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 methods^1–3, HPTLC method⁴, HPLC methods^5-8 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	40ºC
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
S. No	Linearity Level	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

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” 7^th edition CBS Publisher and Distributors, New Delhi, (1991), PP 436-439.

5. Meyer V.R. Practical High-Performance Liquid Chromatography, 4^thEd. 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

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

DOI: 10.52711/0974-360X.2022.00284

Type of degradation	Area of sample		Assay content (% w/w)
Type of degradation	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