Study of Forced Degradation behavior of Atazanavir

 

Angirekula Narendra*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy

GITAM (Deemed to be University), Visakhapatnam-530045, India

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

 

ABSTRACT:

A modified simple and sensitive stability indicating RP-HPLC method has been developedfor the determinationof Atazanavir in presence of its degradant products on isocratic mode. The method was validated and forced degradation studies were performed as per ICH guidelines. Hypersil ODS C-18 Column (250mmx4.6 mm, i.d.5µ particle size) using methanol:tetra butyl ammonium hydrogen sulphatemixture with flow rate 1.0 ml/min are the chromatographic conditions for the present study (UV detection 247 nm) (Isocratic mode). Atazanavir was eluted at 4.86 min within the total run time of 10 min. Atazanavir shows linearity 0.5-200 µg/mL (r2 = 0.999). The method was validated and found to be specific as the degradants were not at allinterferingwith Atazanavir.

 

KEYWORDS: Atazanavir, Stabilityindicating,HPLC, ICH Guidelines.

 

 

 

INTRODUCTION:

Atazanavir (Figure 1) is an antiretroviral drug1. Literature review describes some of the HPLC methods used for the determination of Atazanavir 2-6 and in the present study the authors have studied the forced degradation behavior of Atazanavir in presence of its degradants.

 

 

Figure 1: Chemical structure of Atazanavir

 

 

 

MATERIALS AND METHODS:

Atazanavir was procured as gift sample from HeteroDrugs (India). Methanol(HPLC Grade), tetra butyl ammonium hydrogen sulphate(TBAHS) and hydrogen peroxidewere purchased from MERCK (India). Atazanavir capsules are marketed with brand namesVirataz (Hetero HC (GenX) (Label claim: 300 mg) and ATAZOR (Label claim:100, 150, 200 and 300mg) (Emcure. ARV). The HPLC system is of Model SPD 10A Shimadzu LC-Class-Vp version 6.12 equipped with UV-VIS detector and binary gradient pump (20µl).

 

Chromatographic separation was achieved on Hypersil ODS C-18 column (250mmx4.6 mm; i.d.5µ particle size) using Methanol:10mMtetra butyl ammonium hydrogen sulphate (85:15) as mobile phase. Flow rate was maintained at 1.0 ml/min with UV detection at 247 nm. Atazanavir (1000µg/ml) stock solution was prepared in methanol and diluted with mobile phase. 10 mM tetra butyl ammonium hydrogen sulphate was prepared, sonicated and filtered.

 

Method validation:

The method was validated by evaluating precision, accuracy, linearity, specificity, LOD, LOQ and robustness7. 0.5 to 200 µg/mL Atazanavir solutions were prepared from the stock on dilution with mobile phase and 20 µL of each solution was injected (n=3) in to the HPLC system and the mean peak area of the chromatogram was noted. Calibration graph was plotted with concentration versus mean peak area.Intraday and inter-day precision were performed as per the ICH guidelines same day and on three consecutive days respectively. Accuracy of the method was achieved by standard addition method while robustness wasconducted by introducing deliberate alterations in the optimized conditions such as flow rate (± 0.1mL), mobile phase ratio (± 2%), pH (± 0.2 units) and detection wave length (249 nm& 245 nm).

 

Experimental applicability to Atazanavir capsules:

Atazanavir capsules from the two available brands were bought from pharmacy and content equivalent to 25 mg Atazanavir was extracted with methanol, sonicated, filtered and diluted with mobile phase. These solutions(n=3) were injected and the mean peak area was noted from the respective chromatograms obtained.

 

Forced degradation studies:

Forced degradation experiments were performed by treating Atazanavir with different stress conditions. While performing acidic and alkaline degradations drug solutions were neutralized and then only diluted with mobile phase. 30% v/v H2O2 was opted for oxidative stress.Thermal degradation and photolysis were also performed and 20 µL of each solution was injected in to the system and peak area was noted from the respective chromatograms.

RESULTS AND DISCUSSION:

Method optimization and validation:

A simple stability indicating RP-HPLCwasestablished for the quantification of Atazanavir in capsules using methanol and 10mMtetrabutyl ammonium hydrogen sulphatemixture as mobile phase. During the method development C8 phenomenex column was initially used with a mixture of mobile phases such as acetonitrile and formic acid; methanol and formic acidand different aqueous buffers in combination with acetonitrile and methanol but in some or other manner the system suitability parameters were not satisfied. But usage of Hypersil ODS C-18 Column with methanol and TBAHS (85:15, v/v) mixture with 1 ml/min flow rate finally results acceptable system suitable parameters and Atazanavir was eluted at about 4.8 minFigure 2. The present proposed method was compared with the reported methods in Table 1. The proposed method was validated and a calibration curve was shown in Figure 3 demonstrating that Atazanavir obeys Beer- Lamberts law 0.5 to 200 µg/mL (Table 2) with regression equation y = 20873x + 7458.6 (Correlation coefficient 0.9999). The LOD and LOQ are found to be 0.1342 µg/mL and 0.413 µg/mL respectively. Intraday and inter-day precision were studied and % RSD was found to be 0.13-0.65 (Table 3) and 0.17-0.21 (Table 4) respectively. The % RSD in accuracy was found as 1.01-1.39 (Table 5).

 

 

 

Table. 1. Comparison of previously reported HPLC methods of Atazanavir with the present

Mobile phase (v/v)

Wavelength (nm)

Linearity

Ref

0.02M Ammonium dihydrogen phosphate: Acetonitrile: Methanol (30:25:45)

288

5-50

2

Methanol: Water (90:10) (pH adjusted to 3.55 with Acetic acid)

249

10-90

3

Ammonium dihydrogen phosphate buffer: Acetonitrile (55:45) (pH 2.5)

288

30-600

4

Potassium dihydrogen phosphate buffer: Acetonitrile (55:45)

248

25-150

5

Water: Acetonitrile (20:80) (pH adjusted to 3.0)

255

10-80

6

Methanol: 10 mM Tetra butyl ammonium hydrogen sulphate (85: 15)

247

0.5-200

Present work

 

 

Table. 2. Linearity studyof Atazanavir

Conc. (µg/mL)

*Mean peak area

% RSD

0.5

10851

0.22

1

21901

0.16

2

42446

0.51

5

103527

0.72

10

200733

1.20

20

392991

1.40

50

1014722

0.11

80

1658900

0.60

100

2075264

0.22

120

2490685

0.43

150

3155763

0.55

200

4157282

0.71

*Mean of three replicates

Table. 3. Intraday precision study of Atazanavir

Conc.

(µg/ml)

*Mean Peak Area

Statistical Analysis

*Mean peak area ± SD (% RSD)

20

392991

 

392777±2551.739 (0.65)

20

390125

20

395215

50

1014722

 

1015055±4335.621 (0.43)

50

1019548

50

1010896

100

2075264

 

2072349±2602.653 (0.13)

 

100

2071523

100

2070259

*Mean of three replicates

 

 

 

Table. 4. Inter day precision study of Atazanavir

Conc.

(µg/ml)

*Mean Peak Area

*Mean peak area ± SD (% RSD)

Day1

Day2

Day3

20

382568

381256

381155

381660±788.259 (0.21)

50

1002562

1001211

1004528

1002767±1667.975 (0.17)

100

20506582

20593561

20555769

20551971±43613.726 (0.21)

*Mean of three replicates

Table. 5. Accuracy study of Atazanavir

Conc. (µg/ml)

Conc. (µg/ml)

*Mean conc. (µg/mL)  ± SD (% RSD)

% Recovery

Formulation

Pure drug

Total

Obtained

10

5

15

15.1

15.07 ± 0.1527 (1.01)

101.3

10

5

15

14.9

10

5

15

15.2

10

10

20

19.9

19.97 ± 0.2082 (1.04)

99.8

10

10

20

20.2

10

10

20

19.8

10

15

25

25.6

25.23 ± 0.3512 (1.39)

100.9

10

15

25

24.9

10

15

25

25.2

 

 

 

 

 

 

Figure 2: Typical chromatograms A) Blank B) Atazanavir standard (20 µg/mL) C) Atazanavir capsules (20 µg/mL)

 

 

Figure 3: Calibration curve of Atazanavir

 

 

 

Experimental applicability to Atazanavir capsules (Assay):

The percentage of purity of Atazanavirwas found to be 99.63-99.63 in the marketed formulations and the results were tabulated (Table 6) and there is no interference of excipients (Figure 2c).

 

Table. 6. Assay of Atazanavir capsules

Brands

Label claim (mg)

*Amount found (mg)

*Recovery (%)

I

300

299.53

99.84

II

300

298.89

99.63

* Mean of three replicates

 

Stress degradation studies:

Atazanavir has shown less than 20 % degradationin all the stress studies indicating that Atazanavir is highly resistant and the resulting chromatograms during this study were shown in Figure 4. The system suitability parameters were within the acceptance criteria (Table 7).

 

Table. 7. Stress degradation behavior of Atazanavir

Stress Conditions

% Drug recovered

% Drug decomposed

Tailing factor

Theoretical

plates

Standard drug

100%

-

1.129

5269

Acidic Hydrolysis (0.1N HCl, 30 min at 60°C)

95.8%

4.2%

1.381

5482

Alkaline Hydrolysis (0.1N NaOH_30 min at 60°C)

82.6%

17.4%

1.348

5698

Oxidative degradation (3% H2O2 30 min at 60°C)

96.4%

3.6%

1.143

5487

Thermal degradation (80°C, 12 hrs.)

98.9%

1.1%

1.269

5367

Photolytic degradation (24 hrs.)

99.2%

0.8%

1.367

5984

 

Figure 4: Chromatogram of Atazanavir (20 μg/ml) on A) Acidic degradation B) Alkaline degradation C) Oxidative degradation

D) Thermal degradation E) Photolysis

 

 

CONCLUSIONS:

The proposed stability indicating method for the determination of Atazanavir is specific and there is no interference of the degradants with the drug peak and at the same time there is no interference of the excipients. This method can be applied for the routine assay of Atazanavir in capsules.

 

ACKNOWLEDGEMENT:

The authors are grateful to Hetero Labs Ltd. (India) for providing the gift samples of Atazanavir. The authors do not have any conflict of interest.

 

REFERENCES:

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2.       Charushila HB, Shivanand HH. Stability indicating RP HPLC Method for the determination of Atazanavir in bulk and dosage form. Drug Invention today. 2013; 5: 81-86.

3.       Dey S, SubasisPatro S, NareshBabu N, Murthy PN, Panda SK. Development and validation of stability indicating RP HPLC method for the estimation of Atazanavirsulphate in bulk. Journal of pharmaceutical analysis. 7; 2017: 134-140.

4.       Srinivasu K, Venkateswara Rao J, Appala RajuN and Mukkanti K. A validated RP-HPLC Method for the determinationof Atazanavir in pharmaceutical dosage form.E-Journal of Chemistry. 8(1); 2011:453-456.

5.       JahnaviBandla, S. Ganapaty, Gorja Ashok "Stability indicating method development and validation for the estimation of atazanavir sulfate in pharmaceutical dosage forms by RP-HPLC.Der Pharmacia Lettre. 7 (12); 2015: 271-278.

6.       Sathish Kumar Konidala, K. Sujana and A. Prameela Rani. New validated RP-HPLC method for the determination of AtazanavirSulphate in bulk and dosage form. Der Pharma Chemica. 4 (3); 2012: 1305-1310.

 

 

 

Received on 08.08.2018          Modified on 16.08.2018

Accepted on 23.08.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3693-3697.

DOI: 10.5958/0974-360X.2018.00678.9