Article

Development and Validation of a Single Step GC/MS Method

for the Determination of 41 Drugs and Drugs of Abuse in
Postmortem Blood
Amvrosios Orfanidis 1,2 , Adamantios Krokos 3 , Orthodoxia Mastrogianni 4 , Helen Gika 1,2 ,
Nikolaos Raikos 1 and Georgios Theodoridis 2,3, *

1 Laboratory of Forensic Medicine and Toxicology, School of Medicine, Aristotle University of Thessaloniki,
University Campus, 54124 Thessaloniki, Greece; [email protected] (A.O.); [email protected] (H.G.);
[email protected] (N.R.)
2 Centre for Interdisciplinary Research and Innovation, Bioanalysis and Omics Lab, CIRI-AUTH B1.4,
Aristotle University of Thessaloniki, Thermi, 57001 Thessaloniki, Greece
3 Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,
University Campus, 54124 Thessaloniki, Greece; [email protected]
4 Laboratory of Toxicology, Forensic Service of Ministry of Justice, 56334 Thessaloniki, Greece;
[email protected]
* Correspondence: [email protected]; Tel.: +30-(23)-1099-7718

Abstract: A toxicology laboratory often receives a high number of samples from cases (autopsies or
clinical) that may require the quick delivery of trustworthy, accurate results. Thus, there is a great
need for a fast and reliable method that is capable of identifying and determining a large number of
drugs and drugs of abuse in biological matrices, and especially in blood. In the present study, we
Citation: Orfanidis, A.; Krokos, A.;
describe the development of a fast and simple gas chromatography–mass spectrometry (GC-MS)
Mastrogianni, O.; Gika, H.; Raikos,
method for the determination of 41 drugs and drugs of abuse (DOA) in blood. Sample pre-treatment
N.; Theodoridis, G. Development and
Validation of a Single Step GC/MS
by alkaline liquid–liquid extraction (LLE) was studied through the utilization of different solvents and
Method for the Determination of 41 solvent-to-sample ratios (v/v), which aimed to achieve a greater extraction efficiency and detection
Drugs and Drugs of Abuse in sensitivity with a decreased need for large sample volumes. Butyl acetate with a sample-to-solvent
Postmortem Blood. Forensic Sci. 2022, ratio of 4:1 (1 mL blood: 0.25 mL butyl acetate) was the most efficient. The method was validated for
2, 473–491. https://doi.org/10.3390/ all analytes, and the evaluation parameters were within the acceptance criteria. The coefficient of
forensicsci2030035 determination (R2) was between 0.9934 and 1, the limits of detection (LODs) ranged between 1 ng/mL
Academic Editors: Ricardo
and 113 ng/mL, and the limits of quantification (LOQs) were between 4 ng/mL and 375 ng/mL for
Dinis-Oliveira, Francisca Alves all analytes. The determinations were accurate (accuracy% from 84% to 114%) and precise (RSD%
Cardoso and Pier Matteo Barone from 0.66% to 14.8% for low concentrations). Deconvolution Reporting Software (DRS) for GC-MS
was optimized and applied for data analysis to enhance the identification potential, thereby avoiding
Received: 10 June 2022
false identifications (false positives) and increased productivity. The NIST Automated Mass Spectral
Accepted: 1 July 2022
Deconvolution and Identification Software (AMDIS) and the analytical utility Retention Time Lock
Published: 7 July 2022
(RTL) Database Library assisted in data evaluation. The method was applied to 89 postmortem cases
Publisher’s Note: MDPI stays neutral (history of mental disorders and use of psychiatric pharmaceuticals) in which diazepam (0.13 to
with regard to jurisdictional claims in
4.34 µg/mL), citalopram (0.04 to 0.24 µg/mL), alprazolam (0.01 to 0.12 µg/mL), olanzapine (0.009 to
published maps and institutional affil-
0.083 µg/mL), mirtazapine (0.01 to 0.33 µg/mL), venlafaxine (0.006 to 0.92 µg/mL), haloperidol
iations.
(0.007 to 0.13 µg/mL), and zolpidem (0.01 to 0.16 µg/mL) were successfully quantitated.

Keywords: GC-MS; LLE; DRS; blood; drugs of abuse

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and 1. Introduction
conditions of the Creative Commons In the framework of toxicological analysis, the main groups of drugs and drugs of
Attribution (CC BY) license (https:// abuse (DOA) that have to be investigated include anesthetics, benzodiazepines, antipsy-
creativecommons.org/licenses/by/ chotics, antiepileptics, opiates, cocaine, cannabinoids, and amphetamines [1]. Most of them
4.0/).

Forensic Sci. 2022, 2, 473–491. https://doi.org/10.3390/forensicsci2030035 https://www.mdpi.com/journal/forensicsci

Forensic Sci. 2022, 2 474

have therapeutic applications, while some are used almost exclusively by people addicted
to drugs.
In the literature, there is a large number of reported methods for the determination of
pharmaceuticals and drugs in biological fluids [2–11], and especially in blood [12–19], for the
purposes of a toxicology lab, while the routinely applied methods include either immuno-
chemical methods or methods based on hyphenated techniques, such as GC-MS and liquid
chromatography–mass spectrometry (LC-MS). Most of them focus on the identification of
specific categories of drugs, such as opiates, benzodiazepines, etc. Immunochemical meth-
ods provide quick results, as they do not require sample pretreatment, but confirmation
with a sensitive and selective chromatographic technique is still required; a false positive
result due to cross reactivity with other compounds is possible, and only qualitative results
can be acquired [20,21]. Furthermore, the analysis of different groups of analytes necessi-
tates the use of different immunoassay kits, a fact that increases the cost and complexity of
the analytical process.
The scientific community takes efforts to develop methods able to detect and quantitate
the largest possible number of medicines and drugs. The major aim is the development of a
quick, effective, and sensitive method that would replace immunochemical analysis. The most
common methods applied are based on LC-MS/MS or LC-TOF/MS instrumentation due
to the large number of compounds that can be easily quantitated without time-consuming
sample preparation; usually only protein precipitation is needed [22–24]. The main drawback
of these techniques is the limitation in identifying unknown compounds, as commercial
mass spectral libraries are still under development and only in-house libraries can aid.
Gas chromatography coupled with mass spectrometry (GC-MS) is probably the most
widely accepted technique in forensic toxicology, as it has the advantage of offering drug
identification based on the extended libraries that exist and, at the same time, it can provide
sensitive and accurate determinations. Thus, GC-MS-based methods are considered highly
reliable for the detection and determination of drugs in biological samples, and specifically
in blood. The most notable studies of the last decade that focus on the determination of
drugs in biological fluids by GC-MS all have one common feature that concerns the sample
preparation step. Either solid phase extraction [25–28] or liquid–liquid extraction [29–31]
is applied, and they all require an evaporation step and the majority of them make use
of large volumes of organic solvents. In addition, in almost all cases, the evaporation
step is followed by a derivatization step in order to increase sensitivity [32–37]. Recently,
QuEChERS (Quick Easy Cheap Effective Rugged Safe) methodology has also been applied
for toxicological analysis purposes, however evaporation is still required [38,39].
Here, we report the development, validation, and application of a GC-MS method
for the separation and determination of 41 pharmaceuticals and drugs from different
classes, which includes a single and quick sample preparation step, thereby avoiding the
evaporation and derivatization processes. The developed method has been successfully
applied in a large number of samples and interchangeably for the investigation of several
cases of clinical and forensic interest in the Laboratory of Forensic Toxicology of the
University of Thessaloniki and the Laboratory of Forensic Service of Ministry of Justice
of Thessaloniki; the results obtained in each laboratory were comparable, thus it has been
proven a valuable tool that greatly supports and advances the routine analysis of the two
forensic laboratories.

2. Experimental
2.1. Reagents and Instrumental Conditions
All solvents were of HPLC grade. Methanol was obtained from Fisher Scientific,
(Leicestershire, UK); and butyl acetate, hexane, isooctane, 4-methyl-2-pentanone, and
1-butanol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ethyl acetate was
supplied from Penta (Livingston, NJ, USA). All the reference standards had more than
98.5% purity and were purchased from Lipomed AG (Arlesheim, Switzerland), except for
amantadine, moclobemide, propofol, ropivacaine, biperiden, olanzapine, haloperidol, and
Forensic Sci. 2022, 2 475

nordazepam-d5 (Internal Standard), which were from LGC Standards (Teddington, UK).
Human blood samples were obtained after informed consent from healthy donors and
were screened by GC-MS for the presence of the investigated drugs and DOA.
Analysis by GC-MS was performed on an Agilent Technologies 7890A GC, combined
with a 5975C inert XL EI/CI MSD with a Triple-Axis Detector (Agilent Technologies, Santa
Clara, CA, USA) equipped with a CTC autosampler. The method had a total duration of
21 min with the following temperature program: Initially, 120 ◦ C for 1 min, then increased
to 300 ◦ C with a rate of 15 ◦ C/min, and followed by a backflush program at 300 ◦ C for
10 min. The injection of 1 µL of sample was performed through a PTV injector operating
between 200 ◦ C to 320 ◦ C. The mass spectrometer (MS) was operated at electron impact
ionization mode (EI, 70 eV) in full scan mode at a mass range from 40 to 500 amu. The
initial pressure for this instrument was 19.418 psi with a set flow of 1.2 mL/min. The
second GC-MS system used was also an Agilent Technologies 7890A GC, combined with
an 5975C inrtXL EI/CI MSD with a Triple-Axis Detector (Agilent Technologies, Santa
Clara, CA, USA) equipped with an Agilent G4513A autosampler. The GC-MS analysis
employed an Optima-5-ms, 30 m × 250 µm × 0.25 µm (Scitech Scientific, West Perth,
Western Australia) column. The method had a total duration of 31 min with the following
temperature program: Initially, 120 ◦ C and increasing to 300 ◦ C (15 ◦ C/min). An injection
of 1 µL of sample was done through a split-splitless injector operating at 300 ◦ C. The
mass spectrometer (MS) was operated at electron impact ionization mode (EI, 70 eV) and
the collected mass range was 40–500 amu. The applied flow was also 1.2 mL/min for
this instrument in order to achieve common retention times with an initial pressure at
14.012 psi. On both laboratories GC separations, a 30 m Agilent J&W HP-5ms capillary
column with a film thickness of 0.25 µm and an i.d. of 0.25 µm were performed. Backflash
was performed with a 1.5 m deactivated Agilent column with a film thickness of 0.18 mm.
Both instruments also had the same MS source temperature (230 ◦ C) and MS quadrupole
temperature (150 ◦ C).
For the identification and quantification of the analytes, a full scan mode was selected;
the confirmation ions and the analyte’s retention times (RT) are presented in Table 1 with
the quantification ion for each one shown in bold.

Table 1. Retention times and target ions for the 41 drugs and the IS.

No Compound RT (min) Target Ion Qualifier Ions

1 Methamphetamine 3.18 58 91 65
2 Amantadine 3.69 94 151 108
3 Propofol 4.45 163 178 117
4 MDA 5.51 44 135 136
5 MDMA 5.96 58 135 77
6 MDEA 6.22 72 44 135
7 Bupropion 6.46 44 100 57
8 MBDB 6.65 72 135 89
9 Fluoxetine 8.42 44 104 162
10 Ketamine 8.49 209 180 182
11 Lidocaine 8.53 58 86 120
12 Tramadol 9.10 58 263 135
13 Phenobarbital 9.25 204 232 115
14 Venlafaxine 9.99 58 134 179
15 Methadone 10.33 72 294 309
16 Ropivacaine 10.71 126 84 127
17 Amitriptyline 10.73 58 275 30
18 Cocaine 10.77 82 182 303
19 Atropine 10.81 124 289 140
20 Nortriptyline 10.87 44 202 189
21 Moclobemide 11.13 100 139 113
22 Mirtazapine 11.14 195 208 180
23 Biperiden 11.29 98 218 55
Forensic Sci. 2022, 2 476

Table 1. Cont.

No Compound RT (min) Target Ion Qualifier Ions

24 Phenytoin 11.63 180 104 77
25 Sertaline 11.80 274 262 304
26 Citalopram 11.96 58 324 238
27 Codeine 11.97 299 162 115
28 Clomipramine 12.02 58 85 268
29 Diazepam 12.21 283 256 221
30 Chlorpromazine 12.56 58 318 86
31 Nordazepam 12.61 270 242 269
32 Midazolam 13.04 310 325 163
33 Flunitrazepam 13.14 312 285 266
34 7-AF 13.33 283 255 254
35 Fentanyl 13.72 245 146 189
36 Olanzapine 13.88 242 229 213
37 Zolpidem 14.26 235 307 219
38 Clozapine 14.88 243 256 192
39 Haloperidol 15.56 224 237 42
40 Alprazolam 15.47 204 279 308
41 Quetiapine 18.45 210 239 321
IS Nordazepam-D5 12.61 275 247 274

2.2. Preparation of Standard Solutions and QCs

Stock solutions of all analytes at 1.0 mg/mL were prepared in methanol. Working
solutions were prepared from stock by dilution with methanol. Drug-free blood samples
were used for all method optimization and validation assays. For calibration standards
and quality controls (QCs), 100 µL of the working standards were added to 1 mL of whole
blood after the addition of 25 µL of internal standard (IS). All solutions and QC samples
were stored at −20 ◦ C. Three groups of mixtures were prepared according to the studied
concentration levels.
In order to study the selectivity of the method, blood spiked with drugs and phar-
maceuticals that could be found in real samples were tested for interferences in the de-
termination of the 41 analytes. Thus, blood was spiked at concentrations of 0.125 µg/mL
for flunitrazepam and fentanyl; at 1.25 µg/mL for methamphetamine, propofol, MDA,
MDMA, ecgonine methyl ester, MDEA, MBDB, ketamine, lidocaine, methadone, amit-
ryptiline, nortriptyline, cocaine, atropine, sertraline, codeine, chlomipramine, diazepam,
clozapine, and haloperidol; at 0.4 µg/mL for chlorpromazine and ropivacaine; at 0.5 µg/mL
for bromazepam, nordazepam, citalopram, bupropion, fluoxetine, tramadol, mirtazapine,
biperiden, nordazepam-D5 (IS), midazolam, olanzapine, zolpidem, and alprazolam; and at
5.0 µg/mL for phenobarbital, carbamazepine, phenytoin, and amantadine.

2.3. Biological Samples

The blood samples were collected at the Forensic Service of Greek Ministry of Justice
in Thessaloniki during autopsy 14 to 20 h postmortem from subjects with a history of
pharmaceutical use for mental disorders. The obtained samples were screened in the
frame of the routine toxicological analysis and were analysed by the developed method
for quantification.

2.4. Sample Preparation

To select the most efficient LLE protocol, the extraction recoveries of the analytes
were evaluated using six different commonly used extraction solvents. More specifically,
butyl acetate, ethyl acetate, hexane, isooctane, 4-methyl-2-pentanone, and 1-butanol were
selected. The sample-to-solvent ratio was also evaluated in terms of recovery for the
optimum solvent. Recovery was assessed by the percentage peak ratio of the peak area of
Forensic Sci. 2022, 2 477

the analyte that was spiked before the extraction to the peak area of the standard solution of
the analyte at the same concentration. Finally, the selected applied protocol was as follows:
In 1 mL of blood sample, 25 µL of nordazepam-D5 (IS) solution (10 µg/mL) and 100 µL
of the spiking solution were added. Then, the sample was alkalized by adding 500 µL of a
saturated aqueous solution of K2 CO3 (pH = 12). Finally, 250 µL of butyl acetate were added
and the sample was shaken for two min following centrifugation at 6000× g for ten min.
Finally, 200 µL of the upper-organic phase was collected and 1 µL was directly injected into
the GC-MS system. Calibrators and controls were prepared using the same pretreatment
procedure. At the analysis of real samples, 100 µL of methanol solvent was added before
the extraction solvent.

2.5. Software
Data acquisition and analysis was performed by Agilent Chemstation. Data de-
convolution was performed by AMDIS (Automated Mass Spectral Deconvolution and
Identification Software), which aided in spectrum “cleaning” by correcting the spectral
skew and by determining a more accurate apex retention time (RT). A library search was
then performed either by MSD ChemStation or by AMDIS, combining retention time and
4-ion agreement with the processed spectra and, finally, the target peaks were recognized
among interferences and reported only if the quality match factor exceeds a preset value.
Retention time lock (RTL) was applied in order to avoid RT shifts due to a variety of
reasons such as column trimming, installation of new columns, or other routine procedures.
By this approach, RTs were comparable, and the results were more reproducible between
the two different laboratories. For RTL, an easily identifiable compound with symmetrical
eluting peaks at a crucial point of the chromatogram was chosen and was injected five
times with different pressures (±20%, ±10%, and target pressure). The RTL file was created
by the Chemstation software and used in every analysis. In the present study, lidocaine
was used as the RTL reference compound.
Deconvolution Reporting Software (DRS), which combines AMDIS and MSD Chem-
Station output, was used to aid the identification of co-eluting compounds and to improve
and accelerate the process of the identification of detected peaks.

3. Results
A chromatographic analysis of the 41 analytes was carried out over a 21-min run. All
the compounds of interest were separated efficiently in the first 18 min, as depicted in the
extracted ion chromatograms presented in Figure 1.
A 10-min backflush program at 300 ◦ C was applied after the thermal gradient provided
an adequate purging of the system and aided in the suppression of background noise for
increased detection sensitivity.

3.1. Solvent Extraction Selection

The evaluation of the most appropriate extraction solvent, showing efficient extraction
recovery for all studied analytes, was performed by studying seven different, commonly
used organic solvents. More specifically, butyl acetate, 1-octanol, 4-methyl-2-pentanone,
chlorobutane, hexane, diethyl ether, and ethyl acetate were used for analytes extraction. For
this study, 1 mL of a blood sample spiked with 0.125 µg/mL of flunitrazepam and fentanyl;
0.25 µg/mL of ropivacaine, bupropion, venlafaxine, mirtazapine, biperiden, citalopram,
midazolam, olanzapine, zolpidem, and haloperidol; 1 µg/mL of alprazolam, tramadol,
atropine, moclobemide, 7-AF, propofol, ketamine, lidocaine, methadone, amitriptyline,
cocaine, chlomipramine, diazepam, nordazepam, and clozapine; and 2 µg/mL of metham-
phetamine, MDMA, MDEA, MBDB, nortriptyline, codeine, chloropromazine, fluoxetine,
sertraline, quetiapine, amantadine, MDA, phenobarbital, and phenytoin were used. The
sample was treated as described in the experimental section with the different solvents and
the recoveries were assessed.
 Forensic
Forensic. Sci. 2022,
Sci. 2022, 2 PEER REVIEW
2, FOR 478 6

Figure
Figure1.1.Extracted
Extractedion
ion chromatograms
chromatograms ofofallall drugs
drugs of of interest
interest overlaid
overlaid inapplied
in the the applied conditions.
conditions.
Each number
Each numbercorresponds toaacompound
corresponds to compound following
following thethe numbering
numbering (No)(No) of Table
of Table 1. 1.

ABased
10-minon the experimental observation, ethyl acetate was not considered appropriate
backflush program at 300 °C was applied after the thermal gradient pro-
and was the first to be rejected due to the notable formation of emulsion, which did not
vided an adequate purging of the system and aided in the suppression of background
enable the collection of a clear organic phase. Diethyl ether, hexane, and 1-octanol also
noise for emulsions,
formed increased detection
however tosensitivity.
a lesser extent, thus they were further considered. Besides,
it was observed that diethyl ether, due to its low boiling point, evaporated to some extent
3.1. Solvent
during theExtraction Selection
procedure and this is a fact that should be taken into account, as it can introduce
biasThe
intoevaluation
the method.of the most appropriate extraction solvent, showing efficient extrac-
Regarding
tion recovery forthe
allrecoveries obtained by
studied analytes, waseach solvent, these
performed are depicted
by studying sevenin Figure 2 in com-
different,
a comparative way through a heat map. The darker color corresponds to the increased
monly used organic solvents. More specifically, butyl acetate, 1-octanol, 4-methyl-2- pen-
recovery values of the analytes. It can be understood that based on the various chemical
tanone, chlorobutane, hexane, diethyl ether, and ethyl acetate were used for analytes ex-
structures and properties of the studied analytes, the solvent providing the highest recovery
traction. Formany
differs for this study, 1 mL
analytes. Forofthis,
a blood samplethat
the solvent spiked withincreased
provided 0.125 μg/mL of flunitrazepam
recovery for the
and fentanyl; 0.25 μg/mL of ropivacaine, bupropion, venlafaxine, mirtazapine,
majority of the analytes was selected as the optimum. Such a compromise was necessary biperiden,
citalopram, midazolam, olanzapine, zolpidem, and haloperidol; 1 μg/mL of alprazolam,
tramadol, atropine, moclobemide, 7-AF, propofol, ketamine, lidocaine, methadone, ami-
triptyline, cocaine, chlomipramine, diazepam, nordazepam, and clozapine; and 2 μg/mL
of methamphetamine, MDMA, MDEA, MBDB, nortriptyline, codeine, chloropromazine,
fluoxetine, sertraline, quetiapine, amantadine, MDA, phenobarbital, and phenytoin were
 a comparative way through a heat map. The darker color correspond
recovery values of the analytes. It can be understood that based on the
structures and properties of the studied analytes, the solvent providing
ery differs for many analytes. For this, the solvent that provided incre
Forensic Sci. 2022, 2 479
the majority of the analytes was selected as the optimum. Such a comp
sary in order to be able to simultaneously extract all analytes efficiently
inwas
orderfound that
to be able butyl acetate
to simultaneously extracted
extract 31efficiently
all analytes out of 41 in aanalytes
single step.with
It was a reco
found that butyl acetate extracted 31 out of 41 analytes with a recovery R% > 85%.

Figure 2. Extraction efficacy of each solvent for the 41 compounds. Darker color corresponds to
Figure 2. Extraction efficacy of each solvent for the 41 compounds. Darker c
increased recovery values of the analytes.
increased recovery values of the analytes.
In an attempt to obtain more meaningful conclusions based on the extraction recover-
ies, a trend was observed according to the separate drug categories. For this, the analytes
In an attempt to obtain more meaningful conclusions based on the
were grouped into categories. It can be seen that amphetamines were better extracted with
eries, a trend
chlorobutane, was
opiates, observed according
antidepressants, to by
and anesthetics thebutyl
separate
acetate; drug categories. Fo
benzodiazepines
bywere
butyl acetate
groupedand 4-methyl-2-pentanone;
into categories. Itantiparkinsonians
can be seen that by butyl acetate and diethyl
amphetamines were be
Forensic Sci. 2022, 2 480

ether; and, finally, antipsychotics by butyl acetate, 4-methyl-2-pentanone, and chlorobutane.

Overall, it can be observed that butyl acetate gives the most satisfying result in almost
all categories.
It was seen that diethyl ether provided the lowest recoveries for almost all the analytes
and this could be due to losses during the procedure as evaporation was observed in
the compounds. 1-octanol was also found to provide low recovery values for all the
compounds, with the exception of MDEA, which was extracted with its highest recovery of
64.2% under these conditions. As it concerns the analytes, methamphetamine was the only
case where the recoveries were similar to the solvents. On the other hand, phenobarbital
and phenytoin showed very low extraction rates < 28.1%, with every solvent tested because
of their extremely high pKa values, which are 7.3 and 8.3, respectively. It was also observed
that small-molecular-weight drug analytes such as propofol and bupropion were more
efficiently extracted with hexane and iso-octane, although the recoveries of the majority of
all the other drugs were not favored with these two solvents.
By comparing recovery rates for the whole set of analytes, it can be concluded that
4-methyl-2-pentanone provides a similar extraction efficiency to butyl acetate (25 out of
41 analytes, R% > 85%). However, it was observed that 4-methyl-2-pentanone has a rich
mass spectrum in the m/z 58 fragment. This fragment is very common in many of the
target analytes and there is a great possibility to produce analytical and chromatographical
difficulties, and mainly in cases where the compounds of interest are in low concentrations.
More specifically, it effects a high background noise, thus m/z 58 and 100 are basic ions in
the mass spectrum of the solvent. This makes the quantification of a significant number of
compounds such as some amphetamines, tramadol, and others, which have the ion 58 m/z
in abundance and are used for quantitation, more difficult.
Regarding the chromatographic baseline, the solvents 1-octanol, 1-chlorobutane, and
4-methyl-2-pentanone have a high baseline in the first 9 min of analysis. Among the three
solvents, 1-octanol produces the highest baseline because it elutes slowly from the column
due to its higher molecular weight compared to the others.
Taking into account all of the above-mentioned findings, it can be concluded that butyl
acetate is the most appropriate out of the seven tested extraction solvents for this specific
panel of analytes, as the majority of them were extracted satisfactorily with R% > 85%.
Thus, further optimization was applied for this.

3.2. Sample-to-Solvent Ratio Optimization

In a second step, the solvent-to-blood-sample volume ratio was studied. In general,
for all four tested ratios the recoveries of the analytes were satisfactory, and in all cases
the majority was > 85%. However, a sample-to solvent-ratio of 4:1 (v/v) and 1:1 (v/v)
provided the highest recoveries for the majority of the analytes. In Figure 3, the recoveries
R% for all analytes under the four extraction conditions are presented comparatively
in a heat map. For some cases of analytes, such as amantadine, atropine, codeine, and
alprazolam, their recovery increased as the solvent-to-blood-volume ratio decreased. Under
4:1 and 1:1 ratios, all categories of drugs, e.g., benzodiazepines, antidepressants, etc., were
efficiently extracted. Among these, the 4:1 (v/v) was selected as it has the most satisfying
recovery results for almost all the compounds when using a reasonable volume of blood
sample. In Table S1, all the volumes used in the sample-to-volume ratio experiment
are presented.

3.3. Method Validation

3.3.1. Selectivity
Blood samples from six different drug-free subjects were tested for the presence of
endogenous components, which might interfere with the detection of the 41 drugs and
DOA or the internal standard. It was found that no endogenous blood components were
eluted at the same retention times with the analytes.
 Forensic.
Forensic Sci.Sci. 2022,
2022, 2 2, FOR PEER REVIEW 481 9

Figure
Figure 3. 3.Sample
Sampletotosolvent
solventratio
ratio in
in the absolute peak
peak area
areafor
forthe
the41
41compounds.
compounds.Darker
Darkercolor cor-
color
responds totoincreased
corresponds increasedrecovery
recoveryvalues
valuesof ofthe
theanalytes.
analytes.

3.3.2.
3.3. Carryover
Method Validation
3.3.1. blank
A sample was analysed after six continuous analyses of spiked samples at
Selectivity
the highest concentration, and it was proven that no carryover effect occurred for any of
Blood samples from six different drug-free subjects were tested for the presence of
the analytes.
endogenous components, which might interfere with the detection of the 41 drugs and
DOA
3.3.3. or the internal standard. It was found that no endogenous blood components were
Linearity
eluted at the same retention times with the analytes.
The linearity of the method was studied within the range of the mean therapeutic and
the mean toxic concentrations of each drug, which were found in the literature [40]. Drug-
3.3.2. Carryover
free blood samples were spiked at five concentrations levels with 100 µL of methanolic
A blank
solutions and 25sample
µL of was analysed
IS, then afterEvery
analysed. six continuous
standard analyses of spiked
was analysed samples
in four at the
replicates
highest concentration, and it was proven that no carryover effect occurred for any
and the calibration curves were constructed based on the peak areas ratio of the analyte of the
analytes.
to the IS, which were linear in the studied ranges with a correlation coefficient that was
higher than 0.9934 for all analytes (see Table 2).
3.3.3. Linearity
The linearity of the method was studied within the range of the mean therapeutic
and the mean toxic concentrations of each drug, which were found in the literature [40].
Forensic Sci. 2022, 2 482

Table 2. Figures of merit of the method for 41 studied analytes.

Linear Range LOD LOQ

Analyte Linear Equation R2
(µg/mL) (µg/mL) (µg/mL)
Methampetamine 0.4–10.0 y = 10.101x + 1.4706 0.9978 0.020 0.066
Amantadine 0.8–20.0 y = 4.3684x + 1.559 0.9994 0.010 0.032
Propofol 0.1–5.0 y = 24.617x − 2.6623 0.9983 0.005 0.017
MDA 0.8–20.0 y = 1.6608x − 0.0176 0.9990 0.033 0.109
MDMA 0.4–10.0 y = 11.137x + 0.7223 1.000 0.009 0.031
MDEA 0.4–10.0 y = 20.563x + 1.4997 0.9999 0.002 0.008
Bupropion 0.05–1.00 y = 17.6133 + 0.2203 0.9991 0.013 0.044
MBDB 0.4–10.0 y = 24.32x + 0.8195 0.9997 0.002 0.007
Fluoxetine 0.5–10.0 y = 26.587x − 16.212 0.9968 0.055 0.184
Ketamine 0.1–5.0 y = 2.9372x + 0.3221 0.9957 0.011 0.038
Lidocaine 0.1–5.0 y = 5.7228x + 0.7541 0.9955 0.009 0.029
Tramadol 0.2–5.0 y = 33.57x − 1.1145 0.9995 0.001 0.004
Phenobarbital 0.4–20.0 y = 12.865x + 5.734 0.9987 0.008 0.027
Venlafaxine 0.02–1.00 y = 44.847x − 0.4172 0.9995 0.003 0.011
Methadone 0.1–5.0 y = 37.989x + 0.4546 0.9997 0.003 0.011
Ropivacaine 0.08–2.00 y = 32.713x − 0.4533 0.9998 0.003 0.009
Amitriptyline 0.1–5.0 y = 35.265x − 0.4943 0.9978 0.012 0.040
Cocaine 0.1–5.0 y = 11.402x − 0.6845 0.9976 0.006 0.019
Atropine 0.2–5.0 y = 3.1589x + 0.2277 0.9934 0.011 0.037
Nortriptyline 0.75–10.00 y = 12.427x − 8.7535 0.9996 0.113 0.375
Moclobemide 0.2–5.0 y = 25.376x + 1.6707 0.9996 0.003 0.009
Mirtazapine 0.02–1.00 y = 28.645x − 0.0298 0.9988 0.006 0.020
Biperiden 0.02–1.00 y = 34.649x − 0.5564 0.9995 0.003 0.009
Phenytoin 0.4–20.0 y = 9.9039x − 4.2309 0.9979 0.012 0.041
Sertraline 0.5–10.0 y = 4.3317x − 1.7901 0.9989 0.075 0.251
Citalopram 0.05–1.00 y = 24.7065 − 0.5613 0.9989 0.003 0.010
Codeine 0.4–10.0 y = 4.5406x + 0.8487 0.9997 0.002 0.007
Clomipramine 0.1–5.0 y = 12.546x + 1.8696 0.9981 0.007 0.024
Diazepam 0.1–5.0 y = 8.8033x − 0.5545 0.9987 0.014 0.045
Chlorpomazine 0.4–10.0 y = 16.567x + 1.9159 0.9998 0.003 0.011
Nordazepam 0.1–5.0 y = 10.1663x + 0.6575 0.9986 0.012 0.040
Midazolam 0.02–1.00 y = 30.607x − 0.3161 0.9975 0.002 0.007
Flunitrazepam 0.025–1.000 y = 2.9896x − 0.0529 0.9999 0.003 0.011
7-AF 0.35–5.00 y = 1.5609x − 0.0223 0.9993 0.037 0.128
Fentanyl 0.01–0.50 y = 14.9604x − 0.1360 0.9977 0.003 0.010
Olanzapine 0.02–1.00 y = 6.4316x − 0.2464 0.9974 0.004 0.011
Zolpidem 0.02–1.00 y = 20.3231x + 0.3246 0.9975 0.003 0.011
Clozapine 0.1–10.0 y = 6.9347x − 0.4942 0.9992 0.011 0.037
Haloperidol 0.1–5.0 y = 3.1195x − 0.6406 0.9961 0.028 0.093
Alprazolam 0.05–1.00 y = 4.1498x − 0.1051 0.9986 0.009 0.026
Quetiapine 0.5–10.0 y = 1.4263x − 0.5078 0.9989 0.054 0.174

3.3.4. Limits of Detection and Quantification

The LOD and LOQ were experimentally calculated as a signal-to-noise ratio of 3:1 and
10:1, respectively, for every drug, and were found to range between 1–113 and 4–375 ng/mL,
respectively—as can be seen in Table 2. The reported values for all pharmaceuticals were
lower or close to the lower therapeutic values, whereas for DOA they were quite low.

3.3.5. Accuracy and Precision

The results for accuracy and precision within a batch (intraday) and over a period of a
week (interday) at three quality control levels (LQC, MQC, HQC), which represented the
entire corresponding dynamic range of the calibration curve for each analyte, are presented
in Table 3. The accuracy was found to be between 85% and 114% and the precision was less
than 14.8%.
Forensic Sci. 2022, 2 483

Table 3. Intra- and interassay of the compounds.

Intra-Assay Inter-Assay
Added
Compound Mean Found Accuracy Overall Mean Accuracy
(µg/mL) SD CV % SD CV %
(µg/mL) % Found (µg/mL) %
0.50 0.51 0.03 8.03 103 0.51 0.02 5.84 103
Methamphetamine 2.50 2.47 0.03 1.21 99 2.50 0.04 1.76 100
8.00 8.02 0.21 2.09 100 7.84 0.32 3.28 98
0.90 0.90 0.06 7.36 100 0.89 0.05 6.32 99
Amantadine 5.00 4.94 0.20 4.13 99 4.94 0.17 3.44 99
18.0 18.0 0.63 3.17 100 17.4 1.06 5.47 97
0.40 0.44 0.01 5.10 110 0.45 0.01 5.02 112
Propofol 1.25 1.17 0.08 6.84 94 1.19 0.12 10.4 95
4.00 4.16 0.40 7.60 104 4.20 0.49 9.31 105
0.90 0.94 0.07 8.12 105 0.91 0.06 6.90 102
MDA 5.00 5.25 0.13 2.40 105 5.00 0.35 6.94 100
18.0 18.1 0.52 2.57 100 18.0 0.45 2.27 100
0.50 0.50 0.03 6.50 100 0.50 0.02 4.71 101
MDMA 2.50 2.53 0.05 1.78 101 2.52 0.05 1.90 101
8.00 8.01 0.22 2.20 100 7.98 0.38 3.85 100
0.50 0.52 0.03 6.69 105 0.52 0.02 5.04 104
MDEA 2.50 2.57 0.10 3.89 103 2.57 0.08 2.96 103
8.00 7.96 0.29 2.92 100 7.92 0.31 3.10 99
0.06 0.07 0.01 9.09 110 0.06 0.01 14.8 100
Bupropion 0.25 0.26 0.04 14.5 105 0.25 0.03 11.4 98
0.80 0.75 0.08 7.95 94 0.77 0.10 10.4 96
0.50 0.52 0.02 4.61 103 0.52 0.02 3.86 104
MBDB 2.50 2.58 0.06 2.28 103 2.56 0.05 1.99 102
8.00 7.92 0.24 2.42 99 7.96 0.21 2.06 99
1.30 1.33 0.09 7.19 102 1.31 0.10 7.70 101
Fluoxetine 5.00 5.09 0.51 9.98 102 4.89 0.43 8.81 98
8.00 8.65 1.05 9.69 108 8.26 0.96 9.26 103
0.30 0.28 0.02 9.13 92 0.27 0.03 12.5 90
Ketamine 1.25 0.21 0.09 7.86 90 1.07 0.08 7.68 85
4.00 4.08 0.31 6.00 102 3.93 0.42 8.62 98
0.30 0.30 0.03 9.92 101 0.28 0.03 11.8 95
Lidocaine 1.25 1.31 0.09 7.02 105 1.35 0.12 8.70 108
4.00 3.97 0.27 5.40 99 4.06 0.51 9.98 101
0.30 0.33 0.01 5.07 109 0.33 0.01 5.43 111
Tramadol 1.25 1.29 0.02 1.78 104 1.28 0.03 2.34 103
4.00 3.95 0.07 1.40 99 3.96 0.10 2.10 99
1.25 1.14 0.12 12.7 91 1.19 0.12 13.1 95
Phenobarbital 5.00 5.48 0.31 5.59 110 5.28 0.35 6.55 106
19.0 19.9 1.67 8.00 105 19.7 1.78 8.60 104
0.06 0.07 0.01 12.7 110 0.06 0.01 13.2 106
Venlafaxine 0.25 0.24 0.02 9.92 97 0.24 0.02 8.33 96
0.80 0.77 0.07 7.00 96 0.81 0.10 10.3 101
0.30 0.28 0.02 8.16 93 0.27 0.02 10.2 90
Methadone 1.25 1.28 0.08 6.18 102 1.28 0.07 5.15 103
4.00 4.14 0.40 7.75 103 4.08 0.42 8.23 102
0.09 0.10 0.003 3.33 113 0.10 0.003 3.37 111
Ropivacaine 0.50 0.50 0.02 4.04 99 0.50 0.01 2.63 99
1.90 1.89 0.04 2.11 99 1.88 0.04 1.87 99
0.30 0.33 0.02 5.90 108 0.31 0.03 10.3 105
Amitriptyline 1.25 1.29 0.14 10.9 103 1.25 0.16 12.4 100
4.00 4.07 0.73 14.3 102 3.97 0.51 10.2 99
0.30 0.33 0.01 2.18 110 0.33 0.03 9.49 110
Cocaine 1.25 1.13 0.07 6.31 90 1.12 0.09 7.86 90
4.00 4.25 0.65 12.2 106 4.17 0.57 10.9 104
0.30 0.30 0.01 5.45 101 0.30 0.01 3.96 101
Atropine 1.25 1.42 0.02 1.20 114 1.37 0.07 5.25 110
4.00 3.94 0.14 2.87 98 3.95 0.11 2.31 99
Forensic Sci. 2022, 2 484

Table 3. Cont.

Intra-Assay Inter-Assay
Added
Compound Mean Found Accuracy Overall Mean Accuracy
(µg/mL) SD CV % SD CV %
(µg/mL) % Found (µg/mL) %
0.80 0.81 0.01 0.66 101 0.80 0.01 0.80 100
Nortriptyline 2.50 2.42 0.08 3.30 97 2.40 0.09 3.71 96
9.00 9.85 0.75 6.85 109 9.98 0.63 5.67 111
0.30 0.30 0.01 4.46 101 0.30 0.01 3.96 101
Moclobemide 1.25 1.29 0.09 6.69 103 1.29 0.07 5.34 103
4.00 3.97 0.08 1.69 99 4.00 0.10 2.00 100
0.06 0.07 0.007 12.07 116 0.06 0.007 14.0 100
Mirtazapine 0.25 0.26 0.025 9.80 102 0.26 0.034 13.2 103
0.80 0.73 0.096 10.51 91 0.79 0.105 10.6 99
0.06 0.06 0.003 6.25 96 0.06 0.004 8.00 100
Biperiden 0.25 0.25 0.008 3.20 100 0.25 0.007 2.83 99
0.80 0.75 0.065 6.94 94 0.77 0.053 5.51 96
1.25 1.21 0.117 12.10 97 1.19 0.115 12.0 96
Phenytoin 5.00 5.44 0.222 4.08 109 5.49 0.437 7.97 110
19.0 19.2 2.114 10.45 101 19.5 2.113 10.3 103
1.30 1.01 0.073 6.92 84 1.13 0.093 8.56 87
Sertraline 5.00 5.51 0.490 8.90 110 5.12 0.544 10.6 102
8.00 8.61 1.119 10.40 108 8.27 0.955 9.24 103
0.06 0.06 0.005 10.47 96 0.07 0.007 13.0 108
Citalopram 0.25 0.26 0.036 13.85 104 0.23 0.037 14.5 102
0.80 0.85 0.091 8.58 106 0.81 0.091 8.96 102
0.50 0.49 0.019 4.82 99 0.50 0.021 5.29 99
Codeine 2.50 2.57 0.035 1.36 103 2.57 0.057 2.22 103
8.00 7.86 0.162 1.65 98 7.92 0.221 2.23 99
0.30 0.28 0.022 9.61 92 0.27 0.019 8.37 91
Clomipramine 1.25 1.16 0.069 5.93 93 1.20 0.072 5.99 96
4.00 3.75 0.269 5.74 94 3.85 0.289 6.00 96
0.30 0.33 0.042 15.50 108 0.31 0.039 15.2 103
Diazepam 1.25 1.31 0.061 4.66 105 1.25 0.103 8.26 100
4.00 4.20 0.402 7.60 105 4.06 0.466 9.19 101
0.50 0.50 0.017 4.25 100 0.51 0.016 3.92 102
Chlorpromazine 2.50 2.53 0.042 1.66 101 2.51 0.040 1.59 100
8.00 7.98 0.165 1.66 100 8.03 0.184 1.83 100
0.30 0.31 0.04 12.86 104 0.31 0.04 12.9 103
Nordazepam 1.25 1.24 0.08 6.52 99 1.25 0.09 7.22 100
4.00 4.11 0.26 6.32 103 4.08 0.31 7.60 102
0.06 0.07 0.003 5.36 112 0.07 0.005 8.50 118
Midazolam 0.25 0.21 0.010 4.76 84 0.22 0.020 8.93 88
0.80 0.74 0.070 7.53 93 0.80 0.100 10.0 100
0.025 0.03 0.003 11.11 108 0.03 0.002 7.41 108
Flunitrazepam 0.125 0.12 0.014 12.17 92 0.12 0.011 9.48 93
0.40 0.40 0.030 6.04 99 0.40 0.022 4.41 100
0.40 0.40 0.032 9.20 99 0.40 0.023 6.63 99
7-AF 1.25 1.20 0.037 3.09 96 1.21 0.036 2.98 97
4.00 4.01 0.085 1.69 100 4.01 0.074 1.48 100
0.03 0.03 0.003 11.54 104 0.03 0.003 11.5 104
Fentanyl 0.125 0.11 0.006 5.61 86 0.11 0.008 7.41 86
0.40 0.36 0.046 10.22 90 0.39 0.050 10.4 96
0.06 0.07 0.007 12.07 116 0.07 0.005 8.77 114
Olanzapine 0.25 0.26 0.025 9.80 102 0.23 0.023 10.2 90
0.80 0.73 0.096 10.51 91 0.79 0.091 9.27 98
0.06 0.06 0.007 13.73 102 0.06 0.007 14.6 96
Zolpidem 0.25 0.22 0.021 9.68 87 0.22 0.017 7.83 87
0.80 0.83 0.078 7.57 103 0.79 0.086 8.73 99
0.30 0.26 0.014 6.51 86 0.28 0.023 9.75 94
Clozapine 1.25 1.26 0.046 3.66 101 1.24 0.068 5.49 99
4.00 4.02 0.297 6.18 100 4.02 0.475 9.46 100
Forensic Sci. 2022, 2 485

Table 3. Cont.

Intra-Assay Inter-Assay
Added
Compound Mean Found Accuracy Overall Mean Accuracy
(µg/mL) SD CV % SD CV %
(µg/mL) % Found (µg/mL) %
0.30 0.33 0.016 5.78 111 0.33 0.016 5.80 110
Haloperidol 1.00 1.09 0.110 10.13 87 1.07 0.082 7.66 86
4.00 3.82 0.369 7.72 96 3.94 0.516 10.5 98
0.06 0.06 0.005 10.42 96 0.06 0.009 18.0 100
Alprazolam 0.25 0.21 0.010 4.69 85 0.22 0.020 8.93 90
0.80 0.69 0.070 8.12 86 0.79 0.110 11.2 98
1.30 1.21 0.068 5.84 93 1.23 0.071 5.98 95
Quetiapine 5.008.000 4.90 0.108 2.20 98 5.06 0.367 7.25 101
8.00 8.09 0.618 6.11 101 7.90 0.738 7.48 99

The interlaboratory precision of the method was also examined by the analysis of
twelve real samples, which were found to be positive in more than one drug in our
laboratory (Laboratory of Forensic Toxicology of the University of Thessaloniki) and in the
Laboratory of Forensic Service of Ministry of Justice. Seventeen different compounds were
detected and determined by both labs in all samples. The concentration results were found
to be in good agreement; the correlation coefficient was calculated and found to be 0.9993.
The results can be seen in Table 4.
Table 4. Interlaboratory results.

Concentration Found in Laboratory A Concentration Found in Laboratory B

Sample (S) Compounds
(µg/mL) (µg/mL)
Tramadol 0.271 0.270
Codeine 0.018 0.018
Flunitrazepam 0.289 0.236
S1
7-Aminoflunitrazepam 0.707 0.730
Alprazolam 0.876 0.781
Lidocaine 0.171 0.174
Zolpidem 5.00 4.50
S2
Tramadol 0.253 0.230
Ropivacaine 2.00 2.00
S3
Lidocaine 0.988 0.955
Quetiapine 15.7 15.2
S4
Diazepam 0.028 0.025
Diazepam 0.283 0.273
Propofol 0.901 0.738
S5
Lidocaine 0.049 0.043
Midazolam 0.086 0.083
S6 Sertraline 0.308 0.270
Clozapine 2.50 2.30
S7
Lidocaine 0.051 0.051
Clozapine 4.200 4.100
S8
Lidocaine 78 74
Clozapine 4.20 4.40
S9
Lidocaine 0.066 0.061
Mirtazapine 0.050 0.045
S10
Citalopram 0.206 0.192
Clozapine 4.80 4.90
Zolpidem 0.062 0.059
S11
Diazepam 0.580 0.559
Biperiden 0.142 0.141
Forensic Sci. 2022, 2 486

Table 4. Cont.

Concentration Found in Laboratory A Concentration Found in Laboratory B

Sample (S) Compounds
(µg/mL) (µg/mL)
Diazepam 0.038 0.039
Propofol 1.50 1.60
S12
Midazolam 0.058 0.055
Lidocaine 0.034 0.030

4. Results and Discussion of Real Sample Analysis

The method has been applied for hundreds of samples for the investigation of post-
mortem and clinical cases. It has been proven to be a very valuable, rapid, and low-cost
tool in the routine analysis of our laboratory for the monitoring of various drugs and DOA.
Hundreds of samples have been analysed, however, for the economy of the presentation, a
number of these cases have been chosen. Those with a history of pharmaceutical use for
mental disorders from January to March 2021 have been selected to be presented here for
exemplification purposes and to provide proof of concept.
Postmortem blood samples from 89 cases of forensic interest (history of mental disor-
ders and use of psychiatric pharmaceuticals) were analyzed with the developed method
and provided valuable data.
As an aggregate, the number of positive detections of the monitored drugs and
pharmaceuticals in the 89 samples reached a total sum of 135. Eight pharmaceuticals
out of the forty-one under study were found and determined in the examined samples.
These comprised drugs belonging to different categories (benzodiazepines, antidepressants,
antipsychotics, etc.). In Table 5, the concentrations of the pharmaceuticals in every case are
given, showing the potential and effectiveness of the method. The frequency in which they
were found is outlined schematically in Figure 4. The majority of the identified compounds
were benzodiazepines (74 out of 135), which is in agreement with the metadata, considering
that the samples were obtained by individuals with mental illnesses.

Table 5. Concentration in µg/mL of the determined pharmaceutical in each case.

Case No Diazepam Citalopram Alprazolam Olanzapine Mirtazapine Venlafaxine Haloperidol Zolpidem

1 0.34 0.43
2 1.12
3 0.62 0.17
4 1.30
5 0.52 0.18
6 0.18
7 0.76 0.02 0.013
8 0.13
9 0.74
10 0.25
11 0.14
12 0.26 0.07
13 0.93
14 0.79
15 0.51 0.12
16 0.35
17 1.01
18 1.16
19 4.34 0.24
20 0.66
21 0.59 0.12
22 0.67
23 0.24
24 0.76 0.16
Forensic Sci. 2022, 2 487

Table 5. Cont.

Case No Diazepam Citalopram Alprazolam Olanzapine Mirtazapine Venlafaxine Haloperidol Zolpidem

25 0.56 0.33
26 0.19 0.2
27 0.63 0.024
28 0.21 0.006
29 0.16
30 0.18 0.28
31 0.25 0.04
32 3.50
33 0.69 0.03
34 0.52
35 2.88
36 0.21
37 0.24
38 0.26
39 0.22 0.029
40 0.14 0.19 0.33
41 0.23 0.05 0.13
42 0.20
43 0.14
44 0.27 0.11 0.009
45 0.22
46 0.27 0.12
47 0.41 0.06 0.083
48 0.27 0.21 0.05 0.06
49 0.60
50 0.32 0.17
51 0.22 0.032
52 0.13
53 0.43 0.13 0.03
54 0.31 0.02
55 0.15 0.05
56 0.26 0.05
57 0.32 0.015
58 0.04
59 0.16 0.02
60 0.05
61 0.24 0.08
62 0.15
63 0.14
64 0.05
65 0.01 0.05
66 0.01
67 0.09 0.03
68 0.06
69 0.12 0.035
70 0.028
71 0.010
72 0.022
73 0.017
74 0.05 0.01
75 0.10
76 0.24 0.16
77 0.03
78 0.01
79 0.63 0.015
80 0.92
81 0.01
82 0.22
83 0.06
Forensic Sci. 2022, 2 488

Table 5. Cont.

Case No Diazepam Citalopram Alprazolam Olanzapine Mirtazapine Venlafaxine Haloperidol Zolpidem

84 0.15
85 0.19
86 0.007
Forensic.87
Sci. 2022, 2, FOR PEER REVIEW 0.10 17
88 0.09
89 0.11

60

50

40

30 57

20

10 19
12 12 13 13
4 5
0

Figure 4. Frequency of the compounds found in the 89 cases.

Figure 4. Frequency of the compounds found in the 89 cases.
By comparing the concentration values found with those referred to in the international
By comparing
literature [40], these the concentration
generally appearedvalues
to befound with thoselevels.
at therapeutic referredMoreto specifically,
in the interna- the
tional literatureranges
concentration [40], these generally
of the compoundsappeared
found to in
be the
at therapeutic
studied cases levels. Morefor
ranged specifically,
diazepam
the
fromconcentration
0.13 to 4.34 ranges
µg/mL, offor
thecitalopram
compoundsfrom found 0.04intothe studied
0.24 µg/mL, casesforranged for diaze-
alprazolam from
pam from 0.13 to 4.34 μg/mL, for citalopram from 0.04 to 0.24 μg/mL,
0.01 to 0.12 µg/mL, for olanzapine from 0.009 to 0.083 µg/mL, for mirtazapine from 0.01 tofor alprazolam from
0.01
0.33toµg/mL,
0.12 μg/mL, for olanzapine
for venlafaxine fromfrom 0.009
0.006 to 0.083
to 0.92 µg/mL, μg/mL, for for mirtazapine
haloperidol from from 0.01to
0.007
to0.13
0.33µg/mL,
μg/mL,and for venlafaxine
for zolpidem from 0.006
from to to
0.01 0.92
0.16μg/mL,
µg/mL. for haloperidol
In addition,from 0.007positive
all these to 0.13
μg/mL,
results and forconcentrations
in low zolpidem fromhave 0.01been
to 0.16 μg/mL.
gained with In aaddition,
single LLE all step,
thesewhich
positive results
is in in
contrast
low
withconcentrations
other analytical have been gained
methods that hadwith a single
similar findingsLLEwithstep,much
which is in
more contrast with
time-consuming
steps,analytical
other such as SPE [25,27].
methods that had similar findings with much more time-consuming steps,
The use of
such as SPE [25,27]. sophisticated embedded software such as DRS, where a report of candidate
compounds
The use that combines deconvoluted
of sophisticated peaks and
embedded software suchretention
as DRS,timewheredataa from
reportRTL, is also
of candi-
an important
date compounds feature
that of the methodology.
combines deconvolutedHowever,
peaks analysts should
and retention be extremely
time data from cautious
RTL, is
in the
also aninterpretation
important feature of theofresults; when the concentration
the methodology. However, analysts of the compound is near the
should be extremely
LOD of the
cautious method,
in the the generated
interpretation of thereport may
results; whennot include the specificofcompound,
the concentration the compound thereby
is
leading to false negative results. Thus, aid by the software should
near the LOD of the method, the generated report may not include the specific compound, be wisely used and the
examination
thereby leadingof to
thefalse
rawnegative
data is very important
results. Thus, aidto confirm findings.should be wisely used
by the software
and the examination of the raw data is very important to confirm findings.
5. Conclusions
A rapid, sensitive, and reliable method for the simultaneous detection of 41 drugs and
5. Conclusions
DOA in blood was developed. The method provides detection and accurate quantitation
A rapid, sensitive, and reliable method for the simultaneous detection of 41 drugs
whenever needed. Fast methods that apply LLE without evaporation or derivatization steps
and DOA in blood was developed. The method provides detection and accurate quanti-
are very limited and such a methodology is of high interest for laboratories performing
tation whenever needed. Fast methods that apply LLE without evaporation or derivatiza-
routine drug analysis. Also, mostly all methods used for screening are not validated, thus
tion steps are very limited and such a methodology is of high interest for laboratories
Limits of Detection of the analytes are a blur or not strictly determined. The reported
performing routine drug analysis. Also, mostly all methods used for screening are not
method was validated and figures of merit were determined. A paradigm of application is
validated, thus Limits of Detection of the analytes are a blur or not strictly determined.
presented by providing data for a number of cases showing the utility of the method.
The reported method was validated and figures of merit were determined. A paradigm
of application is presented by providing data for a number of cases showing the utility of
the method.
Forensic Sci. 2022, 2 489

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/forensicsci2030035/s1, Table S1: Volumes used in the sample to volume
ratio experiment.
Author Contributions: Conceptualization, G.T. and N.R.; methodology, A.O., O.M. and A.K.; valida-
tion, A.O., O.M. and A.K.; investigation, N.R.; writing—original draft preparation, A.O., O.M. and
H.G.; writing—review and editing, G.T. and H.G.; visualization, A.O.; supervision, H.G. All authors
have read and agreed to the published version of the manuscript.
Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank Anastasia Chrysovalantou Chatziioannou for her pre-
cious help for Figures 2 and 3.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Schulz, M.; Schmoldt, A. Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics. Pharmazie
2003, 58, 447–474. [PubMed]
2. Janicka, M.; Kot-Wasik, A.; Namieśnik, J. Analytical procedures for determination of cocaine and its metabolites in biological
samples. Trends Anal. Chem. 2010, 29, 209–224. [CrossRef]
3. Persona, K.; Madej, K.; Knihnicki, P.; Piekoszewski, W. Analytical methodologies for the determination of benzodiazepines in
biological samples. J. Pharm. Biomed. Anal. 2015, 113, 239–264. [CrossRef] [PubMed]
4. Cruces-Blanco, C.; García-Campaña, A.M. Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of
forensic interest. Trends Anal. Chem. 2012, 31, 85–95. [CrossRef]
5. Orfanidis, A.; Gika, H.; Mastrogianni, O.; Krokos, A.; Theodoridis, G.; Zaggelidou, E.; Raikos, N. Determination of drugs of abuse
and pharmaceuticals in skeletal tissue by UHPLC-MS/MS. Forensic Sci. Int. 2018, 290, 137–145. [CrossRef]
6. Fernandez-Lopez, L.; Pellegrini, M.; Rotolo, M.C.; Luna Maldonado, A.; Falcon, M.; Mancini, R. Development and validation of a
method for analysing of duloxetine, venlafaxine and amitriptyline in human bone. Forensic Sci. Int. 2019, 299, 154–160. [CrossRef]
[PubMed]
7. Wietecha-Posłuszny, R.; Lendor, S.; Garnysz, M.; Zawadzki, M.; Kościelniak, P. Human bone marrow as a tissue in post-mortem
identification and determination of psychoactive substances—Screening methodology. J. Chromatogr. B Anal. Technol. Biomed. Life
Sci. 2017, 1061, 459–467. [CrossRef]
8. Baciu, T.; Botello, I.; Borrull, F.; Calull, M.; Aguilar, C. Capillary electrophoresis and related techniques in the determination of
drugs of abuse and their metabolites. Trends Anal. Chem. 2015, 74, 89–108. [CrossRef]
9. Di Rago, M.; Chu, M.; Rodda, L.N.; Jenkins, E.; Kotsos, A.; Gerostamoulos, D. Ultra-rapid targeted analysis of 40 drugs of abuse
in oral fluid by LC-MS/MS using carbon-13 isotopes of methamphetamine and MDMA to reduce detector saturation. Anal.
Bioanal. Chem. 2016, 408, 3737–3749. [CrossRef]
10. Odoardi, S.; Valentini, V.; de Giovanni, N.; Pascali, V.L.; Strano-Rossi, S. High-throughput screening for drugs of abuse and
pharmaceutical drugs in hair by liquid-chromatography-high resolution mass spectrometry (LC-HRMS). Microchem. J. 2017, 133,
302–310. [CrossRef]
11. Holler, J.; Levine, B. Confirmation methods for SAMHSA drugs and other commonly abused drugs. In Critical Issues in Alcohol
and Drugs of Abuse Testing; Academic Press: Cambridge, MA, USA, 2019; pp. 189–206.
12. Moeller, M.R.; Steinmeyer, S.; Kraemer, T. Determination of drugs of abuse in blood. J. Chromatogr. B Biomed. Sci. Appl. 1998,
713, 91–109. [CrossRef]
13. Nielsen, M.K.K.; Johansen, S.S. Simultaneous determination of 25 common pharmaceuticals in whole blood using ultra-
performance liquid chromatography-tandem mass spectrometry. J. Anal. Toxicol. 2012, 36, 497–506. [CrossRef]
14. Pouliopoulos, A.; Tsakelidou, E.; Krokos, A.; Gika, H.G.; Theodoridis, G.; Raikos, N. Quantification of 15 psychotropic drugs in
serum and postmortem blood samples after a modified mini-QuEChERS by UHPLC-MS-MS. J. Anal. Toxicol. 2018, 42, 337–345.
[CrossRef] [PubMed]
15. Fisichella, M.; Odoardi, S.; Strano-Rossi, S. High-throughput dispersive liquid/liquid microextraction (DLLME) method for
the rapid determination 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. 2015, 123, 33–41.
Forensic Sci. 2022, 2 490

16. Dulaurent, S.; El Balkhi, S.; Poncelet, L.; Gaulier, J.M.; Marquet, P.; Saint-Marcoux, F. QuEChERS sample preparation prior to
LC-MS/MS determination of opiates, amphetamines, and cocaine metabolites in whole blood. Anal. Bioanal. Chem. 2016, 408,
1467–1474. [CrossRef] [PubMed]
17. Teng, X.; Liang, C.; Wang, R.; Sun, T.; Rao, Y.; Ni, C.; Zeng, L.; Xiong, L.; Li, Y.; Zhang, Y. Screening of drugs of abuse and toxic
compounds in human whole blood using online solid-phase extraction and high-performance liquid chromatography with
time-of-flight mass spectrometry. J. Sep. Sci. 2015, 38, 50–59. [CrossRef]
18. Robin, T.; Barnes, A.; Dulaurent, A.; Loftus, N.; Baumgarten, S.; Moreau, S.; Marquet, P.; El Balkhi, S.; Saint-Marcoux, F.
Fully automated sample preparation procedure to measure drugs of abuse in plasma by liquid chromatography tandem mass
spectrometry. Anal. Bioanal. Chem. 2018, 410, 5071–5083. [CrossRef]
19. Ferrari Júnior, E.; Caldas, E.D. Simultaneous determination of drugs and pesticides in postmortem blood using dispersive solid-
phase extraction and large volume injection-programmed temperature vaporization-gas chromatography–mass spectrometry.
Forensic Sci. Int. 2018, 290, 318–326. [CrossRef]
20. Smith, M.P.; Bluth, M.H. Common interferences in drug testing. Clin. Lab. Med. 2016, 36, 663–671. [CrossRef]
21. Brahm, N.C.; Yeager, L.L.; Fox, M.D.; Farmer, K.C.; Palmer, T.A. Commonly prescribed medications and potential false-positive
urine drug screens. Am. J. Health Syst. Pharm. 2010, 67, 1344–1350. [CrossRef]
22. Maurer, H.H. Multi-analyte procedures for screening for and quantification of drugs in blood, plasma, or serum by liquid
chromatography-single stage or tandem mass spectrometry (LC-MS or LC-MS/MS) relevant to clinical and forensic toxicology.
Clin. Biochem. 2005, 38, 310–318. [CrossRef] [PubMed]
23. Bjørk, M.K.; Nielsen, M.K.K.; Markussen, L.; Klinke, H.B.; Linnet, K. Determination of 19 drugs of abuse and metabolites in
whole blood by high-performance liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2010, 396, 2393–2401.
[CrossRef] [PubMed]
24. Orfanidis, A.; Gika, H.G.; Theodoridis, G.; Mastrogianni, O.; Raikos, N. An UHPLC-MS-MS method for the determination of 84
drugs of abuse and pharmaceuticals in blood. J. Anal. Toxicol. 2020, 45, 28–43. [CrossRef] [PubMed]
25. Langel, K.; Gunnar, T.; Ariniemi, K.; Rajamäki, O.; Lillsunde, P. A validated method for the detection and quantitation of 50
drugs of abuse and medicinal drugs in oral fluid by gas chromatography-mass spectrometry. J. Chromatogr. B 2011, 879, 859–870.
[CrossRef] [PubMed]
26. Adamowicz, P.; Kała, M. Simultaneous screening for and determination of 128 date-rape drugs in urine by gas chromatography-
electron ionization-mass spectrometry. Forensic Sci. Int. 2010, 198, 39–45. [CrossRef] [PubMed]
27. Truta, L.; Castro, A.L.; Tarelho, S.; Costa, P.; Sales, M.G.F.; Teixeira, H.M. Antidepressants detection and quantification in whole
blood samples by GC-MS/MS, for forensic purposes. J. Pharm. Biomed. Anal. 2016, 128, 496–503. [CrossRef]
28. Hara, K.; Waters, B.; Ikematsu, N.; Tokuyasu, T.; Fujii, H.; Takayama, M.; Matsusue, A.; Kashiwagi, M.; Kubo, S.I. Development
of a preparation method to produce a single sample that can be applied to both LC-MS/MS and GC-MS for the screening of
postmortem specimens. Leg. Med. 2016, 21, 85–92. [CrossRef]
29. Papoutsis, I.; Athanaselis, S.A.; Nikolaou, P.D.; Pistos, C.M.; Spiliopoulou, C.A.; Maravelias, C.P. Development and validation of
an EI-GC-MS method for the determination of benzodiazepine drugs and their metabolites in blood: Applications in clinical and
forensic toxicology. J. Pharm. Biomed. Anal. 2010, 52, 609–614. [CrossRef]
30. Huang, Z.; Yu, T.; Guo, L.; Lin, Z.; Zhao, Z.; Shen, Y.; Jiang, Y.; Ye, Y.; Rao, Y. Effects of triglycerides levels in human whole blood
on the extraction of 19 commonly used drugs using liquid-liquid extraction and gas chromatography-mass spectrometry. Toxicol.
Rep. 2015, 2, 785–791. [CrossRef]
31. Versace, F.; Sporkert, F.; Mangin, P.; Staub, C. Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological
screening. Talanta 2012, 101, 299–306. [CrossRef]
32. Feng, Y.; Zheng, M.; Zhang, X.; Kang, K.; Kang, W.; Lian, K.; Yang, J. Analysis of four antidepressants in plasma and urine by gas
chromatography-mass spectrometry combined with sensitive and selective derivatization. J. Chromatogr. A 2019, 1600, 33–40.
[CrossRef] [PubMed]
33. Orfanidis, A.; Mastrogianni, O.; Koukou, A.; Psarros, G.; Gika, H.; Theodoridis, G.; Raikos, N. A GC-MS method for the detection
and quantitation of ten major drugs of abuse in human hair samples. J. Chromatogr. B 2017, 1047, 141–150. [CrossRef] [PubMed]
34. Meatherall, R. GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and
oxymorphone in urine. J. Anal. Toxicol. 1999, 23, 177–186. [CrossRef]
35. Woźniak, M.K.; Banaszkiewicz, L.; Wiergowski, M.; Tomczak, E.; Kata, M.; Szpiech, B.; Namieśnik, J.; Biziuk, M. Development
and validation of a GC–MS/MS method for the determination of 11 amphetamines and 34 synthetic cathinones in whole blood.
Forensic Toxicol. 2020, 38, 42–50. [CrossRef]
36. Pizarro, N.; Ortuño, J.; Farré, M.; Hernández-López, C.; Pujadas, M.; Llebaria, A.; Joglar, J.; Roset, P.N.; Mas, M.; Segura, J.; et al.
Determination of MDMA and its metabolites in blood and urine by gas chromatography-mass spectrometry and analysis of
enantiomers by capillary electrophoresis. J. Anal. Toxicol. 2002, 26, 157–165. [CrossRef]
37. Álvarez-Freire, I.; Brunetti, P.; Cabarcos-Fernández, P.; Fernández-Liste, A.; Tabernero-Duque, M.J.; Bermejo-Barrera, A.M.
Determination of benzodiazepines in pericardial fluid by gas chromatography–mass spectrometry. J. Pharm. Biomed. Anal. 2018,
159, 45–52. [CrossRef] [PubMed]
Forensic Sci. 2022, 2 491

38. Matsuta, S.; Nakanishi, K.; Miki, A.; Zaitsu, K.; Shima, N.; Kamata, T.; Nishioka, H.; Katagi, M.; Tatsuno, M.; Tsuboi, K.; et al.
Development of a simple one-pot extraction method for various drugs and metabolites of forensic interest in blood by modifying
the QuEChERS method. Forensic Sci. Int. 2013, 232, 40–45. [CrossRef]
39. Amorim Alves, E.; Sofia Agonia, A.; Manuela Cravo, S.; Manuel Afonso, C.; Duarte Pereira Netto, A.; Lourdes Bastos, M.;
Carvalho, F.; Jorge Dinis-Oliveira, R. GC-MS method for the analysis of thirteen opioids, cocaine and cocaethylene in whole blood
based on a modified quechers extraction. Curr. Pharm. Anal. 2017, 13, 215–223. [CrossRef]
40. Schulz, M.; Schmoldt, A.; Andresen-Streichert, H.; Iwersen-Bergmann, S. Therapeutic and toxic blood concentrations of more
than 1100 drugs and other xenobiotics. Crit. Care 2020, 24, 195. [CrossRef]