Toxicology Mechanisms and Methods

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/itxm20

Dispersive liquid-liquid microextraction (DLLME)

for determination of tricyclic antidepressants in
whole blood and plasma samples and analysis by
liquid chromatography with diode array detector
(LC-DAD)

Dener Gomes Berlato, André Lucas Bezerra Pacheco, Gustavo Andrade

Ugalde, Fernanda Ziegler Reginato, Geovane de Almeida Saldanha, Tiago
Franco de Oliveira, Sarah Eller & André Valle de Bairros

To cite this article: Dener Gomes Berlato, André Lucas Bezerra Pacheco, Gustavo Andrade
Ugalde, Fernanda Ziegler Reginato, Geovane de Almeida Saldanha, Tiago Franco de Oliveira,
Sarah Eller & André Valle de Bairros (13 Oct 2023): Dispersive liquid-liquid microextraction
(DLLME) for determination of tricyclic antidepressants in whole blood and plasma samples and
analysis by liquid chromatography with diode array detector (LC-DAD), Toxicology Mechanisms
and Methods, DOI: 10.1080/15376516.2023.2269236

To link to this article: https://doi.org/10.1080/15376516.2023.2269236

Published online: 13 Oct 2023.

Submit your article to this journal

Article views: 12

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=itxm20
TOXICOLOGY MECHANISMS AND METHODS
https://doi.org/10.1080/15376516.2023.2269236

RESEARCH ARTICLE

Dispersive liquid-liquid microextraction (DLLME) for determination of tricyclic

antidepressants in whole blood and plasma samples and analysis by liquid
chromatography with diode array detector (LC-DAD)
Dener Gomes Berlatoa, Andre
� Lucas Bezerra Pachecoa, Gustavo Andrade Ugaldea, Fernanda Ziegler Reginatoa,
Geovane de Almeida Saldanhaa, Tiago Franco de Oliveirab, Sarah Ellerb and Andr�e Valle de Bairrosa
a
Nucleous Applied to Toxicology (NAT), Department of Clinical and Toxicological Analysis, Federal University of Santa Maria, Santa Maria,
Brazil; bGraduate Program in Health Sciences, Federal University of Health Sciences of Porto Alegre, RS, Brazil

ABSTRACT ARTICLE HISTORY

Microextractions have been developed for the tricyclic antidepressants (TCAs) analysis in biological matri­ Received 26 July 2023
ces, including dispersive liquid-liquid microextraction (DLLME). The proposed DLLME employed 490 mL of Revised 26 September 2023
biological sample (whole blood or plasma), which were added 15 mg of NaCl, 10 mL of medazepam as Accepted 5 October 2023
internal standard (10 mg/mL) and 100 mL of 2 M NaOH. This mixture was homogenized by vortex
KEYWORDS
(2800 rpm/10 s) and 400 mL of hexane (extractor solvent) with 600 mL of methanol (dispersing solvent) DLLME; antidepressants; LC-
were added to the sample. After the vortex step (2800 rpm/5 s), an ultrasonic bath for 300 s was DAD; whole blood; plasma
employed. Then, this content was centrifuged (10 min/10000 rpm), organic phase was collected and
dried under air flow. After, 30 mL of the mobile phase was used for resuspension and 20 mL is injected
into LC-DAD. This method was optimized and fully validated according to UNODC and SWGTOX guide­
lines, reaching limits of detection equivalent to analytical methodologies that employ mass spectrometry
(MS). Also, it was applied in real cases involving suspected exposure to TCAs. So, the developed DLLME
for the determination of TCAs in whole blood and plasma samples proved to be a simple, reliable,
robust and reproducible method that can be used in toxicology and clinical laboratories.

Introduction concentrations of these molecules are still based on plasma

samples. In addition, hospital laboratories transfer plasma
Medicines are the main agents responsible for the poisoning
samples to measure these xenobiotics in antemortem situa­
in Brazil, responsible for almost 14000 cases registered in the
tions (Perry et al. 1994; Lundstrøm et al. 2022).
country, according to the latest data released by the
Sample handling is an essential step in toxicological analy­
National System of Toxic-Pharmacological Information
ses and dispersive liquid-liquid microextraction (DLLME) has
(SINITOX) (SINITOX (Sistema Nacional de Informaço ~es To
�xico-
been gaining ground in the analyzing of biological samples as
�
Farmacologicas) 2020). Tricyclic antidepressants (TCAs) are
it is a fast, simple, low-cost technique that can be applied to
one of the main classes of medications involved in cases of
any laboratory. DLLME is based on a mixture of solvents in a
poisoning due to the high number of prescriptions and toxi­
ternary system, which extraction solvent and dispersive solvent
cological complications such as myocardial depression and
are injected into the matrix and quickly the microdrops form
cardiac arrhythmias when administered alone or concomi­
the desired cloud effect. Consequently, a large surface area
tantly with other drugs, which can be lethal (Liebelt and
allows almost instantaneously the analyte extraction (Martins
Francis 2002; SINITOX (Sistema Nacional de Informaço ~es
et al. 2012; Tabani et al. 2019; Manousi and Samanidou 2020).
To�xico-Farmacolo�gicas) 2020).
So, this study aimed to the develop a DLLME procedure
These factors can justify toxicological analysis of TCAs in
for TCAs in whole blood and plasma samples employing
biological samples in order to clarify poisoning cases. Among
liquid chromatography with diode array detector (LC-DAD)
the biological matrices used in toxicological laboratories,
and its application in real cases.
whole blood samples stand out, as highly complex matrix,
easily obtainable and can be used both in clinical emergen­
cies and postmortem cases. Whole blood samples reflect the Experimental
concentrations of TCAs and their respective biological effects,
Chemicals
which make it possible to find precursor drugs as well as
their metabolites for TCAs (Titier et al. 2007; Montenarh et al. Reference standards of amitriptyline (AMI), imipramine (IMI),
2014; De Boeck et al. 2018). However, therapeutic doxepin (DOX), nortriptyline (NOR), desipramine (DES) and

CONTACT Andr�e Valle de Bairros andre.bairros@ufsm.br Nucleous Applied to Toxicology (NAT), Department of Clinical and Toxicological Analysis, Federal
University of Santa Maria, Santa Maria, Brazil.
� 2023 Informa UK Limited, trading as Taylor & Francis Group
2 D. G. BERLATO ET AL.
medazepam (internal standard-IS) were purchased from
Sigma-AldrichVR (Saint Louis, USA). Reference standards of
AMI, IMI, DOX, NOR, DES and IS were individually diluted
with methanol (10 mg/mL). The analytes evaluated in this
experiment were prepared to obtain the corresponding
working solutions (1 mg/mL and 100 mg/mL). All stock and
working solutions were stored at −5 � C and kept away from
light. Acetonitrile, methanol, potassium phosphate mono­
basic (KH2PO4), sodium hydroxide (NaOH), phosphoric acid
85% (v/v) and sodium chloride (NaCl) were acquired from
Neon Commercial (Suzano, S~ao Paulo, Brazil).
Instrumentation
It was employed an ultrasonic bath Cristo �foliV
R (Campo
Mour~ao, Brazil), DM22 pHmeter DigimedV R (S~
ao Paulo, Brazil),
centrifuge 5427 R EppendorfV R (AG, Germany), vortex shaker
BiomixerV R QL-901 and analytical balances AX200 ShimadzuV R graphic run. IS was analyzed at a wavelength of 255 nm;
(Kyoto, Japan).
For chromatographic apparatus, it was used column
AgilentVR Zorbax Eclipse Plus C18 3.5 mm x 2.1 mm x 50 mm
(Milford, USA) and column ScharlauV R Kromaphase 100 C18
3.5 mm x 2.0 mm x 50 mm (Barcelona, Spain) with cartridges
PhenomenexV R C18 4.0 mm x 3.0 mm (Torrance, USA), while
TCAs determination occurred in a Nexera - XR - Ultra High-
Performance Liquid Chromatograph (ShimadzuV R, Kyoto,
Japan) – equipped whit a 20ADXR pump; SIL-20AC autosam­
pler system; CTO 10AS Column Oven and SPD-M20A
Photodiode Array Detector – Acquisition data from software
LabSolution (ShimadzuV R , Kyoto, Japan).
Method optimization
Optimization was performed taking into consideration the
choice of mobile phase composition based on the conditions
found in the literature (Queiroz et al. 1995; Theurillat and
Thormann 1998; Karpinska and Starczewska 2002; Alves et al.
2006; Samanidou et al. 2007; Malfar�a et al. 2007; Uddin et al.
2008; Mercolini et al. 2010) and DLLME parameters. The
DLLME parameters studied were extractor solvent (hexane,
toluene; chloroform); essential oil from Eucalyptus (Eucalyptus
globulus) and orange (Citrus sinensis); dispersing solvent
(methanol, acetonitrile, isopropanol, ethanol and acetone);
vortex effect (0-60s) and ultrasonic bath time (0-300s); selec­
tion of alkaline sample solution (1-5 M NaOH); centrifugation
speed and time (4.000-14.000 rpm, 5-10 min); effect of vol­
ume of the extraction and dispersive solvent (300-500 mL;
600-800 mL, respectively). The salting-out effect was also
tested by adding 0, 10 and 15 mg of NaCl (w/v) to the sam­
ple before extraction. The stability of the TCAs in extracts
from the DLLME procedure was evaluated during 24h stor­
age at −10 � C. Stability tests were performed in triplicate. For
the DLLME optimization procedure, drug-free human sam­
ples have been fortified at a concentration of 200 ng/mL for
each analyte and then submitted to the previously described
methods.
Whole blood and plasma samples
Drug-free whole blood and plasma samples containing 2%
sodium fluoride preservative (2 mL) from the blood bank of
the Santa Maria University Hospital (HUSM) (Santa Maria,
Brazil) were employed for optimization and validation of the
method. Blood samples were collected from suspected
human intoxication from HUSM. This study was approved by
the Federal University of Santa Maria Ethics Committee,
Brazil (Ethics Protocol Approval N� . 2.353.688).
Chromatography conditions
The stationary phase was WatersV R XBridge C18 3.5 mm
4.6 � 50 mm column and the mobile phase for the present
study was an aqueous solution containing KH2PO4 100 mM
at pH 2.5 corrected with o-phosphoric acid 85% and metha­
nol (60:40), with a flow rate of 1.2 mL/min for the chromato­
DOX, AMI and NOR at 239 nm; IMI and DES at 249 nm.
DLLME procedure for sample preparation
In a 2 mL microtube were added 15 mg of NaCl, 490 mL of a
biological sample (whole blood or plasma), 10 mL of IS
(10 mg/mL) and 100 mL of 2 M NaOH. This mixture was
homogenized by vortex (2800 rpm) for 10 s. After this step,
the sample was added to a mixture containing 400 mL of
hexane (extractor solvent) with 600 mL of methanol (dispers­
ing solvent). This mixture was homogenized for 5 s on vor­
tex (2800 rpm) and bring it to an ultrasonic bath for 300 s.
Then, this content was centrifuged for 10 min at 10000 rpm.
The organic phase was collected, reallocated in a vial, and
dried under airflow. In the end, 30 mL of the mobile phase
was used for resuspension, and 20 mL was injected into
LC-DAD. .
Validation of the method
After DLLME optimization, the method was validated for
whole blood and plasma samples. Validation parameters fol­
lowed the United Nations Office on Drugs and Crime valid­
ation guide (UNODC (United Nations Office on Drugs and
Crime) 2009). Thus, the parameters of specificity/selectivity,
the limit of detection (LoD), the limit of quantification (LoQ),
recovery, linearity, intra- and inter-day precision, accuracy
and robustness were evaluated (UNODC (United Nations
Office on Drugs and Crime) 2009). To assess both carryover
effect and dilution integrity, the Scientific Working Group for
Forensic Toxicology (SWGTOX) Standard Practices for Method
Validation in Forensic Toxicology guideline was employed
(SWGTOX (Scientific Working Group for Forensic Toxicology)
2013).
Limit of detection (LoD) and limit of quantification (LoQ)
First, the limit of blank (LoB) was established to ensure a reli­
able LoD. . The LoB is the highest apparent concentration

Figure 1. Step-by-step of the proposed DLLME.
expected when 60 replicates of 20 blank samples containing
no analytes are tested. The data is expressed in the following
equation: LoB ¼ mean blank þ 1.645 (standard deviation
blank). With the LoB previously established, LoD can be
defined as the lowest analyte concentration in a sample that
can be distinguished from the LoB with significant reliability
and at which detection is feasible (Armbruster and Pry 2008).
The procedure to determine LoD was performed similarly
to that of LoB. Drug-free whole blood and plasma samples
enriched with decreasing concentrations of the analyte of
interest were used and the results were plotted through the
equation: LoD ¼ LoB þ 1.645 (standard deviation low con­
centration sample). When a value of 1.645 is used as a stand­
ard deviation, no more than 5% of the values should be less
than the LoB. If the observed LoD sample values meet this
criteria, then LoD is considered established or verified
(Armbruster and Pry 2008).
Initially, LoQ was experimentally evaluated in triplicate at
concentrations of 5, 10, 15, 20 and 30 ng/mL for AMI, DOX
and NOR while 5, 10, 15, 20, 30, 40 and 50 ng/mL for IMI and
DES. After reaching the acceptance criteria (relative standard
deviation � 20%), a sextuplicate was performed to confirm
the referred value for each analyte. Acceptance criteria for
the LoQ were coefficient of variation � 20% (relative stand­
ard deviation) for precision and 80-100% for accuracy for
both samples (Armbruster and Pry 2008, UNODC (United
Nations Office on Drugs and Crime) 2009; SWGTOX (Scientific
Working Group for Forensic Toxicology) 2013).
Specificity/selectivity
Whole blood and plasma samples were extracted and ana­
lyzed according to the previously described procedure for
assessing endogenous substances. Peaks at the retention
TOXICOLOGY MECHANISMS AND METHODS 3
time of interest were compared to those from whole blood
samples spiked with the analytes at the LoQ. The method
was also evaluated for potential interfering substances
through the analysis of free-drug whole blood and plasma
samples spiked with caffeine, acetylsalicylic acid, dipyrone,
paracetamol, warfarin, furosemide, atenolol, ibuprofen,
phenobarbital, clonazepam, ephedrine, phenylpropanolamine
and salbutamol at a concentration of 1000 ng/mL.
Acceptance criteria for this assay were based on the absence
of interfering substances at the retention times of the analy­
te(s) of interest and their respective IS (UNODC (United
Nations Office on Drugs and Crime) 2009; SWGTOX (Scientific
Working Group for Forensic Toxicology) 2013).
Linearity
Linearity was performed in whole blood and plasma samples
in sextuplicate for each point of the analytes of interest,
starting with the LQ (10 ng/mL for AMI and DOX; 20 ng/mL
for NOR and 30 ng/mL for IMI and DES) followed by of 50,
100, 200, 300, 400 and 500 ng/mL. These concentrations
were selected according to therapeutic and toxic concentra­
tions of TCAs. For acceptability, the linearity must obtain a
correlation coefficient r � 0.99.
Recovery
Two sets of whole blood and plasma samples were respect­
ively prepared, and set A of samples consisted of the con­
centrations of 45, 250 and 420 ng/mL and it were prepared
as described in the topic DLLME procedure for sample prepar­
ation for both biological samples. For set B, the analytes
were spiked into the samples immediately after the DLLME
4 D. G. BERLATO ET AL.
step, it was added at the same concentration as set A. The
entire procedure was performed in sextuplicate.
Absolute recovery was obtained by comparing the mean
response obtained for both set A (processed) and the
response for set B (unprocessed). The unprocessed response
represented 100% recovery. The UNODC Analytical Methods
Validation Guide (UNODC (United Nations Office on Drugs
and Crime) 2009) does not consider this factor as indicative
of test failure, as long as the other validation parameters
achieve the desired purposes.
Precision and accuracy
Precision was determined by intra- and inter-day replications.
This parameter was assessed by analyzing whole blood and
plasma samples containing three known concentrations of
target analytes in sextuplicate on three consecutive days.
Concentrations were 45 ng/mL (lowest level), 250 and
420 ng/mL. After data collection, a Two-way ANOVA was
performed.
For accuracy, a curve was developed from their respective
LoQ levels (10 ng/mL for AMI and DOX; 20 ng/mL for NOR
and 30 ng/mL for IMI and DES) followed by 200 and 500 ng/
mL for all analytes. Afterward, whole blood and plasma sam­
ples fortified with the analytes at concentrations of 45 ng/mL
(lowest point), 250 and 420 ng/mL were evaluated in sextu­
plicate. Accuracy was expressed as a percentage of the
known concentration, i.e. the acquired mean concentration/
nominal concentration � 100.
Robustness
Robustness is the ability to resist minor and deliberate varia­
tions in analytical parameters, indicating confidence in the
methodology. The following tests were performed: mobile
phase pH variation (± 0.2 units), employment of different
chromatographic equipment, laboratories and laboratory
specialists.
Dilution integrity and carryover effect
Dilution integrity is a parameter that allows the evaluation of
samples with analyte levels above the calibration curve.
When dilution integrity is required, it should be demon­
strated by spiking the matrix with analytes above the highest
concentration of the curve and then diluting this sample
with a blank matrix (at least five determinations per dilution
factor). Accuracy and precision should be within the set crite­
ria, i.e. within ±15%.
First, a curve was constructed from respective LoQ (10 ng/
mL for AMI and DOX; 20 ng/mL for NOR; 30 ng/mL for IMI
and DES), 200 and 500 ng/mL in triplicates. An aliquot of
500 mL of whole blood and plasma samples were contami­
nated with a pool of target analytes with a concentration of
3000 ng/mL. These samples were diluted ten times with
respective biological matrices to obtain a final concentration
of 300 ng/mL. The IS was added to a final concentration of
200 ng/mL, and extraction was performed as described in the
topic DLLME procedure for sample preparation. Diluted
samples were analyzed in sextuplicate. After extraction and
injection of this sample, three blank samples were injected
to evaluate the memory (carryover) effect.
Liquid chromatography-mass spectrometry analysis
It was determined by Franco de Oliveira and collaborators
(Franco de Oliveira et al. 2019) with Nexera X2 ultra high-
performance liquid chromatography (UHPLC), which con­
sisted of a degasser, a binary pump, and an autosampler
coupled to an LC-MS 8050 mass spectrometer (ShimadzuV R,
Kyoto, Japan) with an electrospray source operating in the
positive (ESIþ) and negative (ESI–) ion modes, in two separ­
ate chromatographic runs. The chromatographic separation
was achieved on a Raptor Biphenyl column (50 mm � 3 mm,
2.7 lm; Restek, USA) eluted with a flow rate of 600 lL/min
and 45 � C.
For biological samples analysis, an aliquot of 800 lL of an
acetonitrile/methanol mixture (80:20, v/v) was added to the
biological samples (100 lL) spiked with 20 lL of the internal
standards mix (IS mix; 0.5 lg/mL), and the mixture was
shaken for 30 s. After centrifugation for 6 min at 9000 � g, a
3 lL aliquot was directly injected into the LC-MS/MS system
(Franco de Oliveira et al. 2019).
Gas chromatography-mass spectrometry (GC-MS)
analysis
Biological samples were treated according to the purposed
DLLME. However, these extracts were resuspended with
30 mL of hexane and injected into the equipment. The ana­
lysis was performed using a ShimadzuV R GC-2010 Plus model
gas chromatography coupled with ShimadzuV R single quadru­
pole mass spectrometry GCMS-Q2010 Ultra model (Kyoto,
Japan). The samples were injected by splitless mode (1 lL)
into the GC-MS using the ShimadzuV R autosampler AOC-20is
model (Kyoto, Japan).
Chromatography separation was achieved on a Restek
CorporationV R Rtx-5MS fused-silica capillary column (30 m �
0.25 mm i.d. � 0.25 mm thickness film) (Bellefonte, EUA) con­
taining 5% diphenyl and 95% dimethyl polysiloxane. Helium
was used as carrier gas with 0.8 mL/min at a constant flow
rate mode. Column oven temperature program was as fol­
lows: 125 � C (hold 1 min), then programmed at 50 � C/min to
190 � C; 5 � C/min to 225 � C (hold 3 min); 50 � C/min to 280 � C
(hold 1 min). The total analytical time was 13.40 min. The
injection port and transfer line were set at 220 � C and 280 � C,
respectively. The MS was operated by electron ionization
(70 eV) in scan mode. The following ions (m/z) were chosen
to identify the analytes: DOX (178, 115 and 42); IMI (280, 234
and 220); DES (234, 195 and 193); AMI (58, 91, 202, 189);
NOR (263, 128 and 44); IS (207 and 242).
Real case samples application
Whole blood and plasma samples were collected from TCAs
user volunteers as well as suspected cases of TCAs intoxica­
tion from HUSM. The samples from volunteers were collected

in tubes containing 2% sodium fluoride preservative (2 mL).
In suspected poisoning/exposure by TCAs from the HUSM,
available whole blood and plasma samples were used and
submitted to the developed methodology.
Results and discussion
Mobile phase constitution and stability
The mobile phase that presented the best results for the pre­
sent study is constituted by an aqueous solution containing
100 mM of monobasic potassium phosphate, pH 2.5 cor­
rected with o-phosphoric acid 85% and methanol in the pro­
portion of 60:40. In this condition, AMI and NOR do not co-
elute, which allowed the simultaneous analysis of these
TCAs. We observed that the mobile phase’s acidification
helps stabilize reversed-phase chromatographic columns,
resulting in more symmetrical peaks and, consequently,
higher resolution for weak alkaline substances, as described
by Borges (Borges et al. 2012).
Optimization of DLLME procedure
Salting-out effect
The salting-out effect promoted an improvement in the
extraction because of ionic strength. Our results showed the
best condition with 15 mg of NaCl (w/v), except for DES. It
should be considered that the salting-out effect has variabil­
ities depending on the extraction procedure, matrix sample
and specific analytes (Saldanha et al. 2022). Despite of the
particular behavior of DES, 15 mg of NaCl (w/v) was the most
effective for extraction.
Extraction solvent
Chloroform, toluene and hexane were evaluated as extractor
solvents considering their respective partition coefficient
(Log P: 1.97-3.90). Chloroform did not adequately separate
the organic phase from whole blood sample, so it was not
injected to preserve the chromatographic column.
Considering the characteristics present in biological samples,
especially whole blood samples (high concentration of cells
and consequently high molecular weight endogenous mole­
cules) (Skopp 2004; Peliç~ao et al. 2018), solvents with a dens­
ity greater than water (e.g. chloroform) could cause
difficulties when removing the organic solvent from complex
biological samples such as whole blood samples as well as
the amount of interferences from biological matrix extracted
by the solvent.
As an extracting solvent, toluene could extract the TCAs
in whole blood samples (Chen et al. 2017). However, we
observed the significant of endogenous interferences as well
as waste of solvent volume during the DLLME, similar to
Saldanha e coauthors’ description (Saldanha et al. 2022).
Hexane was the solvent with the higher higher extraction
capacity as expected, considering its high partition coeffi­
cient (Log P: 3.90), a close value to the TCAs evaluated (Log
P: 3.84-4.92). In addition, hexane showed less endogenous
interferences among the tested solvents with little volume
TOXICOLOGY MECHANISMS AND METHODS 5
loss at the end of the process, despite the difficulties in the
formation of the typical cloud effect of DLLME.
An interesting alternative is the employment of low-dens­
ity solvents (e.g. 1-octanol, 1-nonanol, 1-decanol and other
solvents) as extractor solvents because of their respective
coefficient partition (Log P: � 3.0) and ease of handling.
However, during our experiments, we observed an increase
in the HPLC system pressure throughout a vast sequence of
injections, making the chromatographic analysis less viable,
as previously described by Saldanha and collaborators
(Saldanha et al. 2022). To avoid future problems in the chro­
matographic system, we aborted the planned tests with
these solvents.
Dispersive solvent
The dispersing solvent must be soluble both in the aqueous
phase and in the organic phase, and it must guarantee the
dispersion of the solvent in the aqueous phase, in order to
promote the ‘cloud effect’ required by DLLME (Martins et al.
2012; Tabani et al. 2019; Manousi and Samanidou 2020). The
dispersion formed (cloud effect) is directly linked to the
extraction efficiency, so the choice of this solvent must be
carefully evaluated. Methanol, acetonitrile, isopropanol, etha­
nol and acetone were evaluated as dispersing solvents as
well as the absence of dispersing solvent for DLLME.
Acetone and ethanol showed a low rate of enrichment
when observed in the absolute area of the chromatograms.
Indeed, chromatograms generated from the presence of
acetone as a dispersing solvent did not allow the analysis of
DOX due to matrix interferences that coeluted with this ana­
lyte. Isopropanol demonstrated a milky appearance of solv­
ent after extraction. So, in order to prevent the integrity of
the system chromatographic, this specific extract was not
injected. Acetonitrile and methanol were shown to be effi­
cient as dispersing solvents, presenting a low effect of matrix
interferents. In this sense, methanol was the best-dispersing
solvent among all tested solvents, promoting a cloud effect
and, consequently, a higher absolute area of the
chromatograms.
Effect of vortex and ultrasonic bath time
Ultrasonic bath and vortex have been used in several studies
to reduce solvent amounts and increase DLLME extraction
efficiency (Fern�andez et al. 2016; Vaghar-Lahijani et al. 2018;
Fern�andez et al. 2019; Carasek et al. 2021; Tomai et al. 2021;
Saldanha et al. 2022). The extraction efficiency with vortex
homogenization at 2800 rpm was evaluated at times of 0, 15,
30 and 60 s. Vortex homogenization for 30 s showed the best
result and this tested condition was compared with ultra­
sound bath in 30, 60, 150 and 300 s with 40 KHz frequency at
35 � C. Before the ultrasonic bath assay, vortex homogeniza­
tion at 2800 rpm for 5 s was used in order to maximize the
extraction procedure that is promoted by the ultrasound
bath. An ultrasonic bath for 300 s to homogenize the dispers­
ing and extractor solvents in whole blood sample was better
than vortex homogenization.
6 D. G. BERLATO ET AL.
Selection of the pH of sample solution
Because of matrix interference, visualization of the pH value
of the biological matrix was not able to be performed, so we
opted for fixed volume in different concentrations of NaOH.
In this experiment, the extraction efficiency based on pH cor­
rection was evaluated from 1, 2 and 3 M NaOH. The addition
of 100 mL of 2 M NaOH showed greater extraction effective­
ness compared to other NaOH concentrations. Non-addition
of the pH corrector resulted in the non-extraction of TCAs
from the whole blood sample by the purpose of DLLME.
Blood samples pH ranges from 7.35 − 7.45 in antemortem
situations; however, blood matrices from postmortem cases
tend to acidify due to the various processes that occur after
death (Donaldson and Lamont 2013; Peliç~ao et al. 2018). In
both situations, TCAs present in the blood matrices are in
the ionized form due to their respective pKa values (9.4 to
10.02), so the pH must be adjusted to at least 2 points above
the pKa of the molecules (pH > 12.02) to guarantee in non-
ionized form for the mass transfer of said substance from the
biological matrix to the organic solvent to occur (Flanagan
et al. 2007; Dos Santos et al. 2014).
Centrifugation speed and time
Centrifugation speed and time were evaluated in the follow­
ing conditions: 10 min at 4.000 rpm, 10 min at 10.000 rpm and
5 min at 14.000 rpm. The best condition was 10 min at
10.000 rpm showed less matrix interference when centrifuged
for 10 min at 4.000 rpm and there was no difference compared
to centrifuging for 5 min at 14.000 rpm. In addition, during
this optimization step, there was a risk of rupture of the flasks
used with the centrifugation force at 14000 rpm. During this
experiment, we observed that centrifugation helped to break
the emulsion formed by DLLME in whole blood samples,
becoming a vital step for this type of extraction, as described
by Saldanha and coauthors (Saldanha et al. 2022).
Effect of volume on extractor and dispersing solvent
Different volumes of the extractor and dispersing solvents
(300, 400 and 500 mL; 600, 700 and 800 mL, respectively) were
evaluated. Increasing the volume of the extractor solvent with
subsequent may result in a lower pre-concentration factor of
the analytes due to the appropriate ratio between the organic/
aqueous phase (matrix). In order to maximize DLLME extrac­
tion, dispersing solvent volume is studied to reach a constant
volume of the organic phase. So, the appropriate volume of
dispersing solvent for microdroplet formation depends on
both the volume of the aqueous phase and the volume of the
extractor solvent (Martins et al. 2012; Carasek et al. 2021;
Saldanha et al. 2022). In this sense, the best condition was
respectively 400 and 600 mL of extractor and dispersing solvent
while lower volumes showed no increase in DLLME capacity.
Mixture of extracting solvents for DLLME
In an attempt to increase the extraction efficiency, a mixture
of extracting solvents was performed considering the parti­
tion coefficient of TCAs as well as extractor solvents. This
approach is employed successfully for liquid-liquid extraction
(Meatherall 1994; Fu et al. 2010) and hollow-fiber liquid-
phase microextraction (LPME) (De Bairros et al. 2015). So, a
mixture containing hexane:1-octanol was evaluated in the
following proportions: 95:5, 90:10 and 85:15.
However, this approach did not obtain satisfactory results,
in spite of the partition coefficient of 1-octanol (Log P: 3.0).
The drying process of 1-octanol, even in a low volume,
required a time of more than 30 min for total drying. In add­
ition, it was not efficient in the extraction of TCAs in whole
blood samples because of the amount of matrix interferents,
which precluded the chromatographic analysis of DOX and
IS. Such behavior prevented carrying out the chromato­
graphic evaluation of the analytes in question for the opti­
mization step (data not shown).
Stability study from DLLME procedure
Freshly prepared samples from the DLLME procedure were
measured and compared with prepared samples in the day
before and stored at −10 � C in a capped vial. There was a
significant loss of analytes, except DOX, in the storage pro­
cess for one day in the conditions tested. The results are
expressed in the amount of analyte lost in percentage (%)
and it was found 5.5, 23.1, 33.33, 60 and 81% for DOX, IMI,
DES, AMI and NOR, respectively. Probably the oxygen atom
present in the tricyclic ring from DOX allowed greater stabil­
ity of the analyte under the imposed conditions compared
to the other TCAs. However, studies to assess the stability of
extracts from DLLME for TCAs are needed to confirm this
theory.
Traditional freeze-thaw cycle tests assessing the stability
of these analytes in biological samples have already been
related in the literature (Seidi et al. 2013; Mohebbi et al.
2018b). However, our objective was the stability of the
extract from DLLME to support an experiment logistics pro­
posal, which unfortunately did not prove to be viable. So, for
validation of the method, the entire stage of the developed
method was prepared and evaluated on the same day.
Essential oils as extractor solvent
Based on the principles of Green Chemistry, we evaluated
essential oil from Eucalyptus (Eucalyptus globulus) and orange
(Citrus sinensis) in place of the hexane solvent because of
their drying and extraction capacity as described by Silveira
and coauthors (Silveira et al. 2021).
We found some difficulties in promoting the TCAs extrac­
tion with this methodology. In spite of methanol showing
some potential as a dispersing solvent while essential oil
from Eucalyptus (Eucalyptus globulus) is employed as an
extractor solvent, this approach did not demonstrate total
compatibility considering the proposed DLLME technique.
For essential oil from orange (Citrus sinensis), dispersing
solvent did not promote cloud effect as DLLME purpose.
Therefore, the low volume available after extraction and han­
dling of this essential oil presented difficulties. In addition,
both essential oils used in the proposed DLLME technique

demonstrated several matrix interferents, which it did not
allow the evaluation of analytes in whole blood samples.
In an attempt to reduce the interference of whole blood
samples in the extraction with essential oils for the DLLME
technique, a pretreatment step was performed with 10% tri­
chloroacetic acid and 50% NaOH. Despite being able to sep­
arate the phases and facilitate the collection of essential oil,
this strategy was not effective in extracting the TCAs.
Perhaps strong acid and alkaline solutions can lead to the
hydrolysis of the analytes, making the quantification of TCAs
unfeasible. Therefore, the pretreatment step did not elimin­
ate the matrix interferents from the biological sample. A
summary of the data from all optimized parameters can be
seen in Figure 2.
Validation of the method
Based on data from whole blood method optimization in
this study and greater complexity of whole blood samples
compared to plasma samples for the determination of xeno­
biotics, it was considered the possibility of measurement of
TCAs in plasma samples for antemortem situations as well as
the therapeutic concentrations of TCAs are based on the
respective matrix (Perry et al. 1994; Titier et al. 2007;
Montenarh et al. 2014; De Boeck et al. 2018; Lundstrøm et al.
2022). So, validation of the method was applied directly to
plasma samples without the optimization step.
Validation of the method was conducted following UNODC
and SWGTOX guidelines (UNODC (United Nations Office on
Drugs and Crime) 2009; SWGTOX (Scientific Working Group
for Forensic Toxicology) 2013) after optimization of the DLLME
procedure of these analytes and was carried out by
Figure 2. Optimization of DLLME for TCAs in biological samples. AMI, amitriptyline; DES, desipramine; DOX, doxepine; IMI, imipramine; nor, nortriptyline. UB, ultra­
sonic bath. Vortex with 2800 rpm and ultrasonic bath with 40 KHz frequency at 35 � C.
TOXICOLOGY MECHANISMS AND METHODS 7
establishing specificity/selective, linearity, recovery, intra and
inter-day precision, accuracy, limit of detection (LoD), limit of
quantification (LoQ), robustness, dilution integrity and carry­
over effect. No interfering peaks due to endogenous interfer­
ents or exogenous substances (mainly general and prescribed
drugs used concomitantly with TCAs) were observed at the
retention time of the compounds of interest.
Considering the equipment used, extraction technique
and the complexity of the matrices of choice (whole blood
and plasma samples), LoD values as well as the LoD, were
satisfactory within the therapeutic range (2.5-10 and 10-
30 ng/mL, respectively). In fact, the low levels of LoD and
LoQ reached according to this method correspond to con­
centrations found in methodologies that employ liquid chro­
matography or gas chromatography coupled to mass
spectrometry (LC-MS or GC-MS) (Titier et al. 2007; Dos Santos
et al. 2014; Santos et al. 2017).
The linearity parameter was established from their
respective LoQ up to 500 ng/mL, which the phenomenon of
heteroscedasticity, evaluated through the distribution F
(Almeida et al. 2002), was not observed. So, homoscedasticity
behavior was found in all analytes. Therefore, it was not
needed weighted squares linear regression. The respective
calibration curves and coefficients of correlations for whole
blood samples were y ¼ 0.0038x − 0.0314; r ¼ 0,9961 (DOX);
y ¼ 0.0006x − 0.0034; r ¼ 0.9976 (IMI); y ¼ 0.0012x þ 0.0064;
r ¼ 0.9967 (DES); y ¼ 0.0044x − 0.0553; r ¼ 0.9928 (AMI) and
y ¼ 0.0035x − 0.0237; r ¼ 0.9933 (NOR). For plasma samples,
it were found y ¼ 0.0036x − 0.0383, r ¼ 0.9973 (DOX);
y ¼ 0.0007x − 0.0136, r ¼ 0.9987 (IMI); y ¼ 0.0021x − 0.0394,
r ¼ 0.9947 (DES); y ¼ 0.0043x − 0.0204, r ¼ 0.9982 (AMI);
y ¼ 0.006x − 0.1218, r ¼ 0.9966 (NOR).
8 D. G. BERLATO ET AL.
Regarding the intra and inter-day precision, the values for
these analytes at three different concentration levels were
less than 15% in all concentration levels. These data were
considered suitable for all tested substances for intra and
inter-day precision (RSD% < 12.80 and 14.59, respectively).
Accuracy values of the TCAs evaluated at three different con­
centration levels ranged from 85.52 to 113.67% for whole
blood samples while plasma samples ranged from 91.83 to
114.44%. In both validation parameters, the values found are
within the criteria required by the UNODC and SWGTOX val­
idation guides (UNODC (United Nations Office on Drugs and
Crime) 2009; SWGTOX (Scientific Working Group for Forensic
Toxicology) 2013).
Recovery rates showed variations (9.86% to 31.60%; 29.20
to 51.49% for whole blood and plasma samples, respect­
ively), considering the simplicity of the extractive technique
used, the complexity of the biological matrices and the other
results of the validation parameters, the recovery rates for
the TCAs are according to the literature (Dos Santos et al.
2014; Vaghar-Lahijani et al. 2018; Dos Mohebbi et al. 2018).
Whole blood samples showed more complexity than plasma
samples, and it could be observed in the difficulty in forming
the DLLME cloud effect. Consequently, low recovery was
obtained in the proposed method. However, this parameter
is not considered indicative of test failure as long as the
other validation parameters achieve the desired purposes
(UNODC (United Nations Office on Drugs and Crime) 2009;
SWGTOX (Scientific Working Group for Forensic Toxicology)
2013).
For intoxication cases, it is not uncommon to encounter
high TCAs concentrations, possibly above the highest point
of the studied calibration curve (500 ng/mL). So, the inclusion
of the dilution integrity parameter at known levels (e.g.: 1:10;
1:5) is important to ensure reliable results. This parameter
was performed by spiking the blank whole blood and plasma
samples with 3000 ng/mL and diluting them ten times using
the same matrix (six replicates per dilution factor). Whole
blood samples showed 87.30–114.05% for accuracy and 6.93-
9.63% for precision, while plasma samples demonstrated
98.87-114.01% and 2.45-4.30% for accuracy and precision,
respectively. These results are in concordance with the crite­
ria established by SWGTOX (RSD% � 15) (SWGTOX (Scientific
Working Group for Forensic Toxicology) 2013). Also, it was
observed no memory effect (carryover) in the chromato­
graphic runs.
The robustness parameter is a measure of the method’s
capacity to remain unaffected by small and deliberate varia­
tions. The risk of finding out that a given method does not
fulfill these criteria late in the validation process may result
in the need for it to be redeveloped and optimized (Vander
Heyden et al. 2001). So, it was evaluated variation of the pH
of the mobile phase (0.2 units) was not relevant to change
the method as well as chromatographic columns from differ­
ent suppliers (AgilentV R and ScharlauV R ). Also, it was observed
the analysis of TCAs in different equipment (N ¼ 3) and by
laboratory technicians (N ¼ 4) as well as laboratory structures
(N ¼ 3), and the differences were not relevant. Even with the
different conditions, the purpose technique showed
robustness, with satisfactory results, and can be used in
other laboratories.
Furthermore, this methodology can be reproduced in GC-
MS, which the final extract was resuspended with hexane for
posterior injection, allowing the TCAs confirmation. However,
this approach has not been properly validated by following a
validation guideline. The validation parameters of the pro­
posed method are summarized in Table 1.
DLLME studies reported in the literature in urine samples
showed 3-8 ng/mL as LoD for AMI, clomipramine (CLO) and
thioridazine (TIO) (Xiong et al. 2009); 0.6 ng/mL for IMI and
trimipramine (TRI) (Shamsipur and Mirmohammadi 2014);
240-310 ng/mL for AMI, DES, IMI, NOR and CLO (Mohebbi
et al. 2018) employing liquid chromatography with ultraviolet
detector (LC-UV) while gas chromatography with flame ion­
ization detector (GC-FID) detected 2 ng/mL for NOR (Nabil
et al. 2015). For plasma samples, Bazregar and collaborators
(Bazregar et al. 2016) achieved 0.7-1.0 ng/mL for AMI, IMI and
NOR using LC-UV while Vaghar-Lahijania and coauthors
(Vaghar-Lahijani et al. 2018) determined 0.5 ng/mL for AMI
employing LC-DAD. Yazdi and coauthors (Yazdi et al. 2008)
detected 2 ng/mL of NOR through GC-FID.
Considering DLLME studies with the employment of gas
chromatography coupled to mass spectrometry (GC-MS), Ito
and collaborators (Ito et al. 2011) detected 0.5 − 2 ng/mL in
urine samples for AMI, CLO, DES IMI and NOR while concen­
trations between 0.013 − 0.025 ng/mL for AMI, DES and CLO
were considered as LoD by Mohebbi and coauthors
(Mohebbi et al. 2019). Liquid chromatography coupled to
tandem mass spectrometry (LC-MS/MS) was used to deter­
mine CLO, IMI and other psychoactive substances which
2 ng/mL was identified as LoD for TCAs in whole blood sam­
ples (Fisichella et al. 2015). In fact, Fisichella and coauthors
study (Fisichella et al. 2015) is the only published manuscript
that used whole blood samples as biological matrix of
choice.
Considering studies with full validation from DLLME
involving TCAs in biological samples, only Fisichella and col­
laborators (Fisichella et al. 2015) performed a method with
these characteristics. However, their study used five repli­
cates for each concentration for intra and inter-day precision
as well as accuracy, while three replicates were employed for
the linearity parameter. Therefore, this method did not
report the phenomenon of heteroscedasticity as well as dilu­
tion integrity and carryover effect for the target analytes.
Nevertheless, ANOVA was not used to evaluate intra and
inter-day precision as statistical proof. Also, this study
employed LC-MS as equipment to determine TCAs (Fisichella
et al. 2015), a much more expensive and rarer instrument in
laboratories compared to the LC-DAD. Other studies reported
in this manuscript did not carry out an adequate full valid­
ation and/or appropriate statistical analysis in biological sam­
ples. A summary of the procedures that have used DLLME
for the analysis of TCAs in biological samples available in the
scientific literature can be seen in Table 2.
It should be considered that the purposed method is the
only one that evaluates DOX among the other procedures
highlighted in this manuscript. Also, this methodology can
 TOXICOLOGY MECHANISMS AND METHODS 9

Table 1. Validation parameters of the developed method for the determination of TCAs in whole blood and plasma samples (six replicates for each
concentration).
Whole blood Plasma
Parameters
DOX IMI DES AMI NOR DOX IMI DES AMI NOR
Recovery (%)
C1 15.66 14.15 14.42 14.45 18.38 48.44 50.46 31.71 52.80 34.31
C2 11.71 13.40 9.86 13.77 11.32 38.99 44.45 23.20 45.73 29.20
C3 21.60 23.30 17.22 23.99 31.60 42.76 51.49 28.98 51.11 33.96
LD (ng/mL) 2.50 10.00 10.00 2.50 7.50 2.50 10.00 10.00 2.50 7.50
LQ (ng/mL) 10.00 30.00 30.00 10.00 20.00 10.00 30.00 30.00 10.00 20.00
Intra-day Precision (CV%)
C1 10.90 12.80 12.03 12.35 9.83 7.46 11.49 9.84 7.32 8.11
C2 8.59 11.20 7.70 9.78 6.71 7.28 10.66 9.44 7.11 9.11
C3 9.53 8.31 9.98 4.46 8.91 4.29 9.22 9.03 10.13 6.45
Inter-day Precision (CV%)
C1 11.90 13.86 13.06 9.67 5.82 7.84 10.47 8.02 9.15 7.01
C2 9.20 7.56 14.59 10.89 11.87 5.57 9.10 5.67 5.00 5.32
C3 11.62 10.18 12.99 7.23 13.40 8.10 11.10 11.28 11.32 9.43
Accuracy (%)
C1 92.89 85.64 113.78 96.87 113.67 113.33 114.44 94.53 113.40 99.40
C2 100.28 93.45 89.62 85.52 89.95 94.86 95.5 96.86 91.83 93.83
C3 111.16 95.92 97.54 90.19 99.18 100.24 99.78 101.38 98.10 98.33
Dilution integrity
Precision (%)
10 times 8.38 6.93 7.87 9.63 8.42 2.68 4.30 2.60 2.45 3.30
Accuracy (%)
10 times (300 ng/mL) 100.67 87.30 114.05 88.99 105.40 114.01 103.64 106.13 98.87 104.28
AMI, amitriptyline; DES, desipramine; DOX, doxepine; IMI, imipramine; NOR, nortriptyline. C1, 45 ng/mL (lowest point); C2, 250 ng/mL; C3, 420 ng/ml. LoD, limit
of detection; LoQ, limit of quantification. RSD%, relative standard deviation em percentage. 10 times, 3000 ng/mL.

Table 2. Summary of DLLME techniques for the determination of TCAs in biological samples.
Detectability
Matrix Analytes Equipment (ng/mL) Extraction procedure Validation parameters Reference
Whole blood and AMI, DES, DOX, IMI LC-DAD 2.50 − 10 UA-DLLME LoD, LoQ, specificity/selective, Purposed method
plasma and NOR linearity, recovery, intra
and inter-day precision,
accuracy, robustness, dilute
integrity and carryover
effect
Urine AMI, DES and CLO GC-MS 0.013 − 0.025 SSI/DES-DLLME-SFO LoD, LoQ, linearity, selectivity, Mohebbi et al. 2019
accuracy, intra and inter-
day precision, stability,
robustness and extraction
recovery
Plasma AMI and LC-DAD 0.5 UA − IL − DLLME LoD, linearity and recovery Vaghar-Lahijani
psychoactive et al. 2018
drug
Urine AMI, DES,IMI, NOR LC-UV 240 - 310 SI-HLLE followed by LoD, LoQ, specificity/selective, Mohebbi et al. 2018
and CLO DSPE and later linearity, recovery, intra-
DLLME-SFO day precision and accuracy
Plasma AMI, IMI and NOR LC-UV 0.7 − 1.0 Traditional DLLME LoD, linearity, intra and inter- Bazregar et al. 2016
day precision
Urine NOR and GC-FID 2.0 DLLME with in situ LoD, LoQ, linearity, intra and Nabil et al. 2015
psychoactive derivatization inter-day precision and
drugs accuracy
Whole blood CLO, IMI and other LC-MS/MS 2.0 Traditional DLLME LoD, LoQ, specificity/selective, Fisichella et al. 2015
psychoactive linearity, recovery, intra-
drugs day precision and accuracy
Urine IMI and TRI LC-UV 0.6 Traditional DLLME LoD, linearity and intra-day Shamsipur and
precision Mirmohammadi
2014
Urine AMI, CLO, DES, IMI GC-MS 0.5 − 2.0 DLLME with in situ LoD, LoQ, specificity/selective, Ito et al. 2011
and NOR derivatization linearity, intra-day
precision and accuracy
Urine AMI, CLO and TIO LC-UV 3.0 − 8.0 DLLME followed by LoD, LoQ, linearity, intra and Xiong et al. 2009
membrane inter-day precision and
filtration 0.45 lm accuracy
Water and plasma AMI and NOR GC-FID 5.0 – 10 Traditional DLLME LoD, linearity, intra-day Yazdi et al. 2008
precision and recovery
AMI, amitriptyline; CLO, clomipramine; DES, desipramine; IMI, imipramine; TRI, trimipramine; TIO, thioridazine. LoD, Limit of detection; LoQ, Limit of quantifica­
tion. DLLME, dispersive liquid phase microextraction; DLLME-SFO, dispersive liquid-liquid microextraction based on the solidification of floating organic droplet;
DSPE, dispersive solid phase extraction; SSI/DES-DLLME-SFO, sodium sulfate-induced deep eutectic solvent-based solidification of floating organic droplets–dis­
persive liquid phase microextraction; SI-HLLE, salt induced-homogenous liquid-liquid extraction; UA − IL − DLLME, ultrasound-assisted ionic liquid-based dispersive
liquid–liquid microextraction. GC-FID, gas chromatography with flame ionization detector; GC-MS, gas chromatography coupled mass spectrometry; LC-UV, liquid
chromatography with ultraviolet detector; LC-DAD, liquid chromatography with diode array detector.
10 D. G. BERLATO ET AL.
analyze TCAs and their respective metabolites in whole
blood and plasma samples at low concentrations similar to
those achieved by GC-MS and LC-MS (Ito et al. 2011;
Mohebbi et al. 2019; Fisichella et al. 2015) employing a LC-
DAD. Therefore, this is the first method with full validation
following international validation guidelines (UNODC and
SWGTOX) (UNODC (United Nations Office on Drugs and
Crime) 2009; SWGTOX (Scientific Working Group for Forensic
Toxicology) 2013) using DLLME in whole blood samples for
TCAs employing LC-DAD.
Whole blood samples for toxicological analysis allow
greater versatility for drug determination, as it is feasible
to apply a method to antemortem and postmortem sam­
ples (Peliç~ao et al. 2018). Therefore, Amitai and collabora­
tors (Amitai et al. 1993) described a red blood cells/plasma
ratio for TCAs, which AMI and IMI showed < 1, DOX is
close to 1 and the other are > 1 for this ratio, respectively.
Similarly, Montenarh and coauthors (Montenarh et al.
2014) found higher values of AMI in whole blood, while
DES showed lower levels of recovery when compared to
serum/plasma.
Based on this information, this indicates that whole
blood samples should be the preferred biological matrix for
the determination of TCAs (Amitai et al. 1993; Titier et al.
2007; Montenarh et al. 2014; De Boeck et al. 2018).
However, therapeutic levels of these substances are still
based on plasma samples as well as clinical situations such
as hospital laboratories employ this biological matrix to
determine TCAs in antemortem cases (Perry et al. 1994;
Lundstrøm et al. 2022). Thus, the purpose methodology was
validated with success for plasma samples and can be used
for the determination of these analytes in this biological
fluid. So, an analytical methodology with full validation for
both samples improves the capacity of TCAs analysis as
well as diversifies the scope of matrices in suspected expos­
ition for these molecules.
Real case applications
Real case 1
Whole blood sample from a 17-year-old female was sent to
the laboratory. The patient arrived at the hospital with sus­
pected drug poisoning. An immunochromatographic test
was performed, and it was detected methamphetamine and
TCAs. The purpose methodology determined AMI and NOR
in whole blood samples with 68.45 ng/mL and 31.60 ng/mL,
respectively (Figure 3C).
Real case 2
A 23-year-old female was a volunteer to participate in this
research. The volunteer informed that she uses the dosage
of 12.5 mg of AMI and 50 mg of alprazolam to treat temporo­
mandibular disorder. Every morning, she takes her medica­
tion during breakfast. The whole blood sample was
collected, stored at 4 � C for 24 h, and then submitted to the
proposed methodology. It was found 19.33 ng/mL of AMI, as
demonstrated in the Figure 3D.
Real case 3
Postmortem whole blood sample with the suspected pres­
ence of TCAs was submitted to the proposed DLLME. It was
detected IMI and AMI as demonstrated in Figure 3E.
However, below their LQ (30 and 10 ng/mL, respectively).
Real case 4
A plasma sample from a patient from HUSM with suspected
drug poisoning from different classes of medicines, including
exposure to TCAs, was referred to the laboratory. AMI was
detected in the plasma sample, however, below the LQ
(10 ng/mL). An aliquot of the same plasma sample was ana­
lyzed in GC-MS using the DLLME proposal in order to iden­
tify the analyte. Chromatographic conditions and respective
chromatograms are described in Figure 3H and J.
Real case discussion
In real case 1, it was measured AMI and NOR (68.45 ng/mL
and 31.60 ng/mL, respectively) in whole blood sample. The
patient arrived at the hospital, and the whole blood sample
was collected for toxicological analysis 24 h after hospital
intervention. The patient admitted the consumption of AMI
as a psychotherapy drug according to medical prescription.
However, the patient did not describe the posology of AMI.
According to the related from real case 2, the dose of
AMI (12.5 mg/day) is used for the beginning of the temporo­
mandibular disorder treatment. This is considered a lower
dose than that employed at the end of this treatment (75-
100 mg/day) and even smaller than the posology for depres­
sion treatment (25- 300 mg/day) (Diaz et al. 2020). The whole
blood sample was collected from the volunteer 1 h after the
medication was taken. According to a study by Garland
(Garland 1977), oral ingestion of 50 mg of AMI in volunteers
resulted in a peak plasma concentration of 20-40 ng/mL after
2-4 h while, while Edelbroek and collaborators (Edelbroek
et al. 1984) determined serum level between 50-250 ng/mL
after ingestion of 150 mg of AMI. So, the low level of AMI
found in the whole blood sample (19.33 ng/mL) can be
explained by due to the low dosage adopted by the volun­
teer and the period between AMI ingestion and sample
collection.
In the real case 3, postmortem whole blood sample with
the suspected presence of TCAs was submitted to the pro­
posed DLLME. There is no information in this specific case
on the consumption of TCAs, sample collection or storage
time, except the toxicological analysis performed by LC-MS.
However, the proposed methodology was able to detect IMI
and AMI.
Real case 4 demonstrated that AMI concentration was
below the LQ (10 ng/mL). For toxicological analysis, plasma
sample was collected 48 h after hospitalization patient, and
the immunochromatographic test showed negative results
for all drugs, including TCAs. However, the patient’s family
admitted her consumption of TCAs for depression treatment.
Based on the related, toxicological analysis was performed.
However, considering the delay between hospital admission
 TOXICOLOGY MECHANISMS AND METHODS 11

Figure 3. Chromatograms were obtained by the DLLME and LC-DAD and GC-MS analysis of whole blood and plasma samples. AMI, amitriptyline (RT: 13.13 min);
DES, desipramine (RT: 11.05 min); DOX, doxepine (RT: 4.85 min); IMI, imipramine (RT: 9.83 min); is, internal standard (RT: 3.53 min); nor, nortriptyline (RT: 14.55 min).
RT, retention time. A-H, LC-DAD analysis; I-J, GC-MS analysis. (A) Blank whole blood sample spiked with 200 ng/mL of is. (B) Blank whole blood sample spiked with
200 ng/mL of is while 100, 200 and 400 ng/mL for target analytes. (C) Real sample (case 1) containing AMI and NOR in whole blood sample with 68.45 ng/mL and
31.60 ng/mL, respectively. (D) Real sample (case 2) containing 19.33 ng/mL of AMI in whole blood sample. (E) Real sample (case 3) detected IMI and AMI, however,
below respective LQs in postmortem whole blood sample. (F) Blank plasma sample spiked with 200 ng/mL of is. (G) Blank plasma sample spiked with 200 ng/mL of
is while 100, 200 and 400 ng/mL for target analytes. (H) Real sample (case 4) detected AMI, however, below LQ in the plasma sample. (I) Blank plasma sample
spiked with 200 ng/mL of AMI (retention time: 11.32 min). (J) Real sample (case 4) detected AMI (retention time: 11.32 min).

and sample collection, it was not possible to determine
whether the patient was intoxicated with TCAs, in spite of
the detection of AMI in plasma samples.
During the development of the proposed DLLME method­
ology, it was observed difficulty in the dispersion of solvents
in the whole blood sample because of the greater complex­
ity of this biological matrix if compared to plasma samples.
Application of the DLLME method demonstrated different
behavior between antemortem and postmortem whole
blood for TCAs extraction. Experimentally, postmortem whole
blood samples showed lower viscosity when compared to
antemortem samples, which allowed a more prominent
cloud effect characteristic of DLLME. Consequently, extraction
capacity is higher. Only Fisichella and collaborators (Fisichella
et al. 2015) evaluated whole blood samples (50 real cases)
from postmortem cases employing DLLME and LC-MS.
Considering the high capacity of TCAs to bind to plasma
proteins and erythrocytes, the physiological changes caused
by the postmortem phenomenon can release these drugs as
well as decrease the viscosity of whole blood samples.
12 D. G. BERLATO ET AL.
However, further studies should be performed to confirm
this hypothesis. Until this moment, this is the first DLLME
study involving antemortem and postmortem whole blood
samples. Also, the proposed DLLME methodology was fully
validated, and it is able to determine TCAs in whole blood
and plasma samples with success for toxicological analysis.
Conclusion
Proposed DLLME methodology was able to determine TCAs
in whole blood and plasma samples using LC-DAD, and it
proved to be a simple, reliable, robust, and reproducible
method that can be used in toxicology laboratories. Thus, an
analytical approach with full validation for both samples
improves the capacity of TCAs analysis as well as diversifies
the scope of matrices in suspected exposition for these mol­
ecules. Considering the possibilities of DLLME and TCAs
chemical aspects, further studies should be conducted to val­
idate the technique in other biological matrices and chroma­
tographic equipment.
Acknowledgment
The authors would like to acknowledge the financial support from the
Coordination of Improvement of Personal Higher Education – Brazil
(CAPES – Finance Code 001) for the scholarship provided to Dener
Gomes Berlato and Geovane de Almeida Saldanha.
Disclosure statement
The authors declare that they have no known competing financial inter­
ests or personal relationships that could have appeared to influence the
work reported in this paper.
Funding
Coordenaç~ao de Aperfeiçoamento de Pessoal de N�ıvel Superior.
References
Almeida AM, Castel-Branco MM, Falc~ao AC. 2002. Linear regression for
calibration lines revisited: weighting schemes for bioanalytical meth­
ods. J Chromatogr B Analyt Technol Biomed Life Sci. 774(2):215–222.
doi: 10.1016/s1570-0232(02)00244-1.
Alves C, Fernandes C, Dos Santos Neto AJ, Rodrigues JC, Costa Queiroz
ME, Lanças FM. 2006. Optimization of the SPME parameters and its
online coupling with HPLC for the analysis of tricyclic antidepressants
in plasma samples. J Chromatogr Sci. 44(6):340–346. doi: 10.1093/
chromsci/44.6.340.
Amitai Y, Erickson T, Kennedy EJ, Leikin JB, Hryhorczuk DO, Noble J,
Hanashiro PK, Frischer H. 1993. Tricyclic antidepressants in red cells
and plasma: correlation with impaired intraventricular conduction in
acute overdose. Clin Pharmacol Ther. 54(2):219–227. doi: 10.1038/clpt.
1993.133.
Armbruster DA, Pry T. 2008. Limit of blank, limit of detection and limit
of quantitation. Clin. Biochem. Rev. 29(Suppl 1):S49–S52.
Bazregar M, Rajabi M, Yamini Y, Saffarzadeh Z, Asghari A. 2016. Tandem
dispersive liquid-liquid microextraction as an efficient method for
determination of basic drugs in complicated matrices. J Chromatogr
A. 1429:13–21. doi: 10.1016/j.chroma.2015.11.087.
Borges EM, Goraieb K, Collins CH. 2012. O Desafio de analisar solutos
b�asicos por cromatografia l�ıquida em modo reverso: algumas
alternativas para melhorar as separaço ~es. Qu�ım Nova. 35(5):993–1003.
doi: 10.1590/S0100-40422012000500024.
Carasek E, Bernardi G, Morelli D, Merib J. 2021. Sustainable green sol­
vents for microextraction techniques: recent developments and appli­
cations. J Chromatogr A. 1640:461944. doi: 10.1016/j.chroma.2021.
461944.
Chen X, Zheng S, Le J, Qian Z, Zhang R, Hong Z, Chai Y. 2017.
Ultrasound-assisted low-density solvent dispersive liquid-liquid micro­
extraction for the simultaneous determination of 12 new antidepres­
sants and 2 antipsychotics in whole blood by gas chromatography-
mass spectrometry. J Pharm Biomed Anal. 142:19–27. doi: 10.1016/j.
jpba.2017.04.032.
De Bairros AV, de Almeida RM, Pantale~ao L, Barcellos T, e Silva SM,
Yonamine M. 2015. Determination of low levels of benzodiazepines
and their metabolites in urine by hollow-fiber liquid-phase microex­
traction (LPME) and gas chromatography-mass spectrometry (GC-MS).
J Chromatogr B Analyt Technol Biomed Life Sci. 975:24–33. doi: 10.
1016/j.jchromb.2014.10.040.
De Boeck M, Dehaen W, Tytgat J, Cuypers E. 2018. Ionic liquid-based
liquid-liquid microextraction for benzodiazepine analysis in postmor­
tem blood samples. J Forensic Sci. 63(6):1875–1879. doi: 10.1111/
1556-4029.13778.
Diaz D, Vallejos A, Torres S, Hern�andez W, Calvache J, Merch�an J, Latorre
G, Maldonado L. 2020. Detection of potential risks in the prescription
of tricyclic antidepressants through an online clinical alert system.
Rev Colomb Psiquiat. 49(1):9–14.
Donaldson AE, Lamont IL. 2013. Biochemistry changes that occur after
death: potential markers for determining post-mortem interval. PLoS
One. 8(11):e82011. doi: 10.1371/journal.pone.0082011.
Dos Santos MF, Ferri CC, Seulin SC, Leyton V, Pasqualucci CAG, Mun ~oz
DR, Yonamine M. 2014. Determination of antidepressants in whole
blood using hollow-fiber liquid-phase microextraction and gas chro­
matography–mass spectrometry. Forensic Toxicol. 32(2):214–224. doi:
10.1007/s11419-014-0226-9.
Edelbroek PM, Zitman FG, Schreuder JN, Rooymans HG, de Wolff FA.
1984. Amitriptyline metabolism in relation to antidepressive effect.
Clin Pharmacol Ther. 35(4):467–473. doi: 10.1038/clpt.1984.61.
Fern�andez P, Regenjo M, Ares A, Fern�andez AM, Lorenzo RA, Carro AM.
2019. Simultaneous determination of 20 drugs of abuse in oral fluid
using ultrasound-assisted dispersive liquid-liquid microextraction. Anal
Bioanal Chem. 411(1):193–203. doi: 10.1007/s00216-018-1428-5.
Fern�andez P, Taboada V, Regenjo M, Morales L, Alvarez I, Carro AM,
Lorenzo RA. 2016. Optimization of ultrasound assisted dispersive
liquid-liquid microextraction of six antidepressants in human plasma
using experimental design. J Pharm Biomed Anal. 124:189–197. doi:
10.1016/j.jpba.2016.02.041.
Fisichella M, Odoardi S, Strano-Rossi S. 2015. High-throughput dispersive
liquid/liquid microextraction (DLLME) method for the rapid determin­
ation of drugs of abuse, benzodiazepines and other psychotropic
medications in blood samples by liquid chromatography-tandem
mass spectrometry (LC-MS/MS) and application to forensic cases.
Microchem J. 123:33–41. doi: 10.1016/j.microc.2015.05.009.
Flanagan RJ, Taylor A, Watson ID, Whelpton R. 2007. Sample preparation.
In: flanagan RJ, Cuypers E, Maurer HH, Whelpton R, editors.
Fundamentals of analytical toxicology. Chichester: Wiley; p. 49–94.
Franco de Oliveira S, Zucoloto AD, de Oliveira CDR, Hernandez EMM,
Fruchtengarten LVG, de Oliveira TF, Yonamine M. 2019. A fast and
simple approach for the quantification of 40 illicit drugs, medicines,
and pesticides in blood and urine samples by UHPLC-MS/MS. J Mass
Spectrom. 54(7):600–611. doi: 10.1002/jms.4369.
Fu S, Lewis J, Wang H, Keegan J, Dawson M. 2010. A novel reductive
transformation of oxazepam to nordiazepam observed during enzym­
atic hydrolysis. J Anal Toxicol. 34(5):243–251. doi: 10.1093/jat/34.5.243.
Garland WA. 1977. Quantitative determination of amitriptyline and its
principal metabolite, nortriptyline, by GLC-chemical ionization mass
spectrometry. J Pharm Sci. 66(1):77–81. doi: 10.1002/jps.2600660119.
Ito R, Ushiro M, Takahashi Y, Saito K, Ookubo T, Iwasaki Y, Nakazawa H.
2011. Improvement and validation the method using dispersive
liquid-liquid microextraction with in situ derivatization followed by
gas chromatography-mass spectrometry for determination of tricyclic

antidepressants in human urine samples. J Chromatogr B Analyt
Technol Biomed Life Sci. 879(31):3714–3720. doi: 10.1016/j.jchromb.
2011.10.012.
Karpinska J, Starczewska B. 2002. Simultaneous LC determination of
some antidepressants combined with neuroleptics. J Pharm Biomed
Anal. 29(3):519–525. doi: 10.1016/s0731-7085(02)00097-3.
Liebelt EL, Francis PD. 2002. Chapter 57 – Cyclic antidepressants. In:
Goldfrank LR, Flomenbaum NR, Lewin NA, Howland MA, Hoffman RS,
Nelson LS, editors. Goldfrank’s toxicological emergencies, 7th ed. USA:
McGraw-Hill; p. 847–864.
Lundstrøm NH, Holgersen NK, Haastrup MB. 2022. The effect of smoking
on the plasma concentration of tricyclic antidepressants: a systematic
review. Acta Neuropsychiatr. 34(1):1–9. doi: 10.1017/neu.2021.28.
Malfar�a WR, Bertucci C, Costa Queiroz ME, Dreossi Carvalho SA, Pires
Bianchi MdL, Cesarino EJ, Crippa JA, Costa Queiroz RH. 2007. Reliable
HPLC method for therapeutic drug monitoring of frequently pre­
scribed tricyclic and nontricyclic antidepressants. J Pharm Biomed
Anal. 44(4):955–962. doi: 10.1016/j.jpba.2007.04.005.
Manousi N, Samanidou VF. 2020. Recent advances in the HPLC analysis
of tricyclic antidepressants in bio-samples. Mini Rev Med Chem. 20(1):
24–38. doi: 10.2174/1389557519666190617150518.
Martins ML, Primel EG, Caldas SS, Prestes OD, Adaime MB, Zanella R.
2012. Microextraç~ao L�ıquido-L�ıquido Dispersiva (DLLME): fundamentos
e aplicaço~es. SC. 4(1):29–45. doi: 10.4322/sc.2012.004.
Meatherall R. 1994. Optimal enzymatic hydrolysis of urinary benzodi­
azepine conjugates. J Anal Toxicol. 18(7):382–384. doi: 10.1093/jat/18.
7.382.
Mercolini L, Mandrioli R, Finizio G, Boncompagni G, Raggi MA. 2010.
Simultaneous HPLC determination of 14 tricyclic antidepressants and
metabolites in human plasma. J Sep Sci. 33(1):23–30. doi: 10.1002/
jssc.200900493.
Mohebbi A, Farajzadeh MA, Nemati M, Sarhangi N, A, Mogaddam MR.
2019. Development of green sodium sulfate-induced solidification of
floating organic droplets-dispersive liquid phase microextraction
method: application to extraction of four antidepressants. Biomed
Chromatogr. 33(11):e4642.
Mohebbi A, Farajzadeh MA, Yaripour S, Afshar Mogaddam MR. 2018.
Determination of tricyclic antidepressants in human urine samples by
the three-step sample pretreatment followed by HPLC-UV analysis: an
efficient analytical method for further pharmacokinetic and forensic
studies. Excli J. 17:952–963.
Mohebbi A, Yaripour S, Farajzadeh MA, Afshar Mogaddam MR. 2018b.
Combination of dispersive solid phase extraction and deep eutectic
solvent-based air-assisted liquid-liquid microextraction followed by
gas chromatography-mass spectrometry as an efficient analytical
method for the quantification of some tricyclic antidepressant drugs
in biological fluids. J Chromatogr A. 1571:84–93. doi: 10.1016/j.
chroma.2018.08.022.
Montenarh D, Hopf M, Maurer HH, Schmidt P, Ewald AH. 2014. Detection
and quantification of benzodiazepines and Z-drugs in human whole
blood, plasma, and serum samples as part of a comprehensive multi-
analyte LC-MS/MS approach. Anal Bioanal Chem. 406(3):803–818. doi:
10.1007/s00216-013-7513-x.
Nabil AA, Nouri N, Farajzadeh MA. 2015. Determination of three antide­
pressants in urine using simultaneous derivatization and temperature-
assisted dispersive liquid-liquid microextraction followed by gas chro­
matography-flame ionization detection. Biomed Chromatogr. 29(7):
1094–1102. doi: 10.1002/bmc.3396.
Peliç~ao FA, De Martinis BS, Pissinate JF. 2018. Cap�ıtulo 20 – Amostras
Biolo �gicas em An�alises Forenses: matrizes Usuais (Urina e Sangue). In:
Dorta DJ, Yonamine M, Costa JL, Martinis BS, editors. Toxicologia
Forense. S~ao Paulo: Blucher; p. 381–392.
Perry PJ, Zeilmann C, Arndt S. 1994. Tricyclic antidepressant concentra­
tions in plasma: an estimate of their sensitivity and specificity as a
predictor of response. J Clin Psychopharmacol. 14(4):230–240.
Queiroz RH, Lanchote VL, Bonato PS, de Carvalho D. 1995. Simultaneous
HPLC analysis of tricyclic antidepressants and metabolites in plasma
samples. Pharm Acta Helv. 70(2):181–186. doi: 10.1016/0031-
6865(95)00019-6.
TOXICOLOGY MECHANISMS AND METHODS 13
Saldanha GA, Pacheco ALB, Pego AMF, Bairros AV. 2022. Analysis of ben­
zodiazepines in plasma samples by DLLME and LC-DAD: critical
aspects, flaws and issues encountered – a discussion. Quim Nova. 45:
1–8.
Samanidou VF, Nika MK, Papadoyannis IN. 2007. Development of an
HPLC method for the monitoring of tricyclic antidepressants in bio­
fluids. J Sep Sci. 30(15):2391–2400. doi: 10.1002/jssc.200700142.
Santos MG, Tavares IM, Barbosa AF, Bettini J, Figueiredo EC. 2017.
Analysis of tricyclic antidepressants in human plasma using online-
restricted access molecularly imprinted solid phase extraction fol­
lowed by direct mass spectrometry identification/quantification.
Talanta. 163:8–16. doi: 10.1016/j.talanta.2016.10.047.
Seidi S, Yamini Y, Rezazadeh M. 2013. Combination of electromembrane
extraction with dispersive liquid-liquid microextraction followed by
gas chromatographic analysis as a fast and sensitive technique for
determination of tricyclic antidepressants. J Chromatogr B Analyt
Technol Biomed Life Sci. 913-914:138–146. doi: 10.1016/j.jchromb.
2012.12.008.
Shamsipur M, Mirmohammadi M. 2014. High performance liquid chroma­
tographic determination of ultra traces of two tricyclic antidepressant
drugs imipramine and trimipramine in urine samples after their dis­
persive liquid-liquid microextraction coupled with response surface
optimization. J Pharm Biomed Anal. 100:271–278. doi: 10.1016/j.jpba.
2014.08.008.
Silveira GO, Lourenço FR, Fonseca Pego AM, Guimar~aes Dos Santos R,
Rossi GN, Hallak JEC, Yonamine M. 2021. Essential oil-based dispersive
liquid-liquid microextraction for the determination of N,N-dimethyl­
tryptamine and b-carbolines in human plasma: a novel solvent-free
alternative. Talanta. 225:121976. doi: 10.1016/j.talanta.2020.121976.
SINITOX (Sistema Nacional de Informaço ~es To �xico-Farmacolo �gicas). 2020.
Evolution of registered cases of human poisoning by toxic agent (in
Portuguese). [accessed 2023 July 10] Available from: https://sinitox.
icict.fiocruz.br/sites/sinitox.icict.fiocruz.br/files//Brasil3_1.pdf.
Skopp G. 2004. Preanalytic aspects in postmortem toxicology. Forensic
Sci Int. 142(2-3):75–100. doi: 10.1016/j.forsciint.2004.02.012.
SWGTOX (Scientific Working Group for Forensic Toxicology). 2013.
Scientific Working Group for Forensic Toxicology (SWGTOX) standard
practices for method validation in forensic toxicology. J Anal Toxicol.
37:452–474.
Tabani H, Shokri A, Tizro S, Nojavan S, Varanusupakul P, Alexovi�c M.
2019. Evaluation of dispersive liquid-liquid microextraction by cou­
pling with green-based agarose gel-electromembrane extraction: an
efficient method to the tandem extraction of basic drugs from bio­
logical fluids. Talanta. 199:329–335. doi: 10.1016/j.talanta.2019.02.078.
Theurillat R, Thormann W. 1998. Monitoring of tricyclic antidepressants in
human serum and plasma by HPLC: characterization of a simple,
laboratory developed method via external quality assessment. J Pharm
Biomed Anal. 18(4-5):751–760. doi: 10.1016/s0731-7085(98)00263-5.
Titier K, Castaing N, Le-D� eodic M, Le-Bars D, Moore N, Molimard M.
2007. Quantification of tricyclic antidepressants and monoamine oxi­
dase inhibitors by high-performance liquid chromatography-tandem
mass spectrometry in whole blood. J Anal Toxicol. 31(4):200–207. doi:
10.1093/jat/31.4.200.
Tomai P, Gentili A, Curini R, Gottardo R, Franco Tagliaro, Fanali S. 2021.
Dispersive liquid-liquid microextraction, an effective tool for the
determination of synthetic cannabinoids in oral fluid by liquid chro­
matography-tandem mass spectrometry. J Pharm Anal. 11(3):292–298.
doi: 10.1016/j.jpha.2020.11.004.
Uddin MN, Samanidou VF, Papadoyannis IN. 2008. Development and val­
idation of an HPLC method for the determination of benzodiazepines
and tricyclic antidepressants in biological fluids after sequential SPE. J
Sep Sci. 31(13):2358–2370. doi: 10.1002/jssc.200800079.
UNODC (United Nations Office on Drugs and Crime). 2009. Guidance for
de validation of analytical methodology and calibration of equipment
used for testing of illicit drugs in seized materials and biological
specimens. [accessed 2020 December 20] Available from: https://
www.unodc.org/documents/scientific/validation_E.pdf.
Vaghar-Lahijani G, Saber-Tehrani M, Aberoomand-Azar P, Soleimani M.
2018. Extraction and determination of two antidepressant drugs in
14 D. G. BERLATO ET AL.
human plasma by dispersive liquid–liquid microextraction–HPLC. J
Anal Chem. 73(2):145–151. doi: 10.1134/S1061934818020144.
Vander Heyden Y, Nijhuis A, Smeyers-Verbeke J, Vandeginste BG, Massart
DL. 2001. Guidance for robustness/ruggedness tests in method valid­
ation. J Pharm Biomed Anal. 24(5-6):723–753. doi: 10.1016/s0731-
7085(00)00529-x.
Xiong C, Ruan J, Cai Y, Tang Y. 2009. Extraction and determination of
some psychotropic drugs in urine samples using dispersive liquid-
liquid microextraction followed by high-performance liquid chroma­
tography. J Pharm Biomed Anal. 49(2):572–578. doi: 10.1016/j.jpba.
2008.11.036.
Yazdi AS, Razavi N, Yazdinejad SR. 2008. Separation and determin­
ation of amitriptyline and nortriptyline by dispersive liquid-liquid
microextraction combined with gas chromatography flame ioniza­
tion detection. Talanta. 75(5):1293–1299. doi: 10.1016/j.talanta.
2008.01.039.