the treatment of human immunodeficiency virus (HIV-1). It is chemically (4S)-6-chloro -4- (cyclopropylethynyl)-1, 4-dihydro-4- (trifluoromethyl) - 2H-3, 1-benzoxazin-2-one (Figure 1). Efavirenz activity is mediated predominantly by non-competitive inhibition of HIV-1 RT.[1-3]

The drug stability test guidelines Q1A (R2) issued by International Conference on Harmonization (ICH) requires that analytical test procedures for stability samples should be fully validated and the assay should be stability indicating. Dissolution is considered as one of the most important quality control tests performed on pharmaceutical dosage forms and is now developing into a tool for predicting bioavailability. The profiles obtained from dissolution rate studies, have also been used in an attempt to characterize in vitro behavior of drugs with success. It must be performed under precisely specified conditions.[4]

Literature survey revealed that few spectrophotometric, RP-HPLC and LC-MS methods available for stability indicating and quantification of efavireng in pharmaceutical formulations and biological samples.[3-10] Moreover, Efavirenz has not been reported by method development and validation including dissolution studies and related kinetic parameters of tablet dosage form analysis individually. Therefore, the goal of research work was to develop a rapid, simple, sensitive, cost-effective and validated method for direct estimation of efavirenz in bulk and also aimed at the drug dissolution studies.


Figure 1. Structure of efavirenz

 

2. MATERIALS AND METHODS

2.1. Instruments Used: A Shimadzu HPLC-LC-20AD series binary gradient pump with Shimadzu SPD-20A UV detector, LC-solution version software, intersex C18 column (250 X 4.6 mm, 5 am particle size) as stationary phase, a calibrated electronic span balance Shimadzu (AUX-220), a pH meter of Ellice (LI-120) were used during the analysis.

2.2. Reagents and Chemicals: Analytically pure efavirenz has been obtained as a gift sample from Hetero Pharmaceuticals Ltd. (Hyderabad, India). Tablets were purchased from the local market. Sodium acetate, acetic acid of AR grade and HPLC-grade acetonitrile were used.

2.3. Preparation of mobile phase and standard stock solution: Mobile phase was prepared by mixing 125mL of 0.1 M acetate buffer (pH adjusted to 3.4 ±0.05 with sodium acetate or acetic acid) with 375 mL of acetonitrile. The mobile phase was filtered through a 0.45 µ membrane filter paper.

Accurately weighed 50 mg of drug was transferred to 50 mL volumetric flask and was dissolved in acetonitrile. The volume was made with acetonitrile to give 1000 µgmL-1.

2.4. Optimized chromatographic conditions

RP-HPLC analysis was performed by Iso-cratic elution method with a flow rate of 1.5 mL/min. The mobile phase containing 0.1 M acetate buffer (pH 3.4 was adjusted using acetic acid/sodium acetate) and acetonitrile in the ratio 25:75 (% v/v) to obtain well resolved peak of efavirenz (Rt =4.007 min) as shown in figure 2.1 Wavelength of maximum absorption was selected by UV-detector. The drug shows good response at 292 nm.

 

Table 2.1. System Suitability Parameters

Parameters

Value

Regression equation

Y=7780x +11159

Correlation Coefficient

r2 = 0.999

Slope

7780

Intercept

11159

Retention Time

4.007

Theoretical plates

8639

Tailing factor

1.258

LOD (µg/mL)

0.238

LOQ (µg)

0.793

 


Figure 2.1. Chromatogram of efavirenz 100 µgmL-1

 

2.5: Validation of the method

2.5.1. Linearity: Linearity was found for the concentration range of 50-300 µgmL-1.

 2.5.2. Precision: Intra-day and inter-day precision of the assay samples containing efavirenz (50 and 100 µgmL-1) were analyzed three times.

2.5.3. Accuracy: Accuracy was found out by recovery study using standard addition method.

2.5.4. Robustness: By introducing small but deliberate changes in the mobile phase pH (± 0.2 %), mobile phase composition (± 2.0 %) and flow rate (± 10%) robustness of the described method were studied.

2.5.5. Sensitivity: The sensitivity of the method was determined with respect to LOD and LOQ. The LOD and LOQ were separately determined based on the standard calibration curve.[11]

2.6: Degradation studies

In an attempt to develop a stability indicating assay method, the samples of tablet powder were subjected separately for different degradation conditions. Acidic degradation using 10 mL 5N HCl, basic degradation using 10 mL 1N NaOH, oxidative degradation using 10 mL 6%  hydrogen peroxide solution was carried out. The samples were subjected to degradation for 24 hours at ambient temperature and thermal degradation for 24 hours at 105°C. Photo-degradation was performed for the same samples by exposing them to UV light by using photo stability chamber.[4]

2.7: In-vitro dissolution studies

The method developed was used to analyze samples after in-vitro dissolution of efavirenz under standard conditions specified in the Indian pharmacopoeia. The in-vitro dissolution was performed in 1% sodium lauryl sulphate for 30 min at a temperature of 37±5°C by USP apparatus paddle type with 50 rpm.[1,12-17]

 

  1. RESULTS AND DISCUSSION

3.1. Calibration curve for efavirenz: Appropriate aliquots of standard stock solutions were taken in different 10 mL volumetric flasks and diluted up to the mark with mobile phase to obtain final concentrations of 50, 100, 150, 200, 250 and 300 µgmL-1 of efavirenz respectively. The solutions were injected using a 20 µgmL-1 fixed loop system and chromatograms were recorded. Calibration curve was drawn by plotting average peak area versus concentration as shown in figure 3.1.


Figure 3.1. Calibration curve of efavirenz

 

3.2. Analysis of the marketed formulations: Ten tablets (Estiva 600 mg) were weighed accurately and crushed to form fine powder. Accurately weighed quantity of powder equivalent to 50 mg of efavirenz was dissolved in a 50 mL volumetric flask with acetonitrile. The solution was filtered using Whattmann filter paper.

Appropriate volumes of the aliquots were transferred into six different 10 mL volumetric flasks and then volume was made up to the mark with mobile phase to obtain                100 µgmL-1of efavirenz. The chromatographic conditions and peak areas were measured. The tablet analysis results are shown in Table 3.1.

 

 

Table 3.1. Analysis of commercial tablets (Estiva-600 mg)

Analyte

Label Claim (mg)

Average amount found (mg±SD)

% RSD

Efavirenz

600

603.0 ± 1.61

0.267

3.3. Validation of the method

The developed method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantification as per ICH guidelines.

3.3.1. Linearity: The linearity range was found 50-300 µgmL-1. The regression equation for efavirenz was found to be y=7780x+11159 and correlation co-efficient (r2 = 0.999). Table 3.3.1 shows linearity range data.

Table 3.3.1. Linearity

Concentration(µgmL-1)

Area

0

0

50

398388

100

783911.7

150

1194696

200

1571911

250

1994767

300

2303997

3.3.2. Precision: Intra-day and inter-day precision of the assay samples containing efavirenz (50 and 100 µgmL-1) were analyzed three times in the same day (intra day) and for three consecutive days by different analytical methods. The results are shown in the Table 3.3, revealed that the % RSD was less than 2.

Table 3.3.2. Precision

 

Intraday Precision

Interday Precision

Concentration (µgmL-1)

Avg conc. ± SD (n=3)

RSD (%)

Avg conc. ±SD (n=3)

RSD (%)

50

49.76 ± 0.100

0.20

49.81 ± 0.07

0.14

100

99.32 ± 1.67

1.68

98.55 ± 1.08

1.09

3.3.3. Accuracy: Accuracy was found out by recovery study using standard addition method. Known amount of efavirenz was added to pre-analyzed samples at a level from 80% up to 120% and then subjected to the proposed HPLC method. The % recovery was 98-102 and % RSD was found to be less than 2. Results of recovery studies are shown in Table 3.3.3.

Table 3.3.3. Data on accuracy of the method (n=3)

Excess drug added to analyte (%)

Theoretical Content (mg)

Conc. Found (mg ± SD)

Recovery (%)

 % RSD

0

20

19.96 ± 0.223

99.8

1.117

80

36

36.31 ± 0.46

100.8

1.23

100

40

41.04 ± 0.25

102.6

0.609

120

44

43.61 ± 0.05

99.11

0.114

 

3.3.4. Robustness

By introducing small but deliberate changes in the mobile phase pH (± 0.2 %), mobile phase composition (± 2.0 %) and flow rate (± 10%) robustness of the described method were studied. The robustness of the method was assessed for 2 different amounts of calibration plot (50 & 100 µg). The results of the robustness study also indicated that the method is robust and is unaffected by small variations in the chromatographic conditions. Table 3.3.4. shows results of robustness.

 

Table  3.3.4. Robustness data

Parameter

Variation (%) level

System suitability

 

 

Theoretical plates

Peak Tailing Factor

% RSD

S.T.P

-

8709

1.25

1.22

Flow rate

-  10 %

9559

1.26

1.29

+ 10 %

8167

1.27

1.12

% Organic in mobile phase

- 2 %

8816

1.27

0.27

+ 2 %

8404

1.28

0.45

pH of mobile phase

- 0.2 %

8289

1.29

1.12

+ 0.2 %

8501

1.27

0.51

Wavelength

- 5.0 nm

8179

1.28

0.46

+ 5.0 nm

8183

1.28

0.79

 

3.3.5. Sensitivity: The sensitivity of the method was determined with respect to LOD and LOQ. The LOD and LOQ were separately determined based on the standard calibration curve and were found to be 0.238 µgmL-1 and 0.793 µgmL-1 respectively.

3.4. Degradation studies

In an attempt to develop a stability indicating assay method, the samples of tablet powder were subjected separately for different degradation conditions. All the degraded drug solutions after appropriate dilutions (100 µgmL-1) with mobile phase (neutralization for the acidic and basic degradation) were injected in the chromatographic system. Typical chromatograms of all degraded samples are shown in Figures 3.4.1-3.4.3. The degraded samples were compared with a standard efavirenz. The developed method was capable of determining efavirenz in presence of its degradation products.

3.5. In- vitro dissolution studies from tablet dosage form

        The developed method was successfully applied to determine the release rate pattern of the drug form the tablet dosage form in dissolution rate studies. The cumulative percentage of drug released versus time profile showed that more than 90% of efavirenz was dissolved in 1% w/v sodium lauryl sulphate within 30 min, at 50 rpm, 37±5°C by USP apparatus paddle type. The release rate constants for zero order, first order, Higuchi equation and Hixon-Crosswell cube root were calculated. From the in-vitro drug release studies, it was observed that Higuchi Equation was best fitted for the drug release kinetics in comparison to other models (Table-3.5.1.).


Figure 3.4.1. Acid degraded analytical peak 

            

Figure 3.4.2. Basic degraded analytical peak


Figure 3.4.3. Oxidative degraded analytical peak

     

 

\

 

Table 3.4.1. Degradation data

Stress conditions

Degradation (%)

% RSD

Acidic (5N Hcl), 24 hrs

49.08

0.89

Basic (1N NaOH), 24 hrs

85.83

0.25

6% v/v H2O2, 24 hrs

20.23

1.21

Thermal degradation at 105° C, 24 hrs

0

 

Photo-degradation at 254 nm, 24 hrs

decoloration

 

 

Table 3.5.1. Data of  In-vitro dissolution studies

Kinetics

Equation

r2 value

Zero order

Y=2.317x+26.304

0.8063

First order

Y=0.0299+1.0415

0.5077

Higuchi equation

Y=11.564+14.74

0.8063

Hixon-Crosswell

Y=0.0752x+2.2335

0.5757

 

  1. CONCLUSION

Developed RP- HPLC method is precise, reliable and reproducible for the analysis of efavirenz formulation and its dissolution studies. This method has been found to be better because of its wide range linearity and readily available mobile phase, UV detection, lack of extraction procedures, low Rt, and no internal standard. The method can be successfully used for routine analysis of efavirenz in bulk drug, dosage forms and its dissolution studies without interference.

 

ACKNOWLEDGEMENT

The authors are thankful to Prof. C.V.S. Subrahmanyam, Prinicipal, Gokaraju Rangaraju College of Pharmacy, Bachupally.

 

BIBLIOGRAPHY

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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