Received: 16 May 2024 Accepted: 24 January 2025

DOI: 10.1111/arcm.13065

ORIGINAL ARTICLE

Toxicological analyses of the bone matrix: Successes

and challenges

Giordano Gaia 1,2 | Biehler-Gomez Lucie 2 | Cattaneo Cristina 1,2 |

Di Candia Domenico 1

1
Bureau of Legal Medicine and Insurance,
Department of Biomedical Science for Health, Abstract
University of Milan, Milan, Italy This study aimed to conduct an in-depth and systematic
2
LABANOF (Laboratorio di Antropologia e literature review dealing with toxicological analyses on
Odontologia Forense), Department of
human bone tissue and focusing on the forensic toxico-
Biomedical Science for Health, University of
Milan, Milan, Italy logical and archaeotoxicological field. Several studies
have focused their research on medical drugs, drugs of
Correspondence abuse, and trace elements on both human cadavers and
Giordano Gaia, Bureau of Legal Medicine and
Insurance, Department of Biomedical Science
skeletal remains, but a few studies tried to detect some
for Health, University of Milan, 20133 Milan, traces of officinal plants in skeletal remains. The present
Italy. paper illustrates the significant advances made in recent
Email: [email protected]
years in the field of bone toxicology considering all cases
reported in the literature. After the literature review, the
study investigated 40 articles that applied bone toxicol-
ogy to both recent and ancient human remains,
researching the presence of trace elements, stable iso-
topes, medical drugs, drugs of abuse, and active princi-
ples of medical plants. This allowed us to evaluate the
distribution of skeletal remains studied around the world
and throughout history, and to highlight the differences
between trace elements/stable isotopes and medical
drugs/drugs of abuse. Finally, this study permitted us to
gather more information on molecule consumption
(acute/single and/or occasional/chronic drug use) in this
unconventional biological matrix, both in forensic cases
and very ancient human remains, suggesting that the
analytes detected in bones should be referred to occa-
sional or chronic intake of substances.

KEYWORDS
archaeotoxicology, bone toxicology, forensic scenario, forensic
toxicology, unconventional matrices

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
© 2025 The Author(s). Archaeometry published by John Wiley & Sons Ltd on behalf of University of Oxford.

Archaeometry. 2025;1–21. wileyonlinelibrary.com/journal/arcm 1

2 GAIA ET AL.

A WORST-CASE SCENARIO: TOXICOLOGICAL ANALYSES ON

SKELETAL HUMAN REMAINS
Forensic toxicology consists in the detection of xenobiotics from a qualitative and a quantita-
tive point of view for medico-legal purposes with the objective of interpreting the likely role of a
molecule in a specific forensic case. Toxicological investigations can be applied to conventional
and unconventional biological matrices, among which is bone tissue. Here, the challenges of
toxicological analysis and its interpretation in bone are reported and analyzed through an in-
depth literature review.
Forensic toxicological analyses are classically performed on conventional matrices such as
biological fluids or samples of viscera (Biehler-Gomez et al., 2024; Giordano, Mattia, Biehler-
Gomez, et al., 2023; Giordano, Mattia, Boracchi, et al., 2023). When conventional matrices are
no longer available, usually due to the state of preservation of the body, for instance in cases of
advanced decomposition or complete skeletonization, other biological matrices can be used for
the analyses. Indeed, unconventional matrices such as bones, nails, paraffin-embedded blocks
tissues, fabric remains, and teeth can be involved in toxicological analyses, and they have shown
to be valid alternative matrices in toxicological investigation (Baranowska et al., 1995;
Biehler-Gomez et al., 2021; Chang et al., 2018; Giordano, Mattia, Boracchi, et al., 2023; Gon-
z

alez-Reimers et al., 2003; Grattan et al., 2002; Güler & Güçlü Ekinci, 2023; Kepa et al., 2012;
Martínez-García et al., 2005; Vuorinen et al., 1990; Yoshinaga et al., 2013). Here we explore
the most challenging toxicological scenario: having to perform analysis using only skeletal
remains to sample, with no other biological matrix available.
Moreover, in forensic medicine, when an unknown individual is recovered it becomes essen-
tial to reconstruct their biological profile to determine their identity. The first forensic discipline
that should be involved in the reconstruction of a biological profile is forensic anthropology, in
addition to other disciplines capable of providing important information about the identity of a
subject. An example is forensic toxicology, since it can give essential information about the life-
style of an individual by providing data about the medical or recreational drugs that an individ-
ual may have taken during their life. This more exhaustive approach to the biological profile is
now referred to as “biocultural profile” (Biehler-Gomez et al., 2024).
Similarly, the possibility to detect molecules in archaeotoxicological cases is essential to con-
tribute to the reconstruction of the past and the habits of individuals, highlighting the differ-
ences and similarities in different social classes and even, in some cases, to reconstruct the life of
an entire population (Giordano, Mattia, Biehler-Gomez, et al., 2023; Giordano, Mattia,
Boracchi, et al., 2023).
Thus, the objective of this paper was to perform an extensive literature review analyzing the
studies that focused their investigation on bone toxicology of prescription drugs, drugs of abuse,
and trace elements in skeletal tissue.
Through a literature review, this study underlines the bone sampling sites used in the litera-
ture, the molecules detected in bone tissue, the populations utilized as study samples, and the
potential of the analytical interpretation of drugs in bone tissue.

MATERIAL AND METHODS

A literature review was conducted on PubMed and ScienceDirect, searching papers with differ-
ent combinations of keywords including drugs, drugs of abuse, trace elements, bone, bone tis-
sue, skeletal remains, toxicological analyses, forensic toxicology, archaeotoxicology,
archaeotoxicological analyses, and forensic analyses. The inclusion criteria were primarily the
presence of toxicological analyses on bone tissue with the aim to find the presence of medical
drugs, drugs of abuse, and officinal plants. Regarding trace elements and stable isotopes, the
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 3

studies that tended to focus on toxicity thresholds were included; hence, any paper on the inves-
tigation of physiological levels of trace elements in the bone matrix was excluded. Similarly,
papers on hydroxyapatite concentrations of trace elements were not considered. All articles
included in the literature review had to be research papers; thus reviews, books, and book chap-
ters were not included. As a result, a total of 40 papers were selected for this study.
The papers were divided according to the xenobiotics investigated: stable isotopes and trace
elements, medical drugs and drugs of abuse, and medical plants. The papers were then consid-
ered according to state of preservation of human remains, dividing the cases into cadavers, sur-
geries, and skeletons. Finally, the papers including skeletal human remains were further divided
into contemporary and archaeological (Figure 1 and Table 1).

Sampling site and molecules

Various bones were selected as point of sampling in the papers under investigation. Most of the
bones tested in bone toxicology were the rib and femur, followed by cranial bones (Figure 2).
However, there is still no standard reference for bone tissue sampling in the literature.
The systematic literature review revealed the presence of 26 papers in which toxicological
analyses were focused on the detection of stable isotopes and trace elements in bone samples
since the Neolithic and up to the Contemporary age (Figure 1). The analyses were performed
mainly on skeletal remains, but in a few cases the analyses were performed on cadavers and
samples collected during surgeries. Those papers aimed to search barium/calcium ratio (Ba/Ca)
(Martínez-García et al., 2005), cadmium (Cd) (Baranowska et al., 1995; Gonz
alez-Reimers
et al., 2003; Grattan et al., 2002; Martínez-García et al., 2005; Vuorinen et al., 1990), chromium
(Cr) (Chang et al., 2018), cobalt (Co) (Chang et al., 2018), copper (Cu) (Baranowska
et al., 1995; Güler & Güçlü Ekinci, 2023; Martínez-García et al., 2005; Vuorinen et al., 1990),
iron (Fe) (Martínez-García et al., 2005; Vuorinen et al., 1990; Yoshinaga et al., 2013), mercury
(Hg) (Biehler-Gomez et al., 2021; Güler & Güçlü Ekinci, 2023; Kepa et al., 2012; Rasmussen
et al., 2008; Rasmussen et al., 2013; Rasmussen et al., 2015; Yamada et al., 1995), manganese
(Mn) (Chang et al., 2018; Vuorinen et al., 1990), nickel (Ni) (Baranowska et al., 1995), lead
(Pb) (Baranowska et al., 1995; Chang et al., 2018; Corruccini et al., 1987; Gonz
alez-Reimers
et al., 1991; Grattan et al., 2002; Güler & Güçlü Ekinci, 2023; L
opez-Costas et al., 2020; Martí-
nez-García et al., 2005; Reinhard & Ghazi, 1992; Sguazza et al., 2016; Vuorinen et al., 1990),
lead/calcium ratio (Pb/Ca) (Martínez-García et al., 2005; Yoshinaga et al., 2013), lead isotope
ratio (Reinhard & Ghazi, 1992), strontium (Sr) (Vuorinen et al., 1990), thallium (Tl) (Chang
et al., 2018), and zinc (Zn) (Baranowska et al., 1995; Güler & Güçlü Ekinci, 2023; Martínez-
García et al., 2005; Vuorinen et al., 1990) (Figure 1).
Moreover, many classes of medical drugs and/or drugs of abuse were detected in bone tissue
in 19 papers (Figures 1 and 3). Specifically, prescription drugs included anticonvulsants
(Fernandez-Lopez et al., 2020; Fern
andez-L
opez et al., 2022; McIntyre et al., 2000), anesthetics
(Franceschetti et al., 2020), antidepressants (Fernandez-Lopez, Pellegrini, et al., 2019;
Franceschetti et al., 2020; Horak & Jenkins, 2005; McIntyre et al., 2000; Orfanidis et al., 2018;
Vandenbosch et al., 2020; Wiart et al., 2020), antihypertensives (Fernandez-Lopez, Pellegrini,
et al., 2019), antipsychotics (Fernandez-Lopez et al., 2020; Franceschetti et al., 2020; Horak &
Jenkins, 2005; McIntyre et al., 2000; Orfanidis et al., 2018), basic drugs (McGrath &
Jenkins, 2009), benzodiazepines (Franceschetti et al., 2020; Giordano et al., 2021;
Gorczynski & Melbye, 2001; Mancini et al., 2020; McGrath & Jenkins, 2009; McIntyre
et al., 2000; Orfanidis et al., 2018; Orfanidis et al., 2019; Wiart et al., 2020), cannabinoids
(Giordano et al., 2021; Orfanidis et al., 2018), non-benzodiazepine drugs (Franceschetti
et al., 2020), non-steroid anti-inflammatory drugs (Wiart et al., 2020), opioids (Fernandez-
Lopez, Luna-Maldonado, Falcon, Mastrobattista, et al., 2019; Franceschetti et al., 2020;
 4
F I G U R E 1 Chart representing the literature review on bone toxicology. The papers are divided into color sections: detection of trace elements and stable isotopes in green,
detection of medical plants in orange, detection of medical drugs and/or drugs of abuse in light blue. The papers are then divided according to bone sampling: cadavers, surgeries,
and skeletons.

GAIA ET AL.
 TOXICOLOGICAL ANALYSES OF THE BONE MATRIX
T A B L E 1 Summary of data obtained from literature review on medical drugs, drugs of abuse, officinal plants, and trace elements in skeletal human remains. In the first section
of the table are reported the papers that dealt with human samples and medical drugs, drugs of abuse, or medical plants. In the second section of the table are reported the data
obtained for the detection of trace elements in human specimens.
Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Human—drugs, drugs of abuse, medicinal plants

Parsche et al. Neolithic— — Mummies 95 NMen. NMen. NApp. Solub., RIA Drugs/ Cannabinoids, Cocaine, nicotine, delta- Hair, soft
(1993) Middle Ages and GC-MS drugs of stimulants 9-THC tissue,
abuse brain,
teeth

McIntyre Contemp. — Cadavers 36 Femur Midshaft NMac. Solub., LC- Drugs/ Anticonvulsant Carbamazepine, Blood
et al. (2000) Age— MS and GC- drugs of drugs, amitriptyline, dothiepin,
autopsy MS abuse antidepressants, doxepin, mianserin,
antipsychotics, moclobemide, nordoxepin
benzodiazepines (metabolite of doxepin),
chlorpromazine,
clozapine, mesoridazine,
thioridazine, diazepam

Gorczynski Contemp. 3 weeks Skeletons NMen. NMen. NMen. NApp. NMen. Drugs/ Benzodiazepines Midazolam NMen.
and Melbye Age—ASD drugs of
(2001) abuse

Raikos et al. Contemp. 1 year Cadavers 1 Thigh NMen. NMac. LL, FPI and Drugs/ Opioids Morphine Bone
(2001) Age— bone GC-FID drugs of marrow
autopsy + abuse
ASD

Horak and Contemp. — Cadavers 1 NMen. NMen. NMen. LL, GC- Drugs/ Antidepressants, Citalopram Heart
Jenkins Age— NPD and drugs of antipsychotics and
(2005) autopsy GC-MS abuse femoral
blood,
urine,
lung

(Continues)

5
 6
TABLE 1 (Continued)

Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

McGrath and Contemp. — Cadavers 39 Iliac crest, — NMac. LL, GC-MS Drugs/ Opioids, Amitriptyline, citalopram, Blood
Jenkins Age— vertebra drugs of stimulants, diphenhydramine,
(2009) autopsy abuse benzodiazepines, diazepam, nordiazepam,
basic drugs laudanosine (metabolite of
atracurium), codeine,
meperidine,
norpropoxyphene
(metabolite of
propoxyphene),
oxycodone,
propoxyphene,
benzoylecgonine
(metabolite of cocaine)

Vardakou Contemp. — Cadavers 3 Clavicle Midshaft NMac. SPE, GC- Drugs/ Opioids Codeine, morphine Blood
et al. (2014) Age— MS drugs of
autopsy abuse

Orfanidis Contemp. 1 year Cadavers 2 Femur Right. NMac. MeOH extr, Drugs/ Antidepressants, Amphetamine, Bone
et al. (2018) Age— LC-MS drugs of antipsychotics, amitriptyline, marrow
autopsy + abuse benzodiazepines, bromazepam, diazepam,
ASD cannabinoids, nordiazepam, codeine,
opioids, morphine, cocaine,
stimulants benzoylecgonine
(metabolite of cocaine)

Orfanidis Contemp. 3 months Cadavers 1 Femur Thighbone NApp. MeOH extr., Drugs/ Benzodiazepines, Alprazolam, zolpidem —
et al. (2019) Age— UPLC-MS drugs of Z-drugs
decomposed abuse

Fernandez- Contemp. Cadavers 6 Rib Body of the NMac. SPE, GC- Drugs/ Opioids, 6-MAM, methadone, Blood
Lopez, Luna- Age— 5th or 6th MS drugs of stimulants morphine, tramadol,
Maldonado, autopsy ribs abuse benzoylecgonine

GAIA ET AL.
et al. (2019) (metabolite of cocaine)
 TOXICOLOGICAL ANALYSES OF THE BONE MATRIX
TABLE 1 (Continued)
Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Fernandez- Contemp. Cadavers 2 Rib Body of the NMac. SPE, GC- Drugs/ Antihypertensive Atenolol, bisoprolol Blood
Lopez, Age— 5th or 6th MS drugs of drugs
Pellegrini, autopsy ribs abuse
Rotolo,
Luna, et al.
(2019)

Fernandez- Contemp. Cadavers 7 Rib Body of the NMac. SPE, GC- Drugs/ Antidepressants Amitriptyline, duloxetine, Blood
Lopez, Age— 5th or 6th MS drugs of venlafaxine
Pellegrini, autopsy ribs abuse
Rotolo,
Maldonado,
et al. (2019)

Fernandez- Contemp. Cadavers 3 Rib Body of the NMac. SPE, GC- Drugs/ Anticonvulsant Pregabalin, quetiapine Blood
Lopez et al. Age— 5th or 6th MS drugs of drugs,
(2020) autopsy ribs abuse antipsychotics

Mancini et al. Contemp. — Cadavers 20 Rib 5th or 6th NMac. LL, GC-MS Drugs/ Benzodiazepines Nordiazepam, oxazepam, Blood
(2020) Age— ribs drugs of lormetazepam, lorazepam,
autopsy abuse clonazepam, bromazepam
and alprazolam

Vandenbosch Contemp. — Cadavers 12 Clavicle Sternal NMac. Solub., LC- Drugs/ Antidepressant, Citalopram, Blood
et al. (2020) Age— portion MS drugs of opioids desmethylcitalopram,
autopsy abuse clomipramine and
desmethylclomipramine,
methadone, EDDP,
EMDP

(Continues)

7
 8
TABLE 1 (Continued)

Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Wiart et al. Contemp. 1 month Cadavers 1 Femur Cortical and NApp. MeOH extr., Drugs/ BDZ, opioids, Propanolol, oxazepam, Hair
(2020) Age— trabecular LC-MS drugs of non-steroidal lormetazepam, tramadol,
autopsy abuse anti- acetaminophen,
inflammatory, paroxetine, oxetorone,
antidepressants, nordiazepam
serotonin
antagonistic
(antihistamine
and alpha-
blockers)

Rubin et al. Contemp. — Cadavers 1 Femur NMen. Cleaned LL, SPE, Chemical Nerve agents MPA, EMPA, IMPA, -
(2020) Age— LC-MS weapon iBuMPA, CMPA and
autopsy PMPA metabolites,
corresponding to the nerve
agents VX, Russian VX,
sarin, cyclosarin and
soman, respectively

Franceschetti Contemp. 1 month Cadavers 30 Cranium, Occipital Mac/ ASE, LC- Drugs/ Benzodiazepines, Ketamine, citalopram, Blood
et al. (2020) Age— rib bone, sternal NMac. MS drugs of stimulants, trazodone, venlafaxine,
autopsy portion abuse antidepressants, haloperidol, promazine,
antipsychotics, quetiapine, alprazolam,
anesthetics, delorazepam, diazepam,
opioids, non- flurazepam, lorazepam,
benzodiazepine lormetazepam,
drugs, sedative nordiazepam, tramadol,
hypnotic cocaine, benzoylecgonine
(metabolite of cocaine)

Vandenbosch Contemp. — Cadavers 22 Clavicle Proximal NMac. MeOH extr., Drugs/ Opioids Tramadol, O- Blood,
et al. (2022) Age— head LC-MS drugs of desmethyltramadol, bone

GAIA ET AL.
autopsy abuse morphine, fentanyl, marrow
norfentanyl, codeine
TABLE 1 (Continued)

TOXICOLOGICAL ANALYSES OF THE BONE MATRIX

Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Giordano Contemp. 19– Skeletons 7 Cranium, Occipital NApp. ASE, LC- Drugs/ Benzodiazepines, MDA, —
et al. (2021) Age—buried 23 years rib, bone, sternal MS drugs of opioids, methamphetamines
vertebra portion, abuse stimulants, lorazepam, diazepam,
lumbar cannabinoids delorazepam,
vertebral nordiazepam,
body THCCOOH (metabolite
of Delta-9-THC),
buprenorphine,
methadone,
benzoylecgonine
(metabolite of cocaine)

Fern

andez- Contemp. — Cadavers 1 Rib Body of the NMac. SPE, GC- Drugs/ Anticonvulsant Carbamazepine Blood
L

opez et al. Age—autopsy 5th or 6th MS drugs of
(2022) ribs abuse

Giordano, 17th century Skeletons 8 Cranium Occipital NApp. SPE, LC- Plant Opioids Morphine, papaverine, Brain
Mattia, bone MS molecules noscapine, codeine
Biehler-
Gomez, et al.
(2023)

Giordano, 17th century Skeletons 9 Femur Proximal NApp. SPE, LC- Plant Cannabinoids Delta-9-THC, CBD —
Mattia, epiphysis MS molecules
Biehler-
Gomez, et al.
(2023)

Human—trace elements
—
Corruccini — Skeletons 48 Mandible, Ramus, NApp AAS Trace — Pb —
et al. (1987) cranium temporal elements
bone

Reinhard and 18–19th — Skeletons 39 Rib — NApp. ICP-MS Trace — Pb and Pb isotope ratios —
Ghazi (1992) century elements

(Continues)

9
 10
TABLE 1 (Continued)

Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Grattan et al. 4–7th century — Skeletons 36 — — NApp. ICP-MS Trace — Pb and cu —

(2002) elements

Martínez- Neolithic — Skeletons 4 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
García et al. elements and Pb/ca
(2005)
Bronze Age — Skeletons 2 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
elements and Pb/ca

Ancient Age — Skeletons 11 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
elements and Pb/ca

Byzantine — Skeletons 12 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
Age elements and Pb/ca

Islamic Age — Skeletons 37 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
elements and Pb/ca

18th century — Skeletons 6 — — NApp. AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
elements and Pb/ca

Contemp. — Cadavers 8 — — NMen AAS Trace — Pb, cu, Zn, cd, Fe, Ba/ca —
Age— elements and Pb/ca
autopsy

Yoshinaga Copper — Skeletons 57 Rib — NApp. ICP-MS, Trace — Pb/ca and Fe —

et al. (2013) Age— ICP-AES elements
modern age

E. Sguazza 17th century — Skeletons 1 Cranium — NApp. ICP-MS Trace — Pb Hair

et al. (2016) elements

Kepa et al. 15–19th — Skeletons 15 Femur, Zygomatic NApp. LA-ICP-MS Trace — Hg —

(2012) century humerus, arch, elements
rib, sphenoid
phalanx, bone,

GAIA ET AL.
cranium temporal
bone
TABLE 1 (Continued)

TOXICOLOGICAL ANALYSES OF THE BONE MATRIX

Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Biehler- 17th century — Skeletons 2 Cranium Occipital NApp. ICP-MS Trace — Hg —

Gomez et al. bone elements
(2021)

L

opez-Costas 1st–7th — Skeletons 44 Cortical — NApp. ICP-MS and Trace — Pb and hg —
et al. (2020) century bone CV-AAS elements

Gonz

alez- 6–8th century — Skeletons 16 Tibia Diaphysis NApp. AAS Trace — Pb and cd —
Reimers et al. elements
(2003)
Contemp. — Surgeries 8 Tibia Proximal NMen. AAS Trace — Pb and cd —
Age— epiphysis elements
autopsy

Yamada et al. 6–7th century — Skeletons 16 NMen. — NApp. ICP-AES, Trace — Hg Soil
(1995) GF-AAS elements

12–17th — Skeletons 14 NMen. — NApp. ICP-AES, Trace — Hg Soil

century GF-AAS elements

Rasmussen Middle Ages — Skeletons 2 Femur Cortical and NApp. AAS Trace — Hg —
et al. (2013) trabecular elements
bone

Rasmussen Middle Ages — Skeletons 110 Long Cortical NApp. AAS Trace — Hg —
et al. (2008) bone, elements
mainly
femur

Rasmussen Middle — Skeletons 283 Femur, Left (radius, NApp. AAS Trace — Hg —
et al. (2015) Ages— humerus, metatarsal elements
Modern Age tibia, and
radius, rib, metacarpal);
vertebra, epiphysis
1st and
metatarsal diaphysis
and 2nd (femur, tibia,
metatarsal humerus,
radius)

11
(Continues)
TABLE 1 (Continued)

12
Other
Historical ASD/ biological
period (from– Contemp. Corpse Sample Site of Class of samples
Literature to) PMI preservation size Bone type sampling Maceration Methodology Findings molecules Molecules/trace elements analyzed

Güler and Ancient Age — Skeletons 15 Pelvis — NApp. WDXRF, Trace — Zn, cu, Pb, hg —
Güçlü Ekinci RXF, ICP- elements
(2023) MS

Baranowska Contemp. — Cadavers 35 Sternum Manubrium NApp. AAS Trace — Pb, cd, Zn, cu, Ni —
et al. (1995) Age— elements
autopsy

Vuorinen Iron age — Skeletons 19 Long Mainly NApp. PIXE and Trace — Fe, Mn, cu, Pb, Zn, Sr —
et al. (1990) bones, femur PIGE elements

Chang et al. Contemp. — Surgeries 23 Femur, Trabecular NApp. ICP-MS Trace — Cd, Cr, cobalt, Pb, Tl, Mn —
(2018) Age tibia bone elements

Abbreviations: AAS, atomic absorption spectrometry; ASD, autopsy samples decomposed, samples collected during autopsy and then decomposed in control environment; ASE, accelerated solvent extraction; Cd,
cadmium; Co, cobalt; Contemp. Age, contemporary age; Cr, chromium; Cu, copper; CV-AAS, cold vapor–atomic absorption spectrometry; Fe, iron; FPI, fluorescence polarization immunoassay; GC-FID, gas
chromatography–flame ionizer detector; GC-MS, gas chromatography–mass spectrometry; GC-NPD, gas chromatography–nitrogen phosphorus detector; GF-AAS, graphite furnace–atomic absorption spectrometry; Hg,
mercury; ICP-AES, inductively coupled plasma–atomic emission spectroscopy; ICP-MS: inductively coupled plasma–mass spectrometry; LA-ICP-MS, laser ablation–inductively coupled plasma–mass spectrometry; LC–
MS, liquid chromatography–mass spectrometry; LL, liquid–liquid extraction; mac., macerated; MeOH Extr., methanolic extraction; Mn, manganese; NApp., not applicable; Ni, nickel; NMac., not macerated; NMen., not
mentioned; Pb, lead; PIGE, particle-induced gamma emission; PIXE, particle-induced X-ray emission; RIA, radioimmunoassay; Solub., solubilization; SPE, solid-phase extraction; Sr, strontium; Tl, thallium; UPLC-MS,
ultra-performance liquid chromatography–mass spectrometry; WDXRF, wavelength-dispersive X-ray fluorescence; XRF, X-ray fluorescence; Zn, zinc.

GAIA ET AL.
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 13

FIGURE 2 Bone sampling site utilized in the literature.

FIGURE 3 Worldwide distribution of skeletal remains analyzed for trace elements and stable isotopes.

Giordano et al., 2021; McGrath & Jenkins, 2009; Orfanidis et al., 2018; Raikos et al., 2001;
Vandenbosch et al., 2020; Vandenbosch et al., 2022; Vardakou et al., 2014; Wiart et al., 2020),
sedative hypnotic (Franceschetti et al., 2020), serotonin antagonists (Wiart et al., 2020), stimu-
lants (Fernandez-Lopez, Luna-Maldonado, Falcon, Mastrobattista, et al., 2019; Franceschetti
et al., 2020; Giordano et al., 2021; McGrath & Jenkins, 2009; Orfanidis et al., 2018), and
z-drugs (Orfanidis et al., 2019). Additionally, active principles of officinal plants were detected
in two other papers. Precisely, morphine, papaverine, codeine, and noscapine (Giordano,
Mattia, Biehler-Gomez, et al., 2023), and tetrahydrocannabinol and tetrahydrocannabidiol
(Giordano, Mattia, Boracchi, et al., 2023) were detected in human remains from the Modern
14 GAIA ET AL.

age in Italy (Figure 1). Some derivatives of nerve agents were once detected in bone tissue
(Rubin et al., 2020) (Figure 1).

Worldwide scale
A total of 815 skeletal remains from different historical periods around the world were analyzed
for trace elements and stable isotopes. In Denmark (Rasmussen et al., 2008; Rasmussen
et al., 2013; Rasmussen et al., 2015), up to 320 skeletal remains were analyzed (Figure 3),
followed by 87 individuals in Japan (Yamada et al., 1995; Yoshinaga et al., 2013) and 80 indi-
viduals in Tunisia (Martínez-García et al., 2005).
In Germany (Rasmussen et al., 2015), Spain (Gonz
alez-Reimers et al., 2003; L
opez-Costas
et al., 2020), and Poland (Baranowska et al., 1995; Kepa et al., 2012), 75, 68, and 50 skeletal
remains were examined, respectively. Fewer than 50 skeletons were investigated in the USA
(39 cases) (Reinhard & Ghazi, 1992), Jordan (36 cases) (Grattan et al., 2002), China (23 cases)
(Chang et al., 2018), Italy (22 cases) (Biehler-Gomez et al., 2021; Sguazza et al., 2016; Vuorinen
et al., 1990), and, lastly, Turkey (15 cases) (Güler & Güçlü Ekinci, 2023). The extractive
methods and analytical instrumentation for the analysis and detection of stable isotopes and
trace elements in bone tissue are well standardized in the literature, as well as how they are
incorporated into bones by replacement of some hydroxyapatite constituents (Rubin, 2018).
Different is the case of drug detection in bone tissue. Indeed, less is known about the extrac-
tive procedures and analytical instrumentations utilized for the detection of analytes in bones
that varied from solubilization procedures to solid-phase extraction techniques and from fluo-
rescence polarization immunoassay to liquid chromatography–mass spectrometry. Moreover,
there is still a debate in the literature about how drugs are incorporated into bone tissue and, as
a consequence, about the data interpretation of the bone matrix from a toxicological point of
view. A total of 220 skeletal remains from different historical periods were analyzed worldwide.
The majority of the cases were analyzed in Italy with 54 cases (Franceschetti et al., 2020;
Giordano et al., 2021; Giordano, Mattia, Biehler-Gomez, et al., 2023; Giordano, Mattia,
Boracchi, et al., 2023), followed by the USA with 41 individuals (Horak & Jenkins, 2005;
McGrath & Jenkins, 2009; Rubin et al., 2020). Moreover, 39, 36, and 34 subjects were investi-
gated in Spain (Fernandez-Lopez et al., 2020; Fern
andez-L
opez et al., 2022; Fernandez-Lopez,
Luna-Maldonado, Falcon, Mastrobattista, et al., 2019; Fernandez-Lopez, Pellegrini,
et al., 2019; Fernandez-Lopez, Pellegrini, et al., 2019), Australia (McIntyre et al., 2000), and
Belgium (Vandenbosch et al., 2020; Vandenbosch et al., 2022). Lastly, 15 individuals were stud-
ied in Greece (Orfanidis et al., 2018; Orfanidis et al., 2019) and one case in France (Wiart
et al., 2020).

Diachronic scale

In this context, it is evident that the prevailing approach in the field prior to the Modern age
was solely concerned with the detection of trace elements and stable isotopes. There was a
dearth of comprehensive studies that attempted to ascertain the presence of xenobiotics in bone
tissue. Consequently, until the advent of the Modern age, archaeotoxicological findings on skel-
etal remains were constrained to the identification of trace elements, encompassing the results
from the Neolithic period until the Middle Ages. A comparison of trace element records from
the Iron Age to the Middle Ages reveals an increase in the number of records, while a compari-
son from the Modern era to Contemporary times shows a decrease in the number of records.
This may be due to changes and differences in the use of trace elements over time. For example,
studies have shown that lead was used in the pipes of the houses, pots, and cosmetics. Lead was
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 15

even used to treat diseases (Beck et al., 2018; Roberts & Manchester, 2010; Sguazza
et al., 2016): indeed, saturnism, a pathological condition considered pandemic during the
Roman Empire, is a lead intoxication that can leave different signs of diseases (such as anemia
and gout) on bones (Montes-Santiago, 2013). Even mercury was used to treat the lesions due to
tertiary syphilis (Biehler-Gomez et al., 2021).
In contrast, the first evidence that xenobiotics, distinct from trace elements and isotopes,
were identified in Modern Italy was reported by a research group at the University of Milan.
The authors noted the presence of active principles of Papaver somniferum and Cannabis spp.
(Giordano, Mattia, Biehler-Gomez, et al., 2023; Giordano, Mattia, Boracchi, et al., 2023) in
the deceased patients of an important Italian hospital. Those two papers are the testimony of
the first detection of molecules in bone samples before the Contemporary period, recognizing
active principles of plants in skeletal remains of more than 500 years ago. In our contemporary
time, the remaining papers examined various pharmaceutical drugs and substances of abuse in
contemporary skeletal remains (Giordano et al., 2021) and bone samples obtained from
cadavers (Fernandez-Lopez et al., 2020; Fern
andez-L
opez et al., 2022; Fernandez-Lopez,
Luna-Maldonado, Falcon, Mastrobattista, et al., 2019; Fernandez-Lopez, Pellegrini,
et al., 2019; Fernandez-Lopez, Pellegrini, et al., 2019; Franceschetti et al., 2020; Gorczynski &
Melbye, 2001; Horak & Jenkins, 2005; Mancini et al., 2020; McGrath & Jenkins, 2009;
McIntyre et al., 2000; Orfanidis et al., 2018; Orfanidis et al., 2019; Raikos et al., 2001;
Vandenbosch et al., 2020; Vandenbosch et al., 2022; Vardakou et al., 2014; Wiart et al., 2020).
Thus, the data extrapolated from the literature review highlighted that long post-mortem
interval and taphonomic factors do not compromise the success of toxicological analyses.
Indeed, toxicological findings have been identified in contemporary forensic scenarios in which
the biological samples are well preserved, as well as in archaeotoxicological contexts dating
back 500 years. These findings confirm the potential of bone tissue to protect and preserve
xenobiotics for a long time (Figure 4).

The potential of drug interpretation in bone tissue: Bone versus blood and/or bone
marrow analyses
A comparison between the analyses performed on blood and/or bone marrow samples with
bone tissue was reported, with particular attention paid to the detection or non-detection of
molecules in these biological matrices and the differences in drug concentration observed in the
specimens under investigation.

FIGURE 4 Worldwide distribution of skeletal remains analyzed for medical drugs, drugs of abuse, and medical
plants.
16 GAIA ET AL.

After the introduction of xenobiotics into the water layer of bone, drugs are incorporated
into the bone matrix as part of primary or secondary mineralization through bone remodeling
(Rubin, 2018). In the literature (Rubin, 2018), three ways that drugs are incorporated into the
bone mineral matrix are suggested, supposing that drugs may be incorporated inside the bone
tissue in a similar way to stable isotopes and trace elements. First, the authors hypothesized that
the xenobiotics, after passing the layers of the blood–bone interface, can remain entrapped
between the bulk water of the bone and the hydrated layer of hydroxyapatite (low affinity for
bone tissue). The second hypothesis was that the molecules remained temporarily deposited on
the surface of hydroxyapatite (medium affinity for bone tissue). The last hypothesis suggested
that the analytes could be incorporated into the inorganic matrix as part of the primary or sec-
ondary mineralization, during the bone remodeling cycle (high affinity for bone tissue) and
could be only excreted through the same remodeling process. Therefore, the fraction of these
substances may be detected after the death of the individual even after a very long post-mortem
interval (Biehler-Gomez et al., 2021; Giordano et al., 2021; Giordano, Mattia, Biehler-Gomez,
et al., 2023; Giordano, Mattia, Boracchi, et al., 2023) (Figures 1 and 5).
Even though it is important to underline that the detection of molecules in bones does not
exclude that the presence of molecules may be due to acute administration of molecules, our
hypothesis is that bone storage may be due to more than one administration of the substance,
rather than a single and unique intake, in which the occasional or chronic consumption of drugs
corresponds to a sporadic or persistent intake of substances over a variable period of time dur-
ing the life of an individual.
Thus, the results obtained in Franceschetti et al. (Franceschetti et al., 2020) support this
hypothesis. Indeed, on the basis of the results, some molecules were only detected in bone sam-
ples and not in blood specimens (eight times – 6% of the total cases considered), suggesting that
the molecules not detected in blood were not present at the time of death in the organism,
whereas the detection of xenobiotics in bone samples should refer to an accumulation in bones.
Analogously, some molecules were found only in blood samples and not in bone specimens
(52 times – 38% of the total cases considered), suggesting that the single administration of mole-
cules is difficult detect in bone. Other studies achieved similar results: McGrath and Jenkins
(McGrath & Jenkins, 2009) noted in nine cases the presence of molecules in blood only, while
Mancini et al. (Mancini et al., 2020) described it in eight cases; McIntyre et al. (McIntyre
et al., 2000) and Vandenbosch et. al (Vandenbosch et al., 2022) reported four and three cases,

F I G U R E 5 General distribution of centuries targeted for toxicological analyses reported in gray. In orange are
reported the papers that searched for trace elements and stable isotopes, whereas in blue are reported the papers that
described the presence of medical drugs, drugs of abuse, or medical plants.
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 17

F I G U R E 6 Monitoring of the presence of medical drugs or drugs of abuse in blood or bone marrow and bones. In
red, the presence of molecules in blood or bone marrow only; in orange, the detection of molecules in bone tissue only;
in light blue, reports of higher concentrations of molecules in blood and/or bone marrow with respect to bone tissue; in
green, reports of higher concentrations of molecules in bones with respect to blood/bone marrow.

respectively; Horak and Jenkins (Horak & Jenkins, 2005), Vardakou et al. (Vardakou
et al., 2014), Orfanidis et al. (Orfanidis et al., 2018), and Fernandez-Lopez et al. (Fernandez-
Lopez et al., 2020; Fernandez-Lopez, Luna-Maldonado, Falcon, Mastrobattista, et al., 2019)
noted one case each. These results act as further evidence of the accumulation of deposits of
drugs in bone, with some difficulties in detecting the analytes in bones after a single intake
of drugs (Figure 6).
Interestingly, drug concentration in bone was higher than in blood or marrow 30 times
(22% of the total cases considered) (Fernandez-Lopez et al., 2020; Fernandez-Lopez, Pellegrini,
et al., 2019; Fernandez-Lopez, Pellegrini, et al., 2019; McGrath & Jenkins, 2009; Raikos
et al., 2001), whereas the opposite was reported 44 times (33% of the total cases) (Fern
andez-
L
opez et al., 2022; Fernandez-Lopez, Luna-Maldonado, Falcon, Mastrobattista, et al., 2019;
Mancini et al., 2020; Vandenbosch et al., 2020; Vandenbosch et al., 2022; Vardakou
et al., 2014). These results provide evidence of a discrepancy in drug concentrations between
blood/bone marrow and bone. It is suggested that it is not possible to establish any standard dif-
ferences or similarities in drug concentration between these biological matrices. This lends sup-
port to the hypothesis that the detection of drugs in bones after a single consumption may be
improbable. Indeed, higher concentration in bone than blood and/or marrow suggests an accu-
mulation of analytes in bone tissue. Conversely, higher concentration of xenobiotics in blood
and/or marrow than bone tissue shows the limitation of the latter biological matrix in compari-
son to the other two. As such, different toxicological considerations should be done on this
unconventional biological matrix (Figure 6).
Thus, in some cases, it cannot be excluded that the molecules may be detected even after
acute administration in bone samples if these molecules are still present in the body at the time
of death due to its high vascularization. However, these data (Figure 6) suggest that the deposi-
tion of xenobiotics within bone tissue and the detection of substances after the complete
skeletonization of a body should refer to at least an occasional drug consumption. Thus, this
statement does not exclude the consumption of a substance near the time of death but rather
includes the probability that this substance was also taken during life, at least occasionally.

CONCLUSIONS
The present study illustrates the significant advances made in recent years in the field of bone
toxicology. In particular, it showed a wide array of studies performed on bone tissue sampled
18 GAIA ET AL.

from both forensic and archaeological human remains. Yet, little is known about the
archaeotoxicological field—a new discipline that emerged in the scientific community only a
few years ago. Indeed, as seen in this paper, only a few studies have tried to detect analytes in
very ancient human remains. Nonetheless, this limited literature demonstrates that long post-
mortem intervals and taphonomic factors may not compromise toxicological analyses on bones.
This study is important in understanding the differences and similarities of the papers published
in the literature, highlighting the necessity of improving the literature regarding the detection of
molecules in very ancient human remains. The paper describes the population already tested for
bone toxicology throughout history, suggesting the importance of continuing and improving
knowledge about ancient populations still not studied.
This study provides more information regarding the consumption of molecules (acute/single
and/or occasional/chronic intake of drugs) in this unconventional biological matrix in both
forensic cases and very ancient human remains. The achieved results, reported in the
section “The potential of drugs interpretation in bone tissue: Bone versus blood and/or bone
marrow analyses” suggest that the analytes detected in bones should be referred to occasional
or chronic intake of substances.
Hence, it is important to emphasize how this unconventional matrix has the potential to
broaden the biocultural profile of a subject and to shed light on the history of an individual.
Finally, bone toxicology can collaborate with different disciplines, such as forensic medicine,
forensic anthropology, archaeology and even, in some cases, the history of medicine to clarify
and improve knowledge about the life, lifestyle, and health conditions of the skeletal remains
preserved up to today.

A C K NO W L E D G E M E N T S
Open access publishing facilitated by Universita degli Studi di Milano, as part of the Wiley -
CRUI-CARE agreement.

D A T A A V A I L AB I L I T Y S T A T E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the
current study.

PEER REVIEW
The peer review history for this article is available at https://www.webofscience.com/api/
gateway/wos/peer-review/10.1111/arcm.13065.

ORCID
Giordano Gaia https://orcid.org/0000-0002-6081-0596
Biehler-Gomez Lucie https://orcid.org/0000-0001-6674-7850

R E FE RE NC E S
Baranowska, I. Czernickib, K., & Aleksandrowiczb, R. (1995). The analysis of lead, cadmium, zinc, copper and nickel
content in human bones from the upper Silesian industrial district.
Beck, L., Caffy, I., Delqué-Količ, E., Moreau, C., Dumoulin, J. P., Perron, M., Guichard, H., & Jeammet, V. (2018).
Absolute dating of lead carbonates in ancient cosmetics by radiocarbon. Communications Chemistry, 1(34), 1–7.
https://doi.org/10.1038/s42004-018-0034-y
Biehler-Gomez, L., Giordano, G., Sardanelli, F., Di Candia, D., & Cattaneo, C. (2024). Towards an integrative
approach to the biological profile. Legal Medicine, 71, 102499. https://doi.org/10.1016/j.legalmed.2024.102499
Biehler-Gomez, L., Mattia, M., Sala, C., Giordano, G., di Candia, D., Messina, C., Sconfienza, L. M.,
Franchini, A. F., Porro, A., Galimberti, P. M., Slavazzi, F., & Cattaneo, C. (2021). Mercury poisoning in two
patients with tertiary syphilis from the ca’ Granda hospital (17th-century Milan). Archaeometry, 64, 500–510.
https://doi.org/10.1111/ARCM.12721
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 19

Chang, L., Shen, S., Zhang, Z., Song, X., & Jiang, Q. (2018). Study on the relationship between age and the concentra-
tions of heavy metal elements in human bone. Annals of Translational Medicine, 6, 320–320. https://doi.org/10.
21037/atm.2018.08.09
Corruccini, R. S., Aufderheide, A. C., Handler, J. S., & Wittmers, L. E. (1987). Patterning of skeletal lead content in
Barbados slaves. Archaeometry, 29, 233–239. https://doi.org/10.1111/j.1475-4754.1987.tb00416.x
Fernandez-Lopez, L., Luna-Maldonado, A., Falcon, M., Mastrobattista, L., Navarro-Zaragoza, J., & Mancini, R.
(2019). Development and validation of a gas chromatography–mass spectrometry method for opiates and cocaine
in human bone. Journal of Pharmaceutical and Biomedical Analysis, 164, 636–641. https://doi.org/10.1016/j.jpba.
2018.11.015
Fernandez-Lopez, L., Mancini, R., Pellegrini, M., Rotolo, M. C., Luna, A., & Falcon, M. (2020). Postmortem analysis
of quetiapine and pregabalin in human bone. Legal Medicine (Tokyo, Japan), 46, 101717. https://doi.org/10.1016/
J.LEGALMED.2020.101717
Fern
andez-L
opez, L., Mancini, R., Rotolo, M. C., Navarro-Zaragoza, J., Hern
andez del Rinc
on, J. P., & Falc

on, M.
(2022). Carbamazepine overdose after psychiatric conditions: A case study for postmortem analysis in human
bone. Toxics, 10(6), 322. https://doi.org/10.3390/toxics10060322
Fernandez-Lopez, L., Pellegrini, M., Rotolo, M. C., Luna, A., Falcon, M., & Mancini, R. (2019). Development and
validation of a method for the analysis of Bisoprolol and atenolol in human bone. Molecules, 24, 2400. https://doi.
org/10.3390/molecules24132400
Fernandez-Lopez, L., Pellegrini, M., Rotolo, M. C., Maldonado, A. L., Falcon, M., & Mancini, R. (2019). Develop-
ment and validation of a method for analysing of duloxetine, venlafaxine and amitriptyline in human bone. Foren-
sic Science International, 299, 154–160. https://doi.org/10.1016/J.FORSCIINT.2019.04.001
Franceschetti, L., Di Candia, D., Giordano, G., Giordano, G., Carabelli, I., Vignali, G., & Cattaneo, C. (2020). Drugs
in bone: Detectability of substances of toxicological interest in different states of preservation. Journal of Forensic
Sciences, 1–10, 677–686. https://doi.org/10.1111/1556-4029.14636
Giordano, G., Biehler-Gomez, L., Seneci, P., Cattaneo, C., & Di Candia, D. (2021). Detecting drugs in dry bone: A
pilot study of skeletal remains with a post-mortem interval over 23 years. International Journal of Legal Medicine,
135, 457–463. https://doi.org/10.1007/s00414-020-02494-8
Giordano, G., Mattia, M., Biehler-Gomez, L., Boracchi, M., Tritella, S., Maderna, E., Porro, A., Corsi
Romanelli, M. M., Franchini, A. F., Galimberti, P. M., Slavazzi, F., Sardanelli, F., Di Candia, D., &
Cattaneo, C. (2023). Papaver somniferum in seventeenth century (Italy): Archaeotoxicological study on brain and
bone samples in patients from a hospital in Milan. Scientific Reports, 13, 3390. https://doi.org/10.1038/s41598-023-
27953-1
Giordano, G., Mattia, M., Boracchi, M., Biehler-Gomez, L., Cummaudo, M., Porro, A., Caccianiga, M.,
Sardanelli, F., Slavazzi, F., Galimberti, P. M., Di Candia, D., & Cattaneo, C. (2023). Forensic toxicological ana-
lyses reveal the use of cannabis in Milano (Italy) in the 1600’s. Journal of Archaeological Science, 160, 105873.
https://doi.org/10.1016/j.jas.2023.105873
Gonz
alez-Reimers, E., Arnay-de-la-Rosa, M., Galindo-Martin, L., Batista-L
opez, N., Navarro-Mederos, J. F., Castro-
Alem
an, V. V., & Santolaria-Fern
andez, F. (1991). Trabecular bone mass and bone content of diet-related trace
elements among the Prehispanic inhabitants of the western Canary Islands. Human Evolution, 6, 177–190. https://
doi.org/10.1007/BF02435618
Gonz
alez-Reimers, E., Velasco-V
azquez, J., Arnay-de-la-Rosa, M., Alberto-Barroso, V., Galindo-Martín, L., &
Santolaria-Fern
andez, F. (2003). Bone cadmium and lead in prehistoric inhabitants and domestic animals from
gran Canaria. Science of the Total Environment, 301, 97–103. https://doi.org/10.1016/S0048-9697(02)00299-1
Gorczynski, L. Y., & Melbye, F. J. (2001). Detection of benzodiazepines in different tissues, including bone, using a
quantitative ELISA assay. Journal of Forensic Sciences, 46, 916–918. https://doi.org/10.1520/jfs15069j
Grattan, J., Huxley, S., Karaki, L. A., Toland, H., Gilbertson, D., Pyatt, B., & Al Saad, Z. (2002). “Death … More
desirable than life”? The human skeletal record and toxicological implications of ancient copper mining and
smelting in Wadi Faynan, southwestern Jordan. Toxicology and Industrial Health, 18, 297–307. https://doi.org/10.
1191/0748233702th153oa
Güler, H., & Güçlü Ekinci, H. K. (2023). Heavy metals in human bones from the Roman Imperial period. Journal of
Surgery and Medicine, 7, 463–467. https://doi.org/10.28982/josam.7878
Horak, E. L., & Jenkins, A. J. (2005). Postmortem tissue distribution of olanzapine and citalopram in a drug intoxica-
tion. Journal of Forensic Sciences, 50, 1–3. https://doi.org/10.1520/jfs2004067
Kepa, M., Kozłowski, T., Szostek, K., Drozd, A., Walas, S., Mrowiec, H., Stepa
nczak, B., Głab, H., & Grupa, M.
(2012). Analysis of mercury levels in historical bone material from syphilitic subjects - pilot studies (short report).
Anthropologischer Anzeiger, 69, 367–377. https://doi.org/10.1127/0003-5548/2012/0163
L

opez-Costas, O., Kylander, M., Mattielli, N., et al. (2020). Human bones tell the story of atmospheric mercury and
lead exposure at the edge of Roman World. Science of the Total Environment, 710, 136319. https://doi.org/10.1016/
j.scitotenv.2019.136319
20 GAIA ET AL.

Mancini, R., Fernadez-Lopez, L., Falcon, M., Pellegrini, M., Luna, A., & Rotolo, M. (2020). Postmortem analysis of
benzodiazepines in human bone by gas chromatography-mass spectrometry. Journal of Analytical Toxicology, 44,
985–992. https://doi.org/10.1093/jat/bkaa020
Martínez-García, M. J., Moreno, J. M., Moreno-Clavel, J., Vergara, N., García-S
anchez, A., Guillam
on, A.,
Portí, M., & Moreno-Grau, S. (2005). Heavy metals in human bones in different historical epochs. Science of the
Total Environment, 348, 51–72. https://doi.org/10.1016/j.scitotenv.2004.12.075
McGrath, K. K., & Jenkins, A. J. (2009). Detection of drugs of forensic importance in postmortem bone. American
Journal of Forensic Medicine and Pathology, 30, 40–44. https://doi.org/10.1097/PAF.0b013e31818738c9
McIntyre, I. M., King, C. V., Boratto, M., & Drummer, O. H. (2000). Post-mortem drug analyses in bone and bone
marrow. Therapeutic Drug Monitoring, 22, 79–83. https://doi.org/10.1097/00007691-200002000-00017
Montes-Santiago, J. (2013). The lead-poisoned genius: saturnism in famous artists across five centuries. In Progress in
brain research (pp. 223–240). Elsevier B.V.
Orfanidis, A., Gika, H., Mastrogianni, O., Krokos, A., Theodoridis, G., Zaggelidou, E., & Raikos, N. (2018). Determi-
nation of drugs of abuse and pharmaceuticals in skeletal tissue by UHPLC–MS/MS. Forensic Science Interna-
tional, 290, 137–145. https://doi.org/10.1016/j.forsciint.2018.07.004
Orfanidis, A., Gika, H., Zaggelidou, E., Mastrogianni, O., & Raikos, N. (2019). Alprazolam and zolpidem in skeletal
tissue of decomposed body confirms exposure. Journal of Forensic Sciences, 64, 643–646. https://doi.org/10.1111/
1556-4029.13890
Parsche, F., Balabanova, S., & Pirsig, W. (1993). Drugs in ancient populations. Lancet, 341, 503.
Raikos, N., Tsoukali, H., & Njau, S. N. (2001). Determination of opiates in postmortem bone and bone marrow. Foren-
sic Science International, 123, 140–141. https://doi.org/10.1016/S0379-0738(01)00529-1
Rasmussen, K. L., Boldsen, J. L., Kristensen, H. K., Skytte, L., Hansen, K. L., Mølholm, L., Grootes, P. M.,
Nadeau, M.-J., & Flöche Eriksen, K. M. (2008). Mercury levels in Danish medieval human bones. Journal of
Archaeological Science, 35, 2295–2306. https://doi.org/10.1016/j.jas.2008.03.003
Rasmussen, K. L., Skytte, L., Jensen, A. J., & Boldsen, J. L. (2015). Comparison of mercury and lead levels in the bones
of rural and urban populations in southern Denmark and northern Germany during the middle ages. Journal of
Archaeological Science: Reports, 3, 358–370. https://doi.org/10.1016/j.jasrep.2015.06.021
Rasmussen, K. L., Skytte, L., Pilekær, C., Lauritsen, A., Boldsen, J. L., Leth, P. M., & Thomsen, P. O. (2013). The dis-
tribution of mercury and other trace elements in the bones of two human individuals from medieval Denmark -
the chemical life history hypothesis. Heritage Science, 1, 10. https://doi.org/10.1186/2050-7445-1-10
Reinhard, K. J., & Ghazi, A. M. (1992). Evaluation of lead concentrations in 18th-century Omaha Indian skeletons
using ICP-MS. American Journal of Physical Anthropology, 89, 183–195. https://doi.org/10.1002/ajpa.1330890205
Roberts, C., & Manchester, K. (2010). The archeology of disease. Cornell University Press.
Rubin, K. M. (2018). The current state and future directions of skeletal toxicology: Forensic and humanitarian implica-
tions of a proposed model for the in vivo incorporation of drugs into the human skeleton. Forensic Science Interna-
tional, 289, 419–428. https://doi.org/10.1016/j.forsciint.2018.06.024
Rubin, K. M., Rubin, K. M., Goldberger, B. A., & Garrett, T. J. (2020). Detection of chemical weapon nerve agents in
bone by liquid chromatography-mass spectrometry. Journal of Analytical Toxicology, 44, 391–401. https://doi.org/
10.1093/jat/bkz118
Sguazza, E., Gibelli, D., Caligara, M., Di Candia, D., Galimberti, P. M., & Cattaneo, C. (2016). The role of toxicologi-
cal analyses in anthropology: A case report on Lead intoxication. Archaeometry, 58, 152–158. https://doi.org/10.
1111/arcm.12169
Vandenbosch, M., Pajk, S., Van Den Bogaert, W., Wuestenbergs, J., Van de Voorde, W., & Cuypers, E. (2022). Post-
mortem analysis of opioids and metabolites in skeletal tissue. Journal of Analytical Toxicology, 46, 783–790.
https://doi.org/10.1093/jat/bkab095
Vandenbosch, M., Rooseleers, L., Van Den Bogaert, W., Wuestenbergs, J., Van de Voorde, W., & Cuypers, E. (2020).
Skeletal tissue, a viable option in forensic toxicology? A view into post mortem cases. Forensic Science Interna-
tional, 309, 110225. https://doi.org/10.1016/j.forsciint.2020.110225
Vardakou, I., Athanaselis, S., Pistos, C., Papadodima, S., Spiliopoulou, C., & Moraitis, K. (2014). The clavicle bone as
an alternative matrix in forensic toxicological analysis. Journal of Forensic and Legal Medicine, 22, 7–9. https://
doi.org/10.1016/j.jflm.2013.11.012
Vuorinen, H. S., Tappe, U., & Mussalo-Rauhamaa, H. (1990). Trace and heavy metals in infants, analysis of long bones
from Ficana, Italy, 84th century BC.
Wiart, J. F., Hakim, F., Andry, A., Eiden, C., Drevin, G., Lelièvre, B., Rougé-Maillart, C., Decourcelle, M., Lemaire-
Hurtel, A. S., Allorge, D., & Gaulier, J. M. (2020). Pitfalls of toxicological investigations in hair, bones, and nails
in extensively decomposed bodies: Illustration with two cases. International Journal of Legal Medicine, 134, 1339–
1344. https://doi.org/10.1007/s00414-020-02267-3
Yamada, M. o., Tohno, S., Tohno, Y., Minami, T., Ichii, M., & Okazaki, Y. (1995). Accumulation of mercury in exca-
vated bones of two natives in Japan. Science of the Total Environment, 162, 253–256. https://doi.org/10.1016/0048-
9697(95)04435-4
TOXICOLOGICAL ANALYSES OF THE BONE MATRIX 21

Yoshinaga, J., Hisada, A., Yoneda, M., & Ishida, H. (2013). Lead content of bones excavated from archaeological sites
in Hokkaido. Nihon Eiseigaku Zasshi, 68, 53–57. https://doi.org/10.1265/jjh.68.53

How to cite this article: Gaia, G., Lucie, B.-G., Cristina, C., & Domenico, D. C. (2025).
Toxicological analyses of the bone matrix: Successes and challenges. Archaeometry, 1–21.
https://doi.org/10.1111/arcm.13065