Development and Validation of Stability Indicating Related Substances Method for Dolutegravir/Lamivudine/Tenofovir Disoproxil Fumarate (DLT) Tablets using High Performance Liquid Chromatography

 

Saravanan. R1, Somanathan. T1*, Gavaskar. D1, Sriraman. V1, Tamilvanan. M2, Sasieekhumar. A. R2

1Department of Chemistry, Vels Institute of Science, Technology and Advanced Studies (VISTAS),

Pallavaram, Chennai, India.

2Department of Chemistry, VMKV Engineering College, Vinayaka Mission’s Research Foundation,

Salem, Tamilnadu, India.

*Corresponding Author E-mail: soma.sbs@velsuniv.ac.in, soma_nano@yahoo.co.in

 

ABSTRACT:

A novel, accurate, specific, linear, precise and robust RP-HPLC method (stability indicating) has been developed and validated for the related substances (impurities) analysis of Dolutegravir, Lamivudine and Tenofovir Disoproxil Fumarate in tablet formulation. This research paper presents the developed method and the outcome of validation challenges. The RP-HPLC method was developed on a 250 x 4.6 mm, 5 µm, C18 column, with a gradient mode using combination of phosphate buffer and phosphoric acid in methanol and water as mobile phase, the detection was performed at 265nm and 235nm.  The method was subjected to validation challenges of specificity, precision, linearity, accuracy, robustness and is demonstrated to be suitable for testing of stability samples.

 

KEYWORDS: Dolutegravir Sodium, Lamivudine, Tenofovir Disoproxil Fumarate, Liquid chromatography, Stability Indicating, Related Substances.

 

 

INTRODUCTION:

Incidence of HIV infections globally is very high and health agencies have been constantly in pursuit of providing HIV patients with improved therapeutic alternatives.  WHO recommends fixed dose multidrug treatment regimens in an endeavor to enhance the patient compliance (one of the major challenges of multidrug treatment regimens) and thus help achieving key goals of treatment i.e., increase in longevity of patient’s life along with improvement in Quality of life. The WHO 2019 guideline recommends oral triple combination of DLT as first line of treatment for HIV infection in adults.

 

The development of stability indicating related substances method is challenging for this triple combination product as the number of impurities that needs to be separated are high. In this research work a common RP-HPLC method for determination of related substances of all the three active components i.e., Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate in oral solid dosage form was successfully developed and validated.

 

Dolutegravir:

Dolutegravir is an integrase strand transfer inhibitor1 belonging to second generation of antiretrovirals. It is a preferred drug of choice due to no requirement of dose adjustments when combined with the NRTI class2,3. Dolutegravir is chemically (4R,12aS)-N-[(2,4-difluorophenyl)methyl]-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido [1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide (WHO) has a chemical formula of C20H18F2N3NaO5 with Mol. Wt of 419.3788.

 

Lamivudine:

Lamivudine is L-enantiomeric analog of Cytosine having reverse transcriptase inhibitor activity against hepatitis B and Human immunodeficiency virus. It is first line antiretroviral drug in treatment guidelines4. Lamivudine is indicated for the treatment of Human Immunodeficiency Virus and chronic HBV infection5. The chemical name of Lamivudine is 4-amino-1-[(2R, 5S)-2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] pyrimidin-2-one6 and molecular weight of 229.254 with a chemical formula of C8H11N3O3S.

 

Tenofovir Disoproxil Fumarate:

Tenofovir DF is a bis-alkoxyester pro drug and is acyclic nucleotide analog with reverse transcriptase inhibitor activity against Human Immunodeficiency Virus and HBV. Tenofovir is released into systemic circulation by cleavage of promoieties during adsorption7. It is chemically 9- [R- (2[[bis]] isopropoxycarbonyl) oxy] methoxy] phosphonyl] methoxy] propyl] adenine fumarate (1:1) and has molecular weight of 635.52 and a chemical formula of C23H34N5O14P.

 

Development of cost effective, precise, specific, linear, robust and accurate stability indicating quantitative method that simultaneous estimation of 13 known impurities of Dolutegravir, Lamivudine and Tenofovir DF is a challenging task and was accomplished successfully by applying the underlying theoretical concepts and expertise of developers.

 

MATERIALS AND METHODS:

Waters Quaternary pump HPLC equipped with PDA detector, Waters Sunfire C18, 250 x 4.6mm, 5µm analytical column, HPLC grade KH2PO4, o-phosphoric acid, HPLC grade H2O, HPLC grade SD fine methanol and Millipore 0.45µm filter.

 

Several trials were undertaken and the below mentioned method was finalized and subjected to validation challenges as per ICH guidelines8.

 

Method subjected to analytical method validation:

Chromatographic conditions: Mobile Phase A: 10mM Potassium dihydrogen phosphate pH 4.0, Mobile Phase B: 0.1% o-phosphoric acid in methanol and water (75:25) Column: Waters Sunfire C18, 250 x 4.6mm, 5µm, Flow Rate: 1.4 mL/min, Wavelength: 265 and 235 nm, column oven temperature: 30°C±2°C, Sample Cooler Temperature: 10°C±2°C, Injection Volume: 20µL, Injection delay 10 mins, Diluent: 10mM KH2PO4 pH 2.5 and methanol in the ratio of 80:20 respectively.

 

Flow Program:

Time (min)

% MP A

% MP B

0

95

5

9

95

5

27

88

12

45

57

43

55

45

55

82

42

58

100

23

77

108

2

98

116

95

5

120

95

5

 

Table:1 Chemical name of Drug substances and its impurities with its retention times

Name of drug substance/Impurity Name

Drug substance/Impurity Identification Name

Retention time

(in mins)

Lamivudine

Lamivudine

21.20

Tenofovir Disoproxil Fumarate

Tenofovir Disoproxil Fumarate

72.21

Dolutegravir Sodium

Dolutegravir Sodium

94.52

Tenofovir Disoproxil Fumarate Impurities

Adenine Impurity

Impurity A (Imp A)

7.83

Tenofovir Impurity or TDF-II Impurity

Impurity B (Imp B)

17.68

Mono ester impurity

Impurity C (Imp C)

45.89

Mono POC Dimer Impurity

Impurity D (Imp D)

69.71

Mixed Dimer Impurity

Impurity E (Imp E)

101.21

Dimer Impurity

Impurity F (Imp F)

110.43

Lamivudine Impurities

Carboxylic acid Impurity

Impurity G (Imp G)

5.55

Diastereomer Impurity

Impurity H (Imp H)

20.51

Dolutegravir Sodium Impurities

Hydroxy impurity

Impurity I (Imp I)

88.73

Methyl Dolutegravir Impurity

Impurity J (Imp J)

91.14

2-Fluoro impurity

Impurity K (Imp K)

91.73

Des Fluoro or 4-Fluoro impurity

Impurity L (Imp L)

89.62

Isomer-1 and 2

Impurity M (Imp M)

95.32

 

Validation Outcomes:

Linearity:

The Linearity of the test method was established from LOQ to 150% of limit concentration (0.2%) as tabulated below. All the three active pharmaceutical ingredients and its impurities exhibited linear behavior in the specified range. The linearity data is presented below in Table:2.  Refer Fig:2 for corresponding Linearity plots.

 

Table:2 Linearity Data (Dolutegravir, Lamivudine, Tenofovir DF and its impurities).

Component

Concentration (µg/mL)

Regression equation

R2

Impurity A

0.071 – 6.921

y = 66160x+234.94

0.9999

Impurity B

0.092 – 7.044

y = 36521x -491.96

0.9999

Impurity C

0.112 – 7.012

y = 66377x- 2269.7

0.9999

Impurity D

0.106 –7.033

y = 67982x- 1127.9

0.9997

Impurity E

0.104 – 6.982

y = 16441x+ 1146

0.9998

Impurity F

0.114 – 7.013

y = 61661x - 749.2

0.9996

Impurity G

0.091 – 7.084

y = 39493x + 192.5

0.9998

Impurity H

0.102 – 7.023

y = 23770x+706.91

0.9999

Impurity I

0.153 – 7.503

y = 32986x -564.03

0.9999

Impurity J

0.073 – 7.113

y = 66294x -490.95

0.9998

Impurity K

0.076 – 7.234

y = 65911x -907.64

0.9999

Impurity L

0.071 – 7.084

y = 65838x -699.53

0.9999

Impurity M

0.074 – 7.183

y = 65892x -1069.9

0.9998

Tenofovir Disoproxil Fumarate

0.082 – 7.044

y = 36504x + 6015

0.9997

Lamivudine

0.126 – 7.118

y = 73907x -4296.6

0.9994

Dolutegravir

0.108 – 7.243

y = 99770x+123.41

0.9997

 

Accuracy:

The accuracy of an analytical process expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found8.

 

The study was performed at 50%, 100% and 150% (triplicate preparations at each level) of limit concentration (0.2%). The individual and mean accuracy at each level for all the impurities was found to be between 80.0 to 120.0%.  The accuracy data is presented in Table:3

 

Table:3 Accuracy

Criteria

Accuracy Level

Impurities

A

B

C

D

E

F

G

H

I

J

K

L

M

Average % Recovery

50%

87.2

90.2

88.6

89.4

88.2

90.6

100.2

101.1

83.8

95.4

100.4

86.6

93.1

100%

95.4

99.8

92.1

94.3

95.0

94.3

93.1

98.2

97.7

99.3

98.2

96.4

99.7

150%

97.4

99.7

94.0

98.3

96.1

99.8

102.5

103.2

98.3

99.1

100.5

96.3

99.1

RSD (%Recovery)

50%

3.0

3.1

2.8

4.8

2.6

3.8

3.4

4.7

4.0

4.3

1.4

3.6

4.4

100%

2.9

1.8

3.9

4.8

3.0

4.1

1.5

2.3

2.7

3.5

3.2

3.8

3.2

150%

1.2

1.4

4.4

1.9

3.6

2.4

2.9

2.0

1.0

1.9

2.0

4.1

3.7

Specificity: It is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically, these might include impurities, degradants, matrix, etc.8

 

Table:4 Forced Degradation Study

Control sample (No treatment)

Dolutegravir Peak Purity

Lamivudine Peak Purity

Tenofovir Disoproxil Fumarate

 Peak Purity

PA

PT

PA

PT

PA

PT

0.144

0.362

0.221

0.312

0.156

0.329

Forced Degradation Study

Samples

Condition

% Degradation

Dolutegravir Peak Purity

Lamivudine Peak Purity

Tenofovir Disoproxil Fumarate Peak Purity

PA

PT

PA

PT

PA

PT

Acid Degradation

1N HCl for 30 mins at room temperature

10.2

0.208

0.424

0.118

0.301

0.224

0.386

Alkali Degradation

1N NaOH for 30 mins at room temperature

8.3

0.147

0.318

0.281

0.424

0.108

0.264

Peroxide Degradation

30% H2O2 for 90 mins at room temperature

4.6

0.228

0.543

0.192

0.284

0.233

0.414

Thermal Degradation

105°C for 2 hours

9.8

0.486

0.814

0.226

0.374

0.118

0.304

Humidity Degradation

25°C/95%RH/72Hrs

0.6

0.108

0.364

0.174

0.304

0.224

0.364

Water Degradation

60°C for 3 hours

0.3

0.222

0.501

0.443

0.628

0.321

0.484

Photo stability

Exposed to UV light at 200-watt hrs/Sq.mt and white light for about 1.2 million lux hours

0.2

0.133

0.308

0.267

0.474

0.208

0.389

PA=Purity Angle, PT=Purity Threshold

 

Specificity of the method was established by spiking all the available known impurities of all the three active pharmaceutical ingredients in sample. All the peaks were observed to be specific. The peak purity was evaluated, purity angle for Dolutegravir, tenofovir and lamivudine peaks was observed to be less than auto purity threshold in the spiked sample. The method met the validation challenge of specificity.

 

Forced degradation studies were performed. Prominent degradation was observed during acid, alkali and thermal degradation. Since PA is less than the PT for Tenofovir Disoproxil fumarate, Lamivudine and Dolutegravir peaks in all degradation conditions, the method met the criteria of stability indicating for related substances of impurities in Dolutegravir/Lamivudine/Tenofovir Disoproxil Fumarate tablets.  The results from forced degradation study are tabulated in Table: 4

 

Precision:

Precision of the method was established by method precision and intermediate precision (analysis on different day with different lot of analytical column and by using freshly prepared mobile phase and samples) studies. These studies were performed by spiking all impurities at known level. The %RSD for results of individual known impurities and total impurities was found to be below 5.0% for both method precision and intermediate precision studies.

 

Precision at Limit of Quantitation (LOQ) Level:

LOQ of the method is the ability to determine quantitatively the lowest amount of analyte with suitable precision.  Precision at LOQ was performed by spiking all known impurities and three active pharmaceutical ingredients at LOQ level (0.05% level as per ICH guideline) in the placebo and injected six times. %RSD for six replicate injections and signal to noise ratio details are presented below in Table:5

 

Table: 5 Limit of Quantitation (LOQ)

Parameter

Criteria

Impurities

A

B

C

D

E

F

G

H

I

J

K

L

M

Dolutegravir

Lamivudine

Tenofovir

Precision

at LOQ

%RSD

1.1

4.2

3.3

2.6

1.9

3.2

1.8

3.1

2.3

2.6

2.2

1.4

3.6

2.1

1.6

2.3

Signal to Noise ratio

11

13

14

11

15

12

14

11

13

12

15

14

12

11

14

16

 

Robustness study:

Robustness study was performed by evaluating the impact of below mentioned changes on System suitability parameters i.e., tailing factor and %RSD for replicate injections.

i)      Flow rate ± 10%

ii)    Column oven temperature ± 5°C

iii)  Wavelength ± 5nm

 

Robustness study encompassed evaluation of impact of flow rate, column oven temperature and wavelength. All the system suitability criteria were met in robustness study confirming that the method is robust.

 

CONCLUSION:

A Sensitive, Specific, Linear, Precise, Accurate and Robust stability indicating method was developed for simultaneous estimation of 13 known impurities of Dolutegravir, Lamivudine and Tenofovir Disoproxil Fumarate in Tablets. The method met all the validation challenges as per ICH recommendations.

 

ACKNOWLEDGEMENTS:

We are very thankful to the management of VISTAS, Pallavaram, Chennai for all the support provided for this research work.

 

REFERENCES:

1.      Mechanisms of action, pharmacology and interactions of dolutegravir. Esteban Ribera and Daniel Podzamczer. Enferm Infecc Microbiol Clin. 2015 Mar;33 Suppl 1:2-8.

2.      Dolutegravir plus abacavir-lamivudine for the treatment of HIV-1 infection. Walmsley SL, Antela A, Clumeck N, Duiculescu D, Eberhard A, Gutiérrez F, Hocqueloux L, Maggiolo F, Sandkovsky U, Granier C, Pappa K, Wynne B, Min S, Nichols G, Single Investigators. N Engl J Med. 2013 Nov 7; 369(19):1807-18.

3.      Dolutegravir-A review of the pharmacology, efficacy, and safety in the treatment of HIV. Christopher E Kandel, Sharon L Walmsley. Drug Design, Development and Therapy 2015:9 3547-3555.

4.      Bioequivalence of a dolutegravir, abacavir, and lamivudine fixed-dose combination tablet and the effect of food. Weller S, Chen S, Borland J, Savina P, Wynne B, Piscitelli SC. J Acquir Immune Defic Syndr. 2014 Aug 1; 66(4):393-8.

5.      Drugs for HIV Infection. Benjamin J. Eckhardt, Roy M. Gulick, in Infectious Diseases (Fourth Edition), 2017

6.      Laboratory Diagnosis and Therapy of Infectious Diseases. David W. Kimberlin,  Principles and Practice of Pediatric Infectious Diseases (Fourth Edition), 2012

7.      Intracellular Metabolism and In Vitro Activity of Tenofovir against Hepatitis B Virus. William E. Delaney, IV, Adrian S. Ray, Huiling Yang, Xiaoping Qi, Shelly Xiong, Yuao Zhu, and Michael D. Miller. Antimicrob Agents Chemother. 2006 Jul;50(7):2471-7.

8.      https://database.ich.org/sites/default/files/Q2_R1__Guideline.pdf

 

 

 

 

 

Received on 18.01.2021           Modified on 14.05.2021

Accepted on 04.08.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(5):2147-2150.

DOI: 10.52711/0974-360X.2022.00356