Journal of Chromatography B 1061–1062 (2017) 459–467

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Human bone marrow as a tissue in post-mortem identification and T

determination of psychoactive Substances—Screening methodology
⁎
Renata Wietecha-Posłusznya, , Sofia Lendora, Magdalena Garnysza, Marcin Zawadzkib,
Paweł Kościelniaka
a
Laboratory for Forensic Chemistry, Department of Analytical Chemistry, Jagiellonian University, 3 Ingardena St., 30-060 Kraków, Poland
b
Department of Forensic Medicine, Medical University in Wroclaw, 4 Jana Mikulicza-Radeckiego St., 50-345 Wrocław, Poland

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

Keywords: The aim of this study was to apply a new instrumental approach to the analysis of human bone marrow for
Forensics/Toxicology forensic purposes. A new screening method for the detection of more than 30 psychoactive drugs in bone marrow
Biological samples was developed and applied to case samples. The drugs used in this study belonged to the following groups:
Drug monitoring/Drug screening benzodiazepines (BZDs, n = 16), tricyclic antidepressants (TCAs, n = 5), selective serotonin reuptake inhibitors
Human bone marrow
(SSRI, n = 4), serotonin-norepinephrine reuptake inhibitors (n = 1), anticonvulsants (n = 1), imidazopyridines
MAE
Sample pretreatment
(n = 1) and piperazines (n = 3). The sample preparation procedure involved microwave-assisted extraction
(MAE) and the experimental settings were optimized using the simplex method. Separation and detection was
carried out using a UHPLC-TOF system. The method were validated using marrow samples, and further applied
in the analysis of three case samples, in which diazepam, nordiazepam, citalopram, doxepin and paroxetine were
successfully detected. Finally the presented method is a good example of an assay that could potentially find an
application in forensic analysis.

1. Introduction protecting from the destructive postmortem activity of fungi, bacteria,

Bone marrow (BM) is a structurally and functionally diverse con-
nective tissue located within bones. Bone has several functions, in-
cluding acting as a support for muscles, and playing a major role in
hematopoiesis, the immune system response, blood coagulation and the
metabolism of calcium and phosphorus [1]. Bone is the fourth largest
organ in the body, after the skeleton, skin and fat. The weight of bone
marrow within the body of an adult can reach up to approximately 3 kg
in males and 2.6 kg in females [1,2]. Bone marrow quality − along
with the mineral density of the bone − affects bone strength [1].
Bone marrow can be classified into two types, depending on the
main components: red (hematopoietic) and yellow (fatty) marrow. With
age, the red marrow undergoes progressive physiologic conversion to
the yellow type [2]. Chemically, BM consists of lipids, water and pro-
teins; however, the percentage of each component differs between red
and yellow marrow. Similarly, the content of hematopoietic cells and
fat cells (adipocytes) in the two marrow types varies [1,2].
BM is located inside the marrow cavity of solid bone tissue.
Therefore, a natural physical barrier is provided that can prevent ex-
posure to exogenous contaminating factors such as substances con-
tained in the soil or products of soft tissue putrefaction, as well as
animals and plants [3]. Thanks to the bone shield, which provides
mechanical stability, the bone marrow is the most protected tissue
within the body. As a result, despite rich vascularization of bone
marrow, postmortem putrefaction is delayed in this tissue [4]. The
barrier may maintain its role as long as the bone preserves its integrity,
but after bone disintegration, the marrow becomes exposed to exo-
genous contaminating factors in the soil or products of soft tissues’
putrefaction, in addition to the action of micro-organisms, and thus
may no longer be useful as a matrix for analyses [5]. Reported data
suggest that the estimated preservation of bone marrow (on ahistolo-
gical level) is 3 months [6], while its suitability for toxicological ana-
lysis may be preserved for up to 5 years [7]. The initially red or yellow
gelatinous tissue may gradually darken and become a brown oily liquid,
may dry to form brown or yellow powder, or may become an adipo-
cerous substance due to saponification [7].
From the analytical point of view, BM is a complex, heterogeneous
matrix whose cellular and chemical composition depends on both the
site of collection and individual features of the sample source. The
accumulation of xenobiotics in this tissue may also be dependent on
water and fat content. Therefore, to utilize bone marrow as a matrix in
forensic-toxicological analyses of samples collected during an autopsy,

⁎
Corresponding author.
E-mail address: wietecha@chemia.uj.edu.pl (R. Wietecha-Posłuszny).

http://dx.doi.org/10.1016/j.jchromb.2017.08.006
Received 8 February 2017; Received in revised form 31 July 2017; Accepted 3 August 2017
Available online 07 August 2017
1570-0232/ © 2017 Elsevier B.V. All rights reserved.
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

various processes occurring postmortem and affecting the xenobiotics

[10]
[11]

[12]
[13]

[14]

[15]

[16]
Ref.
present in marrow should be taken into account [3,5]. These processes
include postmortem redistribution, transformation, degradation, eva-

Analytical technique
poration and neo-formation. Due to the multitude and complexity of

UHPLC–MS/MS

RP-HPLC-DAD
these phenomena, the interpretation of analytical results may be diffi-

GC–MS/MS
LC–MS/MS

HS-GC–MS
cult or even impossible. This explains why bone marrow is not a ma-

GC–MS

GC–MS
terial collected routinely [6,7]. However, it may be useful in special
cases where reference materials such as blood and urine are unavailable
or unusable due to body decomposition or skeletonization, as well as in

Sample preparation method

exhumation cases or aircraft accidents [7].
The described properties of bone marrow are very advantageous for

incubation at 60 °C for
its use as a specimen for analyses in the field of forensic toxicology.
However, it is not commonly used due to insufficient data about the
pharmacokinetics of bone marrow, as well as the absence of general
knowledge on whether the drug concentrations found in marrow re-

LLE, SPE

39 min
present blood levels at the time of death [7,8].

LLE
LLE

LLE

LLE

LLE
BM is more frequently utilized in other branches of forensic science.
Histopathological analysis may provide a variety of useful data con-

transversal section of the femur, freezing

cerning the postmortem interval and diseases occurring before death

flushing with PBS, freezing at −20 °C

Method of sample collection, storage
[8,9]. Additionally, it has been studied to determine the cause and
manner of death in cases of human remains found in an aquatic context

aspiration, freezing at −20 °C

− by examination, amongst other things, for the presence of diatoms,
which can help to confirm or exclude a diagnosis of drowning [10,12].

squeezing with pliers

However, bone marrow has been most relevant in genetic analyses, as it
provides a valuable source of DNA and RNA, due to the fact that it
remains undecomposed and is available for analyses much longer than

at −20 °C
aspiration

aspiration
any other tissue, except bones [4]. Furthermore, the minimized influ-
ence of body decomposition products that could contaminate genetic

-*
material in bone marrow is another important factor in maintaining the
suitable quality of this matrix [4]. human, proximal and medial femus (left and right), 5th

human, 2724 cases of forensic autopsies, lower thoracic

human, 683 cases of forensic autopsies, lower thoracic

Since the first reported attempt at bone marrow analysis in 1943,
toxicological data have been published concerning determination of
xenobiotics and efforts to find a correlation between blood and marrow

human, 99 cases of forensic autopsy, femur

drug levels in both human and animal specimens obtained postmortem.
human, marrow cells and supernatant

Moreover, there have been several case reports involving toxicological

analysis of BM collected from skeletonized remains. The various reports
and studies on this topic were summarized in an extensive review by
Cartiser et al. in 2011 [7].
Since then, only a few new findings have been published concerning
ribs (left and right),

rat, femur and tibia

determination of various xenobiotics, including psychoactive sub-

stances, in bone marrow. Collected data on the most recently published
human, femur
Sample origin

reports on utilizing BM in toxicological analyses are included in Table 1

vertebrate

vertebrate

[10–16].
Drug screening in specimens obtained during an autopsy is a com-
plex process, due to the possibility of postmortem drug concentration
Studies concerning toxicological analysis of bone marrow reported since 2010.

alterations. These changes may be caused by such phenomena as au-

ethanol, acetone, cyanide, toluene, liquefied petroleum, cresol, ethylene,

tolysis of cells, which can trigger residual enzymatic activity at the

early stages of the postmortem period, leading to the biotransformation
of drugs. During the putrefaction process occurring at subsequent stages
of the postmortem interval, further tissue degradation and the forma-
36 drugs (amphetamines, codeines, sedatives, BZDs, TCAs,

tion of interfering degradation products may be a result of microbial

and fungal invasion. Moreover, the interpretation of postmortem drug
concentrations may be extremely challenging in some cases due to the
anticonvulsants, anesthetics, analgesics)

fact that they do not necessarily reflect blood concentrations at the time
of death, as some substances undergo postmortem redistribution [1,9].
These processes may be further complicated by a variety of environ-
mental factors, such as temperature fluctuations, water exposure, burial
conditions and scavenging [7,9].
Thus, the aim of this paper was to develop and validate, for the first
diazepam nordiazepam

time, a sensitive and effective MAE/UHPLC–MS-TOF method for quick

and simultaneous identification and quantification of psychoactive
*no data available.

substances in human bone marrow. The analytes of interest to this study

meprobamate

clofazimine

involve substances that are routinely screened to determine the cause of

veliparib
Analytes

H2S
caffeine

death or may provide valuable information about the circumstances of

Table 1

death. MAE was used as a green sample preparation technique and the
modified simplex method [17,18] was applied for optimization of the

460
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

extraction parameters. Table 3

Adjusted MS parameters, chosen as optimal.

Parameter (unit) Value for MS coupled with UHPLC

2. Experimental
ESI ionization mode Positive
2.1. Chemicals and reagents capillary voltage (V) −4500
nebulizer pressure (Bar) 2.5
dry gas flow (L/min) 5.5
Drug standards of nordiazepam, nitrazepam, diazepam, oxazepam,
dry gas temperature (°C) 200
tetrazepam, estazolam, temazepam, alprazolam, bromazepam, clona- MS-TOF lens 1 transfer (μs) 45.0
zepam, lorazepam, prazepam, midazolam, lormetazepam, clorazepate, lens 1 pre puls storage (μs) 5.0
sertraline, fluoxetine, citalopram, paroxetine, venlafaxine, venlafaxine- mass range (m/z) 50–800
D5, benzylpiperazine (BZP), 1-(3-chlorophenyl) piperazine (mCPP),
and 1-(3-trifluoromethylphenyl) piperazine (TFMPP) were purchased
from LGC Standards (Teddington, UK). Standards of nortriptyline, de- TOC < 5 ppb) was obtained and filtered through a Milli-Q Plus system
sipramine, amitriptyline, imipramine, clomipramine, clomipramine-D3, (Millipore, Bedford, MA, USA)
carbamazepine, and carbamazepine-D10 were purchased from LGC
Standards (Teddington, UK). Moreover, the hypnotic drug zolpidem, 2.2. Apparatus and conditions
and the deuterated analogues of drugs used as internal standards (lor-
azepam-D4, clonazepam-D4, diazepam-D5, and nordiazepam-D5) were The UltiMate 3000 RS liquid chromatography system (UHPLC;
purchased from Lipomed AG (Arlesheim, Switzerland). Standard drug Dionex, Sunnyvale, CA, USA) equipped with a Hypersil Gold Phenyl
stock solutions (20 μg/mL) prepared in methanol were stored in a column (50 mm × 2.1 mm I.D., particles 1.9 μm; Thermo Scientific,
freezer at −20 °C. All analytes, as well as their abbreviations, are in- Bremen, Germany) was coupled to a mass spectrometer with electro-
cluded in Table 2. spray ionization source (ESI) and MicrOTOF-Q II time-of-flight analyzer
LC–MS–grade acetonitrile, methanol, isopropyl alcohol, and n- (Bruker, Bremen, Germany). The composition of the mobile phase and
hexane were obtained from Sigma-Aldrich (St. Louis, MO, USA); ana- the gradient program used in the UHPLC-based method were in-
lytical grade formic acid and ethanol were purchased from Merck vestigated. A mixture consisting of 0.1% water solution of formic acid
(Darmstadt, Germany). Other reagents: analytical grade ethyl acetate (A) and acetonitrile (B) was chosen as the mobile phase. The best se-
and 30% NaOH water solution were supplied by Avantor Performance paration in the shortest time was achieved by applying the following
Materials (Gliwice, Poland). Ultrapure water (18.2 MΩ cm, gradient program: acetonitrile: 0 min–5%; 14 min–70%; 16.5 min–5%;

Table 2
List of analytes and their selected properties.

Nr Drug Abbrev. [M + H]+ Formula pKa [19,20] LogP [19,20] Protein binding (%) [19,20]

piperazines
1 benzylpiperazine BZP 177.1386 C11H16N2 9.59 1.38 12
2 mCPP mCPP 197.0840 C10H13N2Cl 8.87 2.15 -a
3 TFMPP TFMPP 231.1103 C11H13N2F3 1.45 2.44 -a
anticonvulsants
4 carbamazepine CBZ 237.1102 C15H12N2O 13.94 2.45 75
BZDs
5 nordiazepam NORD 271.0633 C15H11N2OCl 12.00 2.90 97
6 nitrazepam NITR 282.0873 C15H11N3O3 10.80 2.10 85–88
7 diazepam DIA 285.0789 C16H13N2OCl 3.40 2.70 89–99
8 oxazepam OXA 287.0581 C15H11N2O2Cl 11.60 2.20 95
9 tetrazepam TETR 289.1102 C16H17N2OCl 4.30 3.20 30–70
10 estazolam EST 295.0745 C16H11N4Cl 12.33 4.70 93
11 temazepam TEM 301.0738 C16H13N2O2Cl 1.60 2.19 96
12 alprazolam ALP 309.0901 C17H13N4Cl 2.40 2.12 70–80
13 bromazepam BRO 316.0080 C14H10N3OBr 10.50 2.05 70
14 clonazepam CLO 316.0483 C15H10N3O3Cl 11.21 2.41 86
15 lorazepam LOR 321.0192 C15H10N2O2Cl2 11.50 2.40 90
16 prazepam PRA 325.1102 C19H17N2OCl 3.00 3.70 97
17 midazolam MID 326.0855 C18H13N3FCl 5.50 4.30 95–98
18 lormetazepam LORM 335.0348 C16H12N2O2Cl2 11.60 2.35 90
19 clorazepate CLOR 337.0350 C16H11N2O3Cl 12.50 3.00 91
TCAs
20 nortriptyline NORT 264.1746 C19H21N 9.70 1.70 90–95
21 desipramine DESI 267.1855 C18H22N2 10.20 1.40 70–90
22 amitriptyline AMI 278.1903 C20H23N 9.40 4.94 1–97
23 imipramine IMI 281.2012 C19H24N2 9.40 4.80 85–95
24 doxepin DOX 280.1696 C19H21NO 9.76 4.29 80
25 clomipramine CLOMI 315.1622 C19H23N2Cl 9.50 5.20 90–95
SSRI and SNRI
26 venlafaxine VENLA 278.2115 C17H27NO2 10.10 0.43 30
27 sertraline SERT 306.0810 C17H17NCl2 9.50 5.29 98
28 fluoxetine FLUOX 310.1413 C17H18NOF3 10.30 4.05 95
29 citalopram CITA 325.1710 C20H21N2OF 9.50 3.74 50
30 paroxetine PAROX 330.1499 C19H20NO3F 9.90 3.95 95
sedatives/hypnotics
31 zolpidem ZOLP 308.1757 C19H21N3O 6.14 3.85 92

*
no data available

461
R. Wietecha-Posłuszny et al.
20 min–5%. The best parameters were chosen for the mass spectro-
meter based on signal intensity in the range covering [M + H]+ values
of target analytes (ca. 177–337 m/z). The chosen parameters are pre-
sented in Table 3. Before each sequence, cluster mass calibration was
performed using a 10 mM sodium formate solution in a mixture of
isopropanol and water (1:1, v/v). Additionally, the calibration solution
was supplied to the MS throughout the entire run using a valve, al-
lowing calibration solution injection before each measurement.
Data were acquired and processed by HyStar 3.2, micrOTOFcontrol
and Compass DataAnalysis software (Bruker), respectively. The ex-
pected masses of ions of all analytes, [M + H]+, were calculated using
IsotopePattern software (Bruker), in order to obtain extracted ion
chromatograms.
2.3. Sample collection
Drug-free human bone marrow for the method optimization as well
as drugs positive samples collected during an autopsy were provided by
courtesy of the Forensic Medicine Unit at the Department of Forensic
Medicine of Wrocław Medical University (according the Bioethical
Commission Approval no KB 333/2014). Calf bones (femur) that served
as a source of bone marrow used for the method optimization as well
asvalidation experiments were provided by a local meat company from
Tarnowskie Góry. Due to the fact that human BM accumulates com-
monly used, and abused substances, there were difficulties with ob-
taining drug-free/blank samples. Furthermore, the available amounts of
human BM of one type were very limited, not sufficient for the method
development purpose. Thus, animal (calf) BM was used for method
optimization and validation. All BM samples were stored frozen at
−20 °C until analysis.
2.4. Sample preparation and extraction
The MARS5 microwave-assisted system (CEM, Matthews, NC, USA)
equipped with Xpress® PFA vessels (75 mL) was used for bone marrow
preparation. Drug-free human bone marrow and calf bone marrow were
used simultaneously in the preliminary experiments, while only calf BM
was utilized in the optimization and validation experiments. First step
of sample preparation process involved lyophilization for 24 h at
−78 °C using freeze-dry system FreeZone 2.5 (Labconco, Kansas City,
MO, USA). Prior to extraction, BM was thawed, and 45 mg samples
were weighed, transferred into extraction vessels and spiked with
500 μL of a water solution of all analytes at a concentration of 200 ng/
mL. Spiked solutions were prepared daily by appropriately diluting
stock solutions with water. Then 2 mL of matrix modifier (0.6 M solu-
tion of NaOH, pH > 13) was added to achieve the pH value at which
the analytes are non-ionized and therefore are more easily transferred
into the organic phase of the extraction solvent. Samples prepared in
such way were then homogenized by sonication for 10 min.
Subsequently, 3 mL of ethyl acetate was added and the closed vessels
were placed in the microwave oven where extraction at 50 °C for
16 min was carried out. After cooling down to room temperature, the
content of each vessel was transferred into a Falcon tube and additional
2 × 0.5 mL of extraction solvent was used to rinse each vessel. After
centrifugation (10 min, 4000 rpm, 4 °C), 3 mL of organic phase from
each sample was transferred into an Eppendorf tube and evaporated to
dryness under a stream of nitrogen at 40 °C. The next step involved
removing of the residual fat by 30 s vortexing followed by 15 min of
shaking with 4 mL of lipid removal mixture (hexane/ethanol 7:2, v/v
plus 200 μL of water). 500 μL of the ethanolic phase was then trans-
ferred into an Eppendorf tube and evaporated to dryness under a stream
of nitrogen at 60 °C. The residue was dissolved in 110 μL of water/
acetonitrile (1:1, v/v), vortexed for 10 min and centrifuged (10 min,
10000 rpm, 4 °C). Along with the extracted spiked samples, blank
samples prepared from both human and calf BM (using the same pro-
cedure as for spiked samples) were analyzed.
462
Journal of Chromatography B 1061–1062 (2017) 459–467
3. Results and discussion
3.1. Optimization of the sample pre-treatment procedures
The sample preparation procedure for bone marrow involved such
main steps as: lyophilization, homogenization, extraction and lipid re-
moval.
Since bone marrow is a heterogeneous tissue of varying composition
and consistency, the lyophilization process was introduced in order to
investigate the water content within different types of BM and eliminate
this variable, as it could affect the extraction process in different ways,
depending on the origin of BM samples. The freeze-drying process was
carried out at ‐78 °C for 48 h.
The homogenization step was performed to obtain homogenous li-
quid samples suitable for further extraction, as due to the semi-solid
nature of selected types of BM they are less prone to release analytes
during extraction. This process was expected to improve both the ex-
traction efficiency and the precision of the developed methods as it was
expected to further reduce the samples’ heterogeneity.
Homogenization was carried out by means of ultrasonication. In
order to choose the most suitable conditions of ultrasound exposure
needed for the sample homogenization, several process duration times
were tested (0; 0.5; 1; 5; 10 min). Results were evaluated taking into
consideration the number of detected analytes and the relative peak
areas of detected analytes and their internal standards. For the majority
of the detected analytes, the relative peak areas increased with in-
creasing time of ultrasonication. Moreover, increased time did not ne-
cessarily reduce the number of analytes detected. A visual assessment of
the homogenized BM sample consistency also confirmed that longer
homogenization is more beneficial. Ultimately, 10 min of exposure to
ultrasound was chosen as the most suitable time of BM sample homo-
genization.
The extraction step was carried out using the microwave assisted
extraction technique (MAE), which was optimized in terms of extrac-
tion temperature and time using the modified simplex method devel-
oped by Spendley et al. [17,18]. Two factors were further optimized (in
the present study): temperature (X1) and time (X2) of extraction. The
independent factors were regarded as the coordinate axes of a factor
space; every experiment was represented as a point in that space and
the simplex was represented as a triangle. The initial values of factors
X1 and X2 were chosen arbitrarily as 60 °C and 12 min, 80 °C and
12 min, and 70 °C and 14 min respectively. The initial simplex, as well
as further simplex evolution, are shown in Fig. 1S (Supplementary
materials). At each step of optimization, three extractions were per-
formed, using the three sets of factor values corresponding to the sim-
plex vertices. After obtaining the results of analysis, every set of con-
ditions was evaluated, taking into consideration the overall number of
detected analytes and peak intensities. The response function, Y, took
into account the total number of peaks as well as the penalty for low
intensity peaks, according to the equation: Y = n–0.5·l, where n is the
number of peaks and l is the number of peaks of intensity lower than
750 (see Fig. 1). The average Y values for each point are presented in
Table 4.
The best results were obtained in experiment no. 4, as further
simplex progression led back to that point, and therefore the search for
the optimum conditions had ended. The optimal values of temperature
and time of extraction using ethyl acetate as the extraction solvent were
50 °C, and 16 min, respectively. Fig. 1 shown the separation of all
analytes in human and calf marrow. The final separation of 30 psy-
chotropic substances took only 12 min.
After evaporation of the extraction solvent, a relatively large
amount of fat remained on the bottom of the vessel (ca. 20–100 μL,
depending on the BM type). After reconstitution of the residue, an ex-
cess of lipids remained present even after several cycles of centrifuga-
tion. 4 mL of a mixture of n-hexane and ethanol (7:2, v/v) and 200 μL of
water was added [21,22] to the fat remaining after the evaporation of
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

Fig. 1. Chromatogram of screening of 31 analytes extracted from human (A, C) and calf (B, D) bone marrow samples obtained by MAE-UHPLC/MS method in 12 min (where, 1- BZP, 2-
mCPP, 3- VENLA, 4- TFMPP, 5- ZOLP, 6- BRO, 7- MID, 8- TEM, 9-CBZ, 10- NORD, 11- NITR, 12- CITA, 13- DOX, 14- OXA, 15- LOR, 16- EST, 17- DESI, 18-CLO, 19- PAROX, 20- IMI, 21-
NORT, 22- ALP, 23- TETR, 24- AMI, 25- DIA, 26- CLOR, 27- LORM, 28- FLUOX, 29- SERT, 30- CLOMI, 31- PRA).

Table 4 was also analyzed at each concentration level. The consumption of

Values of optimized factors obtained after every operation applied to the simplex for ethyl analytes by hexane was evaluated by calculation of the extraction ef-
acetate as an extraction solvent. ficiency from the hexane layer (the ratio of the analytical signals ob-
Step No. value [°C] value [min] Operation average Y*
tained for the analyte extracted from the spiked sample to the analytical
signals obtained for the analyte from the non-extracted standard solu-
1 60 12 none, initial simplex 28.25 tion). As seen in Table 5, only five analytes were detected at the lower
2 80 12 22.50 concentration and an additional 4 at the higher concentration. As ex-
3 70 16 24.25
4 50 16 reflection of 2 30.50
pected, substances with high values of partition coefficient between the
5 38 17 expansion of 4 23.75 hydrophobic and hydrophilic phase (logP > 2.5) exhibit a higher af-
6 40 12 reflection of 3 23.75 finity to the hexane phase, containing a significant amount of lipids.
7 63 15 contraction of 3 20.25 Among the detected analytes, BZDs and TCAs predominated (see
8 55 14 contraction towards 4 27.00
Table 2). No correlation between the value of recovery from the hexane
9 60 16 contraction towards 4 27.25
10 55 18 reflection of 8 25.50 layer and the concentration of analytes was observed. Despite the fact
that a number of analytes were “consumed” in the process of lipid
* Response function, Y, equals n-0.5*x, where n is total number of peaks and x is a
number of peaks of intensity lower than 750.
Table 5
the extraction solvent. The vessel content was vortexed for 30 s, shaken Results of extraction efficiency (W) of analytes detected in discarded hexane layer of lipid
removal solution.
for 15 min and finally centrifuged (10 min, 10000 rpm, 4 °C). The
process resulted in partition of the solution into two phases. The top n- Drug logP Extraction efficiency from hexane layer (%) at
hexane layer containing an excess of lipids was discarded, while the final extract concentration
bottom ethanolic layer containing analytes was taken for further pre-
200 ng/mL 500 ng/mL
paration, according to the initial procedure.
Since hexane is capable of extracting drugs from biological matrices, amitriptyline 4.49 2.0 2.9
it was suspected that some of the studied substances may remain in the doxepin 4.29 1.2 2.4
discarded hexane layer of the lipid removal solution. In order to test imipramine 4.80 – 2.6
clomipramine 5.20 – 2.5
this hypothesis, two levels of final mix analyte concentration were in-
diazepam 2.70 1.8 0.8
vestigated: 200 and 500 ng/mL, and two extractions of a water stan- tetrazepam 3.20 4.8 3.0
dard drug mixture (without bone marrow) were performed at each prazepam 3.70 0.9 1.0
concentration level. Additionally, a non-extracted water standard drug midazolam 4.30 – 1.0
mixture at a concentration corresponding to 100% extraction efficiency zolpidem 3.85 – 0.2

463
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

removal, the procedure was considered necessary and it was added to Table 7
the initial sample preparation procedure. Summary of MAE/UHPLC-ESI–MS-TOF method validation parameters.

Drug Concentration level Precision, CV (%) Matrix effect

(ng/mg) (%) (n = 4)
3.2. Method validation intraday interday
(n = 4) (n = 12)
The validation procedure of the MAE/UHPLC–MS-TOF method was
BZP and its derivatives
based on the strategy presented in the literature [23–25]. According to
BZP 0.22 -* -* -*
this literature, some more important validation parameters were chosen 1.67 9 16 48
for new screening methodology. Since validation experiments would 3.33 12 10 67
require large amounts of bone marrow of one type, calf BM was chosen mCPP 0.56 10 14 83
1.67 8 9 56
as the specimen for validation experiments (the analysis of blank
3.33 20 18 96
samples showed that calf BM is similar to human BM in terms of lipid TFMPP 0.22 11 11 83
content) − Fig. 1. To test the linearity, analytes were divided into two 1.11 6 10 57
groups of different ranges of concentrations corresponding to the effi- 2.22 8 23 98
ciency with which they were extracted during the preliminary studies. anticonvulsants and sedatives/hypnotics
zolpidem 0.22 7 12 64
The extraction efficiency (W) was calculated as the ratio of the analy-
1.11 2 3 71
tical signals obtained for the analyte extracted from the spiked BM 2.22 8 13 85
sample to the analytical signals obtained for the analyte in the non- carbamazepine 0.22 8 9 84
extracted standard solution. This criterion was selected due to the fact 1.67 4 4 68
that the studied drugs represent a wide range of therapeutic as well as 3.33 8 10 91
TCAs
toxic plasma concentrations, therefore choosing concentration levels as nortriptyline 0.56 7 7 81
the dividing criterion would have resulted in very complicated and 1.67 3 4 70
technically difficult procedures. 3.33 10 14 85
The linearity of the method was tested within the concentration desipramine 0.56 8 8 50
1.67 4 5 63
range: 0.22–4.44 ng/mg (0.22, 0.56, 1.11, 1.67, 2.22, 3.33, 4.44 ng/
3.33 11 12 84
mg) − group of analytes no. 1, and 0.56–6.67 ng/mg (0.56, 1.11, 1.67, amitriptyline 0.22 10 14 78
2.22, 3.33, 4.44, 6.67 ng/mg) − no. 2, respectively. Calibration curves 1.11 6 8 80
were constructed by plotting the peak area ratios (ADRUG/AIS) versus 2.22 12 19 85
drug concentrations. The concentration of each internal standard was doxepine 0.22 12 20 68
1.11 9 10 67
equal to 1.1 ng/mg throughout all the validation studies. The obtained 2.22 6 22 84
results for all tested analytes are summarized in Table 6. The para- imipramine 0.22 11 19 71
meters show agreement with acceptance criteria, and finally was ob- 1.11 4 5 63
tained linear model for each drug. Carryover evaluation showed that 2.22 10 23 85
clomipramine 0.22 7 18 66
1.11 6 6 70
Table 6 2.22 15 19 89
Validation parameters of MAE/UHPLC–MS-TOF method venlafaxine 0.22 9 9 79
1.11 6 10 67
No. Drug R2 range Linear range LOD LOQ 2.22 19 24 106
(ng/mg) (ng/mg) (ng/mg) sertraline 0.56 6 13 77
1.67 7 6 60
1. ALP 0.9946–0.9976 0.65–6.67 0.19 0.65
3.33 12 18 115
2. AMI 0.9971–0.9977 0.32–3.33 0.10 0.32
fluoxetine 0.56 8 11 82
3. BRO 0.9970–0.9989 0.83–6.67 0.25 0.83
1.67 7 7 52
4. BZP 0.9864–0.9953 2.02–4.44 0.61 2.02
3.33 11 17 108
5. CBZ 0.9979–0.9988 0.21–4.44 0.06 0.21
citalopram 0.56 11 21 89
6. CITA 0.9958–0.9987 1.06–4.44 0.32 1.06
1.67 6 12 70
7. CLO 0.9976–0.9997 0.36–3.33 0.11 0.36
3.33 9 19 123
8. CLOMI 0.9966–0.9984 0.33–3.33 0.10 0.33
paroxetine 0.56 9 16 88
9. CLOR 0.9655–0.9862 1.00–2.22 0.30 1.00
1.67 5 6 65
10. DESI 0.9958–0.9981 1.24–4.44 0.37 1.24
3.33 7 14 114
11. DIA 0.9964–0.9989 0.39–3.33 0.12 0.39
BZDs
12. DOX 0.9963–0.9993 0.42–3.33 0.13 0.42
nordiazepam 0.22 5 6 70
13. EST 0.9955–0.9987 0.99–6.67 0.30 0.99
1.11 5 7 69
14. FLUOX 0.9931–0.9984 0.51–4.44 0.15 0.51
2.22 3 5 92
15. IMI 0.9968–0.9987 0.38–3.33 0.11 0.38
nitrazepam 0.22 9 15 68
16. LOR 0.9587–0.9954 0.39–4.44 0.12 0.39
1.11 2 2 64
17. LORM 0.9662–0.9806 3.71–4.44 1.11 3.71
2.22 5 7 82
18. mCPP 0.9636–0.9960 0.57–4.44 0.17 0.57
diazepam 0.22 6 8 66
19. MID 0.9898–0.9988 0.40–4.44 0.12 0.40
1.11 5 6 61
20. NITR 0.9988–0.9992 0.43–3.33 0.13 0.43
2.22 7 7 83
21. NORD 0.9955–0.9966 0.21–4.44 0.06 0.21
oxazepam 0.56 4 4 76
22. NORT 0.9957–0.9967 0.49–4.44 0.15 0.49
1.67 6 5 65
23. OXA 0.9865–0.9959 0.28–3.33 0.08 0.28
3.33 8 9 87
24. PAROX 0.9922–0.9987 0.99–6.67 0.30 0.99
tetrazepam 0.22 7 13 58
25. PRA 0.9953–0.9991 0.24–4.44 0.07 0.24
1.11 4 5 69
26. SERT 0.9964–0.9983 0.84–6.67 0.25 0.84
2.22 8 9 92
27. TEM 0.9812–0.9963 0.57–6.67 0.17 0.57
estazolam 0.56 9 12 74
28. TETR 0.9978–0.9999 0.34–3.33 0.10 0.34
1.67 4 7 66
29. TFMPP 0.9854–0.9983 0.31–2.22 0.09 0.31
3.33 4 10 86
30. VENLA 0.9948–0.9979 0.20–3.33 0.06 0.20
temazepam 1.11 7 8 108
31. ZOLP 0.9954–0.9998 0.28–4.44 0.08 0.28
(continued on next page)

464
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

Table 7 (continued) credible and the results could be burdened with a high risk of error
[23].
Drug Concentration level Precision, CV (%) Matrix effect
(ng/mg) (%) (n = 4)
intraday interday 3.3. Method applicability
(n = 4) (n = 12)
The MAE/UHPLC–MS-TOF method was applied to qualitative ana-
2.22 5 6 75
lysis of samples from three forensic case studies. Two of the samples
4.44 8 14 90
alprazolam 0.56 5 7 75 (denoted #642/15, n = 3 and #643/15, n = 3) originated from cases
1.67 5 5 63 of intoxication with psychoactive drugs, while the other one (#166/14,
3.33 7 7 82 n = 3) was obtained in the case of woman who ingested a big amount
bromazepam 0.56 8 13 89 of drugs (such as: DoxepinTeva, Bellergot, Propranold, Paxtin, Lervon)
1.67 7 10 66
before death. In case #642/15, diazepam (< LOQ = 0.39 ng/mg BM)
3.33 12 10 82
clonazepam 0.56 5 6 76 was detected. In case #643/15, citalopram (< LOQ = 1.06 ng/mg BM)
1.67 6 7 62 was detected. In case #166/14 doxepin was detected
3.33 9 13 81 (< LOQ = 0.42 ng/mg BM) while nordiazepam and paroxetine were
lorazepam 0.56 6 7 75
found at 0.3 ± 0.03, and 1.94 ± 0.06 ng/mg BM, respectively − (see
1.67 5 15 58
3.33 5 8 104 Fig. 2). The results of bone marrow and blood samples analysis for
prazepam 0.22 7 10 65 forensic cases were presented in Table 8.
1.11 5 7 67 The diazepam concentration found in case #642/15 was about 4
2.22 9 9 82 times lower than reported in literature [3], while the nordiazepam
midazolam 0.22 5 14 61
concentration in the same case was much lower (about 40-fold) than
1.11 4 5 71
2.22 12 12 90 reported by McIntyre et al. [3]. The nordiazepam level found in case
lormetazepam 0.56 9 16 72 #166/14 was about four times lower than the lowest finding reported
1.67 7 10 64 in [3]. The doxepin concentration from the same case is within the
3.33 9 9 100
range of concentrations reported in [3]. The BM citalopram level re-
clorazepate 0.56 10 19 72
1.67 6 15 70
ported in [26] was lower than the LOQ of the method presented in this
3.33 9 13 105 paper. Citalopram was detected in case #643/15, however the signal
was below the LOQ of the method.
*
not detected, value below of LOD. Although the data on drug concentrations on bone marrow is lim-
ited, McGrath and Jenkins [27] managed to quantify various drugs,
the signal after injection of standard mixture at the highest con- including citalopram, diazepam and nordiazepam, in human bones. The
centration does not exceed 10% of the lowest concentration signal. bone concentrations of these drugs (in mg/kg) were higher than their
Additionally, the limit of detection (LOD), and limit of quantitation blood levels (in mg/L). Blood and bone concentrations were 0.42 and
(LOQ) were estimated by the analysis of spiked samples for which the 13.2 respectively for citalopram, 0.13–0.39 and 0.6–2.4 (n = 5) for
analytical signal was characterized by S/N values of at least 3 and 10, diazepam, and 0.10–0.67 and 0.32–1.6 (n = 5) for nordiazepam. Some
respectively. For calculation of the LOD, and LOQ, the signal was drugs showed the opposite correlation and others, such as paroxetine
measured at the lowest concentration level of tested group of analytes. and zolpidem, were not detected in bones despite their presence in
The LOD, and LOQ were calculated as the ratios of three times and ten blood. Although these results refer to bone rather than BM, they may
times the standard deviation of the relative analytical signal (σ) to the suggest that some drug concentrations in skeletal tissues may exceed
slope of calibration function (a), respectively [25]. The calculated va- their blood concentrations. In view of these observations, it is very
lues of the LOD, and LOQ were included in Table 6. difficult to speculate about the possible effect of drugs detected in BM
Intra and interday precision and matrix effect were determined for on humans.
three different concentration pools (low, medium, and high) [23]. For Interpreting the analysis results in particular cases is a difficult task.
determination of the precision, four samples at the same concentration There is a very clear lack of literature data [13] and medical papers
level were analyzed for three consecutive days. The results are pre- covering the issue of the potential correlation between xenobiotic
sented in Table 7. The obtained CV values are satisfactory for most of substance levels in blood and bone marrow, as well as reporting con-
the studied compounds and oscillate between 2 and 15%. In some cases centration ranges resulting in therapeutic, toxic and fatal effects. Bone
this critical value was slightly exceeded, especially for psychotropic marrow toxicological analyses results can be used as a basis for drawing
substances present at high concentrations (for mCPP, TFMPP, AMI, conclusions only if interpreted in conjunction with toxicological ana-
DOX, IMI, CLOMI and VENLA, the average value of CV is about ∼20%). lyses of other biological samples, including: blood, vitreous humor or
80% of analytes from the following groups: benzodiazepines, antic- internal organs. This is why the method developed by the authors could
onvulsants and sedatives/hypnotics have met this criterion. According be a very helpful tool complementing toxicokinetic data obtained from
to the literature in validation studies, the value for precision (CV) must the analysis of other tissues, contributing to a better insight into the
not exceed 20% [23,25]. concentrations of drugs in decomposed specimens which is a non-trivial
The matrix effect investigation indicated the existence of significant task due to the multitude of factors that have to be taken into con-
interferences caused by co-eluting matrix components, as the obtained sideration. It can also be used in particularly complicated cases in
values of matrix effect parameter in some cases (mainly for BZP, TFMPP which retrieving typically used biological samples (blood, urine, etc.) is
and lorazepam) were high. Matrix effect (ME) was evaluated according impossible, because of advanced decomposition or charring of a corpse.
to strategy presented by Matuszewski et al. [23]. It was calculated as Taking into consideration all the above mentioned challenges of
the ratio of analytical signal (peak area) measured for the extract from postmortem forensic toxicology, the use of BM seems like a reasonable
blank matrix, and the neat solvent both spiked with the analytes at the choice, as bone marrow is one of the best protected tissues within the
same concentration level. At low concentration levels, the obtained body. Despite the indisputable potential of bone marrow as an alter-
matrix effect parameter values were below 60% in some cases, in- native specimen for forensic-toxicological analyses, our understanding
dicating significant ionization suppression. In both possible cases (io- of the implications, and limitations of drug measurements in this ma-
nization suppression or enhancement, characterized by ME values terial remains poor. To enable a reliable interpretation of toxicological
below or above 100%, respectively), quantitative analysis might not be data obtained from BM samples, appropriate research tools are

465
R. Wietecha-Posłuszny et al.
required. These tools are efficient sample preparation techniques (in-
cluding the essential step of drug isolation from the matrix) and vali-
dated analytical assays that allow the detection of drugs with the
highest possible sensitivity and within a short run time.
4. Conclusions
A new MAE/UHPLC–MS-TOF screening method for 31 psychoactive
drugs in human bone marrow has been developed. The presented ap-
proach introduces numerous advantages compared to strategies for
466
Journal of Chromatography B 1061–1062 (2017) 459–467
Fig. 2. Results of analysis of human bone marrow
samples collected during autopsy − cases: #642/15,
#643/15 and #166/14.
toxicological analysis of bone marrow applied so far. The use of mi-
crowave assisted extraction, as an isolation technique, provides a sig-
nificant decrease in the required time of extraction. Moreover, the se-
paration process of all analytes takes only 12 min. Moreover the MAE/
UHPLC–MS-TOF method was applied to real post-mortem samples in
three forensic cases.
The method developed by the authors can be applied in forensic
toxicology, especially as a screening methodology in cases where re-
trieving a typical sample suitable for testing is difficult, e.g., where a
corpse is decomposed, skeletonized or charred.
R. Wietecha-Posłuszny et al. Journal of Chromatography B 1061–1062 (2017) 459–467

Table 8
Results for analysis of human bone marrow (BM) and blood (B) in forensic cases.

Case no. Concentration (ng/mg) for BM and (ng/mL) for B

CIT DIA DOX EDDP MET NORD PAROX

642/15 BM – < LOQ** – – – < LOD** –

642/15 B – 7.0 ± 0.1 – – – 115 ± 0.9 –
643/15 BM < LOQ** – – – – – –
643/15 B* – – – 57.3 ± 0.6 578.1 ± 9.2 – –
166/14 BM – – < LOQ** – – 0.3 ± 0.1 1.94 ± 0.06
166/14 B – – 8.5 ± 0.1 – – – 12.23 ± 0.12

− no data available in particular case.

* methadone (MET) and (EDDP) were determined only in blood.
** detected below LOQ or LOD.

It should be emphasized that human bone marrow analysis is very 181–198.

difficult to perform, and validation parameters such as intraday preci-
sion and matrix effects showed the presence of matrix interferences that
contribute to decreased method reproducibility. Future research di-
rections include improvement and validation of reliable, quantitative
methods for the analysis of psychoactive drugs in human bone marrow,
based on the developed protocol presented here. In the case of certain
analyte groups, especially SSRI and SNRI, efforts have to be made to
lower the LOD and LOQ, since these drugs tend to be present in very
small concentrations in human blood, and as a consequence in BM as
well.
Acknowledgments
The authors gratefully acknowledge the financial support of the
European Regional Development Fund under the Innovative Economy
Programme (POIG 01.03.01-00-014/08-00).
Magdalena Garnysz has received financial support from Krakowskie
Konsorcjum “Materia-Energia-Przyszłość” under a KNOW subsidy.
The authors gratefully acknowledge the Ministry of Science and
Higher Education for financial support (R. Wietecha-Posłuszny, Sonata
Bis 6, no. 2016/22/E/ST4/00054).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jchromb.2017.08.006.
References
[1] J.S. Blebea, M. Houseni, D.A. Torigian, Ch. Fan, A. Mavi, Y. Zhuge, T. Iwanaga,
S. Mishra, J. Udupa, J. Zhuang, R. Gopal, A. Alavi, Structural and functional ima-
ging of normal bone marrow and evaluation of its age-related changes, Semin. Nucl.
Med. 37 (2007) 185–194.
[2] S. Hwang, D. Panicek, Magnetic resonance imaging of bone marrow in oncology,
part 1, Skeletal Radiol. 36 (2007) 913–920.
[3] L.M. McIntyre, C.V. King, M. Boratto, O.H. Drummer, Postmortem drug analyses in
bone and bone marrow, Ther. Drug Monit. 22 (2000) 79–83.
[4] N.L. van Doorn, A.S. Wilson, E. Willerslev, T.P. Gilbert, Bone marrow and bone as a
source for postmortem RNA, J. Forensic Sci. 56 (2011) 720–725.
[5] N.M. Lafreniere, J.H. Watterson, Detection of acute fentanyl exposure in fresh and
decomposed skeletal tissues part II: the effect of dose-death interval, Forensic Sci.
Int. 194 (2010) 60–66.
[6] W. Grellner, F. Glenewinkel, Ehxumations: synopsis of morphological and tox-
icological findings in relation to the postmortem interval. Survey on a 20-year
period and review of the literature, Forensic Sci. Int. 90 (1997) 139–159.
[7] N. Cartiser, F. Bevalot, L. Fanton, Y. Gaillard, J. Guitton, State-of-the-art of bone
marrow analysis in forensic toxicology: a review, Int. J. Legal Med. 125 (2011)
[8] L. Tattoli, M. Tsokos, J. Sautter, J. Anagnostopoulos, E. Maselli, G. Ingravallo,
M. Delia, B. Solarino, Postmortem bone marrow analysis in forensic science: study
of 73 cases and review of the literature, Forensic Sci. Int. 234 (2014) 72–78.
[9] P. Roll, A. Beham, Ch. Beham-Shmid, Post-mortem histopathological investigations
of the bone marrow in forensic medicine: an important issue for both the forensic
and clinical pathologist, Forensic Sci. Int. 186 (2009) 17–20.
[10] S. Reinhardt, M. Zhao, A. Mnatsakanuan, L. Xu, R.M. Ricklis, A. Chen, J.E. Karp,
M.A. Rudek, A rapid and sensitive method for determination of veliparib (ABT-888)
in human plasma, bone marrow cells and supernatant by using LC/MS/MS, J.
Pharm. Biomed. Anal. 52 (2010) 122–128.
[11] N. Cartiser, F. Bevalot, C. Vhatenay, C. Le Meur, Y. Gillard, D. Malicier, J. Guitton,
L. Fanton, Postmortem measurement of caffeine in bone marrow: influence of
sample location and correlation with blood concentration, Forensic Sci. Int. 210
(2011) 149–153.
[12] T. Delabarde, Ch. Keyser, A. Tracqui, D. Charabidze, The potential of forensic
analysis on human bones found in riverine environment, Forensic Sci. Int. 238
(2013) 1–5.
[13] M. Tominaga, T. Michiue, M.T. Ishikawa, O. Kuwamoto, S. Oritani, K. Ikeda,
M. Ogawa, H. Maeda, Postmortem analysis of drugs in pericardial fluid and bone
marrow aspirate, J. Anal. Toxicol. 13 (2013) 423–429.
[14] M. Tominaga, T. Ishikawa, T. Michiue, S. Oritani, I. Koide, Y. Kuramoto, M. Ogawa,
H. Maeda, Postmortem analyses of gaseus and volatile substances in pericardial
fluid and bone marrow aspirate, J. Anal. Toxicol. 37 (2013) 147–151.
[15] F. Bevalot, M.P. Gustin, N. Cartiser, Y. Gaillard, C. Le Meur, L. Fanton, J. Guitton,
D. Malicier, Using bone marrow matrix to analyze meprobamate for forensic tox-
icological purposes, Int. J. Legal Med. 127 (2013) 914–921.
[16] C.H. Srikanth, P. Joshi, A.K. Bikkasani, K. Porwal, J.R. Gayen, Bone distribution
study of anti leprotic drug clofazimine in rat bone marrow cells by a sensitive re-
verse phase liquid chromatography method, J. Chromatogr. B 960 (2014) 82–86.
[17] W. Spendley, G.R. Hext, F.R. Himsworth, Sequential application of simplex designs
in optimization and evolutionary operation, Technometrics 4 (1962) 441–461.
[18] C. Różycki, Application of the Simplex method for optimization of the analytical
methods, Chem. Anal. (Warsaw) 38 (1993) 681–698.
[19] A.C. Moffat, M.D. Osselton, B. Widdop, Clarke’s Analysis of Drugs and Poisons, third
ed., Pharmaceutical Press, London, 2004.
[20] https://www.drugbank.ca (Accessed 5 August 2015).
[21] L. Ch. Winek, S.E. Westwood, W.W. Wahba, Plasma versus bone marrow desipra-
mine: a comparative study, Forensic Sci. Int. 48 (1982) 49–57.
[22] L. Ch. Winek, E.M. Morris, W.W. Wahba, The use of bone marrow in the study of
postmortem redistribution of nortriptyline, J. Anal. Toxicol. 17 (1993) 93–98.
[23] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment
of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal.
Chem. 75 (2003) 3019–3030.
[24] R. Wietecha-Posłuszny, M. Woźniakiewicz, A. Garbacik, P. Chęsy, P. Kościelniak,
Application of microwave irradiation to Fast and efficient isolation of benzodiaze-
pines from human hair, J. Chromatogr. A 1278 (2013) 22–28.
[25] SWGTOX – A Stand Practices for Method Validation in Forensic Toxicology,
Revision 1, Published May 20, (2013).
[26] N. Cartiser, F. Bevalot, C. Le Meur, Y. Gaillard, D. Malicier, N. Hubert, J. Guitton,
Gas chromatography–tandem mass spectrometry assay for the quantification of four
benzodiazepines and citalopram in eleven postmortem rabbit fluids and tissues,
with application to animal and human samples, J. Chromatogr. B 879 (2011)
2909–2918.
[27] K.K. McGrath, A.J. Jenkins, Detection of drugs of forensic importance in post-
mortem bone, The Am. J. Forensic Med. Pathol. 30 (2009) 40–44.

467