Forensic Toxicol

DOI 10.1007/s11419-016-0325-x

ORIGINAL ARTICLE

Drug-drug interactions and masking effects in sport doping:

influence of miconazole administration on the urinary
concentrations of endogenous anabolic steroids
Amelia Palermo1,2 Francesco Botre`1,3 Xavier de la Torre1 Ilaria Fiacco1

Michele Iannone1,2 Monica Mazzarino1

Received: 4 March 2016 / Accepted: 21 May 2016

Japanese Association of Forensic Toxicology and Springer Japan 2016

Abstract We have investigated the influence of oral
miconazole administration on the urinary concentrations of
endogenous anabolic androgenic steroids of doping relevance,
specifically considering all these compounds routinely moni-
tored in doping control analysis, in the framework of the
steroidal module of the athlete biological passport, and other
steroids, including dehydroepiandrosterone, 5a-dihy-
drotestosterone, and the hydroxylated metabolites recently
proposed as additional markers of the intake of testosterone-
related steroids (16a-hydroxy-androsterone, 16a-hydroxy-
etiocholanolone, 6b-hydroxy-androsterone, 6b-hydroxy-etio-
cholanolone, 7a-hydroxy-dehydroepiandrosterone, and 7b-
hydroxy-dehydroepiandrosterone). Urinary concentrations of
the final metabolic products of the glucocorticoid biosynthetic
pathways (11b-hydroxy-androsterone and 11b-hydroxy-etio-
cholanolone, the formerly used as an endogenous reference
compound for the gas chromatographycombustion-isotope
ratio mass spectrometry confirmation analysis) were also
monitored. Two healthy Caucasian volunteers exhibiting
physiologically high testosterone/epitestosterone ratios and
elevated concentrations of the main target steroids were
selected for the study. Miconazole was administered orally
(500 mg/day) for 1 week. Multiple urine samples were
collected for 1 week before and during the treatment, and
analyzed according to a validated analytical procedure based
on gas chromatographyelectron ionization-mass spectrome-
try in selected ion monitoring mode. Our results indicated that
oral administration of miconazole decreased the urinary con-
centrations of androsterone, and to a lesser extent, of etio-
cholanolone (both detected as the sum of free and glucuronated
steroids), and consequently the androsterone/testosterone and
androsterone/etiocholanolone ratios. Furthermore, the urinary
concentrations of 16a-hydroxy-etiocholanolone, 16a-hy-
droxy-androsterone, 7b-hydroxy-dehydroepiandrosterone,
6b-hydroxy-etiocholanolone, 7a-hydroxy-dehydroepiandros-
terone, 6b-hydroxy-androsterone, 11b-hydroxy-androsterone,
and 11b-hydroxy-etiocholanolone were significantly sup-
pressed. This evidence suggests the potential intake of
miconazole whenever the urinary steroid profile is character-
ized by abnormally low concentrations of the above-men-
tioned steroids.
Keywords Anti-doping analysis
Miconazole

Antifungals
Pseudo-endogenous anabolic androgenic
steroids
Urinary steroid profile
Drug-drug interactions

Introduction
& Francesco Botre`
francesco.botre@uniroma1.it The use of non-prohibited drugs (i.e. compounds not
1
included in the list of prohibited substances and methods,
Laboratorio Antidoping, Federazione Medico Sportiva
Italiana, Largo Giulio Onesti, 1, 00197 Rome, Italy
yearly updated by the World Anti-Doping Agency
2
(WADA) [1]) and nutritional supplements is a relatively
Dipartimento di Chimica e Tecnologia del Farmaco,
common practice among elite athletes. Indeed, based on
Sapienza Universita` di Roma, Piazzale Aldo Moro 5,
00185 Rome, Italy the information reported on doping control forms, 70 %
3 of athletes uses at least one non-banned drug during the
Dipartimento di Medicina Sperimentale, Sapienza
Universita` di Roma, Viale Regina Elena 324, 00161 Rome, 15 days before anti-doping controls and this value rises to
Italy 87 % for females only. Pain relief medicaments are the

123

most commonly declared drugs, followed by medications
for the respiratory tract, glucocorticoids, antidepressants,
antifungals, and antibiotics. Furthermore, very often the
use of non-prohibited drugs is not limited to a single
pharmaceutical preparation, but it involves the co-ad-
ministration of two or more substances. In particular,
30 % of athletes uses three or more medicaments. These
data, gathered from the Italian population [2], are widely
in accordance with those reported for other countries on
the occasion of major international sport events (e.g.,
Summer Olympic Games of Atlanta 1996 and Sydney
2000, FIFA World Cup Japan-Korea 2002 and Germany
2006) [36].
Even if non-banned medicaments are not themselves
eliciting an increase in athletic performance, drug-drug
interactions (DDIs) may occur in the case of the co-ad-
ministration of two or more pharmacologically active
compounds, and in particular in the case of unnoticed
additional intake of doping substances.
DDI assessment is a crucial and routinely performed
step during the pharmaceutical drug development process,
on the basis of significant modifications of pharmacoki-
netic (e.g., absorption, bioavailability, metabolism and
elimination) and pharmacodynamic parameters of co-ad-
ministered substances. In spite of this, DDIs have begun to
be considered in forensic toxicologyincluding anti-dop-
ing scienceonly recently. In particular, from the per-
spective of the anti-doping laboratory, any alteration in the
pharmacokinetics of a prohibited substance may be detri-
mental to the efficacy of the analytical procedure(s) devel-
oped for its detection.
The WADA List International Standard already includes
substances banned precisely for their capacity to activate
DDIs (specifically considered in the class S5, Diuretics
and Masking Agents). The most representative example is
probenecid, an uricosuric agent able to inhibit the renal
tubular transport of performance-enhancing drugs (pri-
marily anabolic androgenic steroids), thus decreasing their
detectability in urine [7]. In addition, the administration of
diuretics (carbonic anhydrase inhibitors, canrenone, fur-
osemide, vaptans, etc.) is also considered a doping offence,
not specifically for their direct effect on the metabolism of
the prohibited drug(s), but rather for their capacity to
increase the urinary flow, with the consequent dilution of
the target compounds [8].
The evaluation of DDI effects in the development of
effective analytical strategies is therefore particularly rel-
evant when dealing with the detection of (1) compounds
undergoing extensive metabolism (e.g. budesonide, and
selective estrogen receptor modulators), (2) threshold
substances (their presence in urine represents an adverse
analytical finding only when the concentration exceeds a
given threshold), and (3) pseudo-endogenous substances,
123
Forensic Toxicol
such as endogenous anabolic androgenic steroids (EAASs)
[911].
Concerning the first group, analytical markers for the
assessment of the intake are usually selected among their
most representative urinary metabolites. Previous in-vitro
studies have shown that DDIs taking place at the level of the
phase I and phase II biotransformation and conjugation
processes can strongly decrease the formation of the selected
target metabolite(s). In particular, it was demonstrated that
the antifungal agents ketoconazole, miconazole, itracona-
zole, and the antidepressant nefazodone, may inhibit the
production of hydroxylated and/or carboxylated metabolites
of toremifene and stanozolol (respectively included in the
classes S4, Hormone and Metabolic Modulators, and S1
Anabolic Agents of the WADA List) [12, 13]. On the
other hand, for the detection of substances that can be (1)
produced also in some physiological conditions (e.g.,
boldenone), or (2) present in the sample as metabolic
products of not forbidden drugs (e.g., morphine from
codeine), threshold values have been set by the WADA [14].
Among them, 19-norandrosterone (19-NA) [quantified in
urine as sum of free and glucuronate form (19-NAG)] is
currently considered the marker of choice for the detection
of doping with 19-norsteroids, i.e. 19-nortestosterone (nan-
drolone) and/or its precursors (19-norandrostenedione and
19-norandrostenediol). Because it has been demonstrated
that 19-NA can be endogenously produced [15], a threshold
of 2 ng/mL (to be normalized for the specific gravity of
urine) has been established. In this regard, the potential
confounding/masking effect of DDIs, based on the in-vitro
alteration of phase II metabolism of 19-nortestosterone by
non-prohibited drugs (e.g., ketoconazole and miconazole),
has also been recently reported [16].
This complicated scenario becomes more intricate when
dealing with the detection of the so-called pseudo-endoge-
nous substances (endogenous compounds administered
exogenously), because the intake of other medicaments may
influence not only their biotransformation, but also their
biosynthetic pathways. In this regard, over the last decades
pseudo-EAASs have constantly been reported by almost all
anti-doping laboratories as being responsible for the greatest
number of adverse analytical findings. Clearly, the most
challenging aspect in their detection is primarily related to
the necessity to discriminate between their physiological
and non-physiological origin. Unfortunately, as stressed in
previous studies [1719], the very high intra- and inter-in-
dividual variability for urinary concentrations of these
steroids calls into questions the applicability of simple
population-based threshold values. For this reason, Donike
et al. [20, 21] proposed the longitudinal studies and the
establishment of subject-based reference ranges for the
parameters of the steroidal profile. Recently, trying to more
comprehensively overcome this issue, the WADA has
Forensic Toxicol
approved an innovative approach that is formalized in the
steroidal module of the athlete biological passport (ABP)
[22, 23]. In detail, the approach is based on the assessment of
the urinary variability, for each individual athlete, of a panel
of selected EAASs [i.e. testosterone (T), epitestosterone (E),
androsterone (Andro), etiocholanolone (Etio), 5a-an-
drostan-3a,17b-diol (5a3a-Adiol), 5b-androstan-3a,17b-
diol (5b3a-Adiol)], and the urinary concentrations (as a sum
of free and glucuronate fractions) have to be measured
together with the values of the specific gravity in all urine
samples. Collected data belonging to the same athlete are
then integrated in a Bayesian algorithm that allows for the
determination of physiological ranges tailored for each
individual, drastically reducing the risk of false atypical
analytical findings and improving the overall detection
capacity of the anti-doping system [24]. In addition to this
approach, several researchers have proposed to consider
also other specific hydroxylated/oxydized metabolites of
EAASs [2528], with the aim of both obtaining supple-
mentary information about the compound administered and
extending its detection window in urine. In particular,
physiological urinary concentrations of most of these EAAS
metabolites (also formed in biotransformations involving
cytochrome P450 enzymes, including CYP3A4 and
CYP2C9 [29]) are normally low [30]. In this regard, it has
been reported that after the intake of either dehy-
droepiandrosterone (DHEA), T and/or its precursors, it is
possible to observe a characteristic increase in the urinary
levels of 7a/b-hydroxy(OH)-DHEA, 16a/b-OH-DHEA,
7-keto-DHEA, 3a,5-cyclo-5a-androstan-6b-ol-17-one,
7-keto-Andro, 6a-OH-androstenedione (ASD), 6b-OH-
Andro, 6b-OH-Etio, 6b-OH-epiandrosterone(EA), 16a-
OH-Andro, 16a-OH-Etio, 16a-OH-ASD, 4-OH-ASD,
4-OH-T, and 6-OH-T [2528].
Despite these efforts to improve the discriminating
power of the threshold values and to incorporate as many
markers as possible for EAAS administration, a number of
exogenous variability factors can occur in a very unpre-
dictable manner and may lead to atypical passport findings
[8, 31]. Among these, several confounding factors are
already considered in a specific WADA Technical Docu-
ment [32]. According to it, the intake of the non-banned
steroidal biosynthesis modulators ketoconazole and finas-
teride, as well as of ethanol, should be monitored.
Finasteride, together with other 5a-reductase inhibitors,
leads to a substantial reduction of 5a-dihydrotestosterone
(5a-DHT), the active metabolite of T [33], and alters the
normal formation of main metabolites of nandrolone (i.e.
19-NA and 19-noretiocholanolone). Ketoconazole is a
widely used antifungal agent. Together with other azolic
antifungals, it acts as an inhibitor of the fungal CYP450
enzyme 14a-lanosterol demethylase (CYP51). Due to a lack
of selectivity, these compounds have demonstrated various
extents of interaction with CYPs involved in steroidogen-
esis and in hepatic metabolism [3436]. In particular,
Touchette et al. [37] reported the increase of serum testos-
terone levels after administration of the triazole antifungal
agent fluconazole; while ketoconazole is a known inhibitor
of T biosynthesis and its administration was proposed as
supportive dynamic test in the detection of doping with
EAASs, because in the absence of any exogenous intake of
T and/or its precursors, it causes a straight/characteristic
decrease in urinary levels of T metabolites [e.g. T glu-
curonide (G), AndroG, EtioG] and of the T/E ratios [38, 39].
This effect was ascribed to the inhibition of the 17a-hy-
droxylase and 17, 20-lyase activities of CYPC17 (conver-
sion of pregnenolone to DHEA and progesterone to ASD)
[40], but recent studies have also shown its inhibitory
potential on steroid glucuronidation in vitro [13, 16]. Fig-
ure 1 schematizes androgen biosynthetic and excretion
metabolic pathways and underlines the sites of metabolic
modulations by ketoconazole and finasteride [41].
In the previous studies we reported that, similarly to
ketoconazole, the azolic antifungal miconazole is also able
to inhibit UDP-glucuronosyltransferase and CYP enzymes
during in vitro steroid metabolism [13, 16], thus stressing
the need to consider this kind of DDI in the evaluation of
the analytical results in anti-doping analysis. Furthermore,
other investigators reported the strong inhibitory effect of
miconazole on CYP2C9 and 2C19 as well as on hepatic
CYP3A4, 1A2, and 2D6 in vitro [42, 43].
Based on the above, we have here preliminarily evalu-
ated the in vivo effects of oral administration of micona-
zole on the markers of the athlete urinary steroid profile.
Two male healthy subjects exhibiting a physiologically
high Caucasian steroidal profile were selected. To obtain a
wider overview on metabolism modifications, urinary
concentrations of DHEA, 5a-DHT, and some of the minor
hydroxylated metabolic products (16a-OH-Andro, 16a-
OH-Etio, 6b-OH-Andro, 6b-OH-Etio, 7a-OH-DHEA, 7b-
OH-DHEA) recently proposed as additional markers for
the steroidal profile of the athlete, were also evaluated,
together with the final metabolites of the glucocorticoid
biosynthetic pathways (11b-OH-Andro and 11b-OH-Etio,
the former being considered as an endogenous reference
compound in the gas chromatographiccombustion-isotope
ratio mass spectrometric (GCC-IRMS) confirmation
analysis of EAAS administration [44]).
Materials and methods
Chemicals and reagents
Nizacol (miconazole, 500 mg tablets) was obtained from
New Research S.r.l. (Aprilia, Italy); T, E, Andro, 17a-
123

Fig. 1 Human androgen biosynthetic and excretion metabolic pathways. Indicators show the sites of metabolic modulation for finasteride and
ketoconazole. UGT UDP-glucuronosyltransferases, SULT sulfotransferase
methyltestosterone (used as internal standard for all the target
analytes, except for T, E, 5a3a-Adiol, and 5b3a-Adiol),
DHEA, 5a-DHT, 16a-OH-Andro, and 16a-OH-Etio from
Sigma-Aldrich (Milano, Italy); Etio, 5a3a-Adiol, 5b3a-
Adiol, 6b-OH-Andro, 6b-OH-Etio, 11b-OH-Andro, 11b-
OH-Etio, 7a-OH-DHEA, and 7b-OH-DHEA from Steraloids
(Newport, RI, USA); deuterated standards (T-d3, E-d3, 5a3a-
Adiol-d3, 5b3a-Adiol-d5, AndroG-d4 (used to monitor the
yield of the enzymatic hydrolysis reaction) from the National
Measurement Institute (NIM, Pymble, Australia).
The preparation of b-glucuronidase from Escherichia
coli was from Roche Diagnostic (Mannheim, Germany).
The derivatizing agent was a mixture of N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA)/ammonium
iodide (NH4I)/dithioerythritol (DTE) (1000:2:4; v/w/w)
stored in screw-cap vials at 4 C for a maximum of 2
weeks. MSTFA was supplied by Chemische Fabrik Karl
Bucher GmbH (Waldstetten, Germany); NH4I and DTE by
Sigma-Aldrich; solvents (tert-butylmethyl ether, ethyl
acetate, methanol) and reagents (potassium carbonate,
sodium phosphate, sodium hydrogen phosphate, sodium
hydrogen carbonate) of analytical or high-performance
liquid chromatography (HPLC) grade by Sigma-Aldrich.
Water was obtained from a MilliQ water purification sys-
tem (Millipore S.p.A., Milano, Italy).
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Forensic Toxicol
Excretion/administration study
The controlled administration study has obtained the
approval of the local ethical committee. Each subject has
been informed on risks and has signed a written informed
consent allowing the use of urine samples for research
purposes. Volunteers were pre-selected subjects with
physiologically high T/E ratios and elevated concentrations
of the main target steroids. The subjects were medically
examined to ensure the absence of diseases and were asked
about the use of drugs, alcohol, and dietary habits. Only
subjects confirmed as not taking other drugs and with a null
to moderate alcohol consumption were included in the
study. Urine samples were finally obtained from two
healthy males, 24 and 45 years, 72 and 83 kg, respectively,
both with normal body mass index. Samples were anon-
ymized and collected under sterile conditions. To obtain a
good representation of the physiological levels of andro-
gens and other metabolites considered in the study, sample
collection was performed for 1 week before starting the
controlled administration study. Subjects were followed for
a period of 2 weeks (including the pre-administration
period). Both subjects took miconazole orally, in a single
daily dose of 500 mg. Urine samples were collected daily
at multiple time points (from five to nine) for 1 week before
Forensic Toxicol
and during treatment. In particular, multiple samples were
collected at different times of the day to contextualize
variations in regard to physiological circadian fluctuations
of selected steroids. The scheme shown in Fig. 2 was fol-
lowed for the in vivo study. All urine samples were stored
at -20 C until analysis.
Gas chromatographymass spectrometry conditions
for steroid analysis
The analysis of target steroids was carried out according
to a previously published method [45], already applied to
assess the influence on the steroid profile of other xeno-
biotics, like anti-estrogenic compounds [46]. Briefly,
quantitative analysis was performed on an Agilent
6890/5973A gas chromatographymass spectrometry
(GCMS) system (Agilent Technologies, Milano, Italy),
in electron ionisation (70 eV) mode, using an HP-1 17-m
fused silica capillary column (cross-linked methyl sili-
cone, i.d. 0.20 mm, film thickness 0.11 lm; Agilent
Technologies). The GC conditions were as follows: the
carrier gas, helium at a flow rate of 1 mL/min; split ratio
of 1:10; the temperature program, 180 C (4.5 min-hold),
3 C/min to 230 C, 20 C/min to 290 C, and 30 C/min
to 320 C; the transfer line and injector temperature,
280 C; the acquisition, the selected ion monitoring
mode. Identification was based on the specific retention
times and on the diagnostic ions listed in Table 1 (frag-
ments for quantification shown in boldface). Quantitative
determination of the urinary concentration was based on
the peak area ratio of the analyte to the corresponding
internal standard (i.e., the corresponding deuterated
compounds, where available, or 17a-methyltestosterone
for all the other target analytes).
Calibration samples were prepared in synthetic urine,
obtained as previously described [47]. The final concen-
trations of target compounds in calibration samples and of
internal standards in all samples are shown in Table 1.
Calibration samples were re-analyzed every 15 injections
along the sequence and used also as quality controls. For
the detection and quantification of steroids currently not
present in the analytical method, they were validated in the
anti-doping laboratory of Rome for the determination of
Fig. 2 In vivo study schematization: samples were collected at multiple time points for 7 days before administration and for 7 days during oral
miconazole administration (single daily dose of 500 mg)
EAASs; derivatized standards were injected in the GCMS
system and analyzed in MS scan mode to obtain retention
times and to select appropriate diagnostic ions. The lin-
earity range was investigated by injection of each steroid at
different concentrations (5, 50, 100, 200, 2000 ng/mL), to
cover the entire range of the expected concentrations.
Different urine samples spiked with increasing steroid
concentrations were also evaluated. For all analytes the
limit of quantification (LOQ) was B5 ng/mL, (calculated
as ten times the standard deviation of the background
noise).
Sample preparation
Sample preparation was performed following the validated
protocol for the detection of EAASs in human urine [45].
Samples collected under sterile conditions were stored at
-20 C until analysis. After thawing out at room temper-
ature, to perform enzymatic hydrolysis of the glu-
curonidated steroids, 1 mL of phosphate buffer (1 M, pH
7.4), 50 lL of the internal standard (see Table 1 for final
concentrations in urine), and 50 lL of b-glucuronidase
from E. coli were added to 2 mL of urine. Samples were
incubated for 1 h at 55 C. After this period, 1 mL of
carbonate/bicarbonate buffer (0.8 M, pH 9) was added and
liquid/liquid extraction was carried out with 7 mL of tert-
butyl methyl ether for 5 min on a mechanical shaker.
Samples were centrifuged and the organic layer was
transferred to a 10 mL tube and evaporated to dryness
under nitrogen stream at 70 C. The residue was recon-
stituted in 50 lL of the derivatizing mixture, and the
sample was maintained at 70 C for 20 min. Then, a 1-lL
aliquot of it was injected into the GCMS system.
Data analysis
For peak integration and analyte quantification, a propri-
etary software of the GCMS system was used. Peak
integration was, however, always double-checked manu-
ally. Specific gravity of urine samples was measured to
normalize the urinary concentrations according to the
WADA Technical Document [30]. All samples with
specific gravity below 1.005 were excluded. All samples
123
 Forensic Toxicol

Table 1 Diagnostic ions for

Target analyte Diagnostic ions (m/z) Final concentration (ng/mL)
trimethylsilyl derivative of all
steroids and internal standards Testosterone 432, 417, 301 40
considered in this study
Epitestosterone 432, 417, 301 10
5a-Androstan-3a,17b-diol 346, 256, 241 100
5b-Androstan-3a,17b-diol 346, 256, 241 100
Androsterone 434, 419, 329 2000
Etiocholanolone 434, 419, 329 2000
16a-Hydroxy-etiocholanolone 522, 507, 417 200
16a-Hydroxy-androsterone 522, 507, 417 200
Dehydroepiandrosterone 432, 327, 237 100
7b-Hydroxy-dehydroepiandrosterone 520, 430, 415 100
7a-Hydroxy-dehydroepiandrosterone 430, 415, 325 100
6b-Hydroxy-androsterone 522, 507, 417 50
6b-Hydroxy-etiocholanolone 522, 507, 417 50
11b-Hydroxy-androsterone 522, 507, 417 240
11b-Hydroxy-etiocholanolone 522, 507, 417 240
5a-Dihydrotestosterone 434, 419, 405 25
Internal standards
17a-Methyltestosterone 446, 431, 301 250
Testosterone-d3 435, 420, 301 100
Epitestosterone-d3 435, 420, 301 25
5a-Androstan-3a,17b-diol-d3 429, 259, 244 50
5b-Androstan-3a,17b-diol-d5 431, 261, 246 50
Diagnostic ions used for quantitation are shown in boldface. The final concentrations of the target com-
pounds in the calibration samples and of internal standards in all samples are shown in column 3

were prepared at least in duplicate and individual values
expressed as average. Data analysis was performed using R
version 3.0.0 (The R Foundation for Statistical Computing,
Vienna, Austria). Data obtained for each subject were
initially explored with the unsupervised multivariate prin-
cipal component analysis (PCA). Because the difference in
the variance of urinary metabolite concentrations appeared
substantial, the inverse of the variance for each metabolite
was used as a weighting factor. The percentage of variation
explained by the method was evaluated for the first seven
principal components (PCs) obtained. The first two PCs
together explained 80 % of the total variance. Score values
were plotted for PC1 and PC2. Metabolites most respon-
sible for group clustering were individuated through com-
parison of score and loading plots.
For each metabolite considered in the study, data dis-
tribution and statistics were assessed. Results were
expressed in terms of median, first, and third quartiles, and
extreme values. In this regard, boxplots gave good repre-
sentation of obtained data. The significance of the variation
between the control and treatment urinary concentration
groups was evaluated using a non-parametric non-paired
MannWhitney test or Students t-test, depending on data
distribution. The p values less than 0.05 were considered
statistically significant.
Results and discussion
In this study we have investigated the effects of oral
administration of miconazole, an antifungal agent report-
edly used by athletes and not included in the WADA
prohibited list, on the urinary concentrations of markers of
the steroidal module of the ABP. In addition, we also
evaluated the influence on the excretion of DHEA, 5a-
DHT, and the hydroxylated steroids (16a-OH-Andro, 16a-
OH-Etio, 6b-OH-Andro, 6b-OH-Etio, 7a-OH-DHEA, 7b-
OH-DHEA) recently proposed as additional markers for
the intake of EAASs, as well as on the final metabolic
products of the biosynthetic pathways of glucocorticoids.
Results for physiological urinary concentrations of
investigated steroids before miconazole administration,
expressed as median, maximum, first and third inter-quar-
tile values, are shown in Table 2. Taking into account the
reference ranges described in the study by Van Renterghem
et al. [30], both volunteers exhibited a typical physiologi-
cally high Caucasian steroidal profile at median T/E values
of 4.8 and 5.7, respectively. According to the WADA
Technical Document that is currently enforced [32], pro-
files with T/E values higher than 4 are considered atypical.
Therefore, after the initial testing procedure, these profiles
would had been submitted to a suspicious steroid profile

123
Forensic Toxicol

Table 2 Urinary concentrations of all target steroids for volunteers 1 and 2, measured before the first administration of miconazole
Volunteer 1 Volunteer 2
Median IQ1 IQ3 Max Min Median IQ1 IQ3 Max Min
(ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)

Compound
Testosterone 55.1 44.2 72.8 93.6 31.6 140 118 182 261 68.3
Epitestosterone 12.4 9.7 14.2 25.7 6.9 26.9 20.1 34.4 50.1 10.3
5a-Androstan-3a,17b-diol 79.5 68.5 94.3 261 42.5 134 104 178 259 51.1
5b-Androstan-3a,17b-diol 352 281 426 676 151 228 163 293 465 97.7
16a-Hydroxy-etiocholanolone 51.4 41.2 59.7 77.6 27.1 81.9 59.8 102 140 29.7
16a-Hydroxy-androsterone 95.0 77.4 114 160 46.2 108 78.7 128 176 37.9
7b-Hydroxy- 38.6 23.9 51.2 81.6 13.1 16.7 12.2 23.4 55.4 \LOQ
dehydroepiandrosterone
7a-Hydroxy- 6.5 5.0 9.1 16.8 \LOD 6.5 5.4 9.4 16.2 \LOQ
dehydroepiandrosterone
Dehydroepiandrosterone 63.7 46.3 79.9 131 25.6 77.0 51.6 99.1 185 32.5
6b-Hydroxy-androsterone \LOQ \LOQ \LOQ 5.7 \LOQ \LOQ \LOQ \LOQ 11.4 \LOQ
6b-Hydroxy-etiocholanolone 50.5 38.1 59.4 92.1 18.3 11.3 8.5 11.3 22.5 \LOQ
Dihydrotestosterone \LOQ \LOQ 6.8 10.7 \LOQ 9.3 6.3 11.9 18.7 \LOQ
11b-Hydroxy-androsterone 970 677 1290 1990 310 477 308 607 998 92.5
11b-Hydroxy-etiocholanolone 451 325 580 1080 130 296 198 365 579 108
Etiocholanolone 2340 1970 3070 4560 1260 3930 2830 4960 7810 1290
Androsterone 2560 1950 3040 3900 1400 4920 3820 6360 8210 1670
Ratio
T/E 4.8 4.1 5.5 13.3 3.1 5.7 5.1 6.4 8.5 3.5
Andro/T 44.1 37.1 50.8 69.1 11.6 33.1 27.6 37.9 54.4 23.1
Andro/Etio 1.0 0.9 1.0 1.3 0.8 1.3 1.2 1.4 1.6 1.0
5a3a/5b3a-Adiol 0.23 0.21 0.26 0.47 0.16 0.6 0.6 0.7 0.8 0.5
5a3a-Adiol/E 6.4 5.9 6.4 28.5 4.2 4.9 4.8 5.5 7.0 4.4
The population of samples considered was composed of 35 and 45 urine samples for volunteers 1 and 2, respectively. Ranges are expressed as
median, maximum, minimum, first, third inter-quartile
IQ1 first inter-quartile value, IQ3 third inter-quartile value, Max maximal value, Min minimal value, T/E testosterone/epitestosterone ratio,
Andro/T androsterone/testosterone ratio, Andro/Etio androsterone/etiocholanolone ratio, 5a3a/5b3a-Adiol 5a-androstan-3a,17b-diol/5b-an-
drostan-3a,17b-diol ratio, 5a3a-Adiol/E 5a-androstan-3a,17b-diol/epitestosterone ratio, LOQ limit of quantification

confirmation procedure and GCC-IRMS confirmation
analysis (WADA TD2016IRMS) [44].
Data obtained for all the steroids included in the study
before and after oral miconazole administration, at the dose
of 500 mg/day, were preliminarily explored with PCA to
obtain a global overview on clustering. The first two PCs
captured 80 % of the total variability. PC1 and PC2 scores
plotted for volunteer 1 (see Fig. 3) clearly show a separa-
tion between control and treatment groups. Only a few
values/points after treatment were distributed along the
border line. These observations represent samples collected
during the first and last days of treatment, this being con-
sistent with what is expected, considering a lag time for the
establishment of miconazole inhibitory activity and adap-
tive mechanisms arising after prolonged/repeated
administrations of the drug. Further examination of loading
plots of PC1 and PC2 suggested a primary role of Andro
and hydroxylated metabolites of Andro and Etio, as well as
hydroxylated DHEA metabolites, in group separation. PCA
score and loading plots for volunteer 2 supported these
findings (data not shown).
The significance of the variation was assessed for each
dataset, comparing normalized concentrations of the
steroids (detected as the sum of free and glucuronate
conjugate) before (control) and after the treatment. The
values of the ratios that are generally considered in the
evaluation of the steroidal profile (T/E, Andro/Etio,
Andro/T, 5a3a-Adiol/5b3a-Adiol, 5a3a-Adiol/E) were
also evaluated. For volunteer 1, the following considera-
tions can be made:

123
 Forensic Toxicol

Fig. 3 Principal component

(PC)1 and PC2 score plots for
volunteer 1 showing separation
between controls (asterisk) and
samples collected after
treatment (asterisk T). The PC
analysis was performed
considering all steroids included
in the study. Each asterisk
represents one urine sample
collected. The first two PCs
captured 80 % of total
variability

1. Among the markers of the steroidal module of the pathways, 11b-OH-Andro and 11b-OH-Etio, were
ABP, statistically significant variation (p \ 0.05) was diminished by 89 and 76 %, respectively (Fig. 4a).
found for the total urinary concentrations of Andro
(median urinary concentration in samples collected
All these observations were confirmed from data
during 1 week of treatment decreased by 50 % of the
obtained for volunteer 2, with an exception made for a
physiological median value) and, to a lesser extent, for
more pronounced decrease of the concentration of Etio
Etio (variation \20 %). Consequently, these modifica-
(\40 % instead of \20 %) and, consequently, a slighter
tions were also reflected in Andro/Etio and Andro/
variation of the Andro/Etio ratio (see Fig. 4b).
T ratios (see Fig. 4a).
These data are consistent with the in vitro evidences
2. Considering specifically the hydroxylated metabolites
reported by previous investigators [40, 48, 49] and with our
recently identified and proposed as additional biomark-
in vivo study about the effect of ketoconazole on the
ers for EAAS administration, after miconazole intake
excretion of methandienone, a synthetic anabolic steroid
the urinary concentrations of 16a-OH-Etio and 16a-
[50]. In particular, miconazole is suspected to exert inhi-
OH-Andro decreased by 42 and 48 %, respectively.
bitory activity on CYP450-dependent enzymes involved in
Also, 7b-OH-DHEA and 6b-OH-Etio median values
steroid metabolic pathways (decrease in hydroxylated
were found to be reduced by 74 and 60 %,
steroid formation) and on phase II metabolic conjugation
respectively.
with glucuronic acid. According to what in vitro demon-
3. Similar inhibitory effects were observed for the minor
strated for other steroids [13, 16], miconazole administra-
metabolic products 7a-OH-DHEA and 6b-OH-Andro.
tion could be responsible for a decrease in Andro and Etio
Indeed, in physiological condition, these metabolites
glucuronidation that reflects in the reduction of concen-
were detected at concentrations below 10 ng/mL, close
trations of these steroids in human urine.
to the LOQ of the analytical method (5 ng/mL) (see
Similarly to ketoconazole, used in the treatment of
Table 2). During 1 week of treatment with the
Cushing syndrome for its inhibitory activity on adrenal
antifungal agent under investigation, urinary concen-
CYP450 enzymes [49], miconazole also appears to have
trations of these compounds decreased to below the
inhibitory activity at the adrenal level, because after its
LOQ [values in the boxplots reported equal to the limit
administration 11b-OH-Andro and 11b-OH-Etio formation
of detection (LOD)] and in some instances became
is strongly suppressed (Fig. 4).
undetectable (peak area below the LOD of the method,
Ketoconazole is a known inhibitor of T biosynthesis due
values in the boxplots reported as zero) (Fig. 4a).
to the inhibition of the 17a-hydroxylase and 17, 20-lyase
4. The urinary median concentrations of the final hydrox-
activities of CYP17 [37]. From our data, urinary levels for
ylated products of the glucocorticoid biosynthetic

123
Forensic Toxicol
Fig. 4 Boxplots showing significantly decreased urinary steroidal
concentrations after oral miconazole administration, 500 mg/day, for
volunteer 1 (a) and volunteer 2 (b). Variations in androsterone/
etiocholanolone (Andro/Etio) and androsterone/testosterone (Andro/
T) ratios are also shown. The statistically significant decrease (p \
0.05) was observed for all pairs of the boxplots shown in Fig. 4a, b
except for the only one pair of 6b-hydroxy-etiocholanolone in the
volunteer 2 (p [ 0.05) (Fig. 4b, the last panel). Concentrations are
T and DHEA were not significantly decreased after the
treatment, indicating no inhibitory activity of miconazole
on CYP17 (data not shown).
shown as the sum of free and glucurono-conjugate steroids, normal-
ized for the specific gravity of each sample. For volunteer 1, the
numbers of urine samples considered in the control and treatment
groups were 43 and 41, respectively; 45 and 35 for volunteer 2.
Andro/Etio androsterone/etiocholanolone, Andro/T androsterone/
testosterone, 7b-OH DHEA 7b-hydroxy-dehydroepiandrosterone.
The values \limit of quantification were plotted at the the limit of
detection (LOD) and values \LOD are plotted as 0
Based on the above, the influence of miconazole
administration on the analytical strategy currently adopted
for the detection of doping with pseudo-endogenous AASs
123
 Forensic Toxicol

Fig. 4 continued

should be carefully taken into account and miconazole
intake should be considered in all cases of atypical ster-
oidal profiles consistent with the findings reported here.
Furthermore, because the concentration of hydroxylated
metabolites was strongly suppressed after the treatment
(Fig. 4), it is of crucial importance to consider this aspect
in any future implementation of the steroidal module of
the ABP with the inclusion of new markers. we believe
that a preliminary, but thoughtful evaluation of their
metabolic stability, also considering the potential intake of
non-banned drugs, should always be undertaken.
Conclusions
We have highlighted that the analytical strategy currently
followed for the detection of the administration of prohib-
ited pseudo-endogenous steroids can be significantly

123
Forensic Toxicol
affected by the presence of miconazole. In particular, our
results also suggest that a careful evaluation of potential
DDIs should be preliminarily considered in the selection of
the most appropriate analytical markers to detect the intake
of prohibited substances in sport doping. More generally,
analytical methods applied in forensic toxicology are usu-
ally developed taking into account the physico-chemical
properties of the analytes, their pharmacokinetic parame-
ters, and the type of sample matrix available. In this context,
the choice of analytical markers is based on data obtained
from in vitro and/or in vivo studies involving healthy vol-
unteers in controlled conditions. Unfortunately, this
approach refers to an ideal situation in which the subject is
using only one particular doping substance/method and
does not consider the possibility of co-administrations, in
particular with other non-banned drugs. In the clinical
practice for the diagnosis of diseases, DDI effects are rou-
tinely considered. In forensic analysis, and in particular in
the anti-doping control field, this aspect has started to be
discussed only recently. Based on the evidences reported
here, we conclude that the inclusion of a comprehensive
evaluation step for potential DDI effects on currently
adopted/newly developed analytical strategies is of high
relevance to a correct interpretation of the analytical data.
Acknowledgments This project has been supported in part by a
research grant of the World Anti-Doping Agency (Project Code:
13D14MM).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
Ethical approval All procedures performed in studies involving
human participants were in accordance with the ethical standards of
the institutional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards. The administration studies were approved by the local
ethical committee (Approval Code: Prot. 1055/2014 CE Lazio 1).
Informed consent was obtained from all individual participants
included in the study.
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