A Validated Stability Indicating RP-HPLC Method for Simultaneous Determination of Lopinavir and Ritonavir in Bulk and Tablet Dosage Form

 

Prasanthi1*, Dannana Gowri Sankar2

1Research Scholar, Department of Pharmaceutical Analysis and Quality Assurance,

University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.

2Department of Pharmaceutical Analysis and Quality Assurance,

University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.

*Corresponding Author E-mail: daveprashanthi@gmail.com

 

ABSTRACT:

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Lopinavir and Ritonavir in its pure form as well as in tablet dosage form. Chromatography was carried out on a Kromasil C18 (4.6 x 250mm, 5µm) column using a mixture of TEA buffer (pH 4.0), Methanol in proportion 65:35 v/v as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 227nm. The retention time of the Lopinavir and Ritonavir were found to be 2.079, 4.045 min respectively. The method produce linear responses in the concentration range of 5-35µg/ml for both Lopinavir and Ritonavir. The method precision for the determination of assay was below 2.0% RSD. The method is useful in the quality control of bulk and pharmaceutical formulations. The optimized method was validated and proved to be suitable for the quality control of the mentioned drugs in their different pharmaceutical dosage forms, according to ICH guidelines. The developed method was found to be fairly precise, rapid and economical for simultaneous estimation of Lopinavir and Ritonavir when compared with the reported method.

 

KEYWORDS: Lopinavir, Ritonavir, RP-HPLC; PDA Detection; ICH validation.

 

 


INTRODUCTION:

Lopinavir (LPV) [1S-[1R*,(R*), 3R*, 4R*]]-N-[4-[[(2,6-dimethyl phenoxy) acetyl] amino]-3-hydroxy-5-phenyl- 1-phenyl methyl) pentyl] tetrahydro –alpha - (1-methyl ethyl) -2-oxo-1(2H) –pyrimidine acetamide. 

Ritonavir (RTV) [10-Hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methyl ethyl)-4-thiazolyl] -3,6-dioxo -8,11-bis (phenyl methyl)-2,4,7,12-tetraaza tridecan-13-oic acid, 5-thiazolyl methyl ester, [5S-5R*,8R*,10R*,11R*)] are anti-HIV (HIV protease inhibitors) drugs. In literature, LPV and RTV have been reported to be quantified individually or in combination by spectrophotometric methods1–3, HPTLC method4, HPLC methods5-8 from bulk drug and dosage forms as well as RP-HPLC/MS methods9–14 for simultaneous determination of LPV and RTV and in combination with other antiviral drugs in the biological matrices which requires very costly instrumentation system for analysis.

 

Chemical Structure of Ritonavir:

 

The availability of an HPLC method with high sensitivity and rapid quantification will be very much useful for the determination of Ritonavir in pharmaceutical formulations. The aim of the study is to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Ritonavir in pharmaceutical dosage form as per ICH guidelines.

 

MATERIALS AND METHODS:

Materials and Reagents:

The reference standards on Lopinavir and Ritonavir were procured from Hetero Labs, Hyderabad, India. Lopimune® tablets nominally containing 200mg of Lopinavir and 50mg of  Ritonavir per tablet were supplied as gift samples from Local Market (Cipla Pvt, Ltd), Hyderabad. All the HPLC solvents and analytical reagent grade chemicals were purchased from S.D. Fine Chemicals, Hyderabad, India

 

Instrumentation:

A Waters HPLC system equipped with a 2695 binary pump, an auto sampler and a 2996 photo diode array detector was employed for the study. The output signal was monitored and processed with Empower software.

 

Chromatographic conditions:

The separation of the drugs was achieved on a Gemini Phenomenex C18 HPLC Column (250 x 4.6mm; 5µ particle size) by running a mobile phase containing a 65:35 v/v mixture of 0.1% TEA orthophosphoric acid in Methanol and Potassium di hydrogen Phosphate at a flow rate of 1.0mL/min. The injection volume was 10 μL. The column temperature was maintained at 30°C and the analytes in the eluates were monitored at 227 nm. The run time was 6.0min. A 50:50 v/v mixture of water and Methanol and Buffer was used as the diluent to prepare drug solutions.

 

Preparation of standard solution:

Accurately weighed and transferred 10mg of Lopinavir and Ritonavir working standard into a 10mL of clean dry volumetric flask add about 7mL of Diluent and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol. Further pipette 0.15 and 0.6ml of the above Lopinavir and Ritonavir stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Tablet sample solution:

Twenty tablets of “Lopimune” (Lopinavir 200mg and Ritonavir 50mg) were accurately weighed and the average weight of the tablet was calculated. The tablets were finely powdered and a quantity of the powder equivalent to one tablet was transferred into a 100mL volumetric flask. 70mL of the diluent was added to it and sonicated for 5 minutes. Then the volume was made up with the diluent and mixed well to prepare the sample stock solution. This solution was filtered through a 0.45 μm nylon filter. 2.0mL of the filtrate was transferred to a 20mL volumetric flask and the volume made up to give final theoretical concentrations of 100μg/mL and 400μg/mL of  Ritonavir  and Lopinavir respectively.

 

Method development and Validation

Different mobile phases were considered for simultaneous separation of the two drugs on a Hypersil C18 HPLC Column. Selection of the mobile phase was done on the basis of ideal resolution among Lopinavir and Ritonavir and also their impurities formed during forced degradation studies. The required chromatographic conditions were optimized. The developed method was validated for precision, specificity, accuracy (recovery), linearity and robustness as per the ICH guideline.

 

Table 1: Optimized Chromatographic condition

Parameters

Chromatographic conditions

Mobile phase ratio

Methanol : Buffer (65:35% v/v)

Column

Gemini Phenomenx  C18 (4.6×250mm) 5µ

Detector

PDA Detector

Column temperature

40şC

Wavelength

227 nm

Flow rate

1.0 ml/min

Injection volume

10 µl

Run time

6 minutes

 

System Suitability:

System suitability was established for initial evaluation of the method before running the sample for the validation parameters. The test was performed according to the USP.10 The standard solutions prepared as per the proposed method were analyzed. The results of the system suitability study are presented in Figure 3. The acceptance criterion is % RSD ≤ 2.0. A percent RSD of 0.8 indicates good system precision of the method. The tailing factor obtained from the standard injection is 1.49 and 1.35 and Theoretical plates obtained from the standard injection are 482499 and 477482 respectively.

 

Specificity:

 

Fig. 1: Typical chromatogram of Lopinavir and Ritonavir

 


Linearity:

The linearity were observed for in the concentration rages 5-35µg/mL for both the Lopinavir and Ritonavir. The Linearity of the method was demonstrated by preparing different concentrations of drug substance and analyzing as per the proposed method. A plot of the area of the peak as a function of analyte concentration was prepared and its regression equation computed. The linearity data and calibration curves of the both the drugs are shown in Table 2.and Fig.2.

 

Fig.2: Calibration Curves of   (a) Ritonavir     b)Lopinavir

 

Table 2: Linearity data of  Ritonavir and Lopinavir

S.

No

Linearity Level

Ritonavir

Lopinavir

Concentration

(µg/mL)

Peak Area

Concentration

(µg/mL)

Peak Area

1

1

5

126626

5

128746

2

2

10

246499

10

246697

3

3

15

364619

15

365060

4

4

20

482268

20

482638

5

5

25

613530

25

615027

6

6

30

724361

30

736431

 

Correlation coefficient

0.9997

0.9995

Limit of detection and limit of quantification:

The LOD values of Ritonavir and Lopinavir were found to be 0.087 and 0.13µg/ml respectively. The LOQ values of Ritonavir and Lopinavir were found to be 0.232 and 0.137µg/ml respectively. Thus the method developed was found to be sensitive.

 

Precision:

In the precision study, %RSD was found to be less than 2% for  Ritonavir  0.6% and Lopinavir 0.2 which indicates the system has a good reproducibility for precision studies 5 replicate studies of  Ritonavir  and Lopinavir formulation (method precision) was performed. %RSD was determined for peak areas of  Ritonavir  and Lopinavir and the acceptance limits should be NMT 2% and the results were found to be within the acceptance limits The chromatograms of precision results were reported in Table:3.

 

Table 3: Data of precision

No. Injections

Ritonavir Peak Area

Lopinavir Peak Area

Injection1

484085

481924

Injection2

485729

480352

Injection3

487158

471534

Injection4

482643

476301

Injection5

483804

477671

Injection -6

485418

483754

Average

484806

479089

S.D

1610.0

3519.3

% RSD

0.33

      0.7

 

Accuracy:

The accuracy studies were shown as % recovery for Ritonavir  and Lopinavir at 50%, 100%,150% ,the limits of recovery should be in range of 98-102% the limits obtained for  Ritonavir  and Lopinavir were found to be within the limits. Hence the method was found to be accurate. The accuracy studies shows % recovery of the  Ritonavir  100% and Lopinavir and the limits of % recovery of drugs were 98-102% and from the above results its indicates that the method was accurate and also revealed that the commonly used exciepients present in the pharmaceutical information do not interfere in the proposed method. The chromatograms of shown in results were shown Tables 4.

 

Table 4: Accuracy Results of Ritonavir and Lopinavir

Ritonavir

% Concentration

(at specification Level)

Peak Area

Amount added

(ppm)

Amount found

(ppm)

% Recovery

Mean Recovery

50%

22938

5

5.01

100.04

99.6%

100%

45426

15

14.9

98.9

150%

70093

20

19.89

98.09

 

Lopinavir

50%

209357

5

4.9

99.8%

99.4%

100%

420697

10

9.8

99.4%

150%

631550

15

14.9

99.2%

 

FORCED DEGRADATION STUDIES:

Acid degradation:

Degradation was observed by the additon of 0.5 N HCl

       

Fig. 5:  Degradation studies chromatograms a) Acid degradation, b) Alkali degradation,  c) Thermal degradation d) Peroxide degradation, e) Photolytic degardation 

 


Table No. 10: Data of degradation studies

Type of degradation

Area of sample

Assay content (% w/w)

Ritonavir

Lopinavir

Ritonavir

Lopinavir

Acid  (0.5N HCl)

424721

457250

89.02

94.6

Base  (0.5N NaOH)

459827

437831

91.4

96.3

Peroxide  (3% H202)

471623

457283

91.2

94.9

Thermal  (at 60-800 c)

460522

466679

93.2

95.3

Photolytic  (sunlight)

460521

468425

92.9

95.7


 

SUMMARY AND CONCLUSION:

Summary:

RP-HPLC method was developed for simultaneous estimation of Ritonavir and Lopinavir in pharmaceutical dosage form. Chromatographic separation was performed on Hypersil C18 (4.6×250mm) 5µ column, with mobile phase comprising of mixture of Acetonitrile: Water in the ratio of 50:50% (v/v), at the flow rate 0.9ml/min. The detection was carried out at 235nm.

 

Table 5: Summary for RP-HPLC Method

S.

No

Parameters

 

Acceptance Criteria

Results Obtained

1

System

suitability

Theoretical Plates- NLT 2000

RIT - 4242

LPV-6515

Tailing factor - NMT 2

RIT -1.15

LPV -1.78

Retention time

RIT -2.033

LPV -3.34

2

Precision

% RSD of  RIT -NLT 2

% RSD of LPV -NLT 2

RIT -0.2

LPV -0.8

4

Linearity

Correlation coefficient NLT 0.999

RIT -0.9997

LPV -0.9995

5

Accuracy

Percentage Recovery 98-102%

RIT -99.6%

LPV -99.4%

6

Limit of Detection

1:3

RIT -0.087µg/ml

LPV – 0.13µg/ml

7

Limit of quantitation

1:10

RIT -0.232µg/ml

LPV- 0.137µg/ml

 

CONCLUSION:

The proposed HPLC method was found to be precise, specific, accurate, rapid and economical for simultaneous estimation of Lopinavir and Ritonavir in tablet dosage form. It was also proved to be convenient and effective for the determination of Lopinavir and Ritonavir in the bulk and combined dosage form. It inferred the method found to be simple, accurate, precise and linear. The method was found to be have a suitable application in routine laboratory analysis with high degree of accuracy and precision.

 

ACKNOWLEDGEMENT:

The authors are very thankful to Management and Principal Nirmala College of Pharmacy, Mangalagiri, Guntur (Dist) Andhra Pradhesh,  for providing literature review and constant support for current Research work, for providing necessary facilities and all standard samples provided for my research work.

 

CONFLICT INTEREST:

Not interested.

 

REFERENCES:

1.      Skoog D A, West D M, Holler FJ: Introduction of analytical chemistry.  Sounder college of publishing, Harcourt Brace college publishers. (1994), PP 1-5.

2.      Sharma B K, Instrumental method of chemical analysis Meerut. (1999), PP 175-203.

3.      Breaux J and Jones K: Understanding and implementing efficient analytical method development and validation. Journal of Pharmaceutical Technology (2003), 5, PP 110-114.

4.      Willard, H. y. Merritt L.L, Dean J.A and Settle F.A “Instrumental methods of analysis” 7th edition CBS Publisher and Distributors, New Delhi, (1991), PP 436-439.

5.      Meyer V.R. Practical High-Performance Liquid Chromatography, 4th Ed. England, John Wiley and Sons Ltd, (2004), PP 7-8.

6.      Dias EM, Donato CL, Rossi, et al. LC method for studies on the stability of lopinavir and ritonavir in soft gelatin capsules, Chromatographia, 2006, 63(9-10): 437-443.

7.      Suneetha A, Kathirvel S, Ramachandrika G, et al. A validated RP HPLC method for simultaneous estimation of lopinavir and ritonavir in combined dosage form, Int. J. of Pharmacy and Pharm. Sci., 2011, 3(1): 49-51.

8.      Marzolini C, Telenti A, Buclin T, Biollaz J, Decosterd LA, et al. Simultaneous determination of the HIV protease inhibitors indinavir, amprenavir, saquinavir, ritonavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitor efavirenz by high-performance liquid chromatography after solid-phase extraction, J. Chromatogr. B, 2000, 740(1): 43-58.

9.      Damaramadugu R, Inamadugu J, Kanneti R, et al. Simultaneous Determination of Ritonavir and Lopinavir in Human Plasma after Protein Precipitation and LCMS-MS, Chromatographia, 2010,71(9/10): 815824.

10.   D'Avolio A, Simiele M, Baietto L, et al. HPLC-MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long-term stability in different conditions, J. of Pharm. and Biomed. Anal., 2010, 52(5): 774-780.

11.   Myasein F, Kim E, Zhang J, et al. Rapid, simultaneous determination of lopinavir and ritonavir in human plasma by stacking protein precipitations and salting-out assisted liquid/liquid extraction, and ultrafast LC-MS/MS, Analytica Chimica Acta, 2009, 651(1): 112-116.

12.   Yadav M, Rao R, Kurani H, et al. Application of a rapid and selective method for the simultaneous determination of protease inhibitors, lopinavir and ritonavir in human plasma by UPLC-ESI-MS/MS for bioequivalence study in Indian subjects, J. of Pharm. and Biomed. Anal., 2009, 49(4): 1115-1122.

13.   International Conference on Harmonization Guidance for Industry, In: Q2A Text on Validation of Analytical Methods, Switzerland, IFPMIA, 1994, 1–4.

14.   International Conference on Harmonization Guidance for Industry, In: Q2B Text on Validation of Analytical Methods, Switzerland, IFPMIA, 1996, 1–8.

 

 

 

Received on 15.11.2020                Modified on 24.05.2021

Accepted on 19.08.2021               © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1696-1700.

DOI: 10.52711/0974-360X.2022.00284