Method Development and Validation for Simultaneous Estimation of Lamivudine and Raltegravir in Pure Drug form by RP-HPLC

 

Shribhumika L, Ramgopal M Dhanwad

Government College of Pharmacy, Department of Pharmaceutical Chemistry, Bengaluru, Karnataka.

*Corresponding Author E-mail: ramgopal.dhanwad@gmail.com

 

ABSTRACT:

A new, simple, accurate, and economical method was developed for simultaneous estimation of Lamivudine and Raltegravir by RP-HPLC. The chromatographic separation was achieved through Phenomenex C18 250mm 4.6mm, 5m as stationary phase and mobile phase was Methanol: Acetonitrile: Millipore Water (60:10:30) v/v ratio with the flow rate of 1ml/min at 292nm. The developed method was validated for the quantitative analysis of commercially available pure drugs. The instrument used was Shimadzu PDA-20AR, Lab solution software. The retention time for Lamivudine and Raltegravir were found to be 2.883 and 4.430 respectively. The linearity study of Lamivudine was performed by using concentration range of 1-20g/ml and for Raltegravir concentration range was at 10-30g/ml and correlation coefficient (r2) was found to be 0.999 and 0.9977 respectively. The developed method was validated in reference to ICH Guidelines. Specificity, Linearity, Accuracy, Robustness, Precision and Ruggedness obtained within the acceptance range.

 

KEYWORDS: Lamivudine, Raltegravir, HIV, RP-HPLC, PDA.

 

 


INTRODUCTION:

The selected drugs for research, Lamivudine and Raltegravir are specifically used to treat HIV. Treatment of HIV started as monotherapy initially, and then multiple drugs with different mechanism of action are used in regimens.

 

A new drug combination regimen, consisting of Raltegravir and Lamivudine used for treatment of HIV-1 infection. This brings new perspectives for the design of multiple drug combination regimens targeting different enzymes involved in replication, which is reverse transcriptase is targeted by Lamivudine and integrase is targeted by Raltegravir. Which shows synergistic activity, reduced likelihood for virus-drug resistance and lesser chance for toxic side effects due to lower dosage levels.

 

Lamivudine (LAM):

Chemically, 4-amino-1-[(2R, 5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl] pyrimidin-2-one. Lamivudine is an anti-retrovirus drug specifically belongs to NRTI. Lamivudine is a deoxycytidine analogue which gets phosphorylated intracellularly and converts to its active metabolite and competes for the incorporation into viral DNA. Inhibit the HIV reverse transcriptase enzyme and chain termination happens1. The molecular formula C8H11N3O3S and molecular weight 229.256g/mol and soluble in Water, Methanol and partially soluble in Acetone.

 

Raltegravir (RAL):

Chemically, N-[(4-fluorophenyl) methyl]-5-hydroxy-1-methyl-2-{2-[(5-methyl-1,3,4-oxadiazol-2-yl)formamido] propan-2-yl}-6-oxo-1,6-dihydropyrimidine-4-carboxamide. Raltegravir inhibits HIV integrase which prevents the viral genome incorporation into the human genome. Raltegravir is used in combination with other antiretroviral agents in the treatment of HIV infection2. The molecular formula C20H21FN6O5 and molecular weight 444.4g/mol and soluble in Water, Methanol and very slightly soluble Ethanol and Acetonitrile.

 

(a) Lamivudine (b) Raltegravir

 

Figure 1: Chemical structure of (a) LAM and (b) RAL

 

The literature review reveals that there are few analytical methods have been reported for the determination of RAL and LAM. Considering all these in the present study an attempt was made to develop a new, simple, accurate, economical method and validation of analytical method for simultaneous estimation of RAL and LAM by RP-HPLC method. Degradation studies was performed. The developed method was validated in reference to the ICH guidelines.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

LAM and RAL were procured as gift sample from Laurus Labs Limited, Hyderabad, India and Hetero Drugs Limited, Hyderabad, India respectively. HPLC grade Methanol (SDFCL), HPLC grade Acetonitrile (Merck), and HPLC grade Water are obtained from the stores of Government College of Pharmacy, Bengaluru. All chemicals were of Analytical grade.

 

Instrumentation:

HPLC experiments were carried out on Shimadzu HPLC-20AR with Photodiode array detector in isocratic mode using manual injection. Data collection and processing was done using LC software solution software. The chromatographic separation was achieved by using Phenomenex C18 (250mm 4.6mm, 5m) column. Other equipment used were Electronic analytical balance, Shimadzu-1800UV Spectrophotometer, Ultrasonic bath Sonicator.

 

Preparation of solutions:

Diluent and Mobile phase:

The mobile phase was prepared using Methanol, Acetonitrile and Millipore water in the ratio of 60:10:30 v/v, filtered with nylon membrane filter of 0.22, degassed and sonicated for 10min.

 

Selection of flow rate of mobile phase:

The optimum flow rate of mobile phase was determined by considering the time factor, retention time and considering the minimum use of mobile phase.

 

Preparation of standards stock solutions:

(a)  LAM: Weigh accurately about 10mg of LAM API in 10ml volumetric flask add 10ml of diluents in the ratio 60:10:30 v/v, sonicate to dissolve and make up the volume with diluent, to get 1000g/ml concentration.

(b)  RAL: Weigh accurately about 10mg of RAL API in 10ml volumetric flask add 10ml of diluents in the ratio 60:10:30 v/v, sonicate to dissolve and make up the volume with diluent, to get 1000g/ml concentration.

 

Further dilute 0.05ml of LAM standard solution in 10ml volumetric flask with diluent to get required concentration of 5g/ml and 1.5ml of RAL solution in 10ml volumetric flask with diluent to get required concentration of 15g/ml.

 

Selection of Detection Wavelength:

Selection of Detection wavelength was done using UV Shimadzu-1800UV-Spectrophotometer. The reference standard 10mg of each standard LAM and RAL were taken and dissolved in HPLC grade Methanol was scanned in the range of 200-400nm and the iso-bestic point of these drugs was found to be 292nm.

 

 

Figure 2: UV Spectrum of LAM and RAL in Mixture

 

There was more than one iso-bestic point hence it was also scanned in PDA detector HPLC instrument and it was confirmed to be 292nm.

 

RESULTS:

Chromatographic condition:

The chromatographic separation was carried out on Shimadzu HPLC-20AR with PDA as detector along with LC solution software. The Phenomenex C18 (250mm 4.6mm, 5m) column was used for separation with Methanol, Acetonitrile and Millipore water 60:10:30 v/v ratio. The detection wavelength was at 292nm with the flow rate of 1ml/min. The 20l volume was used to inject. The retention time of LAM was found to be 2.662 and for RAL was found to be 4.434 (figure 3) and optimized chromatographic condition as shown below,

 

Column: Phenomenex C18 (250mm 4.6mm, 5m)

Flow rate: 1ml/min

Wavelength: 292nm

Column temperature: Ambient

Injection mode and volume: Manual injection mode and 20l

Run time: 10min

Diluent: Methanol, Acetonitrile and Millipore water in the ratio of 60:10:30%v/v

Elution: Isocratic mode

 

Mobile phase:

Methanol, Acetonitrile and Millipore water in the ratio of 60:10:30%v/v

 

 

Figure 3: Chromatogram of LAM and RAL

 

Table 1: Chromatogram of LAM and RAL

Drugs

Retention

Time

Area

Height

Resolution

Tailing Factor

HETP

Theoretical

Plate

LAM

2.883

120400

36671

0.000

1.556

42.84

4110.294

RAL

4.430

216223

16182

7.181

1.553

36.83

4980.128

 

 

Estimation of Retention time of LAM and RAL:

 

 

 

Figure 4: Estimation of Retention time of LAM and RAL

 


Table 2: Estimation of Retention time of LAM and RAL

Drugs

Retention time

Area

Height

Resolution

Tailing Factor

HETP

Theoretical Plate

LAM

2.684

120800

49755

0.000

1.477

38.546

3298.901

Drugs

Retention time

Area

Height

Resolution

Tailing Factor

HETP

Theoretical Plate

RAL

4.127

216245

36792

0.000

1.497

38.591

3854.532

 


Method Validation:

Linearity and range:

Calibration standards of LAM and RAL were prepared at 5 levels within the concentration range of 1-20g/ml and 10-30g/ml respectively. Peak areas were plotted against the corresponding concentration to obtain linearity graphs. Correlation coefficient was found to be 0.999 and 0.9977 for LAM and RAL respectively.

 

Figure 5: Calibration curve of LAM and RAL

Table 3: Linearity results of LAM and RAL

Parameter

LAM

RAL

Concentration range (g/ml)

1-20

10-30

Correlation coefficient

0.999

0.9977

Slope

20334

20143

Intercept

14030

85224

 

Precision:

Precision is of repeatability of a method with normal operation condition. System precision, method precision, intra-day precision and inter-day precision. The peak areas of injections were recorded and then calculated %RSD for all types of precision. The % RSD for system precision was 0.17% and 0.05%, for method precision was 0.24% and 0.25%, for Intraday precision was 0.09% and 0.13% and for inter-day precision was 0.12% and 0.01% respectively for LAM and RAL.

 


 

Table 4: System precision

Replicates

LAM

RAL

 

Area

Concentration

Area

Concentration

1

116210

5.00

216889

15.00

2

115990

5.00

216597

15.00

3

115600

5.00

216695

15.00

4

115982

5.00

216734

15.00

5

116022

5.00

216572

15.00

6

115766

5.00

216783

15.00

Average

115983.3

216711.667

SD

196.2383

118.2939841

%RSD

0.17%

0.05%


 


Table 5: Method precision and Intra-day Precision

Replicates

Method Precision

Intra-day Precision

 

LAM

RAL

LAM

RAL

Peak Area

Conc.

Peak Area

Conc.

Peak Area

Conc.

Peak Area

Conc.

1.

115560

4.9931

216200

14.96

116106

5.0199

216232

14.9655

2.

115566

4.9934

216195

14.9637

115970

5.0132

216290

14.9684

3.

116110

5.0201

216223

14.9651

115860

5.0078

216548

14.9812

4.

115982

5.0138

216245

14.9662

116012

5.0153

216189

14.9662

5.

116022

5.0158

216565

14.9821

115902

5.0099

217109

15.0091

6.

115990

5.0142

218110

15.0588

115896

5.0096

216993

15.0033

Average Conc.

5.0084

14.98265

5.012616667

14.98228333

SD

0.012

0.038

0.004478132

0.019468479

%RSD

0.24

0.25

0.09

0.13

 


Table 6: Method precision and Inter-day Precision

Replicates

LAM

RAL

Area

Concentration

Area

concentration

1st Day

115902

5.0099

216189

14.9662

2nd Day

115896

5.0096

216290

14.9684

3rd Day

116106

5.0199

216232

14.9655

Average (Conc.)

5.01313333

14.9667

SD

0.00586202467

0.001513275

%RSD

0.12

0.01

 

LOD and LOQ:

LOD it is the lowest amount of analyte in the sample which can be detected but not quantify and LOQ it is the lowest amount of analyte in the sample which can quantitatively determine. The LOD of LAM and RAL were 0.008g/ml and 0.02g/ml respectively. The LOQ of LAM and RAL were 0.024g/ml, 0.060g/ml.

 

Accuracy/Recovery:

Accuracy was conducted by using a standard addition technique by recovery studies. The pre analyzed samples were spiked with extra 80%, 100% and 120% of each standard LAM and RAL. The recovery percentage was in the range of 99.944% - 101.95% and 99.722% - 101.04% for LAM and RAL respectively.


 

Table 7: Accuracy results of LAM and RAL

Drugs

%conc

Area

Amount added

Amount found

%recovery

%Mean recovery

 

LAM

80

379975

18

17.99

99.944

 

100.881

100

423934

20

20.15

100.75

120

470310

22

22.43

101.95

 

RAL

80

276512

18

17.95

99.722

 

100.554

100

321367

20

20.18

100.9

120

362715

22

22.23

101.04

 


Robustness:

The chromatographic condition was intentionally altered and degree of reproducibility was evaluated. System suitability parameters were compared with the method precision. The results of LAM and RAL was 99.17 - 101.49% and 99.75 - 100.09% by changing flow rate of mobile phase, 98.99 to 100.88% and 99.68 to 100.422% by changing wavelength of detection and 99.28 to 103.06% and 99.39 to 100.15% by changing the ratio of mobile phase respectively.


 

Table 8: Robustness of LAM and RAL

Drugs

%conc

Area

Amount added

Amount found

%recovery

%Mean recovery

 

LAM

80

379975

18

17.99

99.944

 

100.881

100

423934

20

20.15

100.75

120

470310

22

22.43

101.95

 

RAL

80

276512

18

17.95

99.722

 

100.554

100

321367

20

20.18

100.9

120

362715

22

22.23

101.04


Ruggedness:

The ruggedness parameter was performed by different analyst same instrument or by different instrument. The % assay of LAM and RAL was 101.02% and 100.4046% respectively with respect to instrument-1, analyst-1 and 100.848% and 100.4372% respectively for LAM and RAL with respect to instrument-1 and analyst-2.

 

DISCUSSION AND CONCLUSION:

RP-HPLC method has been developed for the simultaneous estimation of LAM and RAL. The chromatography was carried out by using Phenomenex C18 (250mm 4.6mm, 5m) as stationary phase and mobile phase was Methanol: Acetonitrile: Millipore Water (60:10:30) v/v ratio with the flow rate of 1ml/min at 292nm. The Chromatographic condition was recorded, the runtime was 10 min and the retention time for LAM and RAL was found to be 2.883 and 4.430 respectively. The developed method was found to be optimized method. Validation was carried out in reference to the ICH guidelines. The LAM and RAL concentration range was found 1-20 g/ml and 10-30 g/ml respectively. The % RSD for system precision was 0.17% and 0.05%, for method precision was 0.24% and 0.25%, for Intraday precision was 0.09% and 0.13% and for inter-day precision was 0.12% and 0.01% respectively for LAM and RAL. The LOQ of LAM and RAL were 0.024g/ml, 0.060g/ml. The results were found in acceptance criteria limit. Hence this method was found to precise, accurate, simple, economic.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ABBREVIATIONS:

LAM: Lamivudine, RAL: Raltegravir, Conc: Concentration, ICH: International Conference of Harmonization, RP-HPLC: Reverse Phase High Performance Liquid Chromatography, RSD: Relative standard deviation, SD: Standard deviation, UV: Ultra violet, PDA: Photo Diode Array, LOD: Limit of Detection, LOQ: Limit of Quantification, HIV: Human Immunodeficiency Virus, NRTIs: Nucleoside Reverse Transcriptase Inhibitors, DNA: Deoxyribonucleic acid, HETP: Height Equivalent to a Theoretical Plate.

 

ACKNOWLEDGEMENTS:

The author would like to thank to the Principal, Government College of Pharmacy, Bengaluru for providing all the necessary research facilities to carry out my research work. The author also would like to thank Laurus Labs Limited, Hyderabad for providing LAM gift sample and also for Hetero Drugs Limited, Hyderabad for providing the sample of RAL to carry out my research study.

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Received on 25.07.2022 Modified on 14.04.2023

Accepted on 01.09.2023 RJPT All right reserved

Research J. Pharm. and Tech. 2024; 17(3):1071-1075.

DOI: 10.52711/0974-360X.2024.00167