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Toxicology Mechanisms and Methods
ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: http://www.tandfonline.com/loi/itxm20
Pretreatment of different biological matrices for
exogenous testosterone analysis: a review
Edna Carolina Pizzato, Marcelo Filonzi, Hemerson Silva da Rosa & André
Valle de Bairros
To cite this article: Edna Carolina Pizzato, Marcelo Filonzi, Hemerson Silva da Rosa & André
Valle de Bairros (2017): Pretreatment of different biological matrices for exogenous testosterone
analysis: a review, Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2017.1351015
To link to this article: http://dx.doi.org/10.1080/15376516.2017.1351015
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REVIEW ARTICLE
Pretreatment of different biological matrices for exogenous testosterone
analysis: a review
Edna Carolina Pizzato
a
, Marcelo Filonzi
b,c
, Hemerson Silva da Rosa
d
and Andr
e Valle de Bairros
d,e
a
Setor de Hematologia, Hospital Nossa Senhora da Conceic¸~
ao, Porto Alegre, Brazil;
b
Setor de Qu
ımica Especial, Hospital Israelita Albert
Einstein, S~
ao Paulo, Brazil;
c
Faculdade de Ci^
encias Farmac^
euticas, Universidade de S~
ao Paulo, S~
ao Paulo, Brazil;
d
Laborat
orio de
Desenvolvimento e Controle de Qualidade, Universidade Federal do Pampa, Uruguaiana, Brazil;
e
N
ucleo Aplicado a Toxicologia,
Departamento de An
alises Cl
ınicas e Toxicol
ogicas, Universidade Federal de Santa Maria, Santa Maria, Brazil
ABSTRACT
The presence of exogenous testosterone has been monitored mainly in the urine and blood. However,
other biological matrices such as hair, nail, and saliva samples can be used successfully for in vivo
measurement. Chromatographic analysis requires pretreatment to obtain free testosterone and its
metabolites. Among the pretreatment procedures, digestion, hydrolysis and solvolysis steps are con-
ducted to reach the analytical purpose. Digestion assay is indicated for hair and nail samples. First, it is
recommended to perform the decontamination step. After that, alkaline solution (NaOH), organic sol-
vents and other reagents can be added to the samples and incubated under determined conditions
for the digestion step. Hydrolysis assay is recommended to urine and blood samples. Acid hydrolysis
cleaves conjugated testosterone and its metabolites using HCl or H
2
SO
4
solution at appropriate time
and temperature. However, there is formation of interferent compounds, degradation of dehydroepian-
drosterone and decrease of peak resolution for epitestosterone. Enzymatic hydrolysis is an alternative
technique able to promote free testosterone and its metabolites with low degradation. It is important
to establish the best conditions according to the biological fluid and the amount of the sample.
Sulfatase enzyme is recommended together with b-glucuronidase to cleave sulfoconjugate steroids.
Solvolysis assay is similar to acid hydrolysis, but organic solvents are responsible to promote steroid
deconjugation. Other approaches such as combination of different pretreatments, surface response or
ultrasonic energy have been used to obtain the total of free steroids. So, the biological matrix defines
the best procedure for pretreatment to achieve the analytical purpose, knowing its advantages and
limitations.
ARTICLE HISTORY
Received 31 January 2017
Revised 29 June 2017
Accepted 2 July 2017
KEYWORDS
Testosterone; analysis;
biological samples;
pretreatment;
deconjugation
Introduction
Testosterone is a steroid hormone derived from cholesterol
that is composed of 17 carbon atoms (C-17) and made up of
three 6-membered rings and one 5-membered ring forming
the steroid skeleton (Gr€
oschl et al. 2001; Martinez-Brito et al.
2013; Rosemary 2014). This hormone is responsible for male
characteristics, synthesized by the Leydig cells in the testis;
however, it can be produced by the adrenal cortex in both
genders. Some studies have proved that testosterone dose-
dependently increases muscle mass, maximal voluntary
strength and power. These improvements are correlated with
circulating testosterone concentrations (Oftebro et al. 1994;
Geyer et al. 2014). To obtain these biological effects, syn-
thetic molecules from testosterone, known as exogenous
anabolic androgenic steroids, were synthesized over the past
decades, and its misuse frequently is associated with athletes
(Geyer et al. 2014; Rosemary 2014). However, exogenous
anabolic androgenic steroids are considered as xenobiotic
substances for the human body.
Qualitative presence of exogenous anabolic androgenic
steroids in biological matrices indicates the use of these sub-
stances, whereas quantification of these steroids, mainly tes-
tosterone, is required because of the natural presence of
these molecules in the human body (Thevis et al. 2013;
Geyer et al. 2014; Rosemary 2014). Thus, the introduction of
exogenous testosterone in medical and sport fields revealed
the necessity to develop analytical methods that can detect
them in body fluids.
Since 1960s, techniques have been developed to detect
levels of testosterone and its metabolites in biological sam-
ples. Testosterone levels are measured in blood, specifically
in serum or plasma, which is widely obtained in clinical diag-
nostics for eventual pathologies and/or hormonal treatment.
Urine is one of the most important biological fluids because
it is noninvasive and detects high concentrations of metabo-
lites, useful for doping analysis. However, alternative bio-
logical matrices have been investigated for confirmed
exogenous testosterone administration (Thieme et al. 2000;
Deshmukh et al. 2010; Thieme et al. 2013; WADA 2016).
CONTACT Andr
e Valle de Bairros andrebairros@yahoo.com.br Departamento de An
alises Cl
ınicas e Toxicol
ogicas, Universidade Federal de Santa Maria,
Av. Roraima n1000, Cidade Universit
aria, Bairro Camobi, CEP 97105-900, Santa Maria, RS, Brazil
ß2017 Informa UK Limited, trading as Taylor & Francis Group
TOXICOLOGY MECHANISMS AND METHODS, 2017
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Immunoassays and chromatographic methods are the
best platforms to determinate testosterone and its metabo-
lites in biological matrices. For immunological tests, a com-
plex sample preparation is not necessary to measure this
hormone. However, cross-reaction, endogenous interferences
from biological matrix, limit of detection, accuracy and preci-
sion are limitations for this technique (Campos et al. 2005;
Vieira et al. 2008; Herati et al. 2016). For chromatographic
analysis, mainly gas chromatography, a pretreatment is
required to promote access to the analytes and/or to obtain
free testosterone and its metabolites in the respective sample
(Campos et al. 2005; Vieira et al. 2008; Gomes et al. 2009;
Kotronoulas et al. 2015; Casilli et al. 2016; Pedersen et al.
2017).
In this sense, the objective of this review is to discuss pre-
treatment procedures for exogenous testosterone administra-
tion in different biological matrices for in vivo determination.
Metabolism of exogenous testosterone
administration
Testosterone has a rapid absorption and binds approximately
98% to plasma proteins. However, oral bioavailability is low
(only 2% reaches the muscle). Oil solutions of testosterone
administered in parenteral form has low efficacy because of
fast biotransformation and excretion (Rosemary 2014). In
injection form (no-esterified), the free hormone has 10 min of
half-life, whereas testosterone esters have a longer half-life.
Testosterone esters are absorbed more slowly when adminis-
tered both intramuscularly (oily vehicle) and orally (testoster-
one undecanoate), because they are less polar than free
steroid. Consequently, these molecules show a higher half-
life when compared to no-esterified testosterone (de Boer
et al. 1991; Peng et al. 2000).
Nevertheless, oral administration of testosterone esters
such as testosterone undecanoate shows low bioavailability
because of first-pass metabolism (Walker et al. 1979). In
1990s, transdermal patches applied to the testis became the
first testosterone topical preparation. However, the product
did not have a popular acceptance owing to its application
area and cases of dermatologic reaction, so other nontesticu-
lar method was tried. Thus, gel formulation (2.5% Testop
V
R
)
emerged first in the USA and then in other countries (Walker
et al. 1979).
Owing to its hydrophobic character, steroid hormones are
bound a great extent to carrier proteins (sex hormone-bind-
ing globulin, b-globulin and albumin), and a small fraction
circulates free or unbound to target tissues (Kicman 2010;
Kuurane 2010; Rosemary 2014). The lipophilic character of
hormones enables them to dissociate spontaneously from
the carriers and enter into the target cell by passive diffusion.
Inside the cell, hormones bind to intracellular receptors and
induce structural changes and redesign to active hormone-
receptor complexes (Lundberg 1979; Chen and Farese 1999;
Beato and Klug 2000; Hakansson et al. 2012).
Testosterone is metabolized primarily in the liver where
hydroxylation and reduction (Phase I) occurs followed by
conjugation with glucuronide or sulfate (Phase II). In Phase I
reaction, testosterone is converted to dihydrotestosterone,
the main active metabolite, by 5a-redutase enzyme in the
liver and muscles. This metabolite has a high affinity for the
androgen receptor, and it becomes faster as a hormone-
receptor complex and dissociates more slowly than the tes-
tosterone receptor. Therefore, testosterone can be inactivated
in the liver by oxidation to androstenedione or its 5b-reduc-
tion to dihydrotestosterone from which inactive products are
formed as demonstrated in Figure 1 (Gr€
oschl et al. 2001;
Bresson et al. 2006; Kicman 2010; Kuurane 2010; Thieme
et al. 2013; Forsdahl et al. 2015).
Regarding Phase II reaction, glucuronide and sulfate
metabolites occurring in steroids are available in the
b-hydroxyl group of carbon 17 from perhydrocyclopenta[a]-
phenanthrene ring, common to the majority of its metabo-
lites and precursors. Phase II reaction also occurs in
epitestosterone (an epimer of testosterone) and testosterone
(Figure 2). These conjugates are less toxic, more polar and
hydrophilic, factors that facilitate urinary elimination (Perry
et al. 1997; Vermeulen et al. 1999; Thieme et al. 2000).
Biological matrix for determination of exogenous
testosterone
Blood
In the human blood circulation, testosterone hormone is
available in the serum. There are two different forms: bound
testosterone (nonspecifically bound to albumin and specific-
ally bound to sex hormone-binding globulin), or free testos-
terone in the serum (Vermeulen et al. 1999). Therefore,
conjugated steroids are present in the circulation after
exogenous testosterone administration (Di Luigi et al. 2009).
Reference values for free, bioavailable and total testosterone
are dependent on gender and age. Serum or plasma testos-
terone determination can be used for clinical diagnostics for
pathology and/or monitoring steroid control in hormonal
treatment (Manni et al. 1985; Morley and Perry 2000).
Although serum and plasma samples are important bio-
logical fluids for quantification of testosterone and its metab-
olites, serum is the preferred sample because the
anticoagulant reagents can interfere in the analysis, providing
incorrect results. The negative points of this matrix are inva-
sive procedure, requirement of trained care professionals and
volume available is lower than that of urine. On the other
hand, adulteration is harder to happen when compared with
urine, and the analysis provides information about bioactive
compounds that circulate unmodified (Thomas et al. 2010).
The use of the pharmaceutical form of testosterone ester
by oral or injection administration results in these molecules
diffusing slowly into the blood stream. Esterase enzymes
have a very fast activity on this drug; however, the measure-
ment of testosterone ester in blood is possible. More specific-
ally, propionate, enanthate and undecanoate of testosterone
are detectable in blood until 4–11 days and more than
60 days, respectively, after intramuscular injection. On the
other hand, oral form of administration provides a range of
few hours to quantify, 10 h at maximum (Tretzel et al. 2014;
Forsdahl et al. 2015).
2 E. C. PIZZATO ET AL.
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In the doping monitoring with blood samples, both esters
forms are interesting because the laboratories apply the tests
to confirm other prohibited substances, for instance, human
growth hormone and continuous erythropoietin receptor
activator. Since the human body is unable to produce esters
of testosterone, the presence of these in samples is an irre-
futable evidence of exogenous administration of testosterone
(Forsdahl et al. 2015).
An alternative analysis using this biological matrix is the
use of dried blood spots, in which few volume of blood is
placed and dried on absorbent filter paper. As advantages,
there is a decreased biohazard risk to handlers, and it is eas-
ier to transport and/or store than liquid blood specimens.
However, it requires a complex sample treatment, sophisti-
cated analytical equipment and there is the risk of microbial
contamination (Sharma et al. 2014; Tretzel et al. 2014).
The dried blood spots can apply in monitoring of misuse
of exogenous testosterone. The data provide information of
glucuronidation of testosterone and its main metabolites
(androsterone and etiocholanolone), and this procedure is a
good alternative to plasma as a complement to urinalysis in
doping control. Tretzel et al. (2014) evaluated testosterone
esters using this technique with 20 lL of sample followed by
liquid chromatography-mass spectrometry tandem determin-
ation. Another study evaluated nonconjugated testosterone,
testosterone glucuronide, androsterone glucuronide and etio-
cholanolone glucuronide using this technique for blood sam-
ples comparing oral and intramuscular administration of
testosterone (Peng et al. 2000). According to Peng et al
(2000), there was an increase in nonconjugated testosterone,
androsterone glucuronide and etiocholanolone glucuronide
levels, whereas testosterone glucuronide levels remained
unchanged after intramuscular administration. In this sense,
testosterone glucuronide-to-nonconjugated testosterone ratio
could be a biomarker to identify the type of steroid
application.
The disadvantages are few studies in literature correlating
the reference values in different populations, intraindividual
variations, exogenous testosterone stability and concentra-
tion of the analytes in dried spot blood (Sharma et al. 2014).
Urine
Urine is the main biological fluid for analysis to investigate
doping cases for exogenous testosterone. Considering urine
matrix as a route for steroid elimination, the abundance of
conjugated steroids (Phase II) is predominant if compared
with free-fraction testosterone and other metabolites
(Vermeulen et al. 1999; Thieme et al. 2011). Some intrinsic
advantages are the high volume possible to be collected,
less-invasive procedure than blood and the analytes are
more concentrated owing to slower testosterone metabolism
rate in urine. The reference values are heterogeneous
Figure 2. Testosterone glucuronide and testosterone sulfate.
Figure 1. Metabolism of testosterone and other steroids (adapted from Marques et al. 2003 with minor modifications). DHEA: dehydroepiandrosterone; DHT:
dihydrotestosterone.
TOXICOLOGY MECHANISMS AND METHODS 3
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because of the prevalence of the enzyme uridine diphos-
phate glucuronosyl transferase (UGT2B17), an important ster-
oid metabolizing enzyme (Bresson et al. 2006).
The suitable volume of urine for doping analysis is 90 mL,
and it is divided in two vials, of which bottle A contains
70 mL to be used in screening assays. The remainder is
directed to bottle B for confirmatory tests. Preanalytical pre-
cautions for screening assays are room temperature and no
preservatives are added. Under these conditions, molecular
degradation can occur, and an important marker of undesir-
able physical-chemical change is the pH. Ammonia formation
and/or 5-androstanedione and free steroids metabolized by
glucuronidation pathway can raise the pH (Kicman and
Gower 2003; WADA 2014).
Other important parameters obtained in urine are testos-
terone-to-epitestosterone ratio and testosterone metabolites
(androsterone, etiocholanolone, 5b-androstane-3a,17b-diol
and 5a-androstane-3a,17b-diol). An interesting consideration
in monitoring antidoping program is a threshold to testoster-
one. Unfortunately, most analytical methods are unable to
distinguish testosterone administered exogenously and
endogenous hormone produced in the human body. The first
testosterone-to-epitestosterone ratio was 6:1. However, this
was reduced to a 4:1 ratio according to the World Anti-
Doping Agency (WADA). A good strategy to verify doping
with testosterone is to perform serial testing of testosterone-
to-epitestosterone ratio over at least 30 days after an event.
If the results of individual coefficients of variation are higher
than 60% in three or more urine samples in this period, the
doping can be considered positive (Barceloux and Palmer
2013; WADA 2015).
Some athletes, to circumvent the trials for testosterone
user monitoring and not be detected in doping tests, use
epitestosterone or human chorionic gonadotropin to raise
epitestosterone concentrations and present a more favorable
ratio. Thus, WADA has established a limit of 200 ng/mL for
urinary epitestosterone to detect such attempts to change
this ratio (de Boer et al. 1991). The use of testosterone can
result in suppression of the secretion of luteinizing hormone.
Consequently, testosterone-to-luteinizing hormone ratio is
higher (i.e. 430) (Perry et al. 1997).
The study of two stable carbon isotopes, C
13
/C
12
, with gas
chromatography/combustion/isotope-ratio mass spectrometry
is another tool to prove the use of testosterone. Endogenous
testosterone or precursors contain more C
13
than exogenous
hormones. So, urinary steroids with low C
13
-to-C
12
ratios indi-
cate external source of testosterone (de la Torre et al. 2001;
WADA 2015). This procedure is recommended for a confirmed
use of exogenous testosterone where the testosterone-to-epi-
testosterone ratio 4, androsterone and/or etiocholanolone
>10,000 ng/mL, testosterone and epitestosterone >200 ng/mL;
dehydroepiandrosterone >100 ng/mL (Polet and Van Eenoo
2015; WADA 2015,2016). Coutts et al. (1997) recommended a
confirmatory analysis when urinary values of dihydrotestoster-
one-to-epitestosterone ratio are 2 based on their study,
which shows the use of intramuscular injection of dihydrotes-
tosterone in male athletes.
Some studies used methyltestosterone as the internal
standard for determination of exogenous testosterone
administration (Leinonen et al. 2004; Wang et al. 2008;
Konieczna et al. 2011). However, it was verified that methyl-
testosterone degradation occurs for steroids in urine samples
during menstruation. In fact, there is conversion of methyl-
testosterone into its metabolite 17a-methyl-5b-androstane-
3a,17b-diol by microorganisms present in this sample.
Therefore, methyltestosterone degradation occurs without
showing typical signs of microbial degradation such as tur-
bidity, elevated pH and so forth (Schweizer Grundisch et al.
2014). So, this molecule is not the most appropriate internal
standard for the measurement of exogenous testosterone
administration in urine samples.
Hair
Nowadays, hair analysis has gained attention in toxicological
analysis. This procedure has advantages such as noninvasive
sample, stable at room temperature, pH control, and no
necessary preservatives until the laboratory tests. The main
analytical advantage is the capacity to obtain a broader
detection window for drugs of abuse, until for several weeks
and months. The average human hair growth rate is
0.6–1.5 cm per month, and hair samples of few centimeters
can provide retrospective information on drug intake (Agius
and Kintz 1999). Moreover, the analysis of out-of-competition
control samples, the long-term detection of steroids in hair
could provide complementary information. The steroid esters
are highly lipophilic, and these molecules accumulate in the
adipose tissue for a long time (several weeks) after intramus-
cular injection (Deshmukh et al. 2010).
For cases of exogenous testosterone administration, it is
able to determine this steroid and its metabolites from Phase
I metabolism of hair samples. The study by Kintz, Cirimele,
Sachs, et al. (1999) was the first study on hair analysis for
exogenous testosterone administration, and other studies
with this profile have also been accomplished (Thieme et al.
2000; van de Kerkhof et al. 2000; Strano-Rossi et al. 2013).
However, conjugated steroids with glucuronides or sulfates
were not found in this biological matrix until this moment.
Considering the difficulty to establish an analytical
method to distinguish exogenous testosterone from the nat-
ural physiological hormone in plasma or urine samples, hair
matrix could be an alternative to define confirmation of
exogenous testosterone administration. However, hair ana-
lysis is not suitable for screening tests (Kintz, Cirimele, Sachs,
et al. 1999; van de Kerkhof et al. 2000).
Nails
Another keratinous sample (similar to hair) was evaluated to
obtain an alternative matrix in doping control. Nails provide
a sample collection that can be easily obtained and noninva-
sive, no refrigeration required and low amount of this matrix
is required for analysis. Compared to other biological fluids,
the analytes inserted in nail are stable, not subjected to intra-
individual variations, have slow degradation and a low risk of
cross-reactivity.
Testosterone, testosterone propionate and stanozolol
were also identified in nails. The results confirmed the
4 E. C. PIZZATO ET AL.
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incorporation of steroidal compounds at the proximal nail
fold and nail bed. However, this biological matrix is consid-
ered complex showing very low sensibility, and little amount
of sample is available, impeding its implementation in anti-
doping control (Brown and Perrett 2011; Gosetti et al. 2013).
Similar to hair matrix, conjugated steroids with glucuronides
or sulfates were not found in this biological matrix until this
moment.
Oral fluid
Oral fluid is an alternative biological matrix obtained by a
simple and noninvasive procedure. Saliva samples provide
unbound, unionized parent drugs and/or lipophilic metabo-
lites. Under these conditions, conjugated molecules would
not be present in this biological matrix (Spiehler 2008).
The concentration of salivary steroid levels is more
similar to circulating levels of free steroid than the blood
measurement (Walker et al. 1979;Gr
€
oschl et al. 2001), and
the reference range of testosterone is reported as from 30 to
142 pg/mg in human males.
However, correlation levels between blood and saliva
concentrations are still considered uncertain because of the
binding protein of this hormone (Thieme et al. 2013).
According to Thieme et al. (2013), transdermal administration
of testosterone gel or patches promotes a significant increase
of free hormone (protein unbound) in the oral fluid. More
than 1000 pg/mg of testosterone was found, demonstrating
that saliva samples can be an interesting biological matrix for
the determination of exogenous testosterone adminstration,
mainly for doping analysis.
In spite of low levels of testosterone, oral fluid seems to
be a stable and robust sample, which does not suffer signifi-
cant variations during the storage process. However, it is pos-
sible that a blood contamination in saliva samples and
consequently, the measurement of testosterone can show
false-positive results (Durdiakov
a et al. 2013). In this sense,
conjugated steroids from testosterone and its metabolites
could be present as contaminants in saliva samples.
Pretreatment of the biological matrices for
testosterone analysis
Before the analytical procedure, a pretreatment step of the
biological matrices is required for chromatographic methods.
Digestion, hydrolysis, solvolysis and other approaches are
fundamentals to achieve the analytical purpose. In this
aspect, the importance of the biological sample indicates the
best procedure for pretreatment of the matrix (Flanagan
et al. 2008; Bordin et al. 2015).
Digestion
Digestion step is required when analytes are present in
the internal part of the matrix, intrinsically protected by a
network of macromolecules such as proteins, hindering
the accessibility of solvent and other reagents. Thus, this pro-
cedure promotes the extraction and/or direct analysis of
target analytes. So, for solid matrices such as nail and hair
samples, digestion step is the most appropriate for the pre-
treatment assay (Flanagan et al. 2008; Bordin et al. 2015).
Choi et al. (2001) evaluated testosterone and pregneno-
lone in nails. First, they washed the nails with methanol to
avoid external contamination. Short-length nails (1–2 mm,
100 mg) underwent alkaline digestion with 1 mL of 1 M NaOH
heated at 80 C for 1 h. After this period, 1 mL of 0.1 M phos-
phate buffer (pH 7) was added and pH adjusted to 10–11
with 0.3 mL of 2 M HCl. Another approach to determinate
dehydroepiandrosterone and testosterone is the incubation
of nail samples (10 mg) into a solution containing, respect-
ively, methanol:water (7:3, v/v, 0.3 mL) and ethanol:water (1:1,
v/v, 0.3 mL) during 2 h at 60 C. Later, ultrasonic step (15 min
at 25 C) was performed, and the supernatant was diluted
with water (0.7 and 0.6 mL) to carry out the extraction pro-
cedure (Higashi et al. 2016).
Studies with hair samples showed a similar procedure
when compared to nail samples. One study used 100 mg of
hair powder, and 1 mL of 1 M NaOH was added and incu-
bated for 45 min at 80 C. Then, 1 mL of 0.1 M phosphate
buffer (pH 6) was incorporated and the pH of the sample
was adjusted between 6.5 and 7.5 with hydrochloric acid
(25%) to determine only testosterone (Scherer et al. 1998).
Another study evaluated 8 steroids, and the process started
with hair sample decontamination (acetonitrile and then
methanol). Short lengths of hair (1–2 mm, 200 mg) was incor-
porated with 1 mL of 1 M NaOH, and the solution was heated
at 80 C for 1 h. Later, 1 mL of 0.1 M phosphate buffer (pH 7)
was added, and the pH was adjusted to 10–11 by 0.3 mL of
2 M HCl (Choi and Chung 1999).
For testosterone, Kintz, Cirimele, Jennaeu, et al. (1999)
decontaminated an aliquot of 100 mg of hair samples (4 cm)
twice in 5 mL of methylene chloride for 2 min. The samples
were incubated for 15 min at 95 C in 1 mL of 1 M NaOH as
internal standard; the homogenate was neutralized with 1 M
HCl, and 2 mL of 0.2 M phosphate buffer (pH 7) was added.
The same study evaluated testosterone esters from 100 mg
of hair samples that was cut into small pieces (1 mm) and
incubated overnight at 50 C in 5 mL of methanol as internal
standard after decontamination. Later, the reconstitution of
the dried sample occurred with phosphate buffer (2 mL;
0.2 mL; pH 7; Kintz, Cirimele, Jennaeu, et al. 1999).
Pozo et al. (2009) washed the hair sample with methanol
by agitation for 30 min, dried and finely cut. The hair sample
(200 mg) was mixed with an internal standard tris(2-carboxye-
thyl)phosphine hydrochloride solution (2 mL) and digested
for 1.5 h in ultrasonic bath at 50C for determination of
undecanoate testosterone. Strano-Rossi et al. (2013) used
30 mg of hair samples that was washed three times with
Tween 80 (0.1% v/v in distilled water) for 2 min each time.
After that, it was rinsed twice with 0.5 mL of distilled water,
once with 0.2 mL of acetone for 10 s, dried and cut into small
pieces. Then, there was the addition of internal standard,
300 lL of methanol, and the samples were incubated over-
night under sonication at 40 C.
In spite of the digestion procedure for hair and nail matri-
ces, this assay is not appropriate for biological fluids such as
TOXICOLOGY MECHANISMS AND METHODS 5
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blood and urine. In fact, hydrolysis procedure is the most
common assay for biological fluids.
Hydrolysis
Many drugs and metabolites circulate and/or get excreted in
biological fluids (blood and urine) as conjugated forms,
mainly as glucuronides or sulfates, or both. So, cleavage of
these conjugated molecules, known as hydrolysis, is essential
in toxicological analysis to guarantee better conditions for
the subsequent extraction step and consequently, the deter-
mination of target analyte(s). Hydrolysis can be classified into
chemical or enzymatic hydrolysis (Flanagan et al. 2008). For
testosterone and its metabolites, acid hydrolysis, enzymatic
hydrolysis and solvolysis are the typical procedures to pro-
mote free steroids.
Acid hydrolysis
There are studies that used acid hydrolysis to achieve free
testosterone and/or metabolites for the subsequent extrac-
tion step (Kjeld et al. 1977; Puah et al. 1978; Konieczna et al.
2011). Acid hydrolysis is influenced by the type of mineral
acid (HCl or H
2
SO
4
), molarity, temperature and reaction time
(Ismail and Harkness 1966; Kjeld et al. 1977; Puah et al. 1978;
Gomes et al. 2009; Konieczna et al. 2011).
The study by Puah et al. (1978) started with extraction of
conjugated testosterone in 250 lL and 500 lL of serum and
urine, respectively. After extraction and thin layer chromatog-
raphy tests, pieces of thin layer chromatography were dis-
solved in 1.0 mL of phosphate buffer (0.1 M, pH 7.5
containing 0.2% bovine serum albumin and 0.01% thiomer-
sal) or water, and finally acid hydrolysis was performed using
3MH
2
SO
4
solution for 20 h at 40 C. Kjeld et al. (1977) per-
formed extraction of conjugated testosterone in urine sam-
ples according to another study (Puah et al. 1978) and
evaluated the hydrolytic effect of the mineral acids (H
2
SO
4
and HCl solutions). Their results showed chloride ion had a
stronger hydrolytic effect than sulfate ion at 37 C for 16 h at
pH 1; however, the best condition for complete acid hydroly-
sis was obtained with 3 M H
2
SO
4
solution (24 h at 37 C).
Konieczna et al. (2011) used 0.5 mL of HCl (36%) to each
3.0 mL of urine sample at 95 C for 1 h in a water bath to
analyze testosterone and epitestosterone.
However, acid hydrolysis assay has limitations to obtain
free steroids. The compounds produced during this process
may interfere on chromatographic analysis leading to deg-
radation of the molecular structure of testosterone metabo-
lites such as dehydroepiandrosterone (Ismail and Harkness
1966). Nevertheless, Venturelli et al. (1995) did not report
destruction of the testosterone molecular structure.
Moreover, there is a decrease in the peak resolution in a
chromatographic process for epitestosterone (Venturelli et al.
1995; Ismail and Harkness 1966). Thus, direct application of
acid hydrolysis into urine is not recommended because of
interferences from the reaction between acid and urinary
compounds (Orlov et al. 1996).
To simplify this procedure, Pujos et al. (2004) demon-
strated a one-step hydrolysis –derivatization technique –for
dehydroepiandrosterone sulfate. To analyze testosterone
esters in hair matrix, the digestion step was performed with
2 mL of 25 mM tris(2-carboxyethyl)phosphine in ultrasonic
bath for 1.5 h. Later, samples were extracted twice with 8 mL
of methanol using an overhead shaker. The methanol layer
was separated after centrifuging for 10 min at 2800gand
dried under nitrogen at 40 C. After digestion procedure, the
hydrolysis step was executed by 330 lL of 2.5 M of NaOH
incubated overnight at 35 C. Later, 6 mL of 0.25 M sodium
acetate buffer (pH 4.8) was added, and the pH was adjusted
with HCl until pH 4.8 for the extraction step (Becue et al.
2011).
Acid hydrolysis is able to cleave testosterone and its
metabolites bound to glucuronide and sulfate molecules
(Venturelli et al. 1995). However, enzymatic hydrolysis has
typical aspects to promote free testosterone and its metabo-
lites with low degradation of these molecules when com-
pared with acid hydrolysis (Kent et al. 1965).
Enzymatic hydrolysis
Enzymatic hydrolysis has been used in several studies since
60s and is still the preferred procedure for obtaining testos-
terone and unconjugated metabolites with sulfate or glucur-
onide (Kent et al. 1965; Venturelli et al. 1995; Campos et al.
2005; Gomes et al. 2009; Galesio et al. 2010; Ouellet et al.
2013; Jenkison et al. 2014; Kotronoulas et al. 2015; Casilli
et al. 2016). The appropriate enzyme to cleave conjugated
steroids with glucuronide is b-glucuronidase. This enzyme
can be obtained from bovine liver, Escherichia coli (a gram-
negative bacterium), Helix pomatia (a terrestrial pulmonate
gastropod mollusk), abalone entrails and Patella vulgata
(both marine gastropod mollusks). On the other hand, sulfa-
tase enzyme demonstrated to be absent in E. coli, small
amount in P. vulgata and significant levels in H. pomatia and
abalone entrails (Shackleton 1986; Ferchaud et al. 2000). In
this regard, enzyme activity should be sufficient to promote
complete hydrolysis under specific conditions. Thus, consider-
ing that steroid concentration in a biological sample is
unknown, the correct approach is essential taking into
account the amount of enzyme, incubation time, pH and
temperature (Ferchaud et al. 2000; Hauser et al. 2008; Gomes
et al. 2009).
For enzyme activity, low or excess levels of b-glucuroni-
dase may promote an incomplete hydrolysis for conjugated
testosterone and its metabolites (Ferchaud et al. 2000;
Gomes et al. 2009). Another aspect is steroid-dependence for
temperature of reaction. Higher temperatures improved
deconjugation of dehydroepiandrosterone sulfate, whereas
lower temperatures favored the cleavage of androsterone
and etiocholanolone sulfate (Shackleton 1986). In the case of
incubation time, a short period (few minutes) was required
with b-glucuronidase from bovine liver for conjugated ste-
roids, except for dehydroepiandrosterone glucuronide (1 h;
Ferchaud et al. 2000) until 22 h.
Considering enzyme preparation and absence of artifacts,
an incubation time of 20 h was selected when using abalone
entrails (Ferchaud et al. 2000) and 22 h using E. coli for 23
endogenous steroids that included testosterone and its major
6 E. C. PIZZATO ET AL.
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metabolites (Hauser et al. 2008). However, long periods for
incubation can be a disadvantage especially for diagnosis of
pathology and during sport competitions. Also, hydrolysis is
pH-dependent according to enzyme preparation for which
acidic to neutral conditions are more efficient than alkaline
submission (Wakabayashi and Fishman 1961). According to
Shackleton (1986), hydrolysis is best performed at pH 6–8 for
b-glucuronidase from E. coli, pH 5 for bovine liver and pH
4.5–5.5 for P. vulgata and H. pomatia. Sulfatase activity from
H. pomatia showed a better performance at pH 6.2, and its
activity is interrupted at pH 4.5 in acetate buffer (Gomes
et al. 2009).
Usually, hydrolysis step occurs before extraction to obtain
free steroids. Another approach is direct hydrolysis, a method
used to lead to incomplete hydrolysis as shown for andro-
sterone and etiocholone glucuronides (enzyme from bovine
liver, E. coli and H. pomatia; Graef et al. 1977) or low recovery
of the steroid (Gomes et al. 2009). In fact, microorganisms
may metabolize some steroids during enzymatic hydrolysis
owing to the presence of other enzymes. It was identified
that 3b-hydroxysteroid oxidoreductase and 3-oxosteroid-
4,5-ene isomerase in H. pomatia (Messeri et al. 1984)is
responsible for conversion of dehydroepiandrosterone into
androstenedione, decreasing dehydroepiandrosterone levels
in the biological matrix. In a comparative study of different
enzyme sources on dehydroepiandrosterone, androstene-
dione and androstenediol, only b-glucuronidase from E. coli
did not lead to unwanted products. Androstenedione and
androstenediol were transformed into testosterone (Mass
e
et al. 1989). However, epitestosterone levels remained
unchanged, independent of hydrolysis choice. Therefore,
other enzymes such as 6-hydrolase, 6-dehydroxylase and 6-
hydroxysteroid oxidase were identified in preparation of H.
pomatia (Houghton et al. 1992; Hauser et al. 2008).
Recently, an enzymatic hydrolysis promoted with 30 lLof
E. coli (1 h at 55 C in pH 7) on 2.5 mL of urine samples dem-
onstrated resistance to cleavage for two metabolites from
exogenous testosterone administration (3a-glucuronide-6b-
hydroxyandrosterone and 3a-glucuronide-6b-hydroxyetiocho-
lanolone; Kotronoulas et al. 2015). Considering enzymatic
hydrolysis resistant to these molecules, Kotronoulas et al.
(2016) evaluated the potential of these glucuronides as
markers of oral administration of testosterone undecanoate
by LC-MS/MS without the deconjugation step. The authors
demonstrated a larger detection window when compared to
the testosterone-to-epitestosterone ratio in urine samples.
However, the researchers admitted more studies to include
these markers for anti-doping analysis.
The origin of b-glucuronidase enzyme has no influence on
cleavage of testosterone and the majority of glucuronate
metabolites; however, sulfoconjugate steroids are negligible
when hydrolysis assay is performed only with b-glucuroni-
dase (Venturelli et al. 1995; Gomes et al. 2009) and could
have adverse analytical data. Sulfoconjugate steroid levels
vary according to the individual and the drug administered.
Studies have verified that some individuals demonstrate the
amount of sulfates may exceed that of glucuronides in urine,
and there is a slower elimination of testosterone and sulfate
metabolites compared with glucuronide molecules (Gomes
et al. 2009).
It is possible to use extraction in the first step followed by
hydrolysis to remove interfering compounds in the extraction
step and to improve hydrolysis, even when using low levels
of enzyme. This strategy was used for dehydroepiandroster-
one for which it is necessary to correct enzyme levels to
guarantee the complete cleavage, mainly sulfate-conjugated
metabolites, avoiding insufficient cleavage (less enzyme lev-
els) or low recovery of dehydroepiandrosterone (excess
enzyme levels; Schmidt et al. 1985). In general, testosterone
and metabolites conjugated with sulfate are considered a
challenge for the hydrolysis step because of the resistance to
enzymatic hydrolysis or generation of inappropriate products
(Meng and Sj€
ovall 1997).
An alternate is to use H. pomatia and abalone entrails for
the sufficient presence of sulfatase activity since this specific
enzyme is absent and/or in low levels in other sources
(E. coli, bovine liver or P. vulgata; Shackleton 1986; Ferchaud
et al. 2000). In these cases, chemical hydrolysis can be used
either alone or in combination to reach complete cleavage of
conjugated testosterone and its metabolites (Venturelli et al.
1995).
Nevertheless, WADA puts forth that enzymatic hydrolysis
shall be carried out with purified b-glucuronidase from E.
coli, and it is not acceptable with H. pomatia mixtures.
Therefore, total deconjugation of urinary steroids should
be controlled with isotopically labeled A-glucuronide (or
an equivalent scientifically recognized alternative; WADA
2016).
To avoid typical problems related to acid hydrolysis,
chemical hydrolysis with solvents, known solvolysis, has
been used to promote hydrolysis of conjugated steroids
(Tang and Crone 1989; Meng and Sj€
ovall 1997; Weltring
et al. 2012).
Solvolysis
The first publication on solvolysis occurred in 1963, and this
technique has been used to cleave conjugated steroids with
glucuronide and sulfate. The determined parameters such as
solvents, acid conditions, incubation time and temperature
should be evaluated to guarantee a sufficient cleavage
(Matsuda 1963; Hurwitz 1972; Tang and Crone 1989; Meng
and Sj€
ovall 1997; Higashi et al. 2005; Weltring et al. 2012).
Higashi et al. (2005) used solvolysis in serum samples for
dehydroepiandrosterone, testosterone, androstenedione,
androstenediol and other steroids conjugated with sulfates
with ethyl acetate (0.5 mL) containing 2 lL of 0.5 M H
2
SO
4
and stored in room temperature for 1 h. Later, this mixture
was washed with water (0.5 mL, twice), and the solvent was
evaporated. Another procedure was performed by Hauser
et al. (2008) for 23 endogenous conjugated steroids, but they
used heat for cleavage (55 C) for 1 h, although an incubation
time between 24 and 48 h at 37 C was used by Vestergaard
(1978).
Solvolysis by trimethylchlorosilane diluted in methanol
has been used successfully for steroid deconjugation by
TOXICOLOGY MECHANISMS AND METHODS 7
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generation of hydrochloric acid from trimethylchlorosilane
during the hydrolysis process in urine samples (Dehennin
et al. 1996). Kazihnitkov
a et al. (2004) performed solvolysis
for dehydroepiandrosterone and its metabolites in brain tis-
sues using H
2
SO
4
(20 lL), Na
2
SO
4
(50 mg) and ethyl acetate
(3 mL) frozen with CO
2
bath. The organic layer was sepa-
rated into another glass, incubated overnight at 37 C, and
it was evaporated until dryness. G
omez et al. (2013) used
4 mL of ethyl acetate/methanol/H
2
SO
4
(80:20:0.06, v/v/v) in
urine samples for 2 h at 55 C. After that, extracts were neu-
tralized with 60 lL of 1 M NaOH, evaporated to dryness and
reconstituted in 1 mL sodium phosphate buffer (0.2 M, pH
7) and 250 lLof5%K
2
CO
3
. Later, 6 mL of tert-butyl methyl
ether was used for extraction of the methyltestosterone and
metabolites conjugated with sulfates.
Another approach for solvolysis was applied to aliphatic
steroid sulfates (boldenone sulfate, nortestosterone sulfate
and testosterone sulfate; Pedersen et al. 2017). Pedersen
et al. (2017) first performed an enzymatic hydrolysis and then
the solvolysis procedure using 1 mL of bovine urine sample.
Then, 1 mL of ethyl acetate and 40 lLof4MH
2
SO
4
were
added followed by incubation at 40 C for 30 min. Later,
320 lL of 1 M Tris buffer was added, and the organic phase
was evaporated using nitrogen at 40 C. After that, a total of
1 mL of 20% of methanol was added and mixed to be pre-
pared for extraction step. The recovery of these analytes was
between 50% and 57%, considered acceptable taking into
account the mass equivalence of steroid from its conjugate
forms (Pedersen et al. 2017).
In fact, the solvolysis approach has been used with rela-
tive success to obtain free testosterone and its metabolites
from sulfoconjugate forms. This technique can be used after
enzymatic hydrolysis to obtain complete steroid deconjuga-
tion (Hauser et al. 2008; Blokland et al. 2012; Pedersen et al.
2017). However, it is a further step to promote steroid
hydrolysis, which represents an increase in the cost and time
of analysis.
New approaches for pretreatment of biological
samples for exogenous testosterone
Most of the studies on hydrolysis are based on trial and error
approach, which means labor procedure (Gomes et al. 2009).
Despite the differences among pretreatment types, inter-
changeability of the techniques for different biological matri-
ces is possible with appropriate modifications, since the
effectiveness of the deconjugation of testosterone and its
metabolites is proven as demonstrated by Jenkinson et al.
(2014). These authors adapted an enzymatic hydrolysis step
based on a previous study with nandrolone and stanolozol
over hair samples using alkaline hydrolysis (Deshmukh et al.
2010).
The procedure by Deshmukh et al. (2010) consists of
alkaline hydrolysis with 1 mL of 1 M NaOH at 95 C for
15 min, and the homogenate was neutralized with 1 M HCl
followed by the addition of 2 mL of 0.2 M phosphate buffer
(pH 7) for the hair samples. In this regard, the study by
Jenkinson et al. used 100 lL of rat urine/serum samples
that were spiked with 50 lL of internal standard and pre-
pared with 1 mL of 0.2 M phosphate buffer (pH 7) with
50 lLofb-glucuronidase enzyme from E. coli (Roche
Diagnostics). The samples were incubated for 2 h at 50 C.
The strength of the solution at 37 C is at least
140 U/mL). After that, the samples were cooled on wet ice
and later extracted.
Despite the modifications, typical problems of enzymatic
hydrolysis may persist. Therefore, the risk of failure may be
higher if compared with other types of hydrolysis tests,
increasing costs, number of experiments and time of analysis
to obtain a pretreatment that corresponds to the analytical
proposal.
So, to achieve complete hydrolysis, but avoiding time and
money waste, alternative approaches have been used such
as surface response (Ferchaud et al. 2000; Sanches et al.
2012) and ultrasonic energy (Galesio et al. 2010).
Surface response is based on a mathematical approach
that allows simultaneous optimization of the variables, which
makes it possible to view the interaction between the varia-
bles, reducing the number of experiments and consequently,
decrease time and expenses when compared to the trad-
itional one-variable-at-a-time optimization procedure. The
experimental design depends on the software type disponi-
ble, and how this program may analyze all parameters
(Ferchaud et al. 2000; Sanches et al. 2012).
Ferchaud et al. (2000) used this tool for improvement
of enzymatic hydrolysis of dehydroepiandrosterone, etio-
cholanolone, epitestosterone and other steroids in urine
samples. In this study, different sources of enzymes (aba-
lone entrails, bovine liver, H. pomatia and P. vulgata) and
their parameters (temperature, incubation time, pH, amount
of enzyme and impurities formation/steroid degradation)
were evaluated. The experimental model was composed of
2
4
factorial runs with 2 4 star points to check for pos-
sible response curvature (graphic design to view the
results) and four replicates of the central treatment to esti-
mate error variation. In resume, 28 assays were performed
for each enzyme preparation for which abalone entrails
showed the best results (12,000 U of enzyme activity in pH
5.2 for 20 h at 42 C).
Ultrasonic energy can be an alternative approach to
improve hydrolysis of testosterone and its metabolites.
Galesio et al. (2010) compared energy ultrasonic against trad-
itional enzymatic hydrolysis in urine samples. In this study,
2 mL of urine sample was hydrolyzed by addition of 750 lL
of phosphate buffer (0.8 M at pH 7) and 50 lLofb-glucuroni-
dase from E. coli. From this point, three different approaches
were evaluated: (A) Traditional assay (1 h at 55 C); (B) cup-
horn sonoreactor operating at 60% of sonication amplitude
and (C) ultrasonic probe operating at 60% of sonication amp-
litude. For ultrasonic procedures, incubation time evaluated
was 1, 3, 5 and 10 min. The results showed a high efficiency
of sonoreactor assay during 10 min at 37 C with approxi-
mately 100% of testosterone and mainly metabolites in the
deconjugated form. This developed method reduces total
time for the determination of steroids from around 150 min
compared to the traditional enzymatic hydrolysis with the
same reproducibility.
8 E. C. PIZZATO ET AL.
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Table 1. Summary of pretreatment of testosterone and its metabolites according to the biological matrix.
Biological matrix Pretreatment Procedure Analytes Reference
Hair powder (100 mg) Digestion Incubation of the samples with 1 mL of
1 M NaOH (45 min at 80 C); 1 mL of
0.1 M phosphate buffer (pH 6) was incor-
porated, and the sample pH was adjusted
between 6.5 and 7.5 with hydrochloric
acid (25%).
Testosterone Scherer et al. (1998)
Hair (1–2 mm, 200 mg) Digestion Decontamination was performed with aceto-
nitrile and then with methanol. Later,
1 mL of 1 M NaOH was added (80 C for
1 h). Then, 1 mL of 0.1 M phosphate buf-
fer (pH 7) was added, and the pH was
adjusted between 10 and 11 with 0.3 mL
of 2 M HCl.
Androsterone, androstenedione,
dihydrotestosterone, dihydroe-
piandrosterone, etiocholanolone,
progesterone, pregnenolone and
testosterone
Choi and Chung (1999)
Hair (4 cm, 100 mg) Digestion Decontamination of the sample was per-
formed with 5 mL of methylene chloride
for 2 min (twice) followed by incubation
of the sample with 1 mL of 1 M NaOH as
the internal standard (15 min at 95 C),
and the homogenate was neutralized
with 1 M HCl. Later, 2 mL of 0.2 M phos-
phate buffer (pH 7) was added.
Testosterone Kintz, Cirimele, Sachs,
et al. (1999)
Hair (1 mm, 100 mg) Digestion Decontamination of the sample with 5 mL
of methylene chloride for 2 min (twice)
followed by incubation overnight at 50 C
in 5 mL of methanol as the internal
standard. Later, the dried sample was
reconstituted with phosphate buffer
(2 mL; 0.2 mL; pH 7).
Testosterone acetate, testosterone
benzoate, testosterone cypio-
nate, testosterone decanoate,
testosterone enanthate, testos-
terone isocaproate, testosterone
phenylpropionate and testoster-
one propionate
Kintz, Cirimele, Jeanneau,
et al. (1999)
Nails (1–2 mm,
100 mg)
Digestion Samples were washed with methanol to
avoid external contamination. Alkaline
digestion was performed with 1 mL of
1 M NaOH heated at 80 C for 1 h. After
this period, 1 mL of 0.1 M phosphate buf-
fer (pH 7) was added, and the pH was
adjusted between 10 and 11 with 0.3 mL
of 2 M HCl.
Testosterone and pregnenolone Choi et al. (2001)
Hair (200 mg) Digestion Decontamination with methanol by agitation
for 30 min; tris(2-carboxyethyl)phosphine
hydrochloride solution (2 mL) was added
and the sample was digested for 1.5 h in
ultrasonic bath at 50 C.
Testosterone undecanoate Pozo et al. (2009)
Hair matrix (200 mg) Digestion Digestion was performed with 2 mL of
25 mM tris(2-carboxyethyl)phosphine
hydrochloride solution in ultrasonic bath
for 1.5 h. Samples were then extracted
twice with 8 mL of methanol using an
overhead shaker. The methanol layer was
separated after centrifuging for 10 min at
2800gand dried under nitrogen at
40 C. Hydrolysis was carried out with
330 lL of 2.5 M NaOH and incubated
overnight at 35 C. Later, 6 mL of 0.25 M
sodium acetate buffer (pH 4.8) was
added, and the pH was adjusted with HCl
to pH 4.8 for the extraction step.
Estradiol benzoate, testosterone
cypiate, testosterone decanoate
and 17b-testosterone
Becue et al. (2011)
Hair (30 mg) Digestion Decontamination with Tween 80 (0.1% v/v
in distilled water) for 2 min (three times).
The sample was rinsed twice with 0.5 mL
of distilled water, once with 0.2 mL of
acetone for 10 s and then dried and cut
into small pieces. Then, there was the
addition of internal standard, 300 lLof
methanol, and the samples were incu-
bated overnight under sonication at
40 C.
Boldenone, boldenone propionate,
boldenone undecylenate, bol-
dione, clenbuterol, methandie-
none, mesterolone,
methenolone, 5(10)-estren-3a,
17b-diol, methenolone enan-
thate, methenolone acetate, nan-
drolone, Nandrolone decanoate,
nandrolone laurate, norandroste-
nedione, oxandrolone, stanozo-
lol, testosterone acetate,
testosterone cipionate, testoster-
one decanoate, testosterone
enanthate, testosterone phenyl-
propionate; testosterone propi-
onate; testosterone undecanoate
and trenbolone
Strano-Rossi et al. (2013)
(continued)
TOXICOLOGY MECHANISMS AND METHODS 9
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Table 1. Continued
Biological matrix Pretreatment Procedure Analytes Reference
Nails (10 mg) Digestion Incubation of the samples into a solution
containing, respectively, methanol:water
(7:3, v/v, 0.3 mL) and ethanol:water (1:1,
v/v, 0.3 mL) for 2 h at 60 C. Ultrasonic
step (15 min at 25 C) was performed,
and the supernatant was diluted with
water (0.7 and 0.6 mL) to carry out the
extraction procedure.
Dehydroepiandrosterone and
testosterone
Higashi et al. (2016)
Urine (500 lL) serum
(250 lL)
Acid hydrolysis Extraction of testosterone conjugated in
250 lL and 500 lL of serum and urine,
respectively, followed by evaluation using
thin layer chromatography. Later, pieces
of thin layer chromatography were dis-
solved in 1.0 mL of phosphate buffer
(0.1 M, pH 7.5 containing 0.2% bovine
serum albumin and 0.01% thiomersal) or
water. Acid hydrolysis was performed
using 3 M H
2
SO
4
solution (20 h at 40 C).
Testosterone, testosterone, glucuro-
nide and testosterone sulfate
Puah et al. (1978)
Urine (500 lL) Acid hydrolysis Extraction of testosterone conjugated was
performed as described by Puah et al.
(1978), and the hydrolytic effect of the
mineral acids (H
2
SO
4
and HCl solutions)
was evaluated. The best condition for
hydrolysis is with 3 M H
2
SO
4
solution
(24 h at 37 C).
Dehydrotestosterone, testosterone,
testosterone glucuronide and
testosterone sulfate.
Kjeld et al. (1977)
Urine (3 mL) Acid hydrolysis 0.5 mL HCl (36%) at 95 C for 1 h in a water
bath.
Testosterone and epitestosterone Konieczna et al. (2011)
Urine (10 mL) Enzymatic
hydrolysis
The sample was adjusted to pH 6.5 with
1 mL of buffer, and 60 lLofb-glucuroni-
dase from Escherichia coli was added and
incubated for 25 h at 37 C.
Androsterone, dehydroepiandroster-
one, etiocholanolone, pregnane-
diol and pregnanetriol
Graef et al. (1977)
Urine (50 mL) Enzymatic
hydrolysis
The sample was adjusted to pH 5.2, and
5 mL of acetate buffer was added. After
that, 0.5 mL of Helix pomatia juice
(1.58 nmoL) was incorporated, and it was
incubated for 22 h at 45 C.
5a-androstanediol, 5b-androstane-
diol, 5a-androstenedione,
dihydrotestosterone, estrone and
testosterone
Venturelli et al. (1995)
Heparinized plasma
(1 mL)
Enzymatic
hydrolysis
The sample was diluted with 1.5 mL of
sodium phosphate buffer (0.2 M, pH 7)
and passed through the Detectabuse
V
R
column with methanol and water for pre-
treatment. The column was washed with
water, and the steroids were eluted with
methanol. The solvent was evaporated,
and the residue was reconstituted in
1 mL of sodium phosphate buffer (0.2 M,
pH 7). Nonconjugated steroids were
extracted with 5 mL of tert-butyl methyl
ether, evaporated with N
2
and kept in a
desiccator until derivatization. The
remaining aqueous phase (containing tes-
tosterone glucuronide, androsterone glu-
curonide and etiocholanolone
glucuronide) was then hydrolyzed with
6U of b-glucuronidase and extracted
using the same procedure that was used
for the dried blood-spot samples.
Androsterone, androsterone glucur-
onide, etiocholanolone, etiocho-
lanolone glucuronide, 17a-
hydroxyprogesterone, testoster-
one and testosterone
glucuronide
Peng et al. (2000)
Blood (20 lL–dried
blood spot)
Enzymatic
hydrolysis
The aforementioned extracted potassium
hydroxide-water phase was neutralized
with 1 M hydrochloric acid, incubated
with 6 U of b-glucuronidase at 37 C
overnight after a second addition of the
same amounts of the same internal
standards and then extracted with tert-
butyl methyl ether after the aqueous
solution was adjusted to approximately
pH 10 with 50 g/L potassium carbonate.
Androsterone, androsterone glucur-
onide, etiocholanolone, etiocho-
lanolone glucuronide, 17a-
hydroxyprogesterone, testoster-
one and testosterone
glucuronide
Peng et al. (2000)
Urine (10 mL) Enzymatic hydrolysis One milliliter of acetate buffer (0.2 M; pH
5.2) was added to the sample with
12,000 U of enzyme activity from abalone
entrails. The pH was adjusted with glacial
acetic acid or sodium hydroxide (10 M).
The sample was incubated at 42 C for
20 h.
Dehydroepiandrosterone, etiochola-
nolone, epitestosterone, 17a-
estradiol and estrone
Ferchaud et al. (2000)
(continued)
10 E. C. PIZZATO ET AL.
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Table 1. Continued
Biological matrix Pretreatment Procedure Analytes Reference
Urine (4 mL) Enzymatic hydrolysis Samples were conditioned with 2 mL phos-
phate buffer (pH 6.8) with 40 lLb-glu-
curonidase enzyme from E. coli. The
solutions were incubated at 55 C for 1 h.
Later, 100 mg of NaHCO
3
/K
2
CO
3
(2:1) was
added until pH 9–10 followed by addition
of internal standard.
Epitestosterone, 19-noretiocholano-
lone, 19-norandrosterone, 3-
hydroxyestanozolol, 16b-hydrox-
ystanozolol, 17a-methyl-5b-
androstane-3a-17b-diol, 17a-
methyl-5a-androstane-3a-17b-
diol, 9a-fluoro-17a-methyl-4-
androstene-3a-6b-11b-tetrol, 9a-
fluoro-18-nor-17,17-dimethylan-
drostane-4,13-dieno-11b-ol-3-
ona, 17a-ethyl-5b-estrano-3a-
17b-triol, oxymesterone, oxan-
drolone, metenolone and
testosterone
Campos et al. (2005)
Urine (200 lL) Enzymatic
hydrolysis
Lyophilized b-glucuronidase type VII-A from
E. coli (25,000 U) was dissolved in 5 mL
HPLC water. Each sample was diluted
with 800 lL of 0.25 M potassium phos-
phate buffer (pH 6.9). Then, 40 lL
enzyme (200 U) and 20 lL of an internal
standard mix were added. Samples were
incubated at 37 C for 22 h under gentle
agitation. Potassium carbonate (150 lL,
10%) was added to stop the enzymatic
reaction and to adjust the sample pH to
9.6.
Androstanedione, androsterone, 3b-
androstanediol, a-cortol, cortisol,
dehydroepiandrosterone;
dihydrotestosterone, estriol, 17b-
estradiol, estrone, epitestoster-
one, epiandrosterone; epietio-
cholanolone, etiocholanolone,
11-oxyetiocholanolone, proges-
terone, pregnadiol, pregnadiol
glucuronide, 11b-hydroxy-etio-
cholanolone, testosterone, tes-
tosterone-17b-glucuronide, tetra-
hydrocortisone and
tetrahydrocortisol
Hauser et al. (2008)
Urine (9 mL) Enzymatic
hydrolysis
Samples were collected and frozen in the
refrigerator overnight. For each sample,
9 mL was transferred to 20-mL screw cap
vials containing 9 mL of 2 M sodium acet-
ate buffer (pH 4.6). The total volume in
one vial was 18 mL. Then, 120 lLof
b-glucuronidase from Helix pomatia was
added into the homogenate followed by
incubation at 55 C for 3 h. After the
hydrolysis step, the vials were allowed to
cool to room temperature for thin-film
microextraction. A volume of 5 mL of
hydrolysis mixture aliquots corresponding
to 2.5 mL of urine were transferred to
clean 10-mL sample vials, and one piece
of thin film was added. Later, solid-phase
microextraction procedure was
performed.
Conjugated testosterone and conju-
gated epitestosterone
Zhan et al. (2012)
Urine (5–15 mL
according target
steroid)
Enzymatic
hydrolysis
Samples were preconditioned with C18 Sep
Pak SPE cartridges, rinsed with H
2
O
(5 mL) and hexane (5 mL) and then eluted
with methanol (5 mL). Dry residue was
submitted to enzymatic hydrolysis in
1 mL of phosphate buffer (0.1 M, pH 6.9)
with the addition of approximately
1200 U of b-glucuronidase type 1X-A
from E. coli and incubation (50 C for 1 h).
Androstanol, 5b-androstanediol,
5a-androstanediol, androsterone,
dihydroepiandrosterone, epites-
torone, etiocholanolone, 5b-
pregnanediol, pregnanetriol and
testosterone
Ouellet et al. (2013)
Rat urine and serum
(100 lL)
Enzymatic
hydrolysis
Samples were spiked with 50 lL of internal
standard stanozolol-D3 (2 ng/mL) and
prepared with l mL of 0.2 M pH 7 phos-
phate buffer with 50 lLofb-glucuroni-
dase enzyme from E. coli (Roche
Diagnostics
V
R
). The samples were incu-
bated for 2 h at 50 C. The strength of
the solution 37 C is at least 140 U/mL).
Later, the samples were cooled on wet
ice and then extracted.
Testosterone and epitestosterone Jenkison et al. (2014)
Urine (10 mL) Enzymatic
hydrolysis
First, the sample was prepared by solid-
phase extraction C-18 (SPE), activated
with 5 mL of methanol and 5 mL of water
before and after loading the urine; the
target compounds were then eluted with
3 mL of methanol. The dried residue was
dissolved in 1.5 mL of sodium phosphate
buffer and extracted with 4 mL of tert-
butyl methyl ether (TBME) to separate
Androsterone, 5a–androstenediol,
5b-androstenediol, etiocholano-
lone and pregnanediol
testosterone
Casilli et al. (2016)
(continued)
TOXICOLOGY MECHANISMS AND METHODS 11
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Table 1. Continued
Biological matrix Pretreatment Procedure Analytes Reference
unconjugated steroids. The aqueous resi-
due was hydrolyzed with b-glucuronidase
from E. coli K12 (Roche Diagnostics
V
R
) for
60 min at 50 C and again extracted two
times with 4 mL of TBME. The organic
layer (containing glucuronidated steroids)
was evaporated to dryness and acety-
lated. An aliquot for each steroid to be
injected into the multidimensional gas
chromatography/combustion/isotopic
ratio mass spectrometry (MDGC/C/IRMS)
was prepared according to its urinary
concentration.
Urine (4 mL) Solvolysis First, a sample preparation was based in
solid-phase extraction, and one part of
the eluate was used for solvolysis. The
residue was evaporated, and it was
taken-up in 1 mL of methanol containing
1 M trimethylchlorosilane and then incu-
bated at 55 C for 1 h. Later, the mixture
was evaporated at 60 C, and three drops
of methanol followed by 0.5 mL of phos-
phate buffer (0.1 M, pH 6.5) were added
to the residue. After that, extraction and
the derivatization step were performed to
analyze in gas chromatography–mass
spectrometry.
Androsterone, 5a-androstane-
3a,17b-diol, 5b-androstane-
3a,17b-diol, 5-androstane-3b,17
a-diol. 5-androstane-3b,17b-diol,
dehydroepiandrosterone,
dihydrotestosterone, epiandros-
terone, epitestosterone, etiocho-
lanolone and testosterone
Dehennin et al. (1996)
Serum (100 lL) Solvolysis It was added acetonitrile (0.4 mL) in the
sample, vortex-mixed for 30 s and centri-
fuged at 1500g(4 C for 10 min). The
supernatant was evaporated, and the
residue was dissolved in ethyl acetate
(0.5 mL). After washing with water
(0.5 mL) and solvent evaporation, the resi-
due was dissolved in 10 lL of ethanol,
treated with cholesterol oxidase and
redissolved in 40 lL of ethanol and 10 lL
was injected into the liquid chromatogra-
phy–mass spectrometry. DHEA analysis
was performed without cholesterol oxi-
dase treatment. The supernatant was
diluted with Tris-HCl buffer (1 mL) and
passed through an Oasis HBL cartridge.
After washing with H
2
O (2 ml) and 70%
methanol (2 mL), the steroid was eluted
with ethyl acetate (1 mL), which was
evaporated under an N
2
gas stream. The
residue was dissolved in ethanol (40 lL),
10 lL of which was subjected to the
liquid chromatography–mass
spectrometry.
Sulfates conjugated of androstene-
dione, androstenediol dehydroe-
piandrosterone, testosterone,
and other steroids
Higashi et al. (2005)
Aqueous phase from
enzymatic
hydrolysis
Solvolysis First, remaining tert-butyl methyl ether resi-
dues were evaporated and 1 mL of 0.5 M
sodium acetate buffer (pH 4.7) and 20 lL
internal standards were added. After
solid-phase extraction using C-18 car-
tridges, steroids eluates were evaporated
to a volume of 1 mL and 5 mL ethyl acet-
ate/H
2
SO
4
(250 mL ethyl acetate/200 mg
sulfuric acid, 98%) were added. This solu-
tion was incubated for 1 h at 55 C under
mild agitation, and it was stopped by
adding 250 lL of 1 M NaOH. Samples
were vortexed and centrifuged for 5 min
at 1500 rpm. The ethyl acetate phase was
evaporated, and the residue was dis-
solved in 2 mL deionized water.
Sulfate compounds from 23
endogenous steroids
Hauser et al. (2008)
Urine (5 mL) Solvolysis First, liquid chromatography fraction of the
samples was performed for metabolite
characterization; these fractions were
evaporated, and a solvolysis was per-
formed with 4 mL of ethyl acetate/metha-
nol/sulfuric acid (80:20:0.06, v/v/v) and
incubated at 55 C for 2 h. Extracts were
Methyltestosterone, 17a-methyl-5b-
androstane-3b,17b-diol, 17a-
methyl-5a-androstane-3a,17b-
diol, 17a-methyl-5b-androstane-
3a,17b-diol, 17a-methyl-5a-
androstane-3b,17b-diol, 17b-
methyl-5b-androstane-3b,17a-
G
omez et al. (2013)
(continued)
12 E. C. PIZZATO ET AL.
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Nevertheless, the choice of hydrolysis is steroid-depend-
ent. Gomes et al. (2009) suggest the following approaches
could be used:
a. Enzymatic hydrolysis with H. pomatia
b. Chemical hydrolysis by solvolysis
c. Enzymatic hydrolysis with E. coli followed by solvolysis.
In fact, there is no consensus for steroid deconjugation or
a universal condition. The conversion of some natural steroid
metabolites into testosterone when H. pomatia is used in
enzymatic hydrolysis could promote false-positive results.
Another alternative is addition of antioxidants such as
sodium ascorbate to improve the capacity of hydrolysis,
avoiding oxidation of 5-a-reduced glucuronides, especially
11b-hydroxilated steroids, by protecting cholesterol oxidase
activity from the source of the enzyme (Christakoudi et al.
2008). According to Christakoudi et al. (2008), the best condi-
tion for the hydrolysis of 2 mL of urine sample was the add-
ition of 100 lL of commercial b-glucuronidase from H.
pomatia using 0.5 M of acetate buffer (pH 5), 10 mg of
sodium ascorbate and incubation at 55 C for 3 h.
Degradation of some steroids, increased matrix interfer-
ence and rising of impurities during extraction are considered
disadvantages for acid hydrolysis and solvolysis (Gomes et al.
2009). Enzymatic hydrolysis exclusively using b-glucuronidase
from E. coli neglects sulfoconjugate forms. Therefore, resist-
ance of two glucuronide metabolites (3a-glucuronide-6b-
hydroxyandrosterone and 3a-glucuronide-6b-hydroxyetiocho-
lanolone) was found as demonstrated in the literature
(Venturelli et al. 1995; Kotronoulas et al. 2015).
So, the combination of solvolysis and hydrolysis using
b-glucuronidase from E. coli is indicated to promote
complete cleavage of conjugated steroids (glucuronides and
sulfates; Hauser et al. 2008; Blokland et al. 2012; Pedersen
et al. 2017). However, this procedure requires two different
techniques for one propose, and this strategy could be not
viable and feasible logistically.
Abalone entrails showed interesting results to cleave ste-
roids with glucuronide and sulfate without degradation of
analytes and impurities formation during the procedure.
However, prolonged time of this procedure (20 h) could be a
logistic adversity (Ferchaud et al. 2000). An interesting
approach to promote hydrolysis is by ultrasonic energy. This
technique is able to avoid incomplete deconjugation as
described by Galesio et al. (2010). Therefore, 11 steroids
showed increase in capacity to cleave conjugated forms
using ultrasonic energy. However, steroid sulfate hydrolysis is
unknown because these conjugated molecules were not eval-
uated in this study.
A resume of the methodologies based on digestion,
hydrolysis and solvolysis is demonstrated in Table 1.
Conclusions
Although immunoassays and liquid chromatography proce-
dures allow the determination of testosterone and its metab-
olites in conjugated forms in biological fluids (urine, serum
and oral fluid), other biological matrices need the pretreat-
ment step to be carried out for the measurement of these
molecules. The importance of these procedures is based on
the biological matrix and the equipment available for this
type of measurement.
So, analysis of exogenous testosterone in biological matri-
ces is considered an analytical challenge, and the pretreat-
ment step is critical to obtain reliable results. There are many
Table 1. Continued
Biological matrix Pretreatment Procedure Analytes Reference
neutralized with 60 lL of 1 M NaOH and
evaporated to dryness. The residues were
reconstituted in 1 mL of sodium phos-
phate buffer (0.2 M, pH 7) and 250 lLof
5% K
2
CO
3
solution were added.
diol; 17b-methyl-5a-androstane-
3a,17a-diol, 17b-methyl-5b-
androstane-3a,17a-diol, 17b-
methyl-5a-androstane-3b,17a-
diol and other unknown three
metabolites
Bovine urine (1 mL) Enzymatic hydrolysis
followed by solvolysis
Samples received 1 mL of 3-(N-morpholino)-
propanesulfonic acid (MOPS) buffer
(50 mM, pH 7.0), 250 U of b-glucuroni-
dase, 200 lL of internal standard (20 ng),
and later, it was incubated at 37 C for
1 h. After that, these samples were
applied to the C-18 SPE column (500 mg)
preactivated with 5 mL of methanol and
subsequently by 5 mL of water. The col-
umn was washed with 3 mL of water and
dried with vacuum for 2 min, and the
analytes were eluted from the column
with 3 mL of methanol. The extract was
dried using a N
2
evaporator at 40 C and
reconstituted with 1 mL of ethyl acetate
and 40 lLof4MH
2
SO
4
followed by
incubation at 40 C for 30 min. Later,
320 lL of 1 M Tris buffer, and the organic
phase was evaporated using nitrogen at
40 C. After that, a total of 1 mL of 20%
of methanol was added and mixed to be
prepared for the extraction step.
a-Boldenone, b-boldenone, a-nor-
testosterone, b-nortestosterone;
a-trenbolone; b-trenbolone;
b-boldenone sulfates; a-nortes-
tosterone sulfates; testosterone
sulfates, b-boldenone glucuro-
nide and b-nortestosterone
glucuronide
Pedersen et al. (2017)
TOXICOLOGY MECHANISMS AND METHODS 13
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procedures to obtain free testosterone and its metabolites.
Hydrolysis and digestion are the most used techniques to
obtain the free testosterone and its metabolites in biological
matrices. In fact, the biological sample defines the appropri-
ate pretreatment to be used to achieve the analytical pro-
posal, knowing its advantages and limitations.
Acknowledgement
The authors thank Prof. Dr. Thiago Belarmino de Souza for highlighting
some points on the chemical structure of testosterone and its
metabolites.
Disclosure statement
The authors wish to confirm that there are no conflicts of interest associ-
ated with this publication, and there has been no significant financial
support for this work that could have influenced its outcome.
Funding
The authors thank CAPES for providing doctoral and postdoctoral
scholarships.
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