y excretion ofs conjugated
ogenous administration. Currently, the steroid profile includes the quantification of the
Research articleDrug Testingand Analysis
d: 11 March 2015 Published online in Wiley Online Library: 28 April 2015
110The screening of Tmisuse remained impossible until Donike et al.introduced the ratio between T and its 17-epimer epitestosterone(E) as an indication of T intake. Since then, the use of the T/E ratiohas been the primary marker to distinguish between endogenousconcentrations and those obtained after the exogenous administra-tion of T. A sample is considered suspicious if an abnormal value ofT/E, above the reference limit established by theWorld Anti-DopingAgency (WADA), is detected. However, for the confirmation theuse of gas chromatography coupled to combustion isotope ratiomass spectrometry (GC-C-IRMS) is mandatory in order to discernbetween exogenously administered steroids to those producednaturally in the body by their relative content of carbon isotopes.
following compounds: T, E, Androsterone (Andros), Etiocholanolone(Etio), 5-androstane-3,17-diol (5-Adiol), and 5-androstane-3,17-diol (5-Adiol). The effect of different factors on the ex-creted concentrations of these compounds has been largely stud-ied (see sections Sample preparation, Endogenous factors,Exogenous factors).
* Correspondence to: scar J. Pozo, Bioanalysis Research Group, IMIM, Hospital delMar, Doctor Aiguader 88, 08003 Barcelona, Spain.E-mail: [email protected](
by analogy with -AED, -AED and -T, it is expected that -AD come from a conjugate with cysteine. A method for the
extent to a higher analytical imprecision derived from the lower Tand E concentrations. Variation between genders were reported
Factors affecting cysteinyl testosterone metabolites
Drug Testingand Analysis
11quantification of these compounds in urine was developed andvalidated. Due to the low sensitivity observed for the direct detec-tion of the cysteinyl conjugates, the method includes a cleavageof the cysteine moiety before the analysis. Due to the lack of ref-erence material for 15-AD, a semi quantification method wasperformed using 6-T as reference standard.
The usefulness of these compounds for the detection in the dop-ing control field was also evaluated.[17,18] In particular, it was dem-onstrated that the use of 1-AED/6-AED and 1-AED/6-T ratiosas markers of T abuse improved the detection capabilities of thecommonly employed T/E ratio. The use of the ratios involving15-AD (1-AED/15-AD, 6-AED/15-AD and 6-T/15-AD) showedtheir usefulness when dealing with the topical application of T andother pro-hormones such 5-dihydrotestosterone (DHT). Addi-tionally, the ratios involvingmetabolites 6-AED and6-T exhibitedan increase after the administration of dehydroepiandrosterone(DHEA), being the ratio 6-T/15-AD the best marker for itsdetection. However, it is still unknown how the values of thesemetabolites/ratios are altered by common factors affecting the ste-roid profile.
The aim of this study is to evaluate how the concentrations andratios of the T metabolites conjugated with cysteine are affected bythe factors which are known to influence the steroid profile. Theevaluated parameters are divided in three different categories:sample preservation, endogenous factors and exogenous factors.The influence of these variables in the current steroid profile is fur-ther described below.
Since in most of the cases, the analysis of urine samples for drugtesting is not performed immediately after their collection, it is veryimportant to evaluate the effect of storage conditions.
The fact that the urine samples are collected in non-sterile condi-tions offers the micro-organisms the opportunity to grow, espe-cially when samples are stored for a long time. It is known thatmicroorganisms can cause urinary alteration by hydrolysis of glucu-ronide and sulphate and can cause oxireductase reactions ofsteroids. Therefore, bacterial activities in urine may cause sig-nificant changes in the measured steroid concentrations. The pri-mary reaction that occurs is the deconjugation of glucuronidesand sulphates.[22,23] Since in doping control tests only the combinedglucuroconjugated and free fraction are analyzed, and the influ-The inclusion of the excreted concentrations and ratios of otherminor T metabolites in the steroid profile has been presented as apowerful approach in order to increase the diagnostic specificityof ABP. Before including these metabolites in the ABP, it isnecessary to know how they are altered by common factors affect-ing the steroid profile.
Several T metabolites released after an alkaline treatmentof the urine sample (1,4-androstadien-3,17-dione (1-AED), 4,6-androstadien-3,17-dione (6-AED), 4,6-androstadien-17-ol-3-one(6-T) and 15-androsten-3,17-dione (15-AD)) have been reportedand characterized. These metabolites were shown to originatefrom a phase II cysteinyl conjugate.[14,15] For 15-AD no confirma-tion of the conjugate with cysteine was possible due to the ab-sence of a commercial standard. However, since this compoundhas been only observed after the alkaline treatment of the urine,
1 6 6 15ence of deconjugated sulphate after bacterial hydrolysis is
Drug Test. Analysis 2016, 8, 110119 Copyright 2015 John Win the concentrations of androgens exhibiting lower concentrationin female urine samples. However, no significant changes wereobserved for the T/E ratio between genders.[31,32] Other factors likeextensive exercise can also influence the steroid excretion.
Pregnancy dramatically affects thematernal excretion of steroids.The changes observed in the steroid excretion aremainly due to thefact that products of the feto-placental unit have no other exit thanthe maternal serum and urine. Significant differences wereobserved in the T/E values in the first trimester of pregnancy whencomparing with basal values. Whereas T remained almost unalteredduring gestation, a significant rise was observed for E during thefirst weeks of pregnancy leading to a decrease in the T/E ratio.
At present, pregnancy is the only influencing factor that has beenevaluated for the conjugated steroids with cysteine, and differentbehaviours were revealed. A substantial rise of 1-AED and adecrease of 6-T concentrations were observed during the first tri-mester of pregnancy. Subsequently, all the ratios involving thesecompounds were affected. However, the 15-AD and the ratiosinvolving this compound were not monitored in that study.
Finally, large differences in the steroid excretion between Asianunknown, this primary reaction might influence the measuredsteroid concentrations. In addition to that, this deconjugation isnormally associated to other transformations like uncontrolledoxidoreduction or hydrolysis reactions, whichmight lead to the al-teration of several steroid profile parameters. For instance, Marecket al. reported an increased T/E from 5.3 to 9.8, determined in acombined fraction of conjugated and unconjugated steroids.
Markers of bacterial urine degradation are the accumulation of5-androstan-3,17-dione (5-AD) and 5-androstan-3,17-dione(5-AD) which are the product of Andros and Etio deconjugation re-spectively, followed by a bacterial 3-hydroxysteroid-dehydrogenaseactivity.
Additionally, in anti-doping controls, the urine sample is collectedand divided into two aliquots (A and B). While aliquot A is analyzedimmediately upon arrival to the laboratory and may suffer severalfreeze/thaw cycles for conducting different tests, the B aliquot isstored at 20 C waiting for a possible counter analysis. Knowl-edge about the stability of the steroids to different freeze/thawcycles is critical for a proper interpretation of the results. In thecase of the urinary T/E ratio, a high stability has been observed.
Due to the homeostasis of biosynthesis and metabolism of endog-enous steroids, the amounts of steroids in 24h periods areconstant for a given individual. However, the concentrations ofthe eliminated endogenous steroids may vary with the urinary flow.The use of ratios between steroid compounds minimizes thisfluctuation. Despite this, T/E ratio can vary greater than 30 % inlongitudinal studies.[28,29] Besides, due to the influence of the dailycircadian rhythm in the steroid excretion, plasma variations be-tween 2040% for T have been reported. This circadian rhyth-micity in the production of T is reflected in a moderate circadianrhythm in the urinary T excretion.
Some small differences were reported between genders. For in-stance, while the intra-individual variations in the T/E ratio in malesis expected to be less than 30%, the observed variations in fe-males are higher due to the menstrual cycle, and also in someand Caucasian populations have been widely reported. These
iley & Sons, Ltd. wileyonlinelibrary.com/journal/dta
It is known that several exogenous factors can influence the steroid
Chemicals and reagents
(Darmstadt, Germany). Milli Q water was obtained using a Milli-Q
ence of the mono-trimethylsilyl derivatives of androsterone and
A. Fabregat et al.
Drug Testingand Analysis
112Androsta-4,6-dien-3,17-dione (6-AED) and 17-hydroxy-androsta-4,6-dien-3-one (6-T), 5-androstanedione (5-AD), and 5-androstanedione (5AD) were obtained from Steraloids Inc.(Newport, Rhode Island, USA). Androsta-1,4-dien-3,17-dione (1-AED)was purchased from NMI (Pymble, Australia). Testosterone (T),epitestosterone (E), androsterone (Andros), etiocholanolone(Etio), 5-androstane-3,17-diol (5-Adiol), 5-androstane-3,17-diol (5-Adiol), as well as the internal standardsmet