3 research outputs found
Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid
In humans, conjugation with glucuronic
acid is the most important
phase II metabolic reaction of steroidal compounds. Glucuronoconjugated
metabolites have been conventionally studied by using Ī²-glucuronidase
enzymes to release the phase I metabolites. It is well-known that
hydrolysis with Ī²-glucuronidase presents some limitations that
may result in the underestimation of some conjugates. The aim of the
present work was to develop and to evaluate liquid chromatography-tandem
mass spectrometry (LC-MS/MS) scan methods for the open detection of
steroid glucuronides in urine samples. The mass spectrometric behavior
of thirteen representative steroid glucuronides, used as model compounds,
was studied. Characteristic ionization and collision induced dissociation
behaviors were observed depending on the steroid glucuronide structure.
Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI
of <i>m</i>/<i>z</i> 141, 159, and 177, in positive
mode and <i>m</i>/<i>z</i> 75, 85, and 113, in
negative mode) scan methods were evaluated. The NL scan method was
chosen for the open detection of glucuronoconjugated steroids due
to its sensitivity and the structural information provided by this
method. The application of the NL scan method to urine samples collected
after testosterone (T) undecanoate administration revealed the presence
of two T metabolites which remain conjugated as glucuronides after
an enzymatic hydrolysis of the urine. 3Ī±,6Ī²-Dihydroxy-5Ī±-androstan-17-one
(6Ī²-hydroxyandrosterone) glucuronide and 3Ī±,6Ī²-dihydroxy-5Ī²-androstan-17-one
(6Ī²-hydroxyetiocholanolone) glucuronide were established as
the structures for these metabolites, by comparing the structure of
the steroids released after chemical hydrolysis with reference materials.
An increase of 50ā300-fold of these metabolites after oral
administration of T undecanoate was observed, proving that their determination
can be useful in the doping control field. Moreover, these results
exemplify that significant information might be missed, unless direct
methods for the determination of steroid glucuronides are employed
Constant Ion Loss Method for the Untargeted Detection of Bis-sulfate Metabolites
The
untargeted detection of phase II metabolites is a key issue
for the study of drug metabolism in biological systems. Sensitive
and selective mass spectrometric (MS) techniques coupled to ultrahigh
performance liquid chromatographic (UHPLC) systems are the most effective
for this purpose. In this study, we evaluate different MS approaches
with a triple quadrupole instrument for the untargeted detection of
bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were
synthesized and their MS behavior was comprehensively studied. Bis-sulfates
ionized preferentially as the dianion ([M ā 2H]<sup>2ā</sup>) with a small contribution of the monoanion ([M ā H]<sup>ā</sup>). Product ion spectra generated from the [M ā
2H]<sup>2ā</sup> precursor ions were dominated by the loss
of HSO<sub>4</sub><sup>ā</sup> to generate two product ions,
that is, the ion at <i>m</i>/<i>z</i> 97 (HSO<sub>4</sub><sup>ā</sup>) and the ion corresponding to the remaining
monosulfate fragment. Other product ions were found to be specific
for some structures. As an example, the loss of [CH<sub>3</sub> +
SO<sub>3</sub>]<sup>ā</sup> was found to be important for several
compounds with unsaturation adjacent to the sulfate. On the basis
of the common behavior of the bis-sulfate metabolites two alternatives
were evaluated for the untargeted detection of bis-sulfate metabolites
(i) a precursor ion scan method using the ion at <i>m</i>/<i>z</i> 97 and (ii) a constant ion loss (CIL) method
using the loss of HSO<sub>4</sub><sup>ā</sup>. Both methods
allowed for the untargeted detection of the model compounds. Eight
steroid bis-sulfates were synthesized in high purity in order to quantitatively
evaluate the developed strategies. Lower limits of detection (2ā20
ng/mL) were obtained using the CIL method. Additionally, the CIL method
was found to be more specific in the detection of urinary bis-sulfates.
The applicability of the CIL approach was demonstrated by determining
progestogens altered during pregnancy and by detecting the bis-sulfate
metabolites of tibolone
Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry
The use of untargeted metabolomics
for the discovery of markers
is a promising and virtually unexplored tool in the doping control
field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole
Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure
liquid chromatography, are excellent tools for this purpose. In the
present work, QTOF and Q Exactive have been used to look for markers
for testosterone cypionate misuse by means of untargeted metabolomics.
Two different groups of urine samples were analyzed, collected before
and after the intramuscular administration of testosterone cypionate.
In order to avoid analyte losses in the sample treatment, samples
were just 2-fold diluted with water and directly injected into the
chromatographic system. Samples were analyzed in both positive and
negative ionization modes. Data from both systems were treated under
untargeted metabolomic strategies using XCMS application and multivariate
analysis. Results from the two mass spectrometers differed in the
number of detected features, but both led to the same potential marker
for the particular testosterone ester misuse. The in-depth study of
the MS and MS/MS behavior of this marker allowed for the establishment
of 1-cyclopentenoylglycine as a feasible structure. The putative structure
was confirmed by comparison with synthesized material. This potential
marker seems to come from the metabolism of the cypionic acid release
after hydrolysis of the administered ester. Its suitability for doping
control has been evaluated