Advances in Sample Preparation 6 (2023) 100058

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Advances in Sample Preparation

journal homepage: www.elsevier.com/locate/sampre

Fabric phase sorptive extraction-gas chromatography-mass spectrometry

for the determination of favipiravir in biological and forensic samples
Rajeev Jain a,1,∗, Bharti Jain a,b,1, Abuzar Kabir c,∗, Atul Bajaj a, Ratnasekhar Ch d,
Shweta Sharma b,∗
a
Central Forensic Science Laboratory, Dakshin Marg, Sector – 36A, Chandigarh 160036, India
b
Institute of Forensic Science & Criminology, Panjab University, Chandigarh 160014, India
c
Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA
d
Metabolomics Lab, CSIR—Central Institute of Medicinal & Aromatic Plants (CIMAP), Lucknow 226015, India

a r t i c l e i n f o a b s t r a c t

Keywords: Favipiravir, a pyrazine analog, is proposed as providential antiviral agent against the COVID-19 infection dur-
Fabric phase sorptive extraction ing 2020 pandemic emergency. For the first time, a fabric phase sorptive extraction (FPSE) combined with gas
Favipiravir chromatography-mass spectrometry (GC-MS) has been developed and applied for the determination of favipi-
COVID-19
ravir (FAV) in biological samples (human plasma, blood and urine), pharmaceutical and forensic samples. The
GC-MS
method comprises of extraction of FAV by FPSE followed by its derivatization with N, O-bis (trimethylsilyl) tri-
Anti-viral drugs
Forensic chemistry fluoroacetamide (BSTFA) and GC-MS analysis. Design of experiment-based optimization was performed using
Placket-Burman Design (PBD) and Central Composite Design (CCD) for the screening of significant factors of
FPSE and their optimization, respectively. Among all tested membranes, sol-gel polyethylene glycol (PEG) has
offered the best extraction efficiency for FAV. Under optimum conditions, the proposed method was found to be
linear in the range of 0.01–10 μg mL−1 by GC-MS. The LODs and LOQs were as low as 0.001-0.0026 μg mL−1
and 0.003-0.0086 μg mL−1 , respectively by GC-MS. Intra-day and inter-day precisions were less than 5 and 10%,
respectively, showing good method precision. The proposed method has been successfully applied to detect and
quantify FAV in human urine, whole blood and plasma samples along with seized forensic samples. In addition,
the proposed method has been evaluated for its green character by ComplexGAPI index.

1. Introduction of several antiviral medications such as favipiravir, umifenovir, remde-

Coronaviruses are single-stranded RNA viruses that can infect a wide
range of species such as domestic and wild animals, birds as well as
humans [1]. Since the 1960s, six different human coronaviruses have
been found viz. OC43, 229E, NL63, and HKU1 MERS-CoV SARS-CoV.
SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a new
form of coronavirus, which causes acute respiratory problems referred
as coronavirus disease (COVID-19). The COVID-19 epidemic was dis-
covered in Wuhan (China) in December 2019, and quickly spread over
the globe within a few months [2]. It was declared a pandemic by the
World Health Organization (WHO) in March 2020. Nonetheless, the
rate of infection continues to rise to unprecedented levels [3]. Approxi-
mately 874,151 cases had been recorded as of April 1, 2020 and 43,804
people had died because of the outbreak [4]. As of June 10, 2022,
the virus had infected more than 530 million individuals and caused
more than 6.3 million fatalities worldwide [5]. Despite the approval
sivir, and tocilizumab for different infections, and ongoing research on
new options in various regions, there are currently no specific antiviral
medicines approved for the treatment of SARS-CoV-2. Lately, favipiravir
(FAV) has been added in management guidelines of several countries
including Egypt, Italy, Saudi Arabia, United Arab of Emirates (UAE),
Japan, Russia, India and Turkey as a viable therapeutic option in the
treatment regimen [6].
FAV (6-fluoro-3-oxo-3,4- dihydropyrazine -2-carboxamide), a purine
nucleoside precursor, is a pyrazine carboxamide derivative with antivi-
ral efficacy against a broad array of RNA viruses. In Japan 2014, Fujifilm
Toyama Chemical Company was the first to introduce FAV as a remedy
for influenza [7]. It is transformed into an active form via intracellular
phosphoribosylation that suppresses viral protein synthesis. It has also
been studied for the cure of deadly infections such as Ebola, Lassa, and
most lately, SARS-CoV-2. Numerous clinical studies were conducted to
determine the efficacy of FAV in the treatment of COVID-19 infections,

∗
Corresponding authors.
E-mail addresses: rajeevjaincfsl@gmail.com (R. Jain), akabir@fiu.edu (A. Kabir), 25shweta@pu.ac.in (S. Sharma).
1
Equal first authors.

https://doi.org/10.1016/j.sampre.2023.100058
Received 4 December 2022; Received in revised form 2 March 2023; Accepted 3 March 2023
2772-5820/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
R. Jain, B. Jain, A. Kabir et al.
and it was discovered that FAV increased viral clearance and improved
chest CT scans [8,9]. After ingestion of drug, it is assimilated into cells
where it is ribosylated and phosphorylated by host cellular enzymes to
form the active metabolite T-705-ribofuranosyl-5′ -triphosphate (T-705-
RTP). Then T-705-RTP incorporates into the virus RNA at low quantities,
preferentially it inhibits the transcription and replication of the enzyme
namely RNA dependent RNA polymerase (RdRp) of influenza and many
other RNA viruses [10].
In the view of above, the development of sensitive, green, high
sample throughput sustainable and affordable analytical approaches
for determining FAV, is highly essential not only for quality assessment
in pharmaceutical products but also for metabolic and pharmacokinetic
research in complex biological matrices (CBM) from both humans and
animals.
Liquid chromatography is a method of choice for analytical chemists
for the determination of FAV in different matrices and other applica-
tions, which is evident from very few reports available in the literature.
Liquid chromatographic methods for determining FAV in plasma were
established in five research papers with an emphasis on pharmacoki-
netics and pharmacodynamics studies of drug [11–15]. Another two
methods employed HPLC/UV detection method with isocratic elution
[16,17]. In another method, LC-MS/MS for the bioanalysis of FAV have
been utilized [4]. Recently cyclic voltammetry method was reported for
determination of FAV in urine and pharmaceutical products [18].
In order to establish a novel bioanalytical method, a suitable sample
preparation process is essential prior to the instrumental analysis. Tradi-
tional approaches include solid-phase extraction (SPE) and liquid-liquid
extraction (LLE). Both these methods consume excessive organic sol-
vent and require multiple steps for extraction resulting into error-prone
and time-consuming analysis [19,20]. Keeping these drawbacks in the
mind, principles of green analytical chemistry (GAC) and white analyti-
cal chemistry (WAC) were established where preference has been given
to analytical method which consumes little or zero organic solvent, gen-
erates minimum amount of waste, and leaves little or no impact on the
environment [21].
Fabric phase sorptive extraction (FPSE) developed by Kabir and Fur-
ton is a green, versatile and innovative extraction technique which al-
lows extraction of organic compounds from biological matrices without
tedious sample preparation processes. This method takes advantage of
sol-gel coating technology and does not necessitate any matrix refine-
ment or clean up. The FPSE membrane has a high absorbent capacity,
which allows for rapid extraction kinetics and pH resistance ranging
from 1 to 12 without affecting extraction yield. Additionally, FPSE has
the potential to tune the polarity or selectivity of FPSE membrane by
selecting the appropriate substrate either hydrophobic or hydrophilic
[22,23]. In FPSE, the sorbent is covalently bonded to the substrate sur-
face, which is typically a fabric substrate that provides great chemical,
solvent, and thermal stability. During extraction, FPSE membrane is di-
rectly immersed into the sample and the obtained eluant is analysed by
analytical instrument such as GC, CE, and LC. FPSE combines the su-
perior extraction mechanism of SPE and equilibrium-based extraction
mechanism of SPME in a single membrane [24].
As far as forensic chemical analysis is concerned, it mainly deals
with identification of unknown compounds in complex biological sam-
ples and seized materials routinely received in forensic laboratories. For
this purpose, GC-MS has become a most popular and gold standard an-
alytical instrument which provides combination of separation ability of
GC with the strength of MS as an identification and confirmation ap-
proach. GC-MS also provides the power of unambiguous identification
of unknown compounds by matching their unique mass spectra with
spectral libraries [25]. Additionally, high selectivity, good sensitivity
and high separation efficiency are some of the other benefits of GC-MS
over other analytical instruments in forensic chemistry [26]. Moreover,
the toxic solvents employed in the preparation of mobile phases in liq-
uid chromatographic (LC) analysis intensify the challenge of analyst to
change the process to one that is more environmentally friendly and sus-
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Advances in Sample Preparation 6 (2023) 100058
tainable. Therefore, switching from liquid LC to GC greatly reduces the
consumption of organic solvents and significantly contributes towards
GAC approaches [27].
However, polar analytes need to be chemically derivatized before
GC-MS analysis to enhance their detectability, volatility and minimise
polarity as well as avoid peak tailing. The most prevailing approach of
derivatizing polar analytes is silylation, which involves replacing the
active hydrogens in polar functional groups with trimethylsilyl groups
to form derivatives with a comparatively high level of thermal stability
and volatility [28]. Silylation is commonly achieved using commercially
available silylating reagent such as N, O-bis(trimethylsilyl) trifluoroac-
etamide with 1% trimethylchlorosilane (BSTFA+TMCS).
In view of the above, herein, for the first time we present a
novel combination of FPSE with GC-MS for rapid extraction and
pre-concentration of new antiviral drug FAV in complex biological
and pharmaceutical samples without any sample pre-treatment. FAV
was derivatized with BSTFA+TMCS (99:1 v/v) after FPSE and analysed
by GC-MS. Finally, the greenness of the developed procedure has
been accessed with the help of recently introduced Green Analytical
Procedure Index (GAPI).
2. Experimental
2.1. Materials and methods
Sol-gel sorbent-coated FPSE membranes were synthesized and char-
acterized in the Department of Chemistry and Biochemistry at Florida
International University, Miami, Florida, USA. Unbleached cotton fab-
ric, 100% cellulose was purchased from Joanne Fabrics (Miami,
FL, USA). HPLC grade methanol (MeOH, purity > 99%), acetoni-
trile (ACN, purity > 99%), and acetone were obtained from Thermo
fisher scientific (Waltham, MA, USA). Sodium chloride (NaCl) (pu-
rity > 99%), Poly(tetrahydrofuran) (PTHF), Phenyl triethoxysilane
(PheTES) methyltrimethoxysilane (MTMS), Carbowax 20M (CW 20M),
Poly (ethylene glycol 10,000, trifluoroacetic acid were procured from
Sigma-Aldrich (St. Louis, MO, USA). Standard of Favipiravir (FAV, pu-
rity > 99%) was purchased from Indian Pharmacopoeia Commission
(IPC, Ghaziabad, India) (lot no. IPRS/56/20). Polyethylene glycol (PEG)
was obtained from Alfa Aesar (Ward Hill, MA). Derivatizing reagent i.e.,
N, O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosi-
lane (BSTFA+TMCS) was obtained from Sigma-Aldrich, Switzerland. A
Milli-Q water purification system (Millipore, Bradford, MA, USA) was
used to produce ultrapure water. Stock solution of FAV was prepared
by exactly dissolving 200 mg of FAV in 10 mL of MeOH giving a final
concentration of 20 mg mL−1 . This stock solution was kept at ∼4°C un-
til analysis. All the chemicals and reagents used in this study were of
analytical grade unless otherwise stated.
2.2. Collection of samples
Human blood sample was obtained from the Rotary & Blood Bank
Society Resource Centre, Chandigarh (India) and plasma sample was ex-
tracted from the blood sample using centrifuge for 10 min at 5000 rpm.
Three healthy participants (2 Female and 1 male) provided their urine
samples. After collection of the biological samples, they were stored at
∼4°C until used after gradual thawing. The study has been approved by
Institutional Ethical Committe vide approval no. 1109/20. Two different
tablets of FAV were obtained from local market of Chandigarh (India)
and claimed to contain 800 and 400 mg FAV/tablet, respectively.
2.3. Preparation of real samples
Biological samples such as urine, blood and plasma were fortified
with different concentrations of FAV in the range of 0.01–10 μg mL−1 ,
homogenized by vortex agitation for 5 min and incubated at 37°C for
R. Jain, B. Jain, A. Kabir et al.
Fig. 1. Procedure of fabric phase sorptive extraction.
at least 30 min to mimic drug-protein binding under physiological con-
ditions [29]. For the purpose of method validation, these samples were
used without any pre-treatment such as deproteinization or filtration.
2.4. Fabrication of FPSE membrane
Four different sol-gel sorbent-coated FPSE membranes were pre-
pared using unbleached cotton, 100% cellulose fabric as the sub-
strate. The sol-gel sorbents include sol-gel phenyl triethoxysilane (sol-
gel PTES), sol-gel poly (ethylene glycol) 10,000 (sol-gel PEG 10,000),
sol-gel poly(tetrahydrofuran) (sol-gel PTHF) and sol-gel Carbowax 20M
(sol-gel CW20M). To prepare the sol-gel sorbent coatings on cellulose,
sol-gel solutions were prepared independently using optimized formula-
tion. The sol solution was prepared by sequential addition of 10 mL sol-
gel precursor methyltrimethoxysilane (MTMS), 10 mL methylene chlo-
ride, 10 mL acetone, 5 g polymer/precursor and 4 mL aqueous trifluo-
roacetic acid (95% in water) into a large centrifuge. After vortexing the
mixture for 3 min, centrifugation for 5 min, and sonication for 2 min,
the clear supernatant part of the sol solution was finally transferred to
a clean 3 oz. amber coloured glass reaction bottle. The cellulose fabric
substrate was kept submerged into the sol solution for 4 h to form the
sol–gel coating around the micro fibrils of the substrate.
Following the completion of the residence time of the cellulose fabric
substrate into the sol solution, the coated substrate was removed from
the sol solution and was kept in the desiccator overnight for the evapo-
ration of the solvent and the of aging of the sol–gel coating. The coated
FPSE membrane was subsequently rinsed with methylene chloride: ace-
tone (50:50; v/v) under sonication for 30 min to remove unreacted and
un-bonded residual sol-gel solution ingredients from the coated surface.
The FPSE membrane was then air dried for 1 h and stored in airtight
containers prevented accumulation of unwanted analytes from the en-
vironment.
2.5. Fabric phase sorptive extraction procedure
Initially, sol-gel PEG 10,000 coated FPSE membranes were cleaned
and activated by submerging them in the mixture of 2 mL of MeOH and
ACN (50: 50 v/v) for 5 min. This is followed by submerging the mem-
brane in 2 mL of ultrapure water for 5 min. The organic solvent mixture
diffuses quickly into the porous sol–gel coating and permeable substrate
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Advances in Sample Preparation 6 (2023) 100058
matrix, therefore no shaking, vortexing, or stirring was used. One mL of
biological samples such as whole blood, urine and plasma were diluted
up to 12 mL with ultrapure water in a 15 mL of glass vial and the pH was
adjusted to 5 with 0.1 M HCl and ionic strength was adjusted to 20 %
with the help of NaCl. Now the membrane was immersed in the sample.
Subsequently, the sample was placed on a magnetic stirrer and a Teflon
magnet was introduced into the glass vial. FAV drug was extracted for
30 min with steady stirring at 700 rpm. Thereafter, the sample was dis-
carded; membrane was taken out and immersed in 0.5 mL of MeOH in
an Eppendorf tube for 10 min to elute the target analyte without any ex-
ternal diffusion process. The FPSE extract thus obtained was evaporated
to dryness and mixed with 50 μL of pyridine and 50 μL of BSTFA+TMCS
(99:1; v/v). This mixture was heated in a hot air oven for 30 min at 70°C.
Finally, 1 μL of this mixture was injected into GC-MS for analysis. The
FPSE protocol is shown in Fig. 1. At last, the membrane was rinsed with
a 50:50 v/v blend of MeOH and ACN, dried and stored for further use.
2.6. GC-MS analysis
GC-MS analysis was carried out on Shimadzu Nexis GC – 2030 cou-
pled with QP-2020 NX mass spectrometer in which GC was equipped
with SH-Txi-5Sil MS capillary column (30 m length × 0.25 mm internal
diameter × 0.25 μm film thickness, stationary phase 5% phenyl and 95%
dimethylpolysiloxane). Injection was performed with AOC-20i auto in-
jector at 250°C of injection port temperature with a split value of 10. The
glass liner (95 mm length × 5 mm OD) used in the injection port was a
3.4 mm internal diameter split liner. Helium at a flow rate of 1 mL min−1
was used as carrier gas. Initially, the temperature of oven was kept at
90°C for 4 min and increased up to 300°C at a rate of 20°C min−1 where
it was hold for 2 min. This has resulted in a total run time of 15 min.
The temperature of MS transfer line and ion source was kept at 250°C
and 200°C, respectively. MS was operated in positive electron ionization
(+EI) mode with electron energy of 70 eV. A mass spectrum of the target
analyte was recorded in full scan mode (50–500 amu). GC-MS spectra
of underivatized and derivatized FAV is shown in Fig. 2 (a) and (b).
2.7. Multivariate optimization
Various experimental factors were studied which can affect the ex-
traction efficiency of FPSE such as extraction time, agitation speed, pH,
R. Jain, B. Jain, A. Kabir et al.
Fig. 2. Full scan mass spectra of FAV (a) Underivatized (major ions m/z 157, 114, 86) (b) Derivatized (major ions m/z 301, 286, 270, 257, 147, 73).
elution time, volume of sample, ionic strength and volume of elution sol-
vent. In order to accomplish this, multivariate analysis was performed
to study these factors in two steps: (i) Placket-Burman Design (PBD) to
screen the significant factors, and (ii) a central composite design (CCD)
with desirability profiling (DP) function for exact optimization of signif-
icant factors obtained by PBD. Statistical analysis was carried out using
the TIBCO STATISTICA software (Trial version).
2.8. Method validation
The developed analytical method was validated for its linearity, pre-
cision, sensitivity and accuracy. In order to evaluate linearity, four dif-
ferent calibration curves were produced in ultrapure water, urine, blood
and plasma samples at different concentration levels ranging from 0.01
to 10 μg mL−1 . For this purpose, a linear regression approach was uti-
lized by taking concentration of analyte on x-axis and peak area on y-
axis. Limit of detection (LOD) and limit of quantification (LOQ) were
determined by signal-to-noise ratio at 3 and 10, respectively. The eval-
uation of the relative standard deviation (%RSD) provided the basis for
determining precision. Using FPSE method, three different concentra-
tions of FAV (0.1, 1 and 10 μg mL−1 ) were examined for repeatability
(intraday precision, n=5) and for five consecutive days for reproducibil-
ity (interday precision, n=5). Moreover, the absolute and relative recov-
eries were evaluated at three different concentrations i.e., 0.1 μg mL−1
(low QC), 1 μg mL−1 (medium QC) and 10 μg mL−1 (high QC). The
matrix effect (ME) was assessed using drug-free human plasma, blood,
and urine samples obtained from three different sources. ME was as-
sessed with the following equation in accordance with SWGTOX guide-
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Advances in Sample Preparation 6 (2023) 100058
lines [30]:
Peak ar ea of extr acted sample
%ME = × 100
Peak ar ea of standar d sample
3. Result and discussion
3.1. Optimization of derivatization conditions
The molecule of FAV has one amide group and one hydroxyl group,
which result in peak tailing and decreased sensitivity. The retention time
of underivatized FAV was found to be 8.27 min (Fig. 3a). In order to
overcome this drawback, derivatization of FAV is required to enhance
its chromatographic properties and reduce polarity. Therefore, derivati-
zation of FAV was performed with most widely used silylation reagent
i.e., BSTFA with 1% TMCS. In order to obtain optimum yield of derivati-
zation, factors, which affects the derivatization process such as reaction
time and temperature; and proportion of BSTFA with pyridine, were op-
timized. The retention time of FAV-di-TMS derivative was found to be
9.96 min and no peak tailing was observed (Fig. 3b).
Initially, a set of experiments was conducted where the proportions
of BSTFA and pyridine were studied. FAV in MeOH (50 μg mL−1 ) was
evaporated to dryness and different proportion of pyridine to BSTFA
in the ratio of 30:70, 50:50 and 70:30 μL were added to it. All vials
were sealed and vortexed for one minute and heated in an oven for 60
minutes at 60°C. The resulting derivatives were diluted with 0.5 mL of
hexane and analyzed by GC-MS. From Fig. 4 (a), it is observed that max-
imum peak area of FAV-di-TMS derivative were obtained when volume
of BSTFA and pyridine 50 μL each (1:1 v/v). For further experiments,
R. Jain, B. Jain, A. Kabir et al.
Fig. 3. GC-MS chromatogram of (a) Underivatized FAV (RT 8.27 min.) (b) Derivatized FAV (RT 9.96 min.).
this ratio of BSTFA and pyridine was finalized. The decrease in the peak
area at 30 μL BSTFA is due to not enough derivatizing agent in the start-
ing reaction mixture, and having high quantities of catalyst relative to
derivatizing reagent.
Further, in order to identify the effects of reaction time and temper-
ature, a set of experiment was conducted where different derivatization
reactions were performed with variable reaction times (15, 30, 60 and
90 min). Maximum peak area for FAV-di-TMS derivative was obtained
when reaction was allowed for 30 min. Subsequently, effect of reaction
temperature was also studied on the silylation of FAV in the range of
40 – 100°C. It was observed that maximum derivatization yield was ob-
tained when reaction was allowed at 80°C for 30 min (Fig. 4b & c).
Since, derivatization of FAV results into an increase in the molecular
weight by 144 amu, therefore, it is necessary to find out the best temper-
ature for its vaporization inside the injection port of GC-MS. Different
injection-port temperatures between 150°C to 280°C were examined in
the present study. It was observed that maximum peak area was ob-
tained when injection port temperature was kept at 250°C (Fig. 4d).
Finally, at a fixed concentration (50 μg mL−1 ), sensitivity of under-
ivatized and derivatized FAV were compared. By comparing the S/N
ratios of both compounds, it was observed that sensitivity of GC-MS for
derivatized FAV was 9.2 times greater than underivatized FAV. There-
fore, derivatization not only improved the peak shape, but also resulted
into greater sensitivity for FAV.
3.2. Mass spectra of Favipiravir-di-TMS derivative
FAV (m/z 157) has two functional groups with labile hydrogen i.e.,
one amide group at ortho position and one hydroxyl group at meta po-
sition of benzene ring. When FAV is allowed to react with BSTFA (m/z
366), then one hydrogen from these functional groups is replaced with
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Advances in Sample Preparation 6 (2023) 100058
one trimethylsilyl group [-Si(CH3 )3 ] (m/z 73) resulting into formation
of FAV-di-TMS derivative (m/z 301). The molecular ion of FAV-di-TMS
derivative can be clearly seen at m/z 301 in its mass spectra. Apart from
molecular ion at m/z 301, some other major fragments are also observed
in mass spectra FAV-di-TMS derivative, which are m/z 286 and m/z 270.
The formation of fragment at m/z 286, which is also the base peak; re-
sults due to the loss of one –CH3 (m/z 15) group from m/z 301. Loss of an
oxygen atom (m/z 16) from m/z 286 results into formation of fragment
at m/z 270, whereas loss of O=C-NH2 group (m/z 44) probably due to
McLafferty rearrangement from m/z 301 results into a fragment with
m/z 257. A major fragment is observed with more than 60% of intensity
at m/z 73, which is formed due to dissociation of one TMS group from
m/z 301; whereas dissociation of both TMS groups results in a fragment
at m/z 147.
3.3. Mechanism of extraction on the substrate surface
Unlike classical extraction (e.g., solid phase extraction) and mi-
croextraction (e.g., solid phase microextraction, stir bar sorptive ex-
traction, microextraction by packed sorbent) where extraction and pre-
concentration of the analytes are carried out by the dissolution of the
analytes into the pristine, viscous polymeric coating, immobilized on
the substrate surface, extraction of the analytes in FPSE membrane is
primarily governed by the intermolecular interactions between the an-
alytes and the FPSE membrane. As such, FPSE exploits different inter-
molecular interactions such as London dispersion, hydrogen bonding,
dipole-dipole interactions, and 𝜋-𝜋 interactions. The FPSE membrane
platform can be carefully designed to maximize the intermolecular in-
teractions. Considering the high polarity of favipiravir (log Kow: -0.6)
and the presence of large number of hydrogen bond donors and ac-
ceptors in the molecule, the extraction platform should be hydrophilic
R. Jain, B. Jain, A. Kabir et al.
Fig. 4. Optimization of derivatization conditions: (a) ratio of derivatizing agent (b) effect of reaction time (c) effect of reaction temperature (d) effect of injector
port temperature.
and should possess abundant hydrogen bond acceptors and donors. As
such, 100% cellulose fabric was chosen as the substrate due to its in-
herent hydrophilic nature [31]. The sol solution composition for the
sorbent coating was also designed to maximize the intermolecular in-
teraction sites on the sol-gel polymeric network. To evaluate and ex-
ploit the best extractive platform, four distinct FPSE membranes were
synthesized including sol-gel PheTES, sol-gel PTHF, sol-gel PEG 10,000
and sol-gel CW20M. Among these polymers/precursors, poly (ethylene
glycol) 10,000 was selected as the organic polymer due to its high po-
larity and the possession of hydrogen bond donor and acceptor function
groups. As the experimental data revealed (presented in Section 3.5),
the sol-gel poly (ethylene glycol) 10,000 performed as the best extrac-
tive platform. The observation can be easily justified due to the high
polarity of the target analytes as well as the organic polymer. In addi-
tion, methyl trimethoxysilane (MTMS) was selected as the sol-gel pre-
cursor that serves as the chemical linker to connect the sol-gel sorbent
network and the fabric substrate via strong covalent bond. In addition,
MTMS provides London dispersion type of weak intermolecular inter-
action via its methyl pendant group connected to the Silicon central
atom of the molecule. Sol-gel sorbents are inherently porous and possess
sponge-like porous morphology containing mesopores and micropores
[32]. As such, analytes can easily access and interact with the functional
groups implanted into the sol-gel sorbent network and consequently are
extracted. The porous morphology of the sol-gel sorbent also facilitates
rapid diffusion of the solvent throughout its body, resulting in faster and
exhaustive elution of the extracted analytes. Fabric phase sorptive ex-
traction exploits the extraction mechanism of solid phase extraction (ex-
haustive extraction) and solid phase microextraction (equilibrium-based
extraction) due to its special planer geometry and the permeability of
the sol-gel sorbent coated FPSE membrane. During the analyte extrac-
tion, FPSE membrane behaves like an SPME fiber in its direct immersion
extraction mode. At the same time, the aqueous solution continues per-
meating through the FPSE membrane where the membrane behaves like
an SPE disk. Due to the larger through pores in FPSE membrane com-
pared to an SPE disk, the aqueous sample can permeate through the
FPSE membrane without requiring any external pressure (as in the case
of SPE). Unlike an SPE disk, the aqueous sample permeates through the
FPSE membrane many times during the extraction duration and ensures
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Advances in Sample Preparation 6 (2023) 100058
exhaustive extraction of the analytes from the aqueous sample in a rel-
atively shorter period.
3.4. Characterization of FPSE membranes
The sol-gel poly (ethylene glycol) 10,000 coated FPSE membranes
were characterized by Scanning Electron Microscopy (SEM) and Fourier
Transform Infrared Spectroscopy (FT-IR). The SEM images shed light on
the surface morphology of the fabric substrate and the coated sol-gel sor-
bent. On the other hand, FT-IR spectra provides important information
about the functional makeup of different building blocks of FPSE mem-
brane as well as the information regarding successful integration of the
building blocks in the final product and its functional composition.
3.4.1. Scanning Electron Microscopy (SEM)
Figure S1 represents the scanning electron micrographs (SEM) im-
ages of (a) uncoated cellulose fabric at 100x magnifications; (b) sol-
gel poly(ethylene glycol) 10,000 coated FPSE membrane demonstrat-
ing the thick sorbent coating on cellulose fabric at 100x magnifica-
tions; (c) sol-gel poly(ethylene glycol) 10,000 coated FPSE membrane
at 500x magnifications, demonstrating the through pores on the mem-
brane; (d) demonstration of the uniformity and surface roughness of
sol-gel poly(ethylene glycol) 10,000 coating at 5,000x magnifications.
The sol-gel poly (ethylene glycol) 10,000 coatings are uniformly dis-
tributed on the cellulose fabric substrate. The through pores of the cel-
lulose fabric remained intact even after the sol-gel sorbent coating that
allows rapid permeation of aqueous sample through the FPSE membrane
bed during the analyte extraction. Rapid permeation of the aqueous sam-
ple through the extraction bed facilitates faster extraction kinetic.
3.4.2. Fourier transform infra-red spectroscopy (FT-IR)
Fig. S2 represents the FT-IR spectra of (a) pristine poly (ethylene
glycol) 10,000 polymer, (b) sol-gel poly (ethylene glycol) 10,000 coated
cellulose FPSE membrane. FT-IR spectra of uncoated cellulose fabric and
methyl trimethoxysilane are presented in Supplementary Figs. S3(a),
and S3(b), respectively. Noteworthy features of pristine poly(ethylene
glycol) 10,000 FT-IR spectra include C-O, C-C stretching, CH2 rocking at
R. Jain, B. Jain, A. Kabir et al. Advances in Sample Preparation 6 (2023) 100058

840 cm−1 , CH2 rocking and CH2 twisting at 960 cm−1 , C-O, C-C stretch-
ing at 1096 cm−1 , C-O stretching and CH2 rocking at 1146 cm−1 , CH2
twisting at 1240 cm−1 and 1278 cm−1 , CH2 wagging at 1341 cm−1 and
CH2 scissoring at 1466 cm−1 [33]. The FT-IR spectra of uncoated cellu-
lose fabric have two distinct absorption regions: (a) absorption bands
between 3660 and 2800 cm−1 and (b) absorption bands between 1650
and 400 cm−1 . The band at 2913 cm−1 is attributed to CH stretching
vibration of all hydrocarbon constituent in polysaccharides. The absorp-
tion bands at 1431 cm−1 , 1367 cm−1 , 1314 cm−1 , 1063 cm−1 and 896
cm−1 represents stretching and bending vibrations of –CH2 , CH, -OH,
and C-O bonds in cellulose [34]. Important features of MTMS spectra
(Figure S3b) include characteristic Si-O-C-H bond at 2947 cm−1 and Si-
OCH3 bands at 2839 cm−1 and 1077 cm−1 . The bands at 1264 cm−1
and 789 cm−1 represent the vibrations of –CH3 pendant group of the
precursor. Many features of poly (ethylene glycol) 10,000 and MTMS
FT-IR spectra can be seen in the sol-gel poly (ethylene glycol) 10,000
implicating the successful integration of poly (ethylene glycol) 10,000
and MTMS into the sol-gel poly (ethylene glycol) 10,000 hybrid sorbent.

3.5. Screening of FPSE membrane type

The selection of sol-gel coated FPSE membrane is the fundamental

step in establishing FPSE method as it has a significant impact on the
selectivity of extraction process. In this work, the efficacy of four dis-
tinct sol-gel coated FPSE membrane with cellulose substrate and varied
polarities, namely sol-gel phenyl triethoxysilane (sol-gel PheTES) (po-
lar), sol-gel polyethylene glycol 10,000 (sol-gel PEG 10,000) (polar),
sol-gel polytetrahydrofuran (sol-gel PTHF) (medium polar) and sol-gel
Carbowax 20M (sol-gel CW20M) (polar) were evaluated for the opti-
mum extraction of FAV drug from pharmaceuticals and biological ma-
trices (human urine, blood and plasma). According to the findings, FAV
is best extracted in sol-gel PEG 10,000 followed by sol-gel PheTES, sol-
gel PTHF and sol-gel CW 20M, as depicted in Fig. 5.
3.6. Screening of elution solvent and elution mode
The desorption step for FPSE was optimized once the appropriate
FPSE membrane was selected. Primarily, the type of elution solvent and
elution mode were studied. Three distinct solvents, including MeOH,
EtOH and ACN as well as two solvent mixture such as MeOH: ACN and
EtOH: ACN (50:50 v/v) were explored. As can be seen from Fig. 6a,
MeOH is the best solvent to elute the target analyte from FPSE mem-
brane. Therefore, MeOH was used as the desorption solvent for all fur-
ther experiments.
In a subsequent stage, effect of stirring on elution of FAV from FPSE
membrane was evaluated. As shown in Fig. 6b comparable extraction re-
coveries were attained with and without stirring. Therefore, no stirring
was used during this stage in all subsequent experiments.
Fig. 6. Optimization for FPSE (a) Elution solvent (b) Elution mode.
3.7. Multivariate optimization
3.7.1. Plackett–Burman design (PBD)
PBD is screening design, which is applied to examine most impor-
tant factors among many potential causative factors [35]. Thus, PBD
reduces the number of factors and subsequently further experiments, as
well. Initially, PBD was applied to screen significant factors affecting the
extraction efficiency of FPSE for FVP. In this study, seven independent
factors were considered that influencing the extraction efficacy of FPSE.
The independent variables studied were: (i) volume of sample (mL) (ii)
agitation speed (rpm) (iii) extraction time (min) (iv) ionic strength (%)
(v) pH (vi) elution time (min) and (vii) desorption volume. Factors were
specified in two levels: (-1) for low level and (+1) for high level, as dis-
played in Table (1)S. A 27-4 PBD was applied for identification of signif-
icant factors. All the experiments were carried out in triplicate and in
random manner for 24 runs (7+1 = 8∗ 3 = 24). An analysis of variance
(ANOVA) test was used to analyse the significant factors. Moreover, t-
test was applied with confidence levels greater than 95% (P < 0.05) for
identification of significant factors which are depicted in Pareto chart
shown in Figure S4. In this study, elution time was found to be the most
significant factor followed by pH, ionic strength, and agitation speed.
On the other hand, desorption volume, extraction time and volume of
sample are less significant factors for extraction of FAV by FPSE. There-
fore, these four most significant factors (elution time, pH, ionic strength,
and agitation speed) have been considered for further optimization by
a Central Composite Design (CCD) of experiments.

3.7.2. Central composite design (CCD)

CCD was performed for four significant factors obtained by PBD at
their five different levels, which are summarized in Table S2. Every in-
dependent factor was evaluated on five different levels: factorial points
(-1, +1), axial points (-𝛼, +𝛼), and central point (0). The following equa-
tion is used to calculate the total number of experimental runs:

N = 2k + 2K + Cp

Where k = number of factors and Cp = number of centre points.

Fig. 5. Selection of different sol-gel sorbents. In this way, 16 factorial points (2k ), 8 axial points (2K) and 6 centre

7
R. Jain, B. Jain, A. Kabir et al.
points with 30 runs have been performed. The order of the runs was
randomised to avoid any impact of uncontrolled variables (Table S3).
The peak area of FAV has been considered as response for CCD experi-
ments. ANOVA was used for evaluating the experimental data.
Response surface methodology (RSM) along with desirability pro-
filing (DP) is the two components of CCD, which are used for finding
out an optimum range of tested parameters and their final values [36].
The three-dimensional graphs generated by RSM are known as response
surface graphs and show interaction between two factors while keep-
ing the third factor at their constant value. The RS graphs are shown
in Fig. 7, which clearly shown interaction of elution time vs pH, ionic
strength vs pH and elution time vs agitation speed, respectively. The
highest response for elution time is between 12-18 min, while the max-
imum response for pH is between 5-7. Likewise, the optimum response
for agitation speed and ionic strength were discovered to be in the range
of 600–800 rpm and 18–22%, respectively.
The exact optimum value of all these factors was provided by DP,
which is shown in Figure S5. In DP, the results obtained by RSM are
transformed into a desirability scale which ranges from completely un-
desirable (0) to fully desirable (1) response. For each independent vari-
able, a sequence of graph is produced in which a red line depicts the final
optimum value. The optimum value for pH is found to be 4.9 which can
also be explained by the pKa value of FAV i.e., 5.1, which shows that it
behaves as a weak acid in aqueous solution. The results obtained herein
are in close agreement to previous reports for FAV in similar samples
8
Advances in Sample Preparation 6 (2023) 100058
[37]. The optimum values for other factors are: 15 min (elution time),
711.97 rpm (agitation speed), and 20.38 % (ionic strength). For prac-
tical convenience, the values of agitation speed, pH of the sample and
ionic strength were rounded off to 700 rpm, 5 and 20%, respectively.
3.8. Analytical performance of the method
Under its optimized conditions, the proposed FPSE-GC-MS method
was validated for its linearity, precision, relative recovery (RR%), en-
richment factor (EF), enrichment recovery (ER%), LODs, and LOQs. Ul-
trapure water and biological matrices were spiked with target analyte
at various concentrations ranging from 0.01 to 10 μg mL−1 . A good cor-
relation between concentration and peak area was obtained with an R2
of 0.999 for all matrices by FPSE-GC-MS method. LODs and LOQs were
evaluated at signal-to-noise ratio of 3 and 10, respectively and were
found to be in the range 0.001-0.0026 μg mL−1 and 0.003-0.0086 μg
mL−1 , respectively. Intra-day and inter-day precisions (%RSD) were cal-
culated at three different concentration levels and were less than 5 &
10%, respectively as shown in Table 1. Additionally, the EF, accuracy,
and ER% were also determined and highlighted in Table 2. For the pro-
posed method, accuracy was obtained in the ranged from 86.5 – 100.1
%. Pre-concentration factor (PF) of the proposed method were found to
be 24 as initial volume of sample was 12 mL and final volume of extract
was 0.5 mL. EF was obtained as the ratio of the peak area of the FAV-di-
TMS obtained by FPSE-GC-MS method and the peak area of the standard
Fig. 7. Estimated response surface for FAV using CCD by plot-
ting (a) Elution time vs pH (b) Ionic strength vs pH (c) Extrac-
tion speed vs Elution time.
R. Jain, B. Jain, A. Kabir et al. Advances in Sample Preparation 6 (2023) 100058

Table 1
Analytical characteristics of FPSE-GC-MS method for FAV drug.

Precision (%RSD)
LOD LOQ Linearity
Calibration curve Intra-day (μg mL−1 ) Inter-day (μg mL−1 )
Sample (μg mL−1 ) (μg mL−1 ) R2 (μg mL−1 )
0.01 1 10 0.01 1 10

Pharmaceutical formulation 0.0019 0.0062 0.999 0.01-10 y = 4760x 0.37 0.28 0.12 5.5 8.78 8.9
Urine 0.001 0.003 0.999 0.01-10 y = 9520x 1.3 0.59 0.44 6.3 8.1 6.5
Plasma 0.0015 0.005 0.999 0.01-10 y = 28559x 1.5 0.73 0.51 5.4 6.0 5.5
Whole blood 0.0026 0.0086 0.999 0.01-10 y = 19041x 1.2 2.2 2.2 4.1 7.3 5.0

Table 2
Accuracy and extraction efficiency of proposed method.

Accuracy%
Drug
0.01 μg mL−1 1μg mL−1 10 μg mL−1 EF ER%

Pharmaceutical formulation 92.8 98.3 100.2 23 95.8

Urine 94.5 99.1 99.7 20 83.3
Plasma 90.4 89.3 98.3 18 75
Whole blood 86.3 96.1 98.5 17 70.8

solution. The EF for FAV-di-TMS ranges between 17 and 23 under opti-
mum conditions. On the other hand, ER % was calculated by dividing EF
by PF and multiplying with a factor of 100. This correlates to ER% find-
ings ranging from 70.8 to 95.8%. Moreover, human plasma, blood and
urine samples devoid of drugs that were collected from three different
sources were used to evaluate the matrix effect (ME). It was investigated
by comparing analyte peak area from plasma, blood and urine samples
fortified with FAV at three concentration levels covering the linearity to
samples from standard solutions at the same analyte concentrations. The
matrix effect for human urine, plasma and blood samples were obtained
in the range 0.98-1.05, 0.89-0.95 and 0.85-0.90, respectively, which are
in agreement to the previous studies for same analyte [38]. In addition,
the %RSD of <4% indicated that any ion suppression or enhancement
from the biological samples is almost minimal.
The proposed method has been compared with previously reported
analytical methods for determination of FAV in biological matrices and
the comparison has been shown in Table 3. It is evident from Table 3 that
there are few published methods for determining favipiravir in biolog-
ical matrices. This is the first ever application of GC-MS analysis for
determining FAV. The proposed FPSE-GC-MS approach has achieved
lower LODs and LOQs than previously reported methods [38,39–42].
This could be due to the low enrichment of sample preparation proce-
dures (LLE & SPE) used in these approaches for the extraction of FAV.
Additionally, in some of the reports [38,39–42] the authors used protein
precipitation approach which could have led to loss of analytes result-
ing in lower sensitives. Furthermore, the proposed method due to the
usage of the GC-MS-SIM offered better sensitivity compared to the re-
ported method in ref [43], although the both methods have used similar
sample preparation approaches.
3.9. Reusability of the FPSE membrane
The reusability of the sol-gel PEG membrane was examined to fully
assess its efficiency. For this purpose, a fixed concentration of FAV
(25 μg mL−1 ) was extracted by developed FPSE protocol and eluted in
0.5 mL of MeOH. The same membrane was again re-used to extract same
concentration. A decline in extraction efficiency of 10% in comparison
to the initial efficiency was set as the performance loss threshold. The
findings of the reusability analysis are displayed in Figure S7. It can be
observed from Fig. S6 that, extraction efficiency was slightly declined
with reusability of FPSE membrane and at 10th extraction; the recov-
eries were dipped by 10%. Therefore, the sol-gel PEG membrane can
be used at least ten times to extract FAV without significantly losing its
efficiency.
3.10. Evaluation of the green character of developed procedure
The ComplexGAPI index was utilised to assess the green character
of the proposed method. It was chosen since it enables for the evalua-
tion of the analytical technique in view of its environmental affability
[37,44–48]. Herein, each arm of the hexagon in the GAPI pictogram
corresponds to a distinct part of the process that is being described.
The corresponding guidelines are met if the hexagon is green. Fig. 8
displays the ComplexGAPI pictogram obtained for the proposed proce-

Table 3
Comparison of proposed method with previously reported method for FAV in biological matrices.

Sample Matrix Sample Pre-treatment Technique Linearity Range LOD LOQ Ref.
and Extraction Method

Spiked human plasma Protein precipitation Spectrofluorimetric 40–280 ng mL−1 9.44 ng mL−1 28.60 ng mL−1 39
method
Human serum SPE LC-MS/MS 3291–20790 μg L−1 - 3291 μg L−1 40
Human plasma LLE HPLC/UV 0.5–50 mg L−1 0.15 mg L−1 0.45 mg L−1 41
Human plasma Protein precipitation UPLC–MS/MS 0.25–16 μg mL−1 - 0.25 μg mL−1 38
Human urine - SW-AdSV 1.0-100.0 μg mL–1 0.26 μg mL–1 0.87 μg mL–1 42
Human plasma and FPSE HPLC/UV 0.5-25 μg mL−1 (breast milk) 0.06 μg mL−1 (plasma) 0.2 μg mL−1 (plasma) 43
breast milk 0.2-50 μg mL−1 (plasma) 0.15 μg mL−1 (milk) 0.5 μg mL−1 (milk)
Spiked human urine, FPSE GC-MS 0.01-10 μg mL–1 0.001 μg mL−1 (urine) 0.003 μg mL−1 (urine) Present
plasma and whole 0.0015 μg mL−1 0.005 μg mL−1 work
blood (plasma) 0.0026 μg (plasma) 0.0086 μg
mL−1 (blood) mL−1 (blood)
Abbreviations: LLE- Liquid liquid extraction; SPE- Solid phase extraction; SW-AdSV: square-wave adsorptive stripping voltammetry; GC-MS- Gas chromatography-
mass spectrometry; HPLC- high-performance liquid chromatography; LC-MS- Liquid chromatography-mass spectrometry.

9
R. Jain, B. Jain, A. Kabir et al. Advances in Sample Preparation 6 (2023) 100058

Fig. 8. Evaluating Green Character of proposed method through ComplexGAPI.

dure. Herein, red, yellow, and green pentagrams are used to symbolise
high, medium, and low impacts, respectively on the environment when
evaluating the effects of each step in the stage of analysis. The effect
of pre-analytical processes on the environment is represented by the
hexagon at the bottom of the pictogram. Since most of the pentagrams
in Fig. 8 are either yellow or green, which indicates conformity to the
GAC principles. In view of this, it can be concluded that the suggested
analytical method is sufficiently green and has no unfavourable envi-
ronmental impact. Fig. 8 shows the positions of the red pentagrams,
which were 1, 3, 5, 7, 8, and 12 and correlated to sample collection
(off-line), transportation (required), extraction (required), solvent used
(non-green, i.e., MeOH), additional treatment (advanced treatment, i.e.,
derivatization) and energy consumption (for GC‐MS, >1.5 kWh/hour),
respectively.

3.11. Application to the real samples

The proposed FPSE-GC-MS method has been effectively used to

quantify FAV in fortified biological matrices including human urine,
plasma and blood samples and pharmaceutical formulations under op-
timum conditions. The percentage of FAV relative to labelled content
(400 and 800 mg/tablet) was calculated using the validated methods.
Tables 4 and 5 shows the results of application of the proposed method
for the quantitative analysis of FAV in pharmaceutical product samples
and biological matrices, respectively. GC-MS chromatograms of blank,
pharmaceutical, spiked human urine, plasma and blood samples are
shown in Fig. 9 (a-e), respectively.

Table 4
Determination of FAV in pharmaceutical formulations.

Samples Concentration Recovery Amount of FAV

(claimed Prepared Concentration (%) found (mg)
FAV) (μg/mL) found (FPSE-GC-MS
(μg/mL) method)

800 mg 10 9.3 93 744 ± 1

400 mg 10 9.1 91 364.3 ± 1.3

Table 5
Determination of FAV in biological samples.

Samples Concentration Concentration Recovery RSD%

Prepared (μg/mL) found (μg/mL) (%)
Fig. 9. Total ion chromatogram of blank sample (a); pharmaceutical sample (b);
spiked human urine sample (c); plasma sample (d); and whole blood sample (e).
Urine 10 9.3 93 0.8
Plasma 10 9.1 91 1.2
Whole blood 10 8.7 87 1.5

10
R. Jain, B. Jain, A. Kabir et al.
Table 6
Determination of FAV in seized sample during COVID-19 Pandemic.
Samples Concentration Concentration
(claimed FAV) prepared (μg/mL) found (μg/mL)
400 mg 20 19.2
During second wave of COVID-19 in India, there was a huge demand
of FAV in the market, which resulted into hoarding, manufacturing, and
black-marketing of these tablets. At this point of time, our laboratory
received number of cases for authentication of FAV tablets. In one of
such case, the method was successfully applied for the determination of
FAV in seized tablets received in our laboratory (Table 6).
4. Conclusion
For the first time a novel FPSE–GC–MS technique was developed and
validated for the extraction of FAV from biological matrices and pharma-
ceutical formulations. Fast interaction between the analytes and sorbent
was made possible by the innovative microextraction device with flexi-
ble support, high sorbent loading, and large surface area [49]. This led
to a quick extraction equilibrium time and outstanding recoveries for the
target analyte. Protein precipitation, a potential error-prone step, was
eliminated from the sample preparation procedure by FPSE. The pos-
sibilities of analyte loss along with sources of error are reduced by the
removal of specific sample preparation procedures such as solvent evap-
oration and sample reconstitution in FPSE. Any organic solvent may be
used as the eluant for analyte back-extraction due to the strong chemi-
cal bond that exists between the sorbent and the substrate, which gives
the FPSE membrane great chemical and solvent stability. For the extrac-
tion of FAV from biological matrices and pharmaceutical formulations,
the use of sol–gel short-chain PEG coated FPSE media showed to be an
easy, highly effective, and quick sample preparation method. On top
of that, the validated procedures were evaluated using green metrics
(GAPI complex tool) and were discovered to be easier, more sensitive,
and more environmentally friendly. In conclusion, the current approach
can be routinely used for FAV analysis in clinical and forensic toxico-
logical laboratories as well as therapeutic drug monitoring applications.
This method will also pave the path for the establishment of novel an-
alytical methods for polar analyte employing the FPSE-GC-MS module.
On the other hand, in forensic analysis unambiguous identification of
drugs are of utmost importance, for which GC-MS has been emerged as
a globally acknowledged analytical technique. However, analytes pos-
sessing polar functional groups need to be derivatized prior to GC-MS
analysis in order to improve their chromatographic behaviour. This ad-
ditional step can be avoided if analysis is performed using HPLC or TLC
techniques, but in that case, analysts need to run reference standards at
the same time for identification of analyte.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
The authors are grateful to Dr. Sukhminder Kaur, (Director, Cen-
tral Forensic Science Laboratory, Chandigarh) and Dr. S. K. Jain (Chief
Forensic Scientist, Directorate of Forensic Science Services, Ministry of
Home Affairs, Govt. of India, New Delhi) for their constant encourage-
ment, support and providing necessary infrastructural facilities to carry
11
Advances in Sample Preparation 6 (2023) 100058
Recovery Amount of FAV found (mg)
(%) (FPSE-GC-MS method)
96 384 ± 1
out this research work. Author B. J. is thankful to the Department of
Science & Technology (DST)-Inspire, New Delhi, grant no. DST/INSPIRE
Fellowship/2019/IF190181
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.sampre.2023.100058.
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