Long Term Metabolites: Historical Aspects and the Cologne Strategy

Publication: Chapter in Book/Report/Conference proceedingConference contribution - Article for conferenceResearchpeer-review

452 Downloads (Pure)

Abstract

Extended Abstract

Historically, the wording long term metabolite was first used in the Cologne laboratory in 1995. Unfortunately, this was the year when Manfred Donike, the head of the Cologne laboratory, director of the Institute of Biochemistry of the German Sports University and member of the IOC Medical Commission subgroup Doping and Biochemistry, unexpectedly died. He was the scientific “father” of the anti-doping laboratory in Cologne and published his first scientific articles – dope analysis based on GC separation techniques - in 1966. His respectable work in the fight against doping was a milestone. He untiringly advanced anti-doping science and improved analytical methods to detect doping substances in biological samples of athletes. We all in the Cologne laboratory and also scientists in other laboratories worldwide have learnt from Manfred Donike’s ideas.The wording long term metabolite is related to the detection of exogenous anabolic androgenic steroids (AAS). When in the beginning of the 1970ties the misuse of AAS became aware Manfred Donike and also the laboratory in Montreal focused their analytical investigations on an unambiguous identification of that group of substances by GC-MS. Beside of classical sector field MS systems such as the Atlas MAT CH-5, which was already used during the Olympic Games in Munich 1972, new and less expensive MS instruments became available at that time and in the 1980ties quadrupole separation techniques followed. These instruments were also called single bench top MS-Systems and connected to a gas chromatograph. The first methods could be developed to successfully identify AAS.
But the problem remained with the testing itself as AAS were applied in the training period and doping tests were only conducted at competition events. This was less effective. Since 1988 with the introduction of out-of-competition testing the misuse of AAS could be controlled more effectively. But out-of-competition tests were not uniform and internationally not harmonized.That was one of the reasons why in Cologne Manfred Donike tried to improve the detection of AAS, even for long periods of time after their last administration.

1992 - High resolution mass spectrometry (HRMS)
One possibility to achieve longer detection times was the introduction of HRMS in anti-doping control analysis by Manfred Donike in 1992. With this HRMS technique it was feasible to improve the detection limits of AAS mainly by reducing the biological background compared to MS techniques with a single mass resolution.

1995 - Identification of long term excreted metabolites of AAS
The other possibility to improve detection times was to identify metabolites of AAS which were not so dominant at the beginning of the excretion period but showed prolonged half-lives and were excreted for a longer period of time.
17-Epimerization and 17,17-dimethyl-rearrangement process
In Cologne we mainly focused on 17a-methyl steroids and investigated their metabolism. A sulfate conjugation of the sterically hindered 17ß-hydroxy group became of particular interest. The 17ß-sulfate of 17a-methyl steroids once generated was instable and a 17-epimerization could be identified as a metabolic pathway. In 1989 this new metabolic pathway was described for metandienone in the metabolism of horses by Edlund et al. We investigated this process for a series of 17a-methyl steroids and could identify further water elimination products andrearrangement products with a 18-nor-17,17-dimethyl-13-ene C/D-ring structure.
Especially in the metabolism of metandienone we were able to identify a rearrangement product of metandienone with a 18-nor-17,17-dimethyl-13- ene structure which was further reduced in the A-ring yielding 18-nor-17,17-dimethyl-5ß-androst-1,13-dien-3a-ol (also named 18- norepimetenediol or 18-nor-EMD). In the same excretion urines we could also confirm a 17- epimer 17ß-methyl-5ß-androst-1-ene-3a,17a -diol (epimetenediol, EMD). Both metabolites 18- nor-EMD and EMD could be detected longer than the classical, known metabolites of metandienone at that time.The screening and confirmation of both of these long term metabolites in combination with the use of HRMS analysis led to an increase in the number of adverse analytical findings (AAFs) for metandienone. Furthermore, the detection of stanozolol metabolites was improved when using HRMS. In 1995 the Cologne laboratory could report additional 63 AAFs for metandienone (total number was 78) and additional 9 AAFs for stanozolol (total number was 22) only detected by HRMS. By means of low resolution MS alone we would have missed 80% of the metandienone and 40% of the stanozolol AAFs.

1996 – Long term excreted metabolite of dehydrochloromethyltestosterone (DHCMT)
At the same time, in 1995/96, we also concentrated on the metabolism of DHCMT and identified a long term excreted metabolite but with a 6,16-dihydroxy structure.This was published in 1996 and especially in Germany we were able to report some AAFs on the basis of considering this metabolite. At that time we were of the opinion that DHCMT (Oral- Turinabol®) was only the classical, misused AAS in the East German Democratic Republic (GDR) and after the reunification of Germany we expected that some athletes would continue to misuse this anabolic steroid. About twenty years later, we see an extremely high number of misuse of this substance mainly in Eastern Europe countries.

2006 – Confirmation of a new long term metabolite of metandienone – 18-nor-17ß-hydroxymethyl-17a-methylandrost-1,4,13-trien-3-one (NW, “night watchman”)
The detection of this long term excreted metabolite of metandienone was more or less a discovery by chance. The routinely applied LC/ESI/MS/MS screening method for the detection of dihydroexemestane, the main metabolite of exemestane (a banned aromatase inhibitor), showed in some doping control urine samples an interfering signal for an unknown substance demonstrating a product ion at m/z 135 generated from the protonated molecular ion (M+H)+299. Interestingly, in several samples with this unknown peak we could additionally identify EMD, a long term metabolite of metandienone (see above). Further investigations on this metabolite were successful and contributed to identify the interference as a new metabolite of metandienone with the structure 18-nor-17ß-hydroxymethyl-17a-methylandrost-1,4,13-trien-3- one (NW). This was unexpected and for the first time we confirmed that a rearrangement product with 18-nor-17,17-dimethyl-13-ene C/D-ring structure was hydroxylated at the 17ß- methl group. Based on the history of the discovery we abbreviated this metabolite with the naming “night watchman” (NW). Additionally, but with lower intensity, an epimer of the NW metabolite with the hydroxylation at the 17a-methyl group could be identified. With the introduction of this new metabolite in our screening methods for AAS in 2006, in the same year we were able to report about 53 additional AAFs for metandienone which before this new finding was discovered would have not been detected with the known metabolites.
The investigation of similar metabolic pathways producing long term excreted metabolites of other 17a-methyl steroids was successful and results were published also by other laboratories. I would like to mention two anabolic steroids, oxandrolone and DHCMT, for which the Cologne laboratory was able to report a high number of AAFs by introducing such long term excreted hydroxylated rearrangement metabolites into existing screening methods. For oxandrolone we could identify similar metabolites like for metandienone NW with 18-nor-17ß-hydroxymethyl- 17a-methyl -13-ene structure and an 17-epimer with similar intensity. Comparable to the NW metabolite of metandienone the A-ring remains non-metabolized. The first AAFs for oxandrolone long
term metabolites were reported in Cologne in 2012. The investigations confirming these new
metabolites were published in 2013.

2013 Dehydrochloromethyltestosterone (DHCMT) - New long term metabolites identified by Sobolevsky and Rodchenkov.
A high number of AAFs could be reported for the anabolic androgenic steroid DHCMT when Tim Sobolevsky and Gregory Rodchenkov published several new long term metabolites of DHCMT in 2012 which were similarly metabolized at the C/D-ring as described for metandienone NW but subsequently the A-ring was partially and/or completely reduced. Among these new metabolites especially the fully A-ring-reduced metabolite could be detected for the longest period of time after the last application of DHCMT. Following a single excretion study of an oral application of 20 mg of DHCMT which was performed in Cologne in 2016 this new long term excreted metabolite of DHCMT could be detected for 250 days (approximately eight months).Actually, this metabolite was successfully synthesized as reference material by the working group of Günter Gmeiner in Austria and the exact configuration of the A/B-ring, the 3-hydroxy- and 4-chloro groups will be published soon.
In 2013 we introduced this new metabolite into our screening methods for AAS and were able to report 61 AAFs for DHCMT in the same year compared to only one positive finding in the year before.
2012/2013 - Improved detection of stanozolol metabolites by HRMS orbitrap technology
At the same time, in 2012, we started the investigation to develop an improved method for the detection of stanozolol applying a new high resolution mass spectrometry technique called orbitrap technology. With this new sophisticated MS technique we were able to identify stanozolol and epistanozolol conjugated metabolites as N-glucuronides. The N-glucuronide of epistanozolol was detected up to four weeks after a single oral dose of 5 mg and was therefore confirmed as a long term excreted metabolite. When applying this method for routine doping control samples we increased our reports for stanozolol AFFs from about 12 in 2011 to 64 in 2012 (39 were only detected by the new orbitrap HRMS method) and to 150 AAFs in 2013 (146 were only detected by the new orbitrap HRMS method). However, the pattern of metabolites was changing only in a few samples to the long term metabolite of stanozolol. In most of the analysed samples we obtained a pattern with all metabolites. An explanation for this observation is still missing. Up to now it can only be speculated about the reasons, whether the substance is mainly administered by injection or given orally in low doses, which both may explain the results.
2013 The new Cologne strategy for the detection of long term excreted metabolites of AAS using hydrogen isotope ratio mass spectrometry (HIRMS)
In 2013 my colleagues Mario Thevis and Thomas Piper started a project to identify long term excreted metabolites of AAS by use of hydrogen isotope ratio mass spectrometry (HIRMS) to calculate 2H1/1H1. The isotope 2H1 of hydrogen is also named deuterium (D) and, therefore, the isotope ratio of hydrogen can also be named D/H. As the natural occurrence of D is only 1 out of 6500 H atoms (0.015%), the use of deuterated compounds for excretion studies would substantially increase the signal intensities of the metabolites. As a result concentrations of a deuterium labeled metabolite in the lower ng/mL range will result in a detectable signal above the naturally present D-background in IRMS analyses. This idea was successfully tested by means of an elimination study using trideuterated metandienone (17a-C2H3-17ß- hydroxyandrosta-1,4-dien-3-one, D3-metandienone). For the gas chromatography / thermal decomposition-isotope ratio mass spectrometry (GC/TC-IRMS) analysis selected urine samples were collected following the application (up to 477 h, approximately 20 days). The urine samples (20 ml) were concentrated via a SPE step, glucuronidated and sulfated metabolites
were separated and hydrolyzed. All extracts were further cleaned up by HPLC fractionation and acetylated with acetic anhydride for analysis.After the Gparation of the different fractions the substances are thermally decomposed (TC) generating H2 and DH and the GC/TC-IRMS is measuring the two masses m/z 2 and m/z 3 respectively. For hydrogen isotope ratio (HIR) measurements at natural abundance, the ion chromatogram at m/z 2 shows consistently higher intensities than the corresponding chromatogram at m/z 3, although the abundance of m/z 3 is constantly amplified by a factor of 1000. When a deuterium labeled metabolite is eluting the GC-column the ratio of m/z 3 (DH) to m/z 2 (H2) is obviously increased. This allows the detection of unknown metabolites. In the above-mentioned excretion study with D3-metandienone the metabolite with the longest detection time measured by hydrogen IRMS was the known long term excreted metabolite of metandienone NW (see above). The sensitivity for the actual used method was about 0,25 ng/ml for a deuterium labeled metabolite.
The presented study demonstrates the value of GC/TC-IRMS in metabolism studies of long term excreted metabolites. In general this technique is not limited to AAS and it can be applied to other doping substances when the detection and identification of metabolites are required for a sensitive dope control.

Literature for this new strategy:
Thevis M, Piper T, Horning S, Juchelka D, SchänzerW. Hydrogen isotope ratio mass spectrometry and
high-resolution/high-accuracy mass spectrometry in metabolite identification studies: Detecting
target
compounds for sports drug testing. Rapid Commun. Mass Spectrom. 2013, 27, 1904–1912
Original languageEnglish
Title of host publicationLecture held at the 16th Annual USADA Meeting
Number of pages4
Publication date01.10.2017
Publication statusPublished - 01.10.2017
EventAnnual USADA Symposium on Anti-Doping Science - Orlando, USA/United States of America
Duration: 29.09.201702.10.2017
Conference number: 16

Fingerprint

Dive into the research topics of 'Long Term Metabolites: Historical Aspects and the Cologne Strategy'. Together they form a unique fingerprint.

Citation