INTRODUCTION:

Lopinavir is chemically known as (2S)-N- [(2S, 4S, 5S)-5- [2-(2,6dimethylphenoxy) acetamido] 4-hydroxy-1, 6-diphenylhexan -2-yl] –3-methyl-2-(2-oxo-1, 3-diazinan-1-yl) butanamide and its empirical formula is C₃₇H₄₈N₄O₅, with a molecular weight of 628.80. Lopinavir inhibits the HIV, viral protease enzyme. This prevents cleavage of the gagpolpolyprotein and therefore, improper viral assembly results. Ritonavir is (5S, 8S, 10S, 11S)-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3, 6-dioxo-8, 11-bis (phenyl methyl)-2, 4, 7, 12-tetraazatridecan-13-oic acid 5-thiazolyl methyl ester¹. Lopinavir (LPV) is a protease inhibitor that has been co-formulated with a low dose of Ritonavir (RTV) to improve its pharmacokinetic properties, resulting in substantially increased plasma exposure that maintains high drug levels throughout a 12-h dosing interval^2-4.

Past studies revealed about analytical techniques like ultraviolet spectroscopic technique(UV)^5-7High performance thin layer chromatography (HPTLC), High performance liquid chromatography (HPLC)^8-10 LC-MS for simultaneous determination of Lopinavir and Ritonavir in pharmaceutical dosage forms and biological fluids^11-13 are reported. However, no references are reported so far for the stability indicating simultaneous determination of said drugs by HPLC method. So it was initiated to validate and develop rapid precise and simple stability indicating High performance liquid chromatography (HPLC) method for simultaneous estimation of Lopinavir and Ritonavir in combined dosage form. Few reports were published concerning the degradation behavior of RTV and its forced degradation products, of which, recent report used Stability-indicating HPLC and HPTLC method and LC-MS-MS.

(a)

(b)

Fig. 1: Structure of a) Lopinavir b) Ritonavir

MATERIALS AND METHODS:

Chemicals and Reagents:

Lopinavir and Ritonavir were obtained as gift samples from Mylan Laboratories Ltd., Hyderabad. Methanol AR grade, HCl, NaOH and hydrogen peroxide were procured from SD Fine Chem limited, Mumbai. HPLC water and Acetonitrile were purchased from Merck specialties private limited, Mumbai. The combination tablets RITOCOM 30S (Hetero Health Care Ltd.), 20 were purchased from local pharmacy.

Instrumentation:

Axis Ag N 204-PO digital balance, Elico LI 120 pH meter, 1.5LH Ultrasonic bath sonicator, LAB INDIA 3000⁺ double beam UV-Visible spectrophotometer, Agilent 1120 compact LC system with Kromasil 100-5C18 column (250 x 4.6mm) using Ezchrome Elite Compact software.

Selection of Mobile Phase:

The optimal composition of mobile phase was obtained in the ratio of Phosphate buffer (pH 3): Acetonitrile (30:70).

Method Development:

Preparation of Mobile Phase:

Mobile phase was prepared by mixing Phosphate buffer (pH 3): Acetonitrile (30:70) and filtered through 0.45μm Millipore membrane filter and degassed for 15 min before use.

Preparation of Standard Stock Solution:

The separate stock solutions of Lopinavir and Ritonavir were prepared by accurately weighing 25mg each into a separate 25ml volumetric flasks A and B and made up to the volume with mobile phase to get 1000µg/ml respectively. The stock solution was filtered through 0.45mm Millipore membrane filter, sonicated and degassed.

Selection of Analytical Wavelength:

Each solution was scanned using double beam UV visible spectrophotometer between the range of 200 – 400nm and their spectra were recorded. Lopinavir and Ritonavir 220nm was selected as analytical wavelength.

Optimized Chromatographic Conditions:

Mobile phase consisting of Phosphate buffer: Acetonitrile (30:70v/v) was used in isocratic mode. The flow rate was maintained at 1ml/min and the injection volume was 20μL. UV detection was performed at 220 nm and the separation was achieved at ambient temperature.

Method Validation:

The method was validated according to ICH Q2 B guidelines.

Linearity and Range:

The linearity of the method was determined in concentration range of 20-100µg/ml for Lopinavir and 20-100µg/ml for, Ritonavir. Each solution was injected in triplicate. Chromatogram representing linearity was shown in fig 2. The slope, intercept was reported as required by ICH which were given in table 1.

Fig. 2: Optimized RP-HPLC Chromatogram

Accuracy:

The results of recovery studies by proposed method were validated by statistical evaluation and were given in table 1.

Precision:

The precision of an analytical method was studied by performing intraday and inter day precision. It was determined by analyzing a set of six combined standard solutions and the results were given in table 1.

LOD and LOQ:

The LOD and LOQ values were determined by the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S the results were given in table 1.

Robustness:

The solution containing 100μg/ml of Lopinavir and 100 μg/ml of Ritonavir was injected into sample injector of HPLC three times under different parameters like deliberate variations in flow rate (±0.2ml/min) and detection wavelength (± 2nm). Change in flow rate and the results were given in table 1.

Ruggedness:

The solution containing 100μg/ml of both the drugs was injected into HPLC three times under different parameters like different analysts. The results were given in table 1.

System Suitability:

The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from six replicate injections. The tailing factor (T) and no. of theoretical plates (N) obtained were shown in results were given in table 2.

Table 1: Validation parameters for Lopinavir and Ritonavir

Parameter	Results
Parameter	Lopinavir	Ritonavir
R_t (min)	2.1	4.01
Beer’s Law Range (µg/ml)	20-100	20-100
LOD (µg/ml)	0.316	0.433
LOQ (µg/ml)	0.949	1.28
Assay (% purity) w/w	99.48	98.7
Precision (%RSD)
Intraday Precision	0.14	0.87
Interday Precision	0.93	1.16
Robustness (%RSD)
Flow rate 1.2ml/min	0.36	0.66
Flow rate 1.7ml/min	0.31	0.38
Detection wavelength at 218nm	0.38	0.66
Detection wavelength at 222nm	0.56	0.38
Ruggedness (%RSD)
Analyst 1	0.62	0.41
Analyst 2	0.62	0.62

Table 2: Specificity parameters for Lopinavir and Ritonavir

Parameters	Lopinavir	Ritonavir
Retention Time (min)	2.1	4.01
Resolution (R_s)	2
Tailing Factor (T)	1.2	1.4
Theoretical Plates (N)	11456	10366

Stability studies:

The stability studies were performed as per the ICH guidelines. The samples were stressed under the influence of acid (1N HCl), base (1N NaOH), peroxide (3% Hydrogen peroxide), UV (220nm) and thermal (60^oC) stress for 48 hours. The base stress sample chromatogram was shown in figure 3.

Fig. 3: RP-HPLC Chromatogram of Base stress sample.

RESULTS AND DISCUSSION:

The retention time for Lopinavir and Ritonavir was found to be 2.1 min and 4.01 min respectively. The linearity was observed in concentration range of 20-100µg/ml for both the drugs. Percentage recoveries of Lopinavir and Ritonavir were in the range of 99.6- 100.3 and 99.93-100.8 respectively. Thus the developed method was specific to Lopinavir and Ritonavir respectively.

The robustness of an analytical method was determined by analysis and these values with low %RSD (<2) indicated that the method was quite robust. Ruggedness of the proposed method was determined by analysis and the %RSD reported was found to be less than 2.

The proposed method was validated in accordance with ICH parameters and was applied for analysis of the same in marketed formulations. The content of each component in the formulation was estimated by comparing the peak area of the test sample with that of the peak area of the standard and the results were found to be 99.48% w/w for Lopinavir and 98.7% w/w for Ritonavir respectively.

CONCLUSION:

The developed stability indicating RP-HPLC method was validated according to ICH guidelines and could be used in routine analysis of Lopinavir and Ritonavir in their single and combined dosage forms. The method was economical due to less use of mobile phase and less run time. This may allow the analysis of a large number of samples in a short span of time. This method included the stability analysis of samples, where any degradation of products could be easily detected. Hence above method can be used in quality control for routine analysis of finished products of Lopinavir and Ritonavir simultaneously without any interference.

ACKNOWLEDGEMENTS:

The authors are thankful to AG & SG Degree College and SV University, Tirupati for helping in the completion of project.

REFERENCES:

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Received on 09.11.2020 Modified on 23.04.2021

Research J. Pharm.and Tech 2022; 15(2):661-664.

DOI: 10.52711/0974-360X.2022.00109