A Review on Method Development and Validation of Anti-Viral Drug Using Spectroscopy and Chromatographic Techniques

Monika Gollapalli, Tholichukka Vinay Kumar, Fathima Muskan, Suchithra Rathod, Vishwanath Akshitha, Munnuru Prashanth,

doi:10.5281/zenodo.14786478

Review Paper | Open Access
Volume 03 | Issue 02 | Article Id IJPS/250301344

A Review on Method Development and Validation of Anti-Viral Drug Using Spectroscopy and Chromatographic Techniques
Monika Gollapalli* Tholichukka Vinay Kumar Fathima Muskan Suchithra Rathod Vishwanath Akshitha Munnuru Prashanth
Department of Pharmaceutical Analysis, Pulla Reddy Institute of Pharmacy, Dundigal, Hyderabad, Telangana, India, 502313.

Abstract

This review aims to provide a simple and accurate overview of method development and validation of Anti-Viral drug using Spectroscopic and Chromatographic methods. Ritonavir is an Anti-Retroviral drug used in treatment of HIV/AIDS. Ritonavir alone and in combination is used with many Anti-Retro viral drugs. Methods: This article deals with the study of different analytical techniques such as chromatographic, spectroscopy and their applicability in Analysis of Ritonavir. RP-HPLC is a power full analytical technique for quality assessment of Anti-Viral drug, and it is utilized to design and validate the development parameters such as accuracy, precision, linearity, LOQ, LOD etc. Ultra-Voilet spectroscopy is a physical technique of the optical spectroscopy that uses light in the visible, Ultra-Violet and Near infrared range and is based on Beer’s lambert law. HPLC is a physical separation technique conducted in the liquid phase in which a sample is separated into its constituent components by distributing between the mobile phase and a stationary phase Various method and validations are as per ICH guidelines.

Keywords

Ritonavir, Ultra-Violet Spectroscopy, Chromatography, Spectroscopy, Validation, Method Development.

Introduction

RITONAVIR is an anti-retroviral agent (HIV protease inhibitor); chemically, it is 1,3-thiazole-5-ylmethyl N[(2S,3S,5S)-3-Hydroxy-5-[(2S)-3-Methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazole-4-yl]methyl})carbomyl]amino}-1,6-diphenylhexan-2-yl]carbamate, having molecular formulaC₃₇H₄₈N₆O₅S₂, molecular weight 720.944^[1].It is official in the Indian Pharmacopoeia and the united states pharmacopoeia. Ritonavir is used to treat HIV infection and AIDS. Ritonavir is frequently prescribed with highly active antiretroviral therapy, not for its antiretroviral action, but as it inhibits the same host enzyme that metabolizes other protease inhibitors ^[2]. Ritonavir is extensively metabolized in the liver principally by cytochrome P450 isoenzymes CYP3A and CYP2D6. The major metabolite has anti-viral activity, but concentration in plasma is low, and Ritonavir is excreted in the feaces with a half-life of 3 to 5 hours ^[3]. Ritonavir is freely soluble in methanol and ethanol, isopropanol, and practically insoluble in water ^[4].The mechanism of action of antiviral drugs consists of their transformation to triphosphate following the viral DNA synthesis inhibition, and analysis of the mechanism of action of known antiviral drugs concluded that they can increase the cell’s resistance to a virus (interferons), suppress the virus adsorption in the cell, and its deproteinization process in the cell along with its anti-metabolites that causes the inhibition of nucleic acids synthesis ^[5].The following is the structure of ritonavir.

Fig.1: Chemical Structure Ritonavir

Instrumentation:

HPLC:

High performance liquid chromatography, also known as high pressure liquid chromatography commonly used to separate, identify, quantify each element of a mixture. HPLC is a sophisticated column liquid chromatography technology in HPLC process the solvent is pushed under high pressure of up to 400 atmosphere so that sample can be separated based on difference in relative affinities and HPLC generally comprises a column, that contains packing material (stationary phase), a pump that drives the mobile phase through column and detectors that detect retention time

Table 1: Reported methods to validate active pharmaceutical ingredients as anti-viral components using HPLC.

S. No	Drug	Method		Description		Ref
1	Ritonavir (human plasma)	RP-HPLC		Mobile phase - Sodium Acetate, pH 4.8: Acetonitrile (55:45v/v) Column - C8 (250 mm X 4.6 mm, 5 ?m) Flow Rate - 1.5 ml/min ? max - 212 nm		Abhinandana.Patachala et al.....^[7]
2	Ritonavir	RP-HPLC		Mobile phase - Methanol Acetonitrile (20:80) Column-symmetry C₁₈(250mm×4.6mm,5µm) Flow rate - 1.0ml/min ? max - 210nm		BAJE, S.I., Jyothi, B., et al....^[8]
1	Ritonavir		RP-HPLC		Mobile phase-Acetonitrile: 0.1% OPA (pH 3.5), 80:20 Column-C18 (250mm X 4.6mm) Flow rate -1ml/min ? max - 217nm	Archana B. Chavhan* VSB. 2023..^[17]
12	Ritonavir		RP-HPLC		Mobile phase-Acetonitrile: 0.1M acetate buffer (60:40) v/v pH 4.5 Column - Eclipse C18- 100mm x 2.5mm Flow rate -1ml/min ? max- 250 nm	Rathnasamy R, et al....2018..^[18]
13	Ritonavir		RP-HPLC		Mobile phase-Acetonitrile:Buffer (50:50) Column- SymmetryC18 (4.6 x 100mm, 3.5 ?m) pH -4 flow rate -1ml/min. ? max - 239 nm	Chiranjeevi K et al...2011..^[19]
14	Ritonavir		RP-HPLC		Mobile phase - orthophosphoric acid pH-3 and methanol (40:60) Column -Hyper sill C18 (250 mm×4.6 mm I .d.) 5µm Flow rate - 1.2ml/min ? max - 273nm	Ayeen FQ et al...2019..^[20]
15	Ritonavir		RP-HPLC		Mobile phase - Acetonitrile: water (80:20) pH 3 Column-RP-Purosnosphere C18 Flowrate-1ml/min ? max – 273nm	Yadav Choudary R, [etal]...2022.. ^[21]

U.V Spectroscopy:

Ultra violet spectroscopy is a physical technique of optical spectroscopy that uses light in the visible, ultraviolet, and near infrared ranges and it is based on beer lambert law states that absorbance of a solution is directly proportional to t5he concentration of the absorbing species in the solution and path length.^[22]

Table 2: Different parameters data of Ritonavir by UV method

S.NO	Name of the drug	( ? max )	Linearity range	LOQ	LOD	Ref.
1	Ritonavir	242 nm	10-20 µg/ml	3.1	1.1	23
2	Ritonavir	239 nm 210 nm	10-50 µg/ml	27.6	1.7	24
3	Ritonavir	246 nm	5-30 µg/ml	1.414	4.2	25
4	Ritonavir	217 nm	0-20 µg/ml	5.10	4.6	26
5	Ritonavir	239.4 nm	10-60 µg/ml	2.485	7.5324	27
6	Ritonavir	255 nm	32.210 µg/ml	1.502	0.495	28

HPTLC Method:

High performance thin layer liquid chromatography has been established for determination of Ritonavir in bulk and pharmaceutical formulation. The method showed good recovery in range 98.00-101.11% for ritonavir. The detailed account on development and validation of HPTLC methods for combination of ritonavir with lopinavir is depicted in.^[29]

Table :3 HPTLC method for Ritonavir

Sr. No	Name of the drug	Formulation	Stationary Phase Plates	Mobile phase composition	Detection (nm)	Linearity (ng/band)	Rf	Ref
1.	RTV	Tablet	Silica gel 60 F 254	Toluene: Methanol: Ethyl acetate: Glacial Acetic acid` (7:0.5:0.2:0.5 v/v/v/v)	263	RT -200 -1000	RTV -0.73	30
2.	RTV+ LPV	Capsule	Silica gel 60 F 254	Toluene:Methanol: Ethyl acetate: Glacial Acetic acid (7:0.5:0.2:0.5 v/v/v/v)	263	RTV– 160 – 500 LPV– 660 -2000	RTV – 0.39 LPV – 0.33	31
3.	RTV+ LPV	Tablet	Silica gel 60 F 254	Ethylacetate: ethanol:toluene: diethylamine (7:2.0:0.5:0.5,v/v/v/v)	266	RTV–200 – 1000 LPV-800-2000	RTV-0.41 LPV-0.62	32
4.	RTV+ LPV	Tablet	Silica gel 60 F 254	Toluene:ethyl acetate: methanol:glacial acetic acid 6.5:2.5:0.5:0.5 (v/v/v/v)	266	RTV-400- 2000LPV- 1600-8000	RTV- 0.24 LPV-0.41	33
5.	RTV+ LPV	Tablet	Silica gel 60 F 254	Chloroform: 1, 4 - Dioxane (7:3 v/v)	210	RTV- 40 240 LPV 160-960	RTV-0.78 LP V-0.74	34

Method Development:

Sample preparation
Method optimization
Method validation

Sample preparation: The analyst must complete the sample preparation process as part of the production process. For each analysis technique used for a specific in process sample or dosage type for subsequent HPLC analysis, the sample preparation method should be properly defined. The manufacturer, filter type, and pore size of filter media must be calculated for analytical process.^[35]

Method optimization: The majority of optimization in the development of HPLC methods have focused on optimizing HPLC conditions. As it is necessary to consider the composition of stationary phase and mobile phase, when optimizing liquid chromatography (LC) technique many elements of mobile phase that determine acidity, solvent, gradient, flow rate, solvent type are the main variables.^[36]

Method validation: analytical method validation is a process that meets the requirements of the procedure for its intended use. The validation of analytical method is done as per ICH guidelines.^[37]

Components of method validation

Accuracy: The accuracy of the method id proximity between the true quantity and its test result. The value of method recovery reflect the accuracy of the procedures, and this was done by spiking the active drug to the placebo at three different concentration (5,10and 20µgml^-1)^[38]

Precision: The precision of an analytical procedures expresses the closeness of agreement, precision may be considered at three levels repeatability, intermediate precision and reproducibility.^[39]
Linearity: The linearity of an analytical procedures is its ability to obtain test result which is directly proportional to the concentration of analyte of a sample.^[39]
Range: “RANGE” refers to permissible range of values for a parameters or variables, verifying that a given value is inside a predetermined range of acceptable values is known as range validation.^[40]
Limit of quantitation: Limit of quantitation (LOQ) refers to the minimum concentration of a substance that can be detected and quantified with precision using a particular analytical method.^[40]
Limit of detection: Limit of detection (LOD) it indicates the capacity of measurement method to detect minuscule amount of a substance in a sample and its sensitivity.^[40]

CONCLUSION:

This review provides a comprehensive overview on method development and validation of antiviral drugs using spectroscopy and Chromatographic techniques. By this, we conclude , these methods contribute significantly to ensure the quality, safety and efficacy of antiviral drugs.
UV method is used for simple assay, rapid screening, and cost effectiveness
The HPTLC is only limited to non-volatiles or semi volatile compounds and gives lower resolution and sensitivity compared to HPLC.
So, HPLC methods are more helpful for more sensitivity and accuracy which are widely accepted by regulatory agencies such as the FDA for method development and validation.

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