Toxicology Reports 14 (2025) 101923

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Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep

Highly-sensitive quantification of carbamazepine and identification of its

degradation and metabolism products in human liver by high performance
liquid chromatography – High resolution mass spectrometry
Andrei Pirogov a, Ekaterina Shirokova a, Samvel Barsegyan b, Nikita Gandlevskiy a,c,* ,
Valeriya Akimova b , Alessandro Barge c , Aleksander Nosyrev d
a
Department of chemistry, M.V. Lomonosov Moscow State University, Chair of Analytical Chemistry, Leninskiye gory str. 1/3, Moscow 119991, Russia
b
Federal State Budgetary Institution «Russian Centre of Forensic Medical Expertise» of the Ministry of Health of the Russian Federation, Polikarpov str, 12/13, Moscow
125284, Russia
c
Department of Drug Science and Technology, Turin University, via P. Giuria 9, Turin 10125, Italy
d
FSAEI of Higher Education I.M. Sechenov First Moscow State Medical University (Sechenov University), Trubetskaya str, 8/2, Moscow 119048, Russia

A R T I C L E I N F O A B S T R A C T

Handling editor: Prof. L.H. Lash A method for the qualitative and quantitative determination of carbamazepine in human post mortem liver tissues
using high-performance liquid chromatography coupled with high-resolution mass spectrometry has been
Keywords: developed. Validation has been carried out and the main analytical characteristics of the developed method have
Carbamazepine been determined. The limit of detection (LOD) is 1 ng/g, and the lower limit of quantification (LLOQ) is 5 ng/g.
Liver metabolism
The range of working concentrations for the calibration curve is 5–2000 ng/g. When assessing analyte carryover,
HPLC-HRMS
the analyte signal of the sample does not exceed 20 % of the signal at the LLOQ level. Degradation products of
carbamazepine in model solutions were studied under the presence of hydrochloric acid, sodium hydroxide, and
hydrogen peroxide oxidation. Twenty-two degradation products were identified. It was found that the most
intensive degradation process of carbamazepine, resulting in various degradation products, is observed during its
oxidation with an acidified solution of 3 % hydrogen peroxide at pH= 1–2. The stability of carbamazepine in
liver tissues was studied during storage under ambient conditions over various periods. The maximum con-
centration decline is observed during the first week of storage (on average by 20 %), and then the concentration
approximately halves over 8 weeks. Based on the analysis of forensic samples from human liver, 2 out of the 22
carbamazepine degradation products described in this study were detected.

1. Introduction known. According to the N.V. Sklifosovsky Research Institute of Emer-

gency Medicine, there has been a significant increase in the number of
Carbamazepine (CBZ), or 5H-dibenzo[b,f]azepine-5-carboxamide, is fatal carbamazepine poisonings, including among children, over the past
an antiepileptic and anticonvulsant drug frequently used for neurolog- five years.
ical and psychiatric patients to treat seizures, neuropathic pain, or bi- Additionally, one of the reasons for the forensic toxicology interest in
polar disorder (Fig. 1). CBZ, in its chemical structure, is an iminostilbene antiepileptic drugs is the significant number of sudden unexplained
derivative, contains a carbamyl group, and is structurally similar to deaths among people with epilepsy who take these medications [1]. In
tricyclic antidepressants. This drug is also of interest to forensic medi- conducting forensic investigations of deaths caused by poisoning, it is
cine and chemical-toxicological analysis specialists, as uncontrolled use necessary to accurately identify and quantify the intoxicant and the dose
and overdose of carbamazepine can result in fatal outcomes. Due to the received. For this purpose, a reliable detection method and a validated
close proximity of the toxic dose to the therapeutic values for carba- quantitative determination methodology for pharmaceutical drugs,
mazepine, cases of both intentional and accidental poisonings are their metabolites, and degradation products in biological material are

* Corresponding author at: Department of chemistry, M.V. Lomonosov Moscow State University, Chair of Analytical Chemistry, Leninskiye gory str. 1/3, Moscow
119991, Russia.
E-mail address: nikita.gandlevskiy@unito.it (N. Gandlevskiy).

https://doi.org/10.1016/j.toxrep.2025.101923
Received 20 September 2024; Received in revised form 16 January 2025; Accepted 20 January 2025
Available online 27 January 2025
2214-7500/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Table 1
Conditions for the quantification of carbamazepine in various samples using LC.
Sample Sample Separation Analytical Source
preparation and detection characteristics
conditions

Hair Liquid-liquid Column: LLOQ [4]

extraction ACQUITY < 5.0 pg/mg
Extraction by the UPLC HSS Accuracy
mixture MeOH/ C18 (150 mm 75–125 %
MeCN/ 2 ММ of × 2.1 mm, 1.8
ammonium μm)
formate in 8 % Т = 50◦ C
MeCN, pH= 5,3 Flow rate:
Fig. 1. Structural formula of carbamazepine.
(25:25:50 v/v/v). 0.4 mL/min
18 hours of MP А: 5 ММ
required. The challenge in implementing such methods lies in the high incubation. ammonium
degradation rate of the drug, both under adverse environmental con- formate (pH
3.0)
ditions (during storage) and during sample preparation and analysis. MP В: 0.1 %
The aim of this work is to develop and validate a highly sensitive formic acid in
technique for the quantification of carbamazepine, as well as the iden- MeCN
tification of its metabolites and degradation products in human bio- MS/MS, triple
quadrupole
logical material, using high-resolution chromatographic-mass
(MRM mode,
spectrometry. This methodology is intended to address expert tasks in ESI)
forensic toxicology. Blood serum Blood samples Column: LOD = 0.8 ng/ [5]
After administration, CBZ binds to plasma proteins at a rate of were Phenomenex mL
75–85 %, and its elimination is almost entirely dependent on biotrans- centrifugated, Torrance LLOQ
1 mL of serum has Luna C18 = 2.4 ng/mL
formation in the liver through epoxidation and hydroxylation. It is been sampled. (50 × Recovery:
assumed that carbamazepine-epoxide (CBZ-EP), being an active 30 μL of sample 2,0 mm, 5 μm, 96–98 %
metabolite, contributes to the observed therapeutic and side effects. The were mixed with 100 Å)
study [2] also showed that CBZ is almost completely metabolized in the 150 μL of IS, then Т = 25◦ C
mixed and Flow rate:
liver, with only about 5 % of the drug excreted unchanged. The main
centrifuged. 40 μl 0,4 mL/min
metabolic pathways have been identified [3]. of the supernatant MP А: 0.1 %
Therefore, when working with biological material containing CBZ was placed into a of formic acid
and collected post mortem, it is necessary to consider the time elapsed vial with 160 μL MP В: MeCN
since the presumed intake of CBZ and its elimination pathways. Thus, of 0.1 % formic MS/MS, triple
acid. quadrupole
the liver was chosen as the matrix carrier for this work and for the (ESI+)
development of a quantitative determination method for CBZ. Blood serum Protein Column: LLOQ [6]
The literature presents numerous methods aimed at the quantitative precipitation was Agilent = 50.0 ng/mL
determination of CBZ in various matrices (Table 1). However, due to the carried out in a Eclipse XDB- Recovery
96-well plate. To C18 (2.1 × 74,7–93,5 %
specific requirements of chemical-toxicological forensic examinations,
100 μl of plasma, 50 mm, 1.8
only some of these methods are suitable for forensic purposes. 20 μl of IS μm)
Using HPLC-MS allows for the avoidance of degradation that might (phenacetin, Т = 25◦ C
occur with GC-MS. HPLC-MS provides more accurate results and can 100 ng/mL) and MP А: MeCN
detect lower concentrations of substances in samples. GC has several 300 μl of MeCN MP В: 0,1 %
were added. The formic acid
limitations, including the need for analytes to be volatile and non-polar.
samples were Gradient:
Under normal GC operating conditions, CBZ undergoes thermal degra- mixed, placed in a 15 %A→30 %
dation in the injector, forming iminostilbene (IM) and 9-methylacridine, positive pressure A from 0 to
which sometimes necessitates an additional derivatization step for ac- device (413 kPa, 3 min 30 %
5 min), the A→95 %A
curate analysis [14], [15]. Furthermore, GC is highly sensitive to bio-
supernatant was from 3 to
logical contaminants such as saturated lipids, sterols, and fatty acids, transferred to 5 min
which negatively impact the lifespan of consumables. other tubes and MS/MS, triple
Analysing the data presented in the table, it was found that the pri- evaporated in a quadrupole
mary sample preparation method for the subsequent determination of stream of nitrogen (ESI+)
at 45◦ C.
CBZ in tissues is liquid-liquid extraction (LLE). Based on the physico-
Redissolved in
chemical properties of CBZ and the literature, the optimal solvents for 100 µl of MP.
the extraction of CBZ from biological tissues are acetonitrile and ethyl Blood plasma Liquid-liquid Column: LLOQ [7]
acetate [13]. In modern laboratory practice, solid-phase extraction using extraction Phenomenex = 0.72 ng/mL
To 500 μL of Luna C18 tR:
the QuEChERS method is increasingly employed [16], [17], [18].
plasma, 500 μL of (150 × 2 mm, 2.65–2.84 min
Methodologies utilizing HPLC-MS/MS demonstrate superior LODs 0.1 M NaOH and 5 μm)
and LOQs of CBZ. According to the literature, the LOD using a mass IS (nitrazepam Т = 25◦ C
spectrometric detector is generally an order of magnitude lower. The 800 ng/mL in Flow rate:
influence of the biological matrix is minimized, making this method the H2O) were added. 0.25 mL/min
Then, 3 mL of MP: MeCN/
most relevant for forensic toxicology work.
ethyl acetate was MeOH/ 0.1 %
The relevance of our work is further supported by the fact that there added to the formic acid
are comparatively few scientific studies available to us that propose a mixture, shaken (10:70:20 v/
methodology for the qualitative and quantitative determination of CBZ and centrifuged at v/v)
in biological material (liver) for forensic purposes using the HPLC-MS/ (continued on next page)

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Table 1 (continued ) Table 1 (continued )

Sample Sample Separation Analytical Source Sample Sample Separation Analytical Source
preparation and detection characteristics preparation and detection characteristics
conditions conditions

10◦ C. The MS/MS, triple acetone was

aqueous phase quadrupole added to 2 mL of
was collected and (ESI+) sample, shaken,
frozen at − 30◦ C and centrifuged.
for 3 min. The 1 mL of extraction
organic phase was buffer was added
evaporated under to the
a nitrogen stream supernatant.
at 40◦ C. The dry Afterwards,
residue was extraction was
redissolved in the carried out with a
mobile phase. mixture of 970 mL
Blood plasma Liquid-liquid Column: LLOQ [8] of
extraction Zorbax eclipse = 500 ng/mL dichloromethane
Standard XD8 C8 Accuracy 100 with 30 mL of 2-
solutions were (150 × ± 20 % propanol.
dissolved in 4.6 mm, 4 Recovery 90 Fish liver Liquid-liquid Column: LLOQ [11]
MeCN. 300 μL of μm) ± 6.7 % extraction Alltima С18 = 4.2 ng/g
acetone was Т = 25◦ C 10 mL of hexane (250 × Recovery
added to plasma Flow rate: was added to 2 g 4.6 mm, 5 75–85 %
samples (protein 0.8 mL/min of liver sample, μm)
denaturation). MP А: MeCN mixed, and Т = 25◦ C
Centrifuged and MP В: formate centrifuged. The Flow rate:
evaporated at buffer (2 mM, extraction was 1 mL/min
37◦ C. The dry pH 3) repeated 3 times, MP А: Water
residue was consisted of and the extracts MP В: MeCN
dissolved in a formic acid were combined. MS/MS (MRM
mixture of MeCN: salt (126 mg/ Evaporated under mode, ESI(-).
water (50:50 v/v) L) in purified a stream of
water. nitrogen. The dry
MS/MS, triple residue was
quadrupole dissolved in 2 mL
(ESI+) of 90 % MeCN,
Shellfish Liquid-liquid Column: LOD [9] diluted with
extraction Poroshell 120 = 0.04 ng/g 50 mL of water
10 mL of MeCN SB C8 (50 × LLOQ and purified using
and 5 mL of H2O, 2.1 mm, 2.7 < 5.0 ng/g solid phase
followed by the μm) Accuracy 100 extraction
pack of acetate Т = 60◦ C ± 20 % through a
salts were added Flow rate: cartridge (Oasis
to the tissue 0.6 mL/min HLB) by washing
sample. MP А: 0.01 % with 5 mL of
Centrifuged. The acetic acid in methanol and
supernatant was water 5 mL of water.
evaporated to MP В: MeCN The methanol
dryness at 40 ◦ C MS/MS (MRM fraction was sifted
under N2 flow. mode, ESI(+). out and the
The dry residue residue was
was dissolved in dissolved in 4 mL
50 μL of13C- of 90 % MeCN
phenacetin and Human liver Liquid-liquid Column: LLOQ [12]
diluted to 500 μL (metabolomic extraction Zorbax C8 = 50 µM
with water study) Liver samples of (4.6 ×
Brain tissue Liquid-liquid Column: С18 LOD [10] 10 g were placed 250 mm, 5
extraction ODS (200 × = 870 ng/mL in 10 mM μm)
1 mL of IS solution 2.1 mm, 5 potassium Т = 50◦ C
was added to μm) phosphate buffer Flow rate:
tissue samples Т = 65◦ C (pH = 7.4) 1.5 mL/min
(20–200 μg), Flow rate: containing 0.9 % MP А: MeOH
centrifuged, and 0,3 mL/min NaCl. Then, the MP В: H2O
homogenized. MP А: 0.05 M tissue was UV detection
Extracted with NH4H2PO4 homogenized to (210 nm)
buffer solution buffer obtain
(pH=6.0): 20.0 g (pH=4.4) / microsomes in a
sodium MeCN buffer
dihydrogen (90:10 v/v) supplemented
phosphate 2-hy- MP В: 0.05 M with 0.25 M
drate, 4.5 g di- NH4H2PO4 sucrose.
sodium hydrogen buffer Centrifuged. The
phosphate 2-hy- (pH=4.4) / supernatant
drate, and 1.5 g MeCN (40:60) containing
sodium azide in UV detection microsomal
1 l of deionized (207 nm) protein was
water. 1 mL of carefully
(continued on next page)

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Table 1 (continued ) Table 2

Sample Sample Separation Analytical Source
Chromatographic conditions.
preparation and detection characteristics Parameter Values
conditions
MP А 0.1 % formic acid, 2 ММ ammonium formate, 1 % MeCN in
separated from H2O
the glycogen MP B 0.1 % formic acid, 2 ММ ammonium formate, 1 % H2O in
granules, MeCN
resuspended in a Column 40ºС
mixture of 10 mM temperature
KP buffer (pH = Injection volume 5 μL
7.4): 1 mM EDTA: Flow rate 0,4 mL/min
1.15 % KCI and Run time 15 min
centrifuged again. Gradient time, minMP А, %
The washed pellet programme 0.098
was resuspended 1.098
in either 50 mM 10.02
KP buffer 12.02
(pH=7.4) or 12.198
100 mM Tris HCI 15.098
buffer (pH=7.4).
IS - 2-methyl-CBZ
(11.3 µg/mL) 2.2. Equipment and methods
Fish tissues QuEChERS Column: LLOQ [13]
IS and 10 mL of Hypersil Gold = 2.5 ng/g
MeCN were added C18 (2.1 × Recovery Chromatographic separation was performed on a Vanquish-VH-
to 1 g of 100 mm, 1.9 97 % C10A HPLC system (ThermoScientific, USA) using a Kinetex C18 col-
homogenized μm) umn (50 mm × 3 mm, 2.6 μm, 100 Å, Phenomenex, USA) with an
tissue. Shaked for Т = 35оС
Acquity UPLC BEH C18 1.7 μm precolumn (Waters, USA). The chro-
10 min. A mixture Flow rate:
of salts from the 250 μL/min
matographic conditions are provided in Table 2. Detection of CBZ and its
QuEChERS kit MP А: 0.1 % degradation products in biological samples was conducted using an
(4 g MgSO4/1 g formic acid in Orbitrap Exploris 120 mass spectrometer (ThermoScientific™, USA)
NaCl) was added, water with heated electrospray ionization (H-ESI). Chromatograms were ac-
shaken for MP В: 0.1 %
quired in Full Scan mode with ion fragmentation in the mass range of
1.5 min, and formic acid in
centrifuged MeOH 100–1000 Da. The ionization source parameters are were set as follows:
(5 min, MS/MS, vaporizer temperature – 400 ◦ C, ion transfer tube temperature – 320 ◦ C,
4000 rpm). 2 mL Orbitrap XL transition dwell time – 0.05 sec, spray voltage – positive ion 3500 V,
of organic layer Fourier (full negative ion – 2500 V, Intensity threshold – 1.0e4, HCD collision en-
was collected and scan mode,
transferred to a ESI+).
ergies – 15, 30, 45 eV. Metabolite identification was performed using
test tube Compound Discoverer™ software, Thermo FreeStyle™ (Thermo Scien-
containing 50 mg tific™, USA), as well as the software packages Competitive Fragmen-
of Z-Sep+ Bulk tation Modeling (CMF-ID) and MetFrag (Thermo Scientific™).
salt. Mixed and
Identification of CBZ degradation products relied on parameters such as
centrifuged. The
supernatant layer accurate mass of the hydrated molecular ion (to ten-thousandths of a
was collected and dalton), isotopic distribution (MS), and characteristic ion fragmentation
evaporated to (MS2).
dryness (40◦ C).
Redissolved in
500 μL of 0.1 % 3. Materials and equipment used in sample preparation
formic acid
solution in a Extraction was carried out using the VetexQ-tox IL-5550–4956
water/methanol
extraction kit, 15 mL Pyrex glass tubes with screw caps (Corning, UK),
mixture (10:90 v/
v) 1.5 mL Eppendorf tubes (Eppendorf, Germany), mechanical pipettes
(Eppendorf research plus, Eppendorf, Germany), analytical balances
IS – internal standard, MP – mobile phase, LLOQ – low limit of quantification, (Mettler Toledo XS204, USA), Vortex microcentrifuge (ELMI V-3,
LOD – limit of detection, tR – retention time.
Latvia), orbital shaker (BioSan PSU-10i, Latvia), centrifuge (ELMI CM-
6M, Latvia), centrifuge (Eppendorf AG 22331, Germany), and water
MS method. purification system (Millipore Direct-Q 5 UV, USA).

2. Materials and methods

3.1. Accelerated aging of carbamazepine
2.1. Reagents
Methanol solutions containing 1 mg of CBZ were placed in nine
The following reagents were used during the study and sample Eppendorf-type tubes and evaporated in a concentrator for 15 minutes.
preparation: acetonitrile (Fisher Chemicals Optima LC/MS grade), for- The tubes were divided into sets of three, and 3 mL of 18 % hydrochloric
mic acid (98–100 %, Merck, Germany), ammonium formate (for LC-MS, acid HCl, 2 M sodium hydroxide NaOH solution, and 3 % H2O2 solution
Fluka analytical, Germany), hydrochloric acid HCl (extra pure, Khim- were added to each set, respectively. All samples were heated for various
med, Russia, GOST 3118–77rev1), hydrogen peroxide (37 %, med. durations (10, 30, 60 minutes) in a boiling water bath, followed by
Khimmed, Russia, GOST 177–88rev1), sodium hydroxide NaOH (extra acidification to pH= 1–2 with 0.1 M HCl. Extraction with 2 mL of ethyl
pure, Khimmed, Russia, GOST 4328–77rev.1,2), ethyl acetate (for LC- acetate was performed, and the organic layer was collected. The aqueous
MS, J.T.Baker, USA). phase was basified to pH= 10 with sodium hydroxide solution. Then,
2 mL of ethyl acetate was extracted again, and the supernatant was

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Table 3
Sample preparation for performing of artificial aging.
Samples N◦ 1–3 N◦ 4–6 N◦ 7–9

1 2 3 4 5 6 7 8 9
+ 3 mL 18 % HCl + 3 mL 2М NaOH + 3 mL 3 % solution of H2O2, acidified by HCl to pH= 1–2
Heating in water bath (100 С)
о

10 min 30 min 60 min 10 min 30 min 60 min 10 min 30 min 60 min

Acidification by 0.1 М HCl to pH= 1–2
Extraction by 2 mL of ethyl acetate
Aqueous phase Organic phase
+ 2М NaOH to рН = 10 ​
Extraction by 2 mL of ethyl acetate
Unification of organic extracts
Evaporation 30 min

collected. Acidic and alkaline extracts were combined and evaporated. cadavers FSE_1 and FSE_2 were used for the following purposes:
The dry residues were reconstituted in 150 μL of a mixture of 10 %
acetonitrile - 0.1 % formic acid and transferred to 2 mL glass vials 1. Conducting an experiment to investigate the stability of CBZ under
(Table 3). environmental conditions;
2. Qualitative and quantitative determination of CBZ;
3. Qualitative analysis of CBZ degradation and metabolism products, as
3.2. Liver tissue sampling well as extraneous compounds.

During this study, forensic samples of biological material provided

by the Federal State Budgetary Institution "RCMSE" of the Ministry of 3.3. Preparation of liver tissue samples
Health of Russia were used. Samples not containing CBZ were included
for validation of the quantitative determination method, as well as two A 4.0 g portion of intact liver was homogenized, transferred to an
cadaveric liver samples obtained during pathological autopsies. To extraction tube, and 200 μL standard working solution of CBZ (1 mg/
preserve the confidentiality of the investigation and medical secrecy, mL) and 10 μL of internal standard (amitriptyline, 1 mg/mL) were
these samples will be referred to as FSE_1 and FSE_2 in the future. Blank added. The mixture was shaken and left for 15 minutes at room tem-
liver samples were collected to validate the methodology for quantita- perature. This compound has structural similarity to CBZ and similar
tive determination, with a standard CBZ solution of the corresponding physicochemical properties (M = 277.4 g/mol, LogP = 5, pKa = 9.5),
concentration added to them. Two biological liver samples from thus comparable retention time on the column under the chosen

Fig. 2. Comparison of chromatograms of liver extracts with addition of CBZ and AMT (a) and intact (b).

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

chromatographic separation conditions (tr = 5.6 min). A mixture of salts Table 4

(1000 mg MgSO4 and 250 mg NaCl) was added, followed by 5 mL of Calculated parameters of the accuracy and precision of the method within the
acetonitrile, and vigorously shaken. The mixture was then subjected to fifth analytical cycle. SD – sample standard deviation, RSD – relative standard
ultrasonic bath treatment for 25–30 minutes and centrifuged for deviation (%), E – accuracy (%), (n = 2, P = 0.95).
5 minutes at 3000 rpm. The supernatant (3–3.5 mL) was transferred to a Nominal concentration, Measured concentration, ng/g RSD, Е, %
cleanup tube containing MgSO4 and sorbent. After shaking for ng/g Average ± SD %
5 5.1 ± 0,3 6.15 2.37
15 minutes and centrifuging for 5 minutes at 3000 rpm, 100 μL of the
10 10.9 ± 0,4 3.35 8.58
supernatant was transferred to a chromatographic vial and diluted with 20 19.8 ± 0,3 1.75 − 1.17
900 μL of water. 50 47 ± 6 13.38 − 6.69
100 104 ± 13 13.05 4.55
3.4. Stability assessment 500 524 ± 37 6.99 4.82
750 760 ± 75 9.89 1.17
1000 1040 ± 84 8.10 4.19
To assess the stability of CBZ in liver tissues containing CBZ, samples 2000 2004 ± 138 6.92 0.19
were kept at room temperature (25◦ C) with oxygen and light exposure
for 1–8 weeks. A portion of the samples was collected weekly for anal-
ysis. After the specified time, the samples were frozen at − 20◦ C for therapeutic concentrations of the drug in tissues. Due to the complexity
further storage. After 8 weeks, all frozen samples were thawed, and of working with biological material and its limited accessibility in some
sample preparation was performed according to the general scheme, cases, and because CBZ concentrations in individual expert samples can
except for the addition of CBZ standard. In total, 20 samples were reach toxic or lethal levels exceeding 120.000 ng/g, it is not always
analysed in this manner. feasible to use dilution methods to work within the linear range. It
should be noted that presenting the curve as two linear segments is
4. Results and discussion impractical due to vastly different matrix effects at high and low CBZ
concentrations. Thus, the analytical range of the developed method is
Within the framework of developing a technique for qualitative and 5–2000 ng/g. Concentration deviations of calibration samples from
quantitative analysis of carbamazepine by LC-MS/MS in human liver, nominal values did not exceed acceptable limits ( ± 20 %) across the
the method of sample preparation, internal standard selection, and entire calibration range. The LOD for CBZ was experimentally deter-
analysis conditions were determined. mined to be 1 ng/g (Table S4). Relative standard deviation and accuracy
A reverse-phase chromatography approach with a C18 column and are presented in Table 4.
mobile phases based on water and acetonitrile was chosen. High-
resolution mass spectrometry was essential for retrospective analysis, 4.1. Accuracy and precision
allowing assessment of the drug’s transformation processes over time.
Determining the accurate masses of molecular ions enabled hypotheses The determination of method accuracy and precision was conducted
regarding the compound’s molecular formula, while MS/MS fragmen- by analysing quality control samples at different levels: L, M, H with
tation spectra provided insights into the molecule’s structure. An orbital concentrations of 15.0, 800.0, and 1600.0 ng/g respectively. Accuracy
trap-based mass spectrometric detector was selected to achieve the was assessed using the relative error (E, %) as follows:
analysis objectives. ( )
C − Cknown
Validation and specificity were ensured through method valida- E (%) = ∗ 100%,
Сknown
tion referencing regulatory documents governing validation procedures
in forensic chemical and chemico-toxicological analysis of biological
where С is the average concentration value within or between analytical
material [19], [20]. Parallel measurements were conducted for five
cycles.
analytical cycles, each repeated twice. Following analysis of the cali-
Precision (convergence) was evaluated using the relative standard
bration sample with the highest concentration per cycle, an intact liver
deviation (RSD, %):
sample (Blank) was introduced to control for sample carryover.
Specificity was evaluated by comparing chromatograms obtained SD
RSD(%) = ∗ 100%
from intact liver samples (Blank) with those from samples spiked with C
standard solutions of CBZ and internal standards. Chromatograms and
mass spectra of intact liver samples showed absence of peaks corre- where SD is the standard deviation of the analytical signal.
sponding to CBZ and AMT retention times (Fig. 2). Both parameters must meet the requirements for analytical methods
To construct the calibration model, calibration solutions were pre- used in forensic chemical and chemico-toxicological analysis of biolog-
pared using pure liver samples spiked with CBZ and an internal standard ical materials, not exceeding 20 % for all concentration levels L, M, H.
to the required concentrations. Calibration levels were set at 5, 10, 20, Table 5 presents the calculation of accuracy and precision for the quality
50, 100, 500, 750, 1000, and 2000 ng/g. Control levels were set at 15, control samples within the first analytical cycle. Precision parameters
800, and 1600 ng/g (QCL, QCM, QCH levels). The concentration of the calculated for series of parallel measurements using the one-factor
internal standard was kept constant at 400 ng/g. The concentration dispersion analysis method are presented in Table 6.
level corresponding to the LLOQ of the method should allow for con-
centration determination ≤ 5 % of the maximum concentration. Control 4.2. Sample transfer effect
levels were defined as follows: lower level (L) – three times the LLOQ,
middle level (M) – the average between L and H, approximately According to [21], during method development, it is crucial to
30–50 % of the upper limit of the determined concentrations, upper consider and minimize the transfer of the analyte from sample to sam-
level (H) – 80 % of the upper limit of the determined concentrations. The ple. During validation, the transfer effect was assessed by introducing
calibration curve, the equation and correlation coefficient are presented blank samples after samples with high concentrations or upper-level
in Fig. S4. calibration solutions. To evaluate sample transfer during sequential
The obtained calibration points were well described by a quadratic sample analysis after the maximum concentration calibration sample
relationship. A linear relationship was observed in the low concentra- (2000 ng/g, calibration level 9), and after the quality control sample H
tion range (5–100 ng/g); however, for our forensic medical practice with a concentration of 1600 ng/g, an analysis of a CBZ-free sample
needs, we expanded the analytical range to cover the maximum (Blank) was conducted. Analysis of peak area ratios showed minimal

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Table 5
Calculated accuracy and precision parameters for quality control samples within the first analytical cycle (n = 3, P = 0.95).
Sample Nominal concentration, ng/g Measured concentration, ng/g Average, ng/g SD RSD, % E, %

QC_L_1 15 15 16 1.7 10.6 9.2

QC_L_2 18
QC_L_3 16
QC_M_1 800 962 804 138.8 17.3 0.5
QC_M_2 748
QC_M_3 702
QC_H_1 1600 1774 1744 42.7 2.5 9.0
QC_H_2 1695
QC_H_3 1764

raising concerns about its stability. When working with biological ma-
Table 6 terial collected for forensic medical analysis, understanding the stability
Accuracy and precision for the developed procedure in five analytical runs. AC – of CBZ in tissue during storage under environmental conditions over
acceptance criterion.
different periods of time is crucial.
Concentration level, ng/g 15 800 1600 AC During an 8-week experiment, forensic liver samples (FSE_1, FSE_2)
Convergence within a series of parallel 16.8 16.6 4.1 ±20.0 % were kept under room conditions (25◦ C, oxygen access, light). Upon
quantifications (RSD, %) analysis of liver sample FSE_1, the initial CBZ concentration was found
Convergence between series of parallel 15.5 17.8 3.8 to be 3150 ± 280 ng/g. This concentration exceeds the therapeutic
quantifications (RSD, %)
range for CBZ concentrations in liver tissue, indicating a potential lethal
Accuracy between series of parallel 8.7 18.5 7.1
quantifications (Е, %) poisoning. Two samples were analyzed with two replicates each, and the
average result, error, and relative standard deviation (RSD) were
assessed. The analysis results are presented in Table S1 (Appendix). The
substance transfer, meeting acceptability criteria (AC): the analyte data obtained were plotted graphically (Fig. 3).
signal of the sample did not exceed 20 % of the signal at the LLOQ level, Upon analysis of liver sample FSE_2, the initial CBZ concentration
and the IS signal of the sample did not exceed 5 % of the IS signal. was found to be 7880 ± 820 ng/g. Both analyzed forensic liver samples
initially contained CBZ amounts corresponding to toxic concentrations
4.3. Stability study of CBZ under environmental storage conditions for this compound in tissue. For samples FSE_1 and FSE_2, the upper
limit of the therapeutic concentration range was exceeded by 1.5 and 4
Stability studies are conducted to model situations that may occur in times, respectively. It can be hypothesized that individual or combined
natural and laboratory environments, which can affect the properties of poisoning by the investigated substance could have led to the onset of
the analyte to some extent [19]. It is known that CBZ is highly suscep- death.
tible to thermal and chemical degradation, as well as photodegradation,

Fig. 3. Graph of changes in CBZ concentration in liver samples versus storage time.

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Fig. 4. Chromatograms of model samples of CBZ, obtained after interaction with H2O2 during А) 10 min, B) 30 min, C) 60 min.

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Table 7 Table 8
Relative contents of CBZ and its main degradation products (%) under oxidation Relative contents of CBZ and its main degradation products (%) under acidic
conditions, calculated by the internal normalization method. conditions, calculated by the internal normalization method.
tR, min 3.2 4.0 4.8 5.2 tR, min 3.2 3.4 4.8 5.2 6.9

Exposure to degradation, min AI CBZ-EP acrid¡9-one CBZ Exposure to AI acridine¡9- acrid¡9- CBZ IM
10 45.4 5.0 22.1 27.5 degradation, min carbaldehyde one
30 58.8 8.7 26.9 5.7 10 9.2 7.8 10.3 72.7 0.0
60 58.3 10.6 24.0 7.1 30 7.3 14.6 3.2 43.7 31.3
60 8.0 3.5 8.8 44.1 35.5

4.4. Study of degradation products after artificial aging

Table 9
Accelerated aging tests are conducted in artificial aggressive envi-
Relative contents of CBZ and its main degradation products (%) under basic
ronments and under simulated unfavourable storage conditions of drugs conditions, calculated by the internal normalization method.
to establish their degradation products. Data from artificial aging
tR, min 3.2 5.2 6.9
simplify the process of identifying degradation products in post-mortem
samples and enable forensic experts to draw conclusions about the ante- Exposure to degradation, min AI CBZ IM
10 0.7 93.4 5.9
mortem use of the medicinal product and its approximate dosage. The
30 2.5 89.8 7.7
information on degradation pathways plays a crucial role in cases 60 2.8 86.6 10.6
involving the discovery of bodies in decomposition stages or in the
analysis of exhumed biomaterials.
Table S3 in the Supplementary presents the identified degradation hydrochloric acid during the sample preparation stage to adjust the pH,
products of CBZ, their monoisotopic masses [M], accurate masses of explaining their presence not only in samples exposed to acidic
protonated [M+H]+ and deprotonated [M-H]- ions, structural formulas, conditions.
and mass spectra. In our search for degradants, we relied on literature This detailed analysis highlights the complexity of degradation
data, information from the "HMDB" database [22], and results from pathways and the variety of degradation products formed under
previous work using GC-MS methods [18]. different experimental conditions, essential for forensic and toxicolog-
Upon analysing the obtained data, it was found that the identified ical analyses of CBZ in biological samples.
degradation products were mostly characteristic of all pathways of When analysing chromatograms obtained from the interaction of
artificial aging. However, the concentration of each product varied CBZ with 18 % hydrochloric acid, it was found that CBZ undergoes
significantly in different environments. An exception was iminodiben- minimal degradation under these conditions, with iminostilbene (IM)
zyl, which was present in all conditions except those with the oxidizing being the primary degradation product (Appendix, Fig. S1; Table 8).
agent H2O2. CBZ undergoes conversion to IM by elimination of the CONH2 group
Analysing chromatograms obtained after the experiment involving fragment. This process occurs during aging, metabolism, and thermal
CBZ interaction with hydrogen peroxide H2O2 over 10, 30, and degradation in the ionization source (400ºC). Acridine and its de-
60 minutes (Fig. 4), it is evident that the optimal degradation time for rivatives are also formed, but in smaller quantities. Despite the presence
accumulation of products lies within the 20–30 minute interval during of HCl, the content of chlorinated derivatives is not significant, even
oxidation. Beyond this time, the signal intensity changes insignificantly. compared to oxidative aging.
A similar pattern is observed with other aging agents. In an alkaline medium, the formation of the smallest amount of
Due to the selected mobile phase gradient, the substances of interest diverse degradation products for CBZ was observed. In the chromato-
elute between 2 and 10 minutes. Therefore, signals beyond 10 minutes grams (Appendix, Fig. S2), obtained from the interaction of CBZ with
do not hold significant analytical importance in this experiment. 0.1 M NaOH, there is an intense peak of CBZ along with 10,11-dihydro-
The greatest variety of signals was observed during the oxidation of carbamazepine, formed by hydrogenation of CBZ. The primary product
CBZ using a 3 % H2O2 solution, with the most active transformation of alkaline degradation can be identified as IM. Acridine is also formed,
being the conversion of CBZ to acridine (AI) and its derivatives (tR = 3.2 but to a lesser extent (Table 9).
– 3.4 min) (Table 7). Additionally, acrid-9-one ([M+H]+ = 196.0756) CBZ in its unmodified form was present in all samples, indicating its
emerged as one of the primary degradation products during oxidation, incomplete degradation under our experimental conditions. In identi-
characterized by an intense peak at a retention time of 4.83 min. Be- fying the degradation products, understanding the fragmentation path-
tween 10 and 30 minutes, a sharp decrease in the concentration of CBZ ways of CBZ and its derivatives played a key role (Fig. 5) [23–25].
in the sample indicates that it has been more completely converted into Characteristic ions in the fragmentation of CBZ include 194.0964, cor-
degradation products. Although CBZ continues to be present in its un- responding to IM and formed by the cleavage of the CONH2 group;
modified form. On the chromatogram (Fig. 4 (B)), there is an increase in 220.0757, resulting from the loss of ammonia; and 179.0730, formed by
peak intensity at a retention time of 4.04 min, which corresponds to the cleavage of the NH-CONH2 fragment. The sequential cleavage of
degradation products having an ion mass [M+H]+ = 253.0971. This another methylene group forms a fragment corresponding to the ion
mass corresponds to several possible structural formulas, but CBZ-EP is with m/z = 165.0698. The ion 152.0620 is formed by rearrangement,
hypothesized to contribute significantly to this signal, as one of the main resulting in the formation of a new four-membered ring. The molecular
CBZ metabolites. ion [M+H]+ = 237.10224 is also present in the spectrum, but its in-
In the experimental conditions, a compound elutes first at tR tensity is low and does not exceed 20 % under the selected detection
= 2.38 min with an ion mass [M+H]+ = 224.07061, corresponding to a conditions. In the mass spectra of CBZ degradation products, recurring
compound with the empirical formula C14H9NO2. At tR = 2.97 min, a fragments characteristic of compounds with a common "core" were
signal is formed by an ion with m/z = 210.0912, corresponding to a observed. For example, the ion [M+H]+ = 180.0807, corresponding to
substance with the molecular formula C14H11NO. Further discussion on acridine (AI), is present with quite high intensity in the fragmentation
the separation of isomeric compounds will be addressed below. spectra of all its derivatives (acridine-9-carbaldehyde, 9-oxoacridine-10
Chlorine-containing degradation products elute after 5.5 minutes (9 H)-carbaldehyde, etc.). However, IM derivatives also undergo a
(Fig. 4C), and their quantitative content increases with the reaction rearrangement of the seven-membered ring into a six-membered ring,
time. Their formation is likely associated with the addition of forming a structure similar to AI. Thus, after hydroxylation, CBZ can

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Fig. 5. MS/MS spectrum of CBZ.

Fig. 6. Chromatogram for the [M+H]+ = 224,0706 ion.

undergo further structural changes with the cleavage of the epoxide composition of the compound and assign it a gross formula. At this point
ring, leading to the diminution of the heterocycle and the formation of we can speculate on the structure of the compound, but its structure can
9-oxoacridine-10(9 H)-carbaldehyde, which in turn fragments to IA only be confirmed by analysing the fragmentation spectrum (MS2),
[25]. Under adverse environmental conditions, the most reactive site in taking into account the rearrangement and the fragment ions, which can
the CBZ molecule is likely the double bond between the carbon atoms indicate the presence of substituents and refine the molecular structure.
C10-C11. This bond on the central heterocyclic ring can undergo hy- When identifying degradation products, we encountered the challenge
droxylation, forming the corresponding hydroxyl derivatives such as of determining the structure of isomeric compounds. The difficulty arose
10-OH-CBZ, CBZ-DiOH, or carbamazepine-10,11-epoxide (CBZ-EP). The when attempting to assign structural formulas to degradation products
two external aromatic rings are more inert. In the search for CBZ with the general formula C14H9NO2. In analysing the mass spectra of the
degradation products in model solutions, some compounds could not be ion [M+H]+ = 224.0706, it was necessary to distinguish between
assigned a structural formula due to the absence of a corresponding ion acridine-9-carboxylic acid and 9-oxoacridine-10(9 H)-carbaldehyde
fragmentation spectrum (Table S3 Nos. 7, 9, 16, 18, 21). Based on the (Appendix. Table S1 No. 12). Both compounds share a common core,
mass spectrum containing the ion of interest, we can determine the exact complicating the task. In the fragmentation mass spectrum of the ion
mass of the compound using high-resolution mass spectrometry. By m/z = 224.0706 taken at tR = 2.38 min, we observe intense signals of
analysing the isotopic peak ratios, we can establish the exact elemental ions m/z = 196.0757 and 180.0808, corresponding to the sequential

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Fig. 7. The fragment of chromatogram of CBZ for the m/z = 253,09715 ion, obtained after exposure to HCl for 60 min.

Fig. 8. MS/MS spectrum of m/z = 253,09718 ion fragmentation.

cleavage of the (− CO) and oxygen (− O) groups. By correlating struc- of water (− H2O), followed by sequential cleavage of the (− CO) group
tural features with possible fragmentation pathways, we hypothesized m/z = 178.0651. Thus, we propose that this spectrum corresponds to
that this spectrum belongs to 9-oxoacridine-10(9 H)-carbaldehyde. In acridine-9-carboxylic acid. In Fig. 6, the chromatogram for the selected
the MS/MS spectrum taken at tR = 4.38, an intense peak of ion ion [M+H]+ = 224.0706 shows that the peak at tR = 2.38 is the most
m/z = 206.0601 is observed, formed from the molecular ion by the loss intense, suggesting that the formation of 9-oxoacridine-10

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Fig. 9. Mass spectrum of isotopic distribution of 9-chloroacridine.

Fig. 10. MS/MS spectrum of fragmentation of [M+H]+ = 271,063 ion, corresponding to 10-Cl-CBZ.

(9 H)-carbaldehyde is more favourable during degradation. A large 5.0 minutes. The close retention times again confirm the assumption of
group of degradation products of CBZ includes compounds formed shared structural characteristics among this group of compounds, yet
through hydration, epoxidation, and oxidation processes (Appendix. they exhibit differences in physicochemical properties and represent
Table S1, No. 17): Carbamazepine-10,11-epoxide (CBZ-EP), distinct degradation products. The mass spectrum fragmentation of the
carbamazepine-2,3-epoxide, hydroxy-5H-dibenzazepine-5-carbox- ion m/z = 253.0972 is depicted in Fig. 8, revealing a structure charac-
amide, 10-hydroxy-5H-dibenzazepine-5-carboxamide, 10-oxo-10, teristic of CBZ fragmentation. Therefore, the separation of these isomers
11-dihydro-5H-dibenzazepine-5-carboxamide, and 9-formylacridine-10 is only feasible with specially tailored chromatographic conditions,
(9 H)-carboxamide. All these compounds share the same empirical for- which may be an area of interest for future research.
mula C15H12N2O2 and, of course, ion mass [M+H]+ = 253.0972. Identification of chlorinated compounds began with analysing the
Consequently, their separation and identification solely through mass isotopic distribution in mass spectra (MS) and identifying a unique
spectrometric methods are nearly impossible [26,27]. Fig. 7 presents a pattern of intensity ratios between peaks A and A+ 2 (3:1), which is
selective chromatogram for the ion m/z = 253.0972 obtained during characteristic of chlorine atoms in the molecule. With knowledge of the
aging in acidic conditions. It is evident that compounds with this pro- natural abundance of isotopes, the presence of chlorine atoms can be
tonated ion mass predominantly elute within the time range of 3.3 – easily detected by the characteristic multiplet signal for this element.

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Table 10
Compounds (CBZ and products of its degradation) found in the human liver samples.

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Table 11 mass corresponding to the empirical formula C15H12N2O2 and a series of

Relative content of CBZ и CBZ-EP (%) in the liver samples, calculated by internal structures described previously. Based on available literature sources,
normalization method. we speculate that the ion with this m/z value may correspond to
FSE_1 FSE_2 carbamazepine-10,11-epoxide (CBZ-EP), the most expected metabolite
Weeks CBZ CBZ-EP CBZ CBZ-EP
of CBZ. CBZ-EP was also present in intact, unpreserved liver samples,
0 98.9 1.1 99.2 0.8 indicating its metabolic origin. However, when assessing the relative
1 98.8 1.2 98.7 1.3 content of CBZ and CBZ-EP in samples exposed to environmental con-
3 98.2 1.8 98.3 1.7 ditions for 8 weeks (Table 11), we observe a gradual increase in the
5 98.0 2.0 98.1 1.9
percentage content of CBZ-EP over time, indicating ongoing degradation
8 97.2 2.8 97.8 2.2
of CBZ.
During the analysis of human liver samples (FSE_1, FSE_2), com-
Fig. 9 shows the mass spectrum for 9-chloroacridine; the typical isotopic pounds unrelated to the metabolism and degradation products of CBZ
distribution of compounds containing a chlorine atom is evident. were detected. Identification was performed using the TraceFinder.
Fragmentation analysis of the chlorine-containing degradation Forensic software (Thermo Scientific™), designed for chemical-
products shows the loss of one chlorine atom (loss of m/z = 34.968), toxicological and forensic analysis, along with the NIST Mass Spectral
while maintaining a general fragmentation pattern characteristic of Search Program database and a database of narcotic and toxic sub-
CBZ. Fig. 10 shows the fragmentation spectrum of 10-chlorocarbazepine stances provided by the Federal State Budgetary Institution "RCME" of
(10-Cl-CBZ) (see Appendix Table S3, No. 20). the Ministry of Health of Russia.
During the study, 22 degradation products of CBZ were identified in In the human liver sample FSE_1, in addition to CBZ, amiodarone
model solutions subjected to artificial aging using 18 % HCl, 0.1 M was detected (Fig. 11). This compound is a part of the antiarrhythmic
NaOH, and 3 % H2O2. It was found that oxidation of CBZ leads to sig- drug of the same name and possesses antianginal properties.
nificant molecule degradation, indicating its instability under oxidative In sample FSE_2, four compounds were found in addition to CBZ: 6-
conditions. However, all identified degradation products, except imi- beta-naltrexone (a metabolite of naltrexone, an opioid receptor antag-
nodibenzyl, were present in all model solutions, indicating the overall onist used for opioid and alcohol dependence), venlafaxine – (an anti-
instability of CBZ in adverse environmental conditions and its tendency depressant belonging to the group of selective serotonin and
towards chemical modifications and degradation. norepinephrine reuptake inhibitors, prescribed for depressive and anx-
iety disorders), its metabolite desmethylvenlafaxine and chlorprothix-
ene (a neuroleptic with sedative and antipsychotic effects) (Fig. 12).
4.5. Analysis of samples of human liver tissue
5. Conclusions
Forensic samples of human liver autopsy evidence provided by the
Federal State Budgetary Institution "Research Centre for Medical Ge- A methodology for qualitative and quantitative determination of
netics" of the Ministry of Health of Russia were analysed under the same carbamazepine (CBZ) in human liver tissue using HPLC-HRMS on a
conditions as the model samples. Analysis of the two forensic samples Vanquish chromatographic system (Thermo Scientific™, USA) coupled
containing CBZ (FSE_1, FSE_2) revealed 2 of the 22 degradation products with an Orbitrap Exploris 120 mass spectrometer (Thermo Scientific™,
described above (Table 10). USA) has been developed. Validation of the developed method has been
Iminostilben (IM) were identified in the two forensic samples both conducted, and key analytical characteristics have been determined.
when analysed immediately and after air aging for 1, 3, 5 and 8 weeks. It The limit of detection (LOD) is 1 ng/g, the lower limit of quantitation
can be hypothesized that IM may have formed in the tissue through (LLOQ) or minimum detectable concentration is 5 ng/g (Fig. S7). The
degradation processes as well as natural metabolism. The second working range of calibration covers concentrations from 5 to 2000 ng/g.
detected degradation product had an ion [M+H]+= 253.0973, with a

Fig. 11. The section of the chromatogram, MS spectrum of fragmentation and library spectrum of amiodarone, found the sample FSE_1.

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Fig. 12. The section of the chromatogram, MS spectrum of fragmentation and library spectrum of compounds (6-beta-naltrexone (С20Н25NO4), Venlafaxine
(С17H27NO2), Dimethylvenflaxine (С16H25NO2), Chlorprothixene (С18H18ClNS) found the sample FSE_2.

Method accuracy meets the requirements for analytical methods different periods of time was investigated. The experiment showed that
used in forensic chemistry and chemico-toxicological analysis of bio- CBZ tends to degrade under environmental conditions. The maximum
logical materials, with deviations not exceeding ± 20 % for all control decrease in concentration was observed during the first week of storage
concentration levels. Signal recovery evaluation showed that the analyte (on average by 20 %), with a subsequent approximate halving of con-
signal in samples does not exceed 20 % of the LLOQ signal level, and the centration over 8 weeks. This relationship allows estimating the possible
internal standard signal in samples does not exceed 5 % of the back- lethal dose in poisoning and the time of death.
ground signal. The developed method complies with the validation The presented data, when carrying out forensic medical examina-
guidelines of the Federal State Budgetary Institution "RCM&E" of the tion, will allow to estimate the level of toxicant concentration in bio-
Ministry of Health of Russia. Degradation products of carbamazepine in logical material shortly before death. This is relevant in case of finding a
model solutions were investigated under the influence of hydrochloric corpse 1–2 months after death.
acid, sodium hydroxide, and hydrogen peroxide oxidation. Twenty-two Analysis of two post-mortem forensic liver samples provided by the
degradation products were identified. It was found that the most Federal State Budgetary Institution "RCM&E" of the Ministry of Health of
intensive degradation of carbamazepine with the formation of various Russia (FSE_1 and FSE_2) revealed CBZ concentrations corresponding to
degradation products occurs during oxidation in an acidic solution toxic levels in tissue. The measured concentration was 3150 ± 280 ng/g
adjusted to pH= 1–2 with 3 % hydrogen peroxide. The stability of car- for FSE_1 and 7880 ± 820 ng/g for FSE_2. The upper limits of thera-
bamazepine in human liver tissues under environmental conditions over peutic concentration ranges were exceeded by 1.5 and 3.9 times,

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Fig. 12. (continued).

respectively. Based on the analysis of the forensic liver samples (FSE_1 Declaration of Competing Interest
and FSE_2), two out of the 22 CBZ degradation products described in this
study were detected. Additionally, amiodarone was found in FSE_1, and The authors declare that they have no known competing financial
four compounds representing pharmaceutical substances or their me- interests or personal relationships that could have appeared to influence
tabolites were identified in FSE_2. Therefore, combined poisoning could the work reported in this paper.
have been a possible cause of death.
Acknowledgments
CRediT authorship contribution statement
The study was carried out using the equipment of the Central Col-
Barge Alessandro: Writing – review & editing, Validation. Nosyrev lective Use Center of Moscow State University “Technologies for
Aleksander: Funding acquisition. Barsegyan Samvel: Writing – review Obtaining New Nanostructured Materials and Their Comprehensive
& editing, Supervision, Resources, Methodology. Akimova Valeriya: Study”, acquired by Moscow State University under the program for
Writing – review & editing, Supervision, Data curation. Gandlevskiy updating the instrumentation base within the framework of the national
Nikita: Writing – review & editing, Formal analysis, Data curation, project “Science” and the Development Program of Moscow State
Conceptualization. Shirokova Ekaterina: Investigation, Formal anal- University.
ysis, Data curation. Pirogov Andrei: Writing – original draft, Project
administration, Conceptualization.

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A. Pirogov et al. Toxicology Reports 14 (2025) 101923

Appendix A. Supporting information [12] B.M. Kerr, K.E. Thummel, C.J. Wurden, S.M. Klein, D.L. Kroetz, F.J. Gonzalez,
RenéH. Levy, Human liver carbamazepine metabolism, Biochem. Pharmacol. 47
(11) (1994) 1969–1979, https://doi.org/10.1016/0006-2952(94)90071-x.
Supplementary data associated with this article can be found in the [13] A.G. Kalogeropoulou, I.K. Christina, A.A. Triantafyllos, Simultaneous
online version at doi:10.1016/j.toxrep.2025.101923. determination of pharmaceuticals and metabolites in fish tissue by QuEChERS
extraction and UHPLC Q/Orbitrap MS analysis, Anal. Bioanal. Chem. 413 (2021)
7129–7140, https://doi.org/10.1007/s00216-021-03684-y.
Data Availability [14] H.J. Kupferberg, GLC determination of carbamazepine in plasma, J. Pharm. Sci. 61
(N 2) (1972) 284–286, https://doi.org/10.1002/jps.2600610235.
Data will be made available on request. [15] C.J. Least, G.F. Johnson, H.M. Solomon, Therapeutic monitoring of anticonvulsant
drugs: gas-chromatographic simultaneous determination of primidone,
phenylethylmalonamide, carbamazepine, and diphenylhydantoin // clinical
References chemistry, Ther. Monit. Antico Drugs 21 (1975) 1658–1662. PMID: 1164796.
[16] G. Ferro, M.I. Polo-López, A.B. Martínez-Piernas, P. Fernández-Ibáñez, A. Agüera,
[1] S. Deeb, D.A. McKeown, H.J. Torrance, F.M. Wylie, B.K. Logan, K.S. Scott, L. Rizzo, Cross-contamination of residual emerging contaminants and antibiotic
Simultaneous analysis of 22 antiepileptic drugs in postmortem blood, serum and resistant bacteria in lettuce crops and soil irrigated with wastewater treated by
plasma using LC–MS-MS with a focus on their role in forensic cases, J. Anal. sunlight/H 2 O 2, Environ. Sci. Technol. 49 (18) (2015) 11096–11104, https://doi.
Toxicol. 38 (2014) 485–494, https://doi.org/10.1093/jat/bku070. org/10.1021/acs.est.5b02613.
[2] R.E. Pearce, W. Lu, Y. Wang, J.P. Uetrecht, M.A. Correia, J.Steven Leeder, [17] B. Martínez-Piernas, M.I. Polo-López, P. Fernández-Ibáñez, A. Agüera, Validation
Pathways of Carbamazepine Bioactivation in Vitro. III. The role of human and application of a multiresidue method based on liquid chromatography-tandem
cytochrome p450 enzymes in the formation of 2,3-dihydroxycarbamazepine, Drug mass spectrometry for evaluating the plant uptake of 74 microcontaminants in
Metab. Dispos. 36 (2008) 1637–1649, https://doi.org/10.1124/dmd.107.019562. crops irrigated with treated municipal wastewater, J. Chromatogr. A 1534 (2018)
[3] F.T. Peters, A.E. Steuer, Antemortem and postmortem influences on drug 10–21, https://doi.org/10.1016/j.chroma.2017.12.037.
concentrations and metabolite patterns in postmortem specimens, WIREs Forensic [18] A.V. Pirogov, N.A. Gandlevskiy, A.A. Vasil’eva, S.S. Barsegyan, A.E. Nosyrev,
Sci. 1 (2019) e1297, https://doi.org/10.1002/wfs2.1297. Highly sensitive determination of carbamazepine and oxcarbazepine and
[4] X. Wang, S.S. Johansen, M.K.K. Nielsen, K. Linnet, Targeted analysis of 116 drugs identification of their degradation products in material evidences and human
in hair by UHPLC-MS/MS and its application to forensic cases, Drug Test. Anal. 9 cadaveric liver by gas chromatography–mass spectrometry, J. Anal. Chem. 78 (6)
(8) (2017) 1137–1151, https://doi.org/10.1002/dta.2130. (2023) 783–793, https://doi.org/10.1134/S1061934823060096.
[5] L. Lionetto, B. Casolla, M. Cavallari, P. Tisei, C. Buttinelli, M. Simmaco, High- [19] Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices
Performance liquid chromatography–tandem mass spectrometry method for for Method Validation in Forensic Toxicology, J. Anal. Toxicol. 37 (7) (2013)
simultaneous quantification of carbamazepine, oxcarbazepine, and their main 452–474.
metabolites in human serum, Ther. Drug Monit. 34 (1) (2012) 53–58, https://doi. [20] World Health Organization. Quality assurance of pharmaceuticals: a compendium
org/10.1097/FTD.0b013e3182425168. of guidelines and related materials: vol. 2: Good manufacturing practices and
[6] W. Jiang, T. Xia, Y. Yun, M. Li, F. Zhang, S. Gao, W. Chen, UHPLC-MS/MS method inspection / Text: electronic // Good manufacturing practices and inspection. –
for simultaneous determination of carbamazepine and its seven major metabolites 2007. – Quality assurance of pharmaceuticals. – URL: https://iris.who.int/handle/
in serum of epileptic patients, J. Chromatogr. B 1108 (2019) 17–24, https://doi. 10665/43532 (date accessed: 19.02.2024).
org/10.1016/j.jchromb.2018.12.016. [21] https://sudact.ru/law/reshenie-soveta-evraziiskoi-ekonomicheskoi-komissii-ot-
[7] G.F. Van Rooyen, D. Badenhorst, K.J. Swart, H.K.L. Hundt, T. Scanes, A.F. Hundt, 03112016_17/pravila-provedeniia-issledovanii-bioekvivalentnosti-
Determination of carbamazepine and carbamazepine 10,11-epoxide in human lekarstvennykh/prilozhenie-n-6/ii_4/2_7/vliianie-effekt-perenosa/ (Date of
plasma by tandem liquid chromatography–mass spectrometry with electrospray reference 20.03.2024). – Text: electronic.
ionisation, J. Chromatogr. B 769 (1) (2002) 1–7, https://doi.org/10.1016/s1570- [22] TMIC: The Human Metabolome Database (HMDB). – URL: https://hmdb.ca/ (Date
0232(01)00590-6. of reference: 14.03.2024). – Text: electronic.
[8] H. Breton, M. Cociglio, F. Bressolle, H. Peyriere, J. Blayac, D. Hillairebuys, Liquid [23] Y. Li, Y. Yang, J. Lei, W. Liu, M. Tong, J. Liang, The degradation pathways of
chromatography–electrospray mass spectrometry determination of carbamazepine, carbamazepine in advanced oxidation process: a mini review coupled with DFT
oxcarbazepine and eight of their metabolites in human plasma, J. Chromatogr. B calculation // science of the total environment, Degrad. Pathw. carbamazepine
828 (1-2) (2005) 80–90, https://doi.org/10.1016/j.jchromb.2005.09.019. Adv. Oxid. Process 779 (2021) 146498, https://doi.org/10.1016/j.
[9] G. Daniele, M. Fieu, S. Joachim, A. Bado-Nilles, R. Beaudouin, P. Baudoin, scitotenv.2021.146498.
A. James-Casas, S. Andres, M. Bonnard, I. Bonnard, A. Geffard, E. Vulliet, [24] V. Calisto, M.R.M. Domingues, G.L. Erny, V.I. Esteves, Direct photodegradation of
Determination of carbamazepine and 12 degradation products in various carbamazepine followed by micellar electrokinetic chromatography and mass
compartments of an outdoor aquatic mesocosm by reliable analytical methods spectrometry, Water Res. 45 (3) (2011) 1095–1104, https://doi.org/10.1016/j.
based on liquid chromatography-tandem mass spectrometry, Environ. Sci. Pollut. watres.2010.10.037.
Res. 24 (20) (2017) 16893–16904, https://doi.org/10.1007/s11356-017-9297-6. [25] J. Li, L. Dodgen, Q. Ye, J. Gan, Degradation kinetics and metabolites of
[10] B. Rambeck, R. Schnabel, Th May, U. Jürgens, R. Villagran, Postmortem carbamazepine in soil, Environ. Sci. Technol. 47 (8) (2013) 3678–3684, https://
Concentrations of phenobarbital, carbamazepine, and its metabolite doi.org/10.1021/es304944c.
carbamazepine-10,11-epoxide in different regions of the brain and in the serum: [26] A. Bahlmann, W. Brack, R.J. Schneider, M. Krauss, Carbamazepine and its
analysis of autoptic specimens from 51 epileptic patients, Ther. Drug Monit. 15 (2) metabolites in wastewater: analytical pitfalls and occurrence in Germany and
(1993) 91–98, https://doi.org/10.1097/00007691-199304000-00004. Portugal // water research, Carbamazepine its Metab. Wastewater 57 (2014)
[11] J.-W. Kwon, K.L. Armbrust, D. Vidal-Dorsch, S.M. Bay, K. Xia, Determination of 17- 104–114, https://doi.org/10.1016/j.watres.2014.03.022.
ethynylestradiol, carbamazepine, diazepam, simvastatin, and oxybenzone in fish [27] M. Soufan, M. Deborde, A. Delmont, B. Legube, Aqueous chlorination of
livers, J. AOAC Int. 92 (1) (2009) 359–370. PMID: 19382594. carbamazepine: Kinetic study and transformation product identification // water
research. Aqueous chlorination carbamazepine 47 (2013) 5076–5087, https://doi.
org/10.1016/j.watres.2013.05.047.

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