The
SARS-CoV-2 virus is the source of the 2019 coronavirus pandemic. The majority
of those infected with this illness had mild to moderate symptoms prior to it
becoming well known. One derivative that shows antiviral activity against RNA
viruses is pyrazine carboxamide. We call it Favipiravir.
The
medication can lessen the intensity of SARS-CoV-2 symptoms in people. In
certain cases, it has also been demonstrated to shorten the length of illness.
A new antiviral medication called favipiravir (FVP) is being used in Japan to
treat a pandemic influenza infection. After numerous clinical trials to
evaluate the suitability for potential repurposing of FVR in Covid 19, it was
recommended to use FVP as a medication for the treatment of SARS-CoV2 because
the selectivity index against SARS-CoV2 was 6.46.
Degradation
of the active ingredients and verification of their acceptable safety limits
according to ICH guidelines, are of great importance (CPMP, 2007; ICH Q1A –––(R2),
2003). Thus, the stability of drug substances and pharmaceutical dosage forms
are evaluated and tested under drastic conditions by studying the effect of
different factors like heat, pH of the medium, and light to understand the
behaviour of drug molecules. Predicting the drug molecule's degradation mechanism
under different conditions, like hydrolysis, oxidation, and photolysis, can be
aided by the identification of the degradation products, which should be
followed by isolation and characterization steps. Moreover, toxicity
assessments in vitro and in vivo can be used to assess the toxicity of isolated
degradation products.
The
global outbreak of coronavirus disease (COVID-19) prompted most researchers to
work hard at creating treatment plans to slow the disease's rapid progression.
One of the possible medications for the treatment of COVID-19 infection is
favipiravir (FAV). Adenovirus, SARS Coronavirus, influenza A virus, and other
RNA viruses were effectively inhibited by FAV, a derivative of pyrazine
carboxamide.
The Fujifilm Toyama
Chemical Company was the first to report on the innovation of FAV.
Subsequently, Japan approved its use for treating influenza. FAV is
phosphoribosylated intracellularly to become FAV-RTP (favipiravir
ribofuranosyl-5′-triphosphate), the active form that can interfere with viral
replication and is recognised as a substrate by RNA-dependent RNA polymerase
(RdRp). It also acts as a competitor with purine nucleosides and suppresses the
activity of RNA polymerase. Chemically speaking,
5-fluoro-2-oxo-1H-pyrazine-3-carboxamide is known as FAV. Currently, FAV is
approved in many countries, including Japan, Italy, Bangladesh, Turkey, Egypt,
India, Russia, KSA, and UAE, and is used for mild to moderate SARS-CoV-2
infections caused by the coronavirus, or COVID-19 outbreak. The chemical
stability of novel drug molecules is crucial for the safety and effectiveness
of the drug product; it helps choose the right formulation and packaging and
offers ideal shelf life and storage conditions. Drug products are forced to
degrade under extreme conditions in order to identify possible degradation
products. This process also aids in the establishment of degradation pathways
and the validation of stability-indicating techniques.
Table
1. Drug Profile
|
Drug Name
|
Favipiravir
|
|
Chemical name
|
6-fluoro-3-oxo-3,4-dihydro-2-pyrazine-carboxamide
|
|
Molecular formula
|
C5H4FN3O2
|
|
Mass
|
157.104 amu.
|
|
Chemical Structure
|
|
2.
MATERIALS AND METHOD:
2.1
Chemicals and Reagents:
The
99.9% pure favipiravir standard was supplied, along with impurities A, B, and C
that are known to be associated with it. Film-coated tablets containing
favipiravir (sample sold). For this study, a more compatible buffer was chosen.
Phosphate dihydrogen potassium. Using acetonitrile and orthophosphoric acid,
Halite Forced degradation studies were conducted in order to determine the
primary compound behaviour in various stress reagents, including acid, base,
and peroxide solution. We bought peroxide solution, sodium hydroxide (NaOH)
pellets, and concentrated hydrochloric acid (HCl) solution.
2.2
Equipment and software:
An
RP-HPLC was used for the development and analysis of the liquid chromatographic
method. Development and analysis of liquid chromatographic methods were carried
out on an RP-HPLC fitted with a photodiode array (PDA) detector and a
quaternary pump. Empower-3 software was used for both data processing and
acquisition. An XP4002S precision balance, an AX205 Delta Range analytical
balance, an XP205 Delta Range analytical balance, or an MX5 microbalance were
used for the weighing. A pH metre made by SevenMulti was used to measure the
pH.
Table 2. Comparison of
existing and proposed methods:
|
|
Sample
name/details
|
Mobile
phase/pump mode
|
Column
|
Observations/disadvantages
|
|
1
|
Quantification of favipiravir as COVID-19 management
in spiked human plasma
|
Methanol/acetonitrile/20
mM phosphate buffer pH 3.1 (30:10:60 v/v/v), isocratic mode.
|
Symmetry
C18-(250 4.6 mm, 5 μm)
|
1.
This method did not explain the degradation study 2. It is helpful for
bioanalytical samples, not for the finished product and API samples
|
|
2
|
Quantification of favipiravir in human plasma:
application to a bioequivalence study
|
mobile
phase A: 10 mM ammonium formate + 0.1% formic acid, B: methanol/gradient mode
|
Acquity
UPLC HSS C18 (100 2.1 mm, 1.8 μm)
|
3.
This method did not explain the degradation study 4. It is helpful for
bioanalytical samples, not for the finished product and API samples
|
|
3
|
Quantification of COVID-19 drug favipiravir by a
two-dimensional isotope dilution LC-MS/MS method in human serum
|
Mobile
phase A: water, B: acetonitrile: formic acid (99.9:0.01, v/v); gradient mode
|
HP
column (30 2.1 mm, 20 M, Waters) online solid-phase extraction
|
5.
This technical method is helpful for bioanalytical samples of seven
repurposed COVID-19 drugs 6. It is not helpful for finished product and API
samples
|
|
4
|
This method has been developed for green micellar
solvent-free HPLC and spectrofluorimetric determination of favipiravir as one
of COVID-19 antiviral regimen
|
The
mobile phase consisting of 0.02 M Brij35, 0.15 M Sodium Dodecane Sulfonate,
and 0.02 M disodium hydrogen phosphate adjusted to pH 5.0, isocratic mode
|
VDSpher
150 C18-E column (5 μm, 250 4.6 mm
|
.
This method has been developed for green micellar solvent-free 8. It is not a
useful method for finished product analysis
|
|
5
|
Development and validation of a sensitive, fast, and
simple LC-MS/MS method for the quantitation of favipiravir in human serum
|
Mobile
phase A: 0.1% formic acid in water, B: 0.1% formic acid in methanol; gradient
mod
|
Phenomenex
C18 column (50 4.6 mm, 5 μm, 100 Å)
|
9.
This was developed for human plasma, not for finished products
|
2.3
Chromatographic conditions:
A
suitable stationary phase (Inert sustain AQ-C18, 250 4.6 mm, 5-μm particle) was
used to achieve chromatographic separation, and the gradient programme was set
as time/%B: 0.0/0, 40/30, 60/55, 62/00, and 70/00. Phosphate buffer (pH 2.5)
and acetonitrile were combined in mobile phase A in a ratio of 98:2 (v/v),
while acetonitrile and water were combined in mobile phase B in a ratio of
50:50 (v/v). The injection volume was 20 μL, the flow rate was 0.7 mL/min, the
column temperature was 33°C, and the UV detection wavelength was 210 nm.
Acetonitrile and water were combined in a ratio of 98:2 (v/v) to create the
diluent. In these circumstances, all impurities were well separated.
2.4
LC-MS conditions
A
triple quadrupole mass spectrometer Waters TQD was used for the LC–MS
investigations. The capillary temperature was maintained at 400C and the source
voltage at 5000 V. The mass range in positive ionisation mode was maintained at
m/z 90–500. In mobile phase B, acetonitrile and water were combined in a 1:1
(v/v) ratio, while in mobile phase A, 0.01 M ammonium acetate (pH 2.5) and
acetonitrile were combined in a 98:2 (v/v) ratio.
2.5
Standard solution preparation (0.5%, with respect to sample concentration)
5
mL of this solution was transferred into a 100-mL volumetric flask and made up
to volume with the diluent. Favipiravir standard 60 mg was transferred into a
200-mL volumetric flask, to which 120 mL of the diluent was added and sonicated
for a few minutes. 5 mL of this solution were put into a 50 mL volumetric flask
for the final concentration (0.5%), and the remaining volume was diluted with
the diluent.
2.6
Impurity stock solution preparation
After
precisely weighing about 25 mg of impurities A, B, and C, 120 mL of the diluent
was added, the flask was sonicated for a short while, and the diluent was used
to bring the mixture up to volume. Five millilitres of this solution were put
into a 200 millilitre volumetric flask and diluted further with the diluent to
reach volume.
2.7
Sample solution preparation (0.3 mg/mL concentration)
A
250 mL volumetric flask was filled with a prepared 0.3-mg/mL concentration of
the sample solution, such as 750 mg of favipiravir-equivalent sample powder
(1031 mg). A suitable volume of the diluent (120 mL) was then added, sonicated
for 20 minutes, and the residual volume was diluted with the diluent. The
sample solution was then passed through a 0.45-micron filter for filtering.
Five millilitres of the filtered sample solution were put into a fifty
millilitre volumetric flask and diluted further with diluent until the flask
was filled to the brim.
2.8
Placebo solution preparation (without favipiravir API)
A
250 mL volumetric flask was filled with approximately 281.25 mg of favipiravir
placebo powder. A suitable volume of diluent (120 mL) was then added, sonicated
for 20 minutes, and the remaining volume was diluted with the diluent. The
sample solution was then passed through a 0.45-micron filter for filtering.
Five millilitres of the filtered sample solution were put into a fifty millilitre
volumetric flask and diluted further with the diluent until the flask was
filled to the brim.
3.
Method development and optimization
Using
the LC system, the associated contaminants associated with favipiravir were
found in the completed dosage form. But first, we gathered more important
details about the compound from the literature: its pKa of 5.1, its solubility
polarity (in water: 8.7 μg/mL), its melting point (187–193C), whether or not it
is hygroscopic, and its polymorphism (it exists in two forms). The technique of
reversed-phase chromatography was chosen based on the characteristics of the
compound. The mobile phase has a big impact on LC development. Based on the pKa
value of the compound, its pH 2.5 was optimised. Compound polarity is typically
a crucial component of the stationary phase; accordingly, a suitable stationary
phase (C18) was selected based on the compound's polarity. The LC–MS technique
was used to identify the mass value of two unknown degradation impurities found
during forced degradation studies. The liquid chromatography with mass
spectrometer used with Electrospray positive ionization source with required
nebulization gas, drying gas with source temperature. The mass of degradation
impurities was identified in MS Scan mode, and the structures of major
degradation impurities were identified. The proposed method was validated per
the current ICH guidelines (ICH Q2 (R1), 2005).
4.
Analytical method validation:
4.1
Specificity
The
finalisation of the method, which can detect interference at the retention time
of known and degradation impurities, is greatly influenced by the specificity
parameter. Verification samples, such as blank, placebo, test, and all
impurities-spiked sample solutions, were injected in the current LC method in
order to identify the interference test. After reviewing all of the
chromatography data, it was discovered that the peaks of favipiravir and known
impurities were spectrally pure, and no interference was seen at the retention
time of known or degradation impurities.
4.2
Precision
The
precision of the current method was proven by its ruggedness and repeatability.
The reproducibility results of the target impurities could be found with this
experiment. In order to assess repeatability, six recently made drug product
sample solutions with 0.3 μg/mL of every known impurity were injected on the
same day, and the subjects' recoveries were tracked. Six recently made sample
solutions with the same concentration of each known impurity were injected on
different days, using various LC instruments, and by different scientists in
order to determine ruggedness. The overall percentage relative standard
deviation (RSD) values for each <3% attest to the good precision and
suitability of the developed test method for various laboratory conditions.
4.3
Accuracy
The
standard addition method was used to test this study, which involved spiking
known impurities at 50, 100, and 150% of the specification concentrations, or
0.09, 0.15, 0.3, and 0.45 μg/mL, with the corresponding test concentrations.
4.4
Linearity
This
experiment demonstrates the method's ability, which varies from low to high
levels. As a result, injections of all known impurities (impurities A, B, and
C) were made at various concentrations ranging from the specification's 200%
(0.60 μg/mL) to LOQ (0.09 μg/mL).
4.5
Determination of LOD and LOQ
This
work is crucial to the development of the related substances method, which will
allow for the identification of the sensitivity method. The signal-to-noise
ratio method was used to calculate the limit of detection (LOD) and LOQ. The
impurity standard stock solution was used to prepare the LOQ and LOD solutions.
To get a limit of quantification (LOQ), the concentrations of each impurity solution
were gradually lowered during the procedure. The LOQ solution was diluted three
times, and the LOD solution was then injected into the HPLC apparatus. LOD was
computed using the formula LOD = LOQ/3.3. It's good that the experimental and
theoretical LOD values are comparable.
4.6
Robustness
This
research may attain method sensitivity. Intentional adjustments to the mobile
phase pH, column temperature, and flow rate were used to evaluate the optimised
test procedure.
5.
Forced degradation studies
This
work could reveal the molecular behaviour in the various stress reagents and is
important in determining the stability-indicating method. This study is a
prerequisite for any LC method to be reviewed by regulatory bodies such as the
European Union, the US Food and Drug Administration, and others. Studies on
forced degradation attest to the method's stability-indicating characteristics.
Prior to the experiment, we used regulatory guidelines to gather data on the
solubility and polarity of the molecules as well as the concentrations and
conditions of some stress reagents.
·
Acid hydrolysis degradation sample
·
Base hydrolysis degradation sample
·
Peroxide degradation sample
·
Water hydrolysis degradation sample
·
Solid-state degradation studies
·
Thermal degradation sample
·
Humidity degradation Sample
·
UV light degradation sample
6.
CONCLUSION
To
ascertain favipiravir degradation and pinpoint contaminants in the tablet
dosage form, an extremely sensitive, precise, linear, specific, and robust
analytical method was created and verified. Using the LC–MS method, the mass
values of degradation impurities were determined.
As per the most recent ICH guidelines, the
suggested approach has been validated. Three unidentified degradation
impurities were found in the forced degradation studies when water, oxidation,
acid, and base were present. It is therefore susceptible to chemical stress
situations.
As
per the findings of the solid-state degradation study, there was no
degradation. It was discovered that the molecule was stable in UV light,
humidity, and heat. This method is generally able to estimate the amount of
favipiravir impurities in the drug substances and finished dosage forms, based
on method validation results. In the current pandemic scenario, this medication
is more beneficial for treating SARS-CoV-2 patients. Consequently, this
research work adds more to the development of an effective favipiravir drug
product in the current pandemic situation. Therefore, this approach can be
applied to high-quality products. The current quality control analysis method
is easy to use and reasonably priced.
7. ACKNOWLEDGMENT
Authors
are thankful to Principal and Management J.I.I.U' S Ali Allana College of
Pharmacy Akkalkuwa, Dist. Nandurbar for providing moral support and necessary
facilities for completion of this work.
8.
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