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Excretion of 19‐norandrosterone after consumption of boar meat



The consumption of the offal of noncastrated pigs can lead to the excretion of 19‐norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the δ¹³C values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. δ¹³C values ranged from −18.5‰ to −23.5‰, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that δ¹³C values for wild boar's steroids are unpredictable and may vary seasonally.
Excretion of 19-norandrosterone after consumption of
boar meat
Frank Hülsemann
| Gregor Fußhöller
| Christine Lehn
| Mario Thevis
Institute of Biochemistry, German Sport
University Cologne, Cologne, Germany
Institute of Legal Medicine, University of
Munich, Munich, Germany
Frank Hülsemann, Institute of Biochemistry,
German Sport University Cologne, Cologne,
The consumption of the offal of noncastrated pigs can lead to the excretion of
19-norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the
method of choice to differentiate between an endogenous or exogenous origin of
urinary NorA. In some cases, after the consumption of wild boar offal, the δ
values of urinary NorA fulfill the criteria of an adverse analytical finding due to differ-
ing food sources of boar and consumer. However, consumption of wild boar's offal is
not very common in Germany, and thus, the occurrence of such an analytical finding
is unlikely. In contrast, the commerce with wild boar meat has increased in Germany
within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In
order to probe for the probability of the occurrence of urinary NorA after consump-
tion of wild boar meat, human urine samples were tested following the ingestion of
commercially available game. In approximately half of the urine samples, traces of
NorA were detected postadministration of 200 to 400 g boar meat. The highest uri-
nary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h
after the meal. δ
C values ranged from 18.5to 23.5, which would have led
to at least two adverse analytical findings if the samples were collected in an
antidoping context. IRMS analysis on German boar tissue samples showed that δ
values for wild boar's steroids are unpredictable and may vary seasonally.
boar meat, carbon isotope ratios, norandrosterone
In doping control analysis, isotope ratio mass spectrometry (IRMS) is
used to distinguish between an endogenous or exogenous origin of the
urinary 19-norsteroid norandrosterone (NorA).
In humans, NorA can
be naturally found in urine in small amounts (<2 ng/ml) formed, for
example, by demethylation of androsterone (A) and, at higher concen-
trations, during pregnancy.
NorA can also be formed in urine by in
situ microbial degradation of A.
In addition, NorA is the main
urinary metabolite of the therapeutic nortestosterone (NT, nandrolone)
as well as of prohormones such as 4-norandrostenediol,
5-norandrostenediol, or 4-norandrostenedione (NAED), which are all
prohibited in sports.
A source of so-called pseudo-endogenousuri-
nary NorA can be the consumption of the offal of noncastrated pigs.
In boars, the highest concentrations of 19-norsteroids can be
found in testicles (NAED up to 84 μg/kg, NT up to 172 μg/kg), liver,
and kidney.
Much lower but nevertheless significant amounts have
been found in boar meat (NAED up to 0.9 μg/kg, NT up to 3.6 μg/
Although the consumption of boar offal is not particularly com-
mon in Germany, the consumption of wild boar meat is increasing
Received: 30 June 2020 Revised: 28 September 2020 Accepted: 17 October 2020
DOI: 10.1002/dta.2958
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.
© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd
Drug Test Anal. 2020;16. 1
within the last years with around 600,000 wild boars harvested per
hunting season,
corresponding to approximately 10,000 to 20,000
tons of wild boar meat being consumed in Germany every year.
We have reported previously that the carbon isotopic composition
of free-ranging (wild) boar tissue and steroids in Germany may vary
seasonally and can result in δ
Cvaluesfrom13to 24.
forage of free ranging boars is usually dominated by C
-plants (wheat,
barley, acorns, etc.) in winter and spring, but as soon as maize crops are
available, the boars rely elusively on this C
-plant as primary forage.
Thus, the consumption of offal but also potentially meat can lead to the
urinary excretion of NorA with a carbon isotopic signature different
from the consumer. This pseudo-endogenousorigin of NorA may lead
to adverse analytical findings in doping control testing if δ
consumer and consumed animal differ more than 3.
It has been stated improbable that concerning antidoping tests,
significant amounts of urinary NorA in humans may originate from the
consumption of boar meat,
and if so, the corresponding δ
C values
of such urinary NorA would be endogenous-like.
As these assump-
tions are yet to be corroborated, an excretion study was conducted to
verify or falsify the possibility of an adverse analytical finding after
consumption of boar meat.
2.1 |Test meals and participants
The meals were prepared using varying amounts of boar meat prod-
ucts (Table 1), with weights ranging from 187 to 491 g (prepared
weight). The products were randomly selected and obtained from
butcheries or online distributors. Each volunteer consumed one meal,
with time and side dishes being arbitrary. Nine male volunteers with
an average body weight of 90.4 ± 8.9 kg and three female volunteers
(67.0 ± 5.6 kg) were included in the study. The participants were
requested to collect one urine sample prior to the meal and all urine
samples for a period of 24 h after meat consumption. Samples were
stored at +4C until analysis. The participants gave written informed
consent prior to the study. Test meals were not checked for NorA
content or carbon isotope composition.
2.2 |Sample preparation of urine for GC/MS/MS
The samples were prepared according to the laboratory internal stan-
dard operating procedure for anabolic steroids.
Conjugated and
unconjugated steroids were extracted from urine at pH 9.6 with tert-
butylmethyl ether (TBME, in-house purified by distillation) following
enzymatic hydrolysis of the glucuronides at pH 7 (β-glucuronidase,
Roche). After centrifugation, the organic layer was transferred and
evaporated to dryness. The dry residue was derivatized with 100 μlof
N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA, Macherey-
Nagel)/ammonium iodide (Sigma-Aldrich)/ethanethiol (Merck, for syn-
thesis) (v:w:v, 1000:2:3).
2.3 |GC/MS/MS
The GC/EI-MS/MS experiments were performed in accordance to
earlier protocols
using a Trace 1310 gas chromatograph interfaced
to a TSQ 8000 triple quadrupole mass spectrometer (all Thermo Sci-
entific). The GC system was equipped with an Ultra1 capillary column
(length 17 m, i.d. 0.2 mm, film thickness 0.1 μm, Agilent) in split (1:10)
mode. The initial GC oven temperature was 184C, increasing at 3C/
min to 232C and at 40C/min to a final temperature of 310C.
TABLE 1 Type and amount of test meals including maximum urinary NorA concentrations and δ
C values after consumption for all
participants (m = male, f = female)
Type Amount/g [NorA]
/ng/ml NorA/NorE δ
1 (m) Roast
187 <1 3.5 22.9 19.7 3.2
2 (m) Roast
3 (m) Roast
410 2.9 7.6 24.3 18.5 5.8
4 (m) Roast
5 (f) Roast
6 (m) Ham
300 2.5 4.9 23.3 23.5 0.2
7 (f) Canned meat
220 <1 4.0 22.8 21.4 1.4
8 (f) Canned meat
215 <1 4.3 23.8 21.9 1.9
9 (m) Canned meat
10 (m) Canned meat
11 (m) Jerky
12 (m) Jerky
Obtained from a meat market, Northern Germany.
Local supplier, Southern Germany.
Online distributor, Estonian origin.
Number in brackets represents the corresponding raw weight of the dried meat.
Helium (4.6, Linde) was used as carrier gas (0.9 ml/min, constant pres-
sure) and argon (5.0, Linde) as collision gas. The injector and interface
temperatures were both set to 300C, and the ion source was oper-
ated at 250C. Ionization was accomplished using electron ionization
(EI) (70 eV).
2.4 |Sample preparation of urine for GC/C/IRMS
Preparation of the urine samples (2030 ml) followed the laboratory
internal standard operating procedure
that comprised the following
steps: solid-phase extraction (Chromabond C18, 500 mg, 6 ml,
Macherey-Nagel) with methanol (LC grade, J.T. Baker) followed by a
liquidliquid extraction with TBME (GC grade, Merck), an enzymatic
hydrolysis with β-glucuronidase (Roche) at pH 7, and a second liquid
liquid extraction with n-pentane (pa, Merck) at pH 9.6. By means of
two different HPLC runs, the steroids of interest were separated and
fractionated. The first HPLC was a reversed-phase purification on a
XBridge RP18 5 μm column, followed by a second HPLC employing a
XBridge C18 column 5 μm (both columns from Waters). The fractions
were dried and acetylated for IRMS.
2.5 |GC/C/IRMS of urinary NorA
Samples were measured per GC/C/IRMS on a Trace 1310 gas
chromatograph equipped with an HP-5MS chromatographic column
and coupled via a ConFlo IV to a MAT 253 isotope ratio mass spec-
trometer (all Thermo Scientific). Carbon isotope ratios are expressed
in per mill relative to VPDB. Monitoring gas (CO
, purity 4.5, Linde)
was scale calibrated using acetylated steroid mixtures USADA 33-1
(Cornell University, Ithaca, NY) comprising 5α-androstan-3β-ol
acetate, 5α-androstan-3α-ol-17-one acetate, 5β-androstan-3α-ol-11,-
17-dione acetate, and 5α-cholestane
and CU/PCC 44-1 (Cornell
University, Ithaca, NY), which comprises 5α-androstan-3α-ol-17-one
acetate, 5β-androstan-3α,17β-diacetate, 5α-cholestane, and
5β-pregnan-3α-20α-diacetate. Accuracy of the instrument was
checked using quality control charts for all steroids analyzed in every
sequence, as well as using the internal working standard
5α-androstan-3β-ol acetate. Sample preparation was checked using
negative and positive control samples according to the WADA
technical document.
2.6 |EA/IRMS of bristles
Bristles were washed using distilled water and soaked for 30 min
in methanol (Roth)/chloroform (pa, Merck; v:v, 2:1) in an ultrasonic
bath (Bandolin Sonorex). After drying, they were cut into 10 mm
segments. As a multi-isotopic analysis (CHNS) was performed, and
due to the limited sample volume, only two to three segments per
animal were analyzed for δ
C. Between 1.8 and 2.0 mg of bristles
were weighed into tin capsules in quadruplicate. Samples were
analyzed at isolab GmbH, Schweitenkirchen, Germany, using a
Vario EL Cube elemental analyzer (Elementar Analysensysteme)
connected to a mass spectrometer (Isoprime). Internal standards
used during analysis were casein (Kremer Pigmente) and two
different horse tail hair samples (both from local suppliers). Scale
calibrations were performed with NBS 22 (30.03, IAEA) and
IRMM-BCR 657 (10.76, IRMM). The analytical precisions using
at least quadruplicate measurements were δ
C = ±0.1. The data
were drift corrected using repeated measurement of laboratory
standard after 30 samples.
3.1 |Urinary concentrations of NorA
No NorA was detected in the blankurine samples sampled prior
to ingestion of the boar meat. Therefore, endogenous production of
NorA of the volunteers can be excluded. NorA was detected in
urine samples of five of the 12 volunteers (Table 1). Two of them
exhibited significant amounts of NorA with maximum concentrations
of 2.9 and 2.5 ng/ml (1.9 and 2.1 ng/ml after adjustment for
specific gravity),
whereas in the urine samples of the other three
volunteers, only traces of NorA lower than 1 ng/ml were found
(Figure 1). No NorA was detected in the urine samples of the
remaining seven volunteers. Peak NorA concentrations were
reached after 3.3 to 6.0 h after consumption of the boar meat. All
urine samples from volunteers that showed traces of urinary NorA
tested negative 24 h after the meal.
For all urine samples, also the concentration of nor-
etiocholanolone (NorE) was determined. The NorA/NorE ratio, which
is an indicator of an exogenous administration of 19-norsteroids,
increased (>3) in all urine samples with NorA concentrations greater
than 0.5 ng/ml. The results for NorA concentrations and NorA/NorE
ratios of this study were similar to those found in another studies
after ingestion of boar's offal.
FIGURE 1 Excretion profiles of urinary NorA after consumption
of boar testicles (white circles),
mixed meals of boar meat and offal
(grey circles),
and boar meat (black circles). The horizontal line
reflects the 2.5 ng/ml cut-off level for mandatory IRMS analyses
3.2 |δ
C of urinary NorA
For each volunteer those urine samples with the detectable NorA
concentrations were further analyzed by GC/C/IRMS. Pregnanediol
(PD) was analyzed as endogenous reference compound (ERC) with
C values around 24.3and 22.8and, thus, in good agree-
ment with typical values observed for German inhabitants.
NorA showed δ
C values between 23.5and 18.5(Table 1).
Accordingly, the absolute differences between ERC and NorA as
defined in the relevant technical Document of WADA ranged from
jΔδj= 0.2to 5.8for the volunteers. Hence, urine samples of two
study participants yielded jΔδj> 3, which would be considered as
adverse analytical findings (AAF) according to currently enforced
All urine samples with traces of NorA resulted from the ingestion
of boar meat obtained from the same meat market in Northern Ger-
many; however, not all meals prepared from meat originating from
that market (n= 7) led to an excretion of urinary NorA. It is assumed
that the meat was derived from different animals, as the products
were either purchased at different times and/or the best-before dates
were not identical. Further, deviating δ
C values of the urinary NorA
indicate different sources of the metabolite's precursor.
3.3 |δ
C of boar's bristles
Bristles of seven wild boars hunted in Germany were analyzed per
EA/IRMS. Two wild boars originated from Northern Germany, one
boar was from in North-Rhine Westphalia (data have already been
published before
), and four boars were from southern Germany
(Figure 2). The wide range of δ
C values within the bristles of one
individual, published earlier,
were confirmed by the herein conducted
additional analyses, although the intraindividual variation differed
between the animals. There was neither any spatial difference in δ
detectable nor any difference according to age.
Almost all boars showed a dietary shift from C
to C
-plant based
diets (or vice versa) within their bristles. It is known that the δ
values of mammalian (and human) body protein, either hair keratin or
muscle protein, rapidly adapts toward a change in the
C content of
the diet.
According to these studies, δ
C values for mammalian
FIGURE 2 Range of δ
C values of wild
boar's bristles in Germany. Each circle with
corresponding δ
C values represents one
individual at its origin. Values for individual
marked with an asterisk (*) are from a previous
hair of 15can be attributed to an almost completely corn-based
On the other hand, δ
C values of 24and lower are
attributed to exclusively C
-plants in the diet.
The wide range of δ
C values of about 10found in boar's
bristles correspond to values found for urinary NorA after consump-
tion of boar meat or offal, which range from 13to 24. From
diet experiments, it is known that human protein (hair keratin) and
endogenous steroids adapt concurrently to dietary changes (C
-plant based food or vice versa).
The absolute δ
C values of
steroids and hair protein are comparable, although individual offsets
up to 2.5exist, with the urinary steroids being, in most of the cases,
more depleted in
C than hair protein.
3.4 |Interpretation of human urinary NorA
Our results confirm the hypothesis that low urinary NorA concentra-
tions in humans can be the result of the consumption of meat from
uncastrated male pigs. Although the concentration of 19-norsteroids
in wild boar meat is lower than in offal such as testicles or liver, the
consumption of a typical meal of wild boar meat can lead to urinary
concentrations around 2 ng/ml (after adjustment for specific gravity).
In order to distinguish between an endogenous or exogenous origin
according to TD2019NA for urinary concentrations of NorA greater
than 2.5 ng/ml (after adjustment for specific gravity), IRMS is manda-
tory; below 2.5 ng/ml, IRMS is optional. Due to the varying and
unpredictable diets of wild boars (in Germany) δ
C values of urinary
NorA can present values from 15to 24. As typical endoge-
nous values for steroids of a German population are around
urinary NorA deriving from wild boar meat may be
interpreted as endogenous,if the boar's diet was C
conversely, it can be identified as exogenousif the animal was on a
-based diet. According to TD2019NA, an absolute difference
between ERC and NorA greater than 3has to be reported as an
adverse analytical finding.
To our knowledge, synthetic pharmaceutical preparations of
19-norsteroids exhibit δ
C values between 33and 21.
Thus, to date, urinary NorA presenting more enriched δ
C values is
more likely an indication for boar meat (or offal) ingestion than for the
administration of a synthetic 19-norsteroid.
The fact of varying δ
C values of wild boar could also be prob-
lematic for people living in countries with a high consumption of
-plants like the United States or Southern Africa. Human endoge-
nous δ
C values in these countries are enriched in
C compared
with Germany, and for these, there is the possibility of adverse analyt-
ical findings after the consumption of the meat of 19-norsteroid
producing C
-fed boars.
Moreover, it appears unlikely that after the consumption of boar
meat urinary concentrations of NorA rise above 15 ng/ml, which is
the upper cut-off level for a GC/C/IRMS target analysis of suspicious
The highest urinary concentration of NorA after consump-
tion of boar meat in our study was 2.9 ng/ml (410 g of prepared
meat). Higher urinary concentrations of NorA may be found after con-
sumption of offal, or mixed meals of meat and offal.
The herein presented results show that detectable amounts of NorA
may occur in human urine after ingestion of wild boar meat. As it has
been shown previously, it is unpredictable which δ
urinary NorA can be expected after such a meal. It is still advisable to
avoid the term endogenousfor δ
C values of 19-norsteroid metabo-
lites of a free ranging animal in comparison with δ
C values of human
urinary steroids. It has been confirmed that the δ
C values for animal
19-norsteroids vary between 13and 25and do not necessarily
correlate with δ
C values found for human individuals of the same
geographical region. Not only the consumption of wild boar's offal in
the hours preceding a doping control test but also the consumption of
wild boar meat may result in an atypical or even positive test result,
albeit the urinary NorA concentrations are expected to be lower than
after consumption of wild boar's offal. Both athletes as well as anti-
doping laboratories and authorities should still be aware of this aspect.
The authors thank the Manfred Donike Institute for Doping Analysis
(Cologne, Germany), the Doping Authority Netherlands (Capelle aan
den Ijssel, The Netherlands), and the Federal Ministry of the Interior
of the Federal Republic of Germany (Berlin, Germany) for supporting
the presented work. Open access funding enabled and organized by
Projekt DEAL.
Frank Hülsemann
Mario Thevis
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How to cite this article: Hülsemann F, Fußhöller G, Lehn C,
Thevis M. Excretion of 19-norandrosterone after consumption
of boar meat. Drug Test Anal. 2020;16.
... For this aspect, the concomitant steroid profiles of testosterone, estradiol, and progesterone, determined in pig serum samples according to a fit-for-purpose validated method compliant to Commission Regulation (EU) 2021/808, allowed further verification in a real field context of the specificity of selected biomarkers. The developed multi-residue method will need to be extended to other steroids like nandrolone, which is known to occur in both domestic and wild boars [10,34,35]. ...
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The use of anabolic–androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon®) and other nine with nandrolone esters (Myodine®). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller subcluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.
19-norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/ml, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/ml were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well-known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. 2 volunteers produced 19NA findings above 2.5 ng/ml and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.
Most core areas of anti‐doping research exploit and rely on analytical chemistry, applied to studies aiming at further improving the test methods’ analytical sensitivity, the assays’ comprehensiveness, the interpretation of metabolic profiles and patterns, but also at facilitating the differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds and comprehending the athlete’s exposome. Further, a continuously growing number of advantages of complementary matrices such as dried blood spots has been identified and transferred from research to sports drug testing routine applications, with an overall gain of extremely valuable additions to the anti‐doping field. In this edition of the annual banned‐substance review, literature on recent developments in anti‐doping published between October 2020 and September 2021 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified in the World Anti‐Doping Agency’s 2021 Prohibited List.
Similar to the general population, elite athletes are exposed to a complex set of environmental factors including chemicals, radiation, but also biological and physical stressors, which constitute an exposome that is, unlike for the general population, subjected to specific scrutiny for athletes due to applicable anti‐doping regulations and associated (frequent) routine doping controls. Hence, investigations into the athlete’s exposome and how to distinguish between deliberate drug use and different contamination scenarios has become a central topic of anti‐doping research, as a delicate balance is to be managed between the vital and continually evolving developments of sensitive analytical techniques on the one hand, and the risk of the athletes’ exposome potentially causing adverse analytical findings on the other.
Probing for evidence of the administration of prohibited therapeutics, drugs and/or drug candidates as well as the use of methods of doping in doping control samples is a central assignment of anti-doping laboratories. In order to accomplish the desired analytical sensitivity, retrospectivity, and comprehensiveness, a considerable portion of anti-doping research has been invested into studying metabolic biotransformation and elimination profiles of doping agents. As these doping agents include lower molecular mass drugs such as e.g. stimulants and anabolic androgenic steroids, some of which further necessitate the differentiation of their natural/endogenous or xenobiotic origin, but also higher molecular mass substances such as e.g. insulins, growth hormone, or siRNA/anti-sense oligonucleotides, a variety of different strategies towards the identification of employable and informative metabolites have been developed. In this review, approaches supporting the identification, characterization, and implementation of metabolites exemplified by means of selected doping agents into routine doping controls are presented, and challenges as well as solutions reported and published between 2010 and 2020 are discussed.
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Anabolic-androgenic steroids are some of the most frequently detected drugs in amateur and professional sports. Doping control laboratories have developed numerous assays enabling the determination of administered drugs and/or their metabolic products that allow retrospectives with respect to pharmacokinetics and excretion profiles of steroids and their metabolites. A new metabolite generated from metandienone has been identified as 18-nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one in excretion study urine samples providing a valuable tool for the long-term detection of metandienone abuse by athletes in sports drug testing. The metabolite was characterized using gas chromatography/(tandem) mass spectrometry, liquid chromatography/tandem mass spectrometry and liquid chromatography/high-resolution/high-accuracy (tandem) mass spectrometry by characteristic fragmentation patterns representing the intact 3-keto-1,4-diene structure in combination with typical product ions substantiating the proposed C/D-ring structure of the steroid metabolite. In addition, structure confirmation was obtained by the analysis of excretion study urine specimens obtained after administration of 17-CD(3)-labeled metandienone providing the deuterated analogue to the newly identified metabolite. 18-Nor-17beta-hydroxymethyl,17alpha-methyl-androst-1,4,13-trien-3-one was determined in metandienone administration study urine specimens up to 19 days after application of a single dose of 5 mg, hence providing an extended detection period compared with commonly employed strategies.
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RationaleNatural stable carbon (δ13C) and nitrogen isotope ratios (δ15N) of humans are related to individual dietary habits and environmental and physiological factors. In forensic science the stable isotope ratios of human remains such as hair and nail are used for geographical allocation. Thus, knowledge of the global spatial distribution of human δ13C and δ15N values is an essential component in the interpretation of stable isotope analytical results.Methods No substantial global datasets of human stable isotope ratios are currently available, although the amount of available (published) data has increased within recent years. We have herein summarised the published data on human global δ13C andδ15N values (around 3600 samples) and added experimental values of more than 400 additional worldwide human hair and nail samples. In order to summarise isotope ratios for hair and nail samples correction factors were determined.ResultsThe current available dataset of human stable isotope ratios is biased towards Europe and North America with only limited data for countries in Africa, Central and South America and Southeast Asia. The global spatial distribution of carbon isotopes is related to latitude and supports the fact that human δ13C values are dominated by the amount of C4 plants in the diet, either due to direct ingestion as plant food, or by its use as animal feed. In contrast, the global spatial distribution of human δ15N values is apparently not exclusively related to the amount of fish or meat ingested, but also to environmental factors that influence agricultural production.Conclusions There are still a large proportion of countries, especially in Africa, where there are no available data for human carbon and nitrogen isotope ratios. Although the interpretation of modern human carbon isotope ratios at the global scale is quite possible, and correlates with the latitude, the potential influences of extrinsic and/or intrinsic factors on human nitrogen isotope ratios have to be taken into consideration. Copyright © 2015 John Wiley & Sons, Ltd.
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Stable isotope measurements are increasingly being used to gain insights into the nutritional ecology of many wildlife species and their role in ecosystem structure and function. Such studies require estimations of trophic discrimination factors (i.e. differences in the isotopic ratio between the consumer and its diet). Although trophic discrimination factors are tissue-and species-specific, researchers often rely on generalized, and fixed trophic discrimination factors that have not been experimentally derived. In this experimental study, captive wild boar (Sus scrofa) were fed a controlled diet of corn (Zea mays), a popular and increasingly dominant food source for wild boar in the Czech Republic and elsewhere in Europe, and trophic discrimination factors for stable carbon (Δ 13 C) and nitrogen (Δ 15 N) isotopes were determined from hair samples. The mean Δ 13 C and Δ 15 N in wild boar hair were –2.3 ‰ and +3.5 ‰, respectively. Also, in order to facilitate future derivations of isotopic measurements along wild boar hair, we calculated the average hair growth rate to be 1.1 mm d-1. Our results serve as a baseline for interpreting isotopic patterns of free-ranging wild boar in current Euro-pean agricultural landscapes. However, future research is needed in order to provide a broader understanding of the processes underlying the variation in trophic discrimination factors of carbon and nitrogen across of variety of diet types.
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Across Europe wild boar numbers increased in the 1960s-1970s but stabilised in the1980s; recent evidence suggests that numbers and impact of wild boar grew steadily since the 1980s. As hunting is the main cause of mortality for this species, we reviewed wild boar hunting bags and hunter population trends in 18 European countries from 1982 to 2012. Hunting statistics and numbers of hunters were used as indicators of animal numbers and hunting pressure. The results confirmed that wild boar increased consistently throughout Europe whilst the number of hunters remained relatively stable or declined in most countries. We conclude that recreational hunting is insufficient to limit wild boar population growth and that the relative impact of hunting on wild boar mortality had decreased. Other factors, such as mild winters, reforestation, intensification of crop production, supplementary feeding and compensatory population responses of wild boar to hunting pressure might also explain population growth. As populations continue to grow, more human-wild boar conflicts are expected unless this trend is reversed. New interdisciplinary approaches are urgently required to mitigate human-wild boar conflicts that are otherwise destined to grow further.
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The carbon-isotope composition of hair and feces offers a glimpse into the diets of mammalian herbivores. It is particularly useful for determining the relative consumption of browse and graze in tropical environments, as these foods have strongly divergent carbon-isotope compositions. Fecal δ13C values reflect the last few days consumption, whereas hair provides longer term dietary information. Previous studies have shown, however, that some fractionation occurs between dietary δ13C values and those of hair and feces. Journal Article
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For the first time in the field of steroid residues in humans, demonstration of 19-norandrosterone (19-NA: 3α-hydroxy-5α-estran-17-one) and 19-noretiocholanolone (19-NE: 3α-hydroxy-5β-estran-17-one) excretion in urine subsequent to boar consumption is reported. Three male volunteers agreed to consume 310 g of tissues from the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine samples before and during 24 h after meal intake. After deconjugation of phase II metabolites, purification and specific derivatisation of target metabolites, the urinary extracts were analysed by mass spectrometry. Identification was carried out using measurements obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-NE concentrations in urine reached 3.1 to 7.5 µg/L nearby 10 hours after boar tissue consumption. Levels returned to endogenous values 24 hours after. These two steroids are usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT: 17β-hydroxyestr-4-en-3-one), especially in the antidoping field. We have thus proved that eating tissues of non-castrated male pork (in which 17β-nandrolone is present) might induce some false accusations of the abuse of nandrolone in antidoping.
Anabolic androgenic steroids (AAS) are the most widely abused class of drugs by athletes, and thus represent a significant problem to the anti‐doping community. Confirmation of a doping violation for AAS cannot always be based on their presence alone due to the endogenous production of some steroids. Both testosterone (and its metabolites) and the major diagnostic metabolite of nandrolone (19‐norandrosterone) are produced endogenously. Gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) is used in such cases to differentiate between the administration of a synthetic preparation and endogenous steroid production, by measurement of their differing carbon isotope (13C/12C) ratio. The availability of synthetic steroid preparations with a 13C content analytically indistinguishable from that of endogenous steroids would prevent the confirmation of a doping violation. The purpose of this study was to investigate the potential availability of such steroid preparations in the UK. Fourteen preparations containing nandrolone (n = 9) and testosterone (n = 5) were analysed. The δ 13C values were determined using GC‐C‐IRMS, and the identity of the steroid preparations was confirmed using gas chromatography mass spectrometry (GC‐MS). Ten steroid preparations displayed δ 13C values within the range expected for synthetic steroids (less than ‐27 ‰). However, four nandrolone preparations displayed δ13C values that overlap with the values considered to be endogenous in origin (range ‐26 to ‐16 ‰). Misuse of these preparations could prevent the confirmation of nandrolone administration using GC‐C‐IRMS in anti‐doping cases.
Isotope‐ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19‐norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in‐situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ13C values of synthetic 19‐norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ13C values of such a scenario, urine samples collected after consumption of a wild boar's testicle meal were analyzed. IRMS revealed highly enriched δ13C values for urinary NorA, which could be related to a completely corn‐based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C3‐based forage, probably in winter and spring, to a C4‐based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ13C values for NorA of ‐18 ‰, most probably caused by the consumption of a wild boar's ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar's edible parts may result in atypical or even adverse analytical findings in sports drug testing.
Determining the origin of anabolic androgenic steroids (AAS) that also are produced endogenously in the human body is a major issue in doping control. In some cases, the presence of nandrolone and boldenone metabolites might result from endogenous production. The GC-C-IRMS technique (gas chromatography-combustion-isotope ratio mass spectrometry) enables the carbon isotopic ratio (CIR) to be measured to determine the origin of these metabolites. The aim of this study was to use GC-C-IRMS to determine the δ(13) CVPDB values of seized boldenone and nandrolone preparations to decide if the steroids themselves were depleted in (13) C, compared to what is normally seen in endogenously produced steroids. In addition, several testosterone preparations were analyzed. A total of 69 seized preparations were analyzed. The nandrolone preparations showed δ(13) CVPDB values in the range of -31.5 ‰ to -26.7 ‰. The boldenone preparations showed δ(13) CVPDB values in the range of -32.0 ‰ to -27.8 ‰, and for comparison the testosterone preparations showed δ(13) CVPDB values of -31.0 ‰ to -24.2 ‰. The results showed that the values measured in the nandrolone and boldenone preparations were in the same range as those measured in the testosterone preparations. The study also included measurements of CIR of endogenously produced steroids in a Norwegian/Danish reference population. The δ(13) CVPDB values measured for the endogenous steroids in this population were in the range of -21.7 to -26.8. In general, most of the preparations investigated in this study show (13) C-depleted delta values compared to endogenously produced steroids reflecting a northern European diet. Copyright © 2014 John Wiley & Sons, Ltd.