Critical Assessment of the Current WADA Approach for the Detection of 4-Chlorodehydromethyltestosteron

This paper is focused on application of method for detection of novel metabolites of 4-chlorodehydromethyltestosterone (known as oral turinabol, OT), which were postulated as long-term metabolites. The method started to be applied without rigorous validation, verification and excretion assay assisted as evidences for existence of the declared detectable compounds as true metabolites derived after OT biotransformation. This method has been started to use almost in its original form which arose in 2012 year for the very first time and never been revised and re-confirmed in peer-reviewed journals. Usually WADA encourages accredited laboratories to publish their results of methods development, validation and specific excretion studies in peer-reviewed journals. However, an incorrect method with neglected validation design and implicit data contradictions is currently widely applied. The author of this presented paper was athletes’ representative on repeated occasion and has been provided with several full documentation packages of OT novel metabolites analysis. Thus, the author makes its own conclusion based on its own evidences of that currently employed method and data analysis is exactly the same declared in a wrong way as emerged from the original paper.


Introduction
In summer of 2016 year WADA initiated re-tests of urinary samples obtained from the Olympic Games 2008 and 2012. The re-tests aimed to survey the newly discovered metabolites of turinabol (chemical name: 4-chlorodehydromethyltestosterone, CID ref. number 98521), which are characterized as long-term compounds retained in human body for up to 45 -50 days after administration of turinabol. These proposed long-term metabolites are 4-chloro-17α-methyl-5β-androstan-3α,16,17β-triol (M1), 4-chloro-18-nor-17β-hydroxymethyl,17αmethyl-5β-androsta-1,13-dien-3α-ol (M2), 4-chloro-18-nor-17β-hydroxymethyl, 17α-methyl-5β-androst-13-en-3α-ol (M3) and 4-chloro-18-nor-17β-hydroxymethyl,17α-methylandrosta-4,13-dien-3α-ol (M4) (Figure 1). Such properties allow probably unmasking those athletes which avoided punishment due to imperfection of previously employed analytical approach based on the detection of short-term metabolites. Several professional athletes over the world were banned or suspended due to the presence of the prohibited substance of winners of the Olympic Games. Almost all of these athletes have never been tested as positive before. However, rules violation left them only very strict margin of possible actions. If test of the sample has been performed according to International Standards for Laboratories (ISL), the only way for athlete is to demonstrate that WADA accredited laboratories made a significant departure from the ISL and that this departure reasonably caused erroneous adverse analytical finding (AAF). Basically, WADA inclines to believe that result of a test was conducted in a regular manner since the method applied for analysis was perfect and impeccable. Nevertheless, there was an extremely severe point of view in Nature [1] journal delivering shortages in WADA's guidelines for prohibited substances identification criteria. This critical paper opened a few significant problems in data analysis and decision criteria accomplished by scientist associated with WADA. The main conclusion was accommodated in "Nature believes that accepting legal limits of specific metabolites without such rigorous verification Figure 1. Structure of 4-chlorodehydromethyltestosterone (DHCMT) and proposed structures of long-term metabolites (M1, M2, M3 and M4) of DHCMT. Assumed structure of metabolites is reproduced from the manuscript [2]. goes against the foundational standards of modern science, and results in an arbitrary test for which the rate of false positive and false negatives can never be known" [1].
The newly discovered stable metabolites of turinabol were declared for the very first time in manuscript entitled "Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine", which was published in 2012 in Journal of Steroid Biochemistry and Molecular Biology by Tim Sobolevsky and Grigory Rodchenkov [2]. Among the proposed metabolite, the M3 (Figure 1) was the most promising one and depicted broad detection window as assumed. The method used by WADA laboratories for detection of these metabolites is based exactly on this publication. For the past four years, since the manuscript has been published until nowadays, it gained totally 25 citations in different journals [3]. As demonstrated on the SCOPUS preview page [3], most of the authors citing the manuscript are affiliated with WADA laboratories or institutes in one way or another. Only small portion of authors are not related to WADA, which implies that interest in the provided information outcomes from the WADA-affiliated researchers preferably, although anabolic steroids are extensively investigated in cell signaling, drugs development, inhibitory activity, etc. Apparently, this information is not as intriguing for other researchers as expected. It could be seen that the context of citing this manuscript is restricted to referring on either derivatization procedure [4] [5] or strategies for research completely different steroids in blood or urinary matrices [6] [7]. Therefore, the proposed method and observed metabolites may accommodate questions for their validity.
Specifically, it should be noted that WADA technical Document TD2015IDCR [8] (which is actual document when all analysis were being made) requires three diagnostic ions to be used. However, as we will see later, the mass shift for most of ions is −90 u or −103 u which corresponds to loss of TMS (trimethylsilanol) or CH 2 O (TMS) from polyhydroxylated steroids with vicinal or primary hydroxyl group, respectively. It should be specifically pointed that these ions do not provide much diagnostics information; notwithstanding, there is nothing in WADA's technical documentation to prevent the analysis from using such ions. A precedent of employment invaluable diagnostic ions (elimination of methyl group, [M-CH 3 ] + ) has already been in WADA method development story and related to detection of 3'-OH-stanozolol molecule [9]. That time WADA again allows itself to relax identification criteria. Hence, the ions used for discrimination of turinabol-derived molecules do not carry valuable information. Below we will try to review some explicit aspects of the established analytical method and information provided in the manuscript.

Time Elapsed from Discovering of New Metabolites and Absence the Confirmation of Their Existence
As mentioned before, four years passed from the date of publication and disco- Sports University in Cologne), published a manuscript about routs of metabolism of turinabol [11]. This is especially interesting point due to the fact that Prof.
Thevis and co-authors conducted complete study of OT metabolism by using in vitro whole-cell system of cytochromes P450. This group of scientists revealed several metabolites of OT, made suggestion of their structures based on massspectrometry data, and confirmed the assumed structures by nuclear magnetic resonance spectroscopy. Another important point in this publication, dedicated to metabolism of turinabol, is lack of mentioning of the stable metabolites discovered in 2012 by Rodchenkov et al. [2], which are currently extensively used by WADA-accredited labs as markers of turinabol ingestion. Instead, Prof. Thevis and co-authors listed several metabolites (11β-OH-OT, 11β, 18-diOH-OT, 11β-OH-OT-18-al, 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT) produced by either of cytochromes 11B1, 11B2 and 11A1 [11], but no one of the mentioned metabolites is related to those stated and proposed in 2012 year publication [2] and used by WADA-accredited laboratories. Among other findings, the authors concluded that "these findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies" [11]. Thus, still the metabolism of this prohibited substance is not completely studied.
For years since the publication about newly discovered stable metabolites came out, some sufficient time has passed to provide good experimental evidences for the existence of the proposed molecules. There are numerous possibilities among mentioned above, and one of them is administration of deuterated steroids followed by the identification of the respective deuterated urinary metabolites using hydrogen isotope ratio mass spectrometry (IRMS), as stated, for example, by Dr. Hans Geyer from Center for Preventive Doping Research in Cologne in his publication of 2014 year [5]. So, both WADA-affiliated researchers provided evidences of any newly discovered molecules passing through specifically designed experimental work and routine analysis, in order to convert assumptions into evidence. Any new molecules were recovered and confirmed in many works by (1) in vitro experiments, (2) synthetic substances, (3) other alternative approach, such as NMR spectroscopy, and this line was honored everywhere except for the method discussed. A complete assay of metabolic molecules with rigorous study, excretion assay and structures confirmation, for example androgen receptor modulator, is mandatory practice when findings to be applied in doping control [12]. Nevertheless, no one confirmation experiment has been conducted for all this time. Unfortunately, it leaves the new metabolites as an illusion in which merely want to believe. In this way, the discovered structures needed to be re-confirmed because evidences of their existence and their origination from OT are not represented till nowadays. If WADA accredited laboratories do have data of method validation and excretion study, they must represent it in open access peer-reviewed publications.

Lack of Negative Controls and Enormous Number of Candidate Molecules
The design of experimental workflow for survey and characterization of new stable metabolites of OT consisted of several steps. Initially, the researchers looked for the candidate molecules that probably could be originated from the prohibited substance. For this purpose, the researchers used 10 biological samples (urine samples) available as laboratory reference library [2]. These samples were previously confidently confirmed for the presence of well-known "old" metabolites of turinabol, so-called short-term metabolites. Thus, these samples [13] [14], giving specific signal for isolation and recognition of turinabol-derived molecules. In addition, this data could also be packed as evidence of absence of C3-oxo function which would be overshadowed by string absorbance of C=C-C=O system in A-ring of steroid if it presented [14]. This advantage (presence of chlorine) has not been employed by researchers for the preliminary survey of candidates and routine has not been facilitated. Additionally, presence of double bound C=C near to tertiary carbon atom for some metabolites (like M4 and its epi-M4) grants additional advantage for fast recognition by increasing absorbance (hyperchromic effect) in region near to 270 -280 nm. Unfortunately, no UV spectrum scanning has been performed and only detection at the certain wavelength of 197 nm was accomplished for fractions collection. Probably, low selectivity of the adjusted UV irradiation detector may be due to the fact of avoidance of possible losses the information during survey procedure, but selective adjustment was enabled. It would be interesting to see such UV spectrum scans for the selected fractions, that contained these stable metabolites, but now it seems impossible. It is merely very convenient to use only single common wavelength of λ = 197 nm, which is absolutely unselective and more appropriate in combination with other UV waves, because in this case many questions and doubts cannot be addressed.
This question will turn back our attention further, when we will go to discus-

Allegation about Detection Window of Novel Stable Metabolites
In the section "Results and discussion", subsection 3 days …" [2]. The term "could be estimated" does not mean "validated" or "confirmed". These authors wrote later in the text: "An additional controlled excretion study is needed to fully evaluate the time at which novel metabolites can be detected" [2]. In summary, it is clearly demonstrated that the detection window of 40 -50 days for so-called novel metabolites is simply assumed to be in so way, and based on the assumption of relative concentration. It is also typical misleading error to suggest the detection window of a new metabolite based on the known data for resemble structure. For example, detection window for betamethasone and its epimere dexamethasone are differed up to 3 times. Only correctly designed excretion assay may provide evidences. There were no any experiments of post-administration analysis to reveal time of biotransformation of turinabol into the certain metabolites, to detect exactly these metabolites in the urine samples after designed administration of turinabol and confirm their detection window, elimination rate as well as structures. We also should mind, that till nowadays there is no any experimental and scientific data regarding to independent observation of these metabolites after administration of turinabol, and no data available regarding real, but not "estimated", observation of detection window of novel metabolites. According to literature search, no one laboratory over the world, even among WADA-associated laboratories [11], has still seen these molecules except for authors. Again, authors stated that additional controlled excretion study is needed, but such study has not been conducted by any independent researcher and by WADA-accredited laboratories, since information of the results is not available anywhere.
It is very dangerous game which some laboratories try to play, because they employ a method which is not validated through specific assay with standards or alternative approaches, not fully controlled due to absence of correct negative control samples collection, not examined on presence of interference from other non-prohibited substances. The method is still not confirmed by specific post-administration excretion assay to reveal the declared metabolites from the living system, in vivo. Eventually, it is confusing that WADA-accredited laboratories rely to half-stuff incomplete method that requires severe and rigorous re-vision: found some confusing molecule in probably suspicious samples (that were clear in previous analysis) using non-validated method for detection of unconfirmed metabolite for which detection window was only estimated based on subjective assumption. That is what we get eventually now. The situation bears similarity to the "meldonium saga". After disclosure that the pharmacokinetic data of meldonium elimination and accumulation were obscured, WADA has decided to carry special investigation and research studies, and two best laboratories (in Lausanne and Cologne) were implicated. Apparently, these metabolites need to be taken for careful prior investigation before introducing in routine laboratory screening.

Contradiction of Presented Findings
Deep study for the details of the method revealed several contradictions of the presented data and their description. We should mention here, that these con- and their believability on corresponding novel metabolites, more data of this experimental workflow is necessary to be analyzed and examined. The provided spectra [2] and experimental data were obtained on GC-MS triple-quadrupole mass spectrometers (QqQ). Obviously, it means that data analysis and processing as well as structures prediction were performed using only low-resolution spectra. It is true, that QqQ is a conventional way for routine analytical research, but usage of low-resolution and low mass accuracy spectra for spectrum match structures elucidation is absolutely wrongdoing way. The low mass accuracy MS (QqQ) cannot distinguish a very narrow mass difference like, for example, between phosphorylation and sulfonation. Neutral loss of hydrogen chloride can successfully be confused with sequential elimination of hydrogen fluoride (which is included in many pharma) and methyl radical, for example. Thus, obtaining of high resolution spectra of the proposed structures in the isolated fraction would be highly desirable to elucidate full fragmentation spectra and mass accuracy estimation.
We will start from the metabolite M2 ( Figure 2) which spectrum is represented on Figure 4 of the manuscript (page 125 of the [2]). This metabolite (M2) is especially discussed by authors due to its high resemblance to another metabolite, M4 ( Figure 1). In authors' proposition, their structures differ only by location of double bond in A-ring of steroidal core, which causes spectra similarity for these molecules, however M2 is likely featured by some specific diagnostic ions for its detection and discrimination.
In the text of the manuscript the authors wrote: "At the same time for M2, the ion at m/z 377 sequentially eliminates trimethylsilanol and hydrogen chloride followed by the cleavage of the A-and B-rings between C-2/C-3, C-5/C-10 and C-7/C-8 to form the fragments at m/z 287, 251 and 185, respectively" [2]. A closer look on the Figure 2, to which this statement refers, gives us contradictive information ( Figure 2).
In the reality, the fragment ion 287 and 251 (Figure 2 and Figure 3) cannot be the products of sequential cleavage of A-and B-ring as postulated by the authors. As shown in the Figure 2, the ion 287 is the product of TMS (trimethylsilanol) group elimination. At this stage of fragmentation, the A-ring still remains intact and no cleavage between C2/C3 atoms can be inspected. There is additional detail in the spectrum giving evidence that this ion is a product of TMS group loss and this ion cannot appear in spectra after elimination of hydrogen  chloride: the mass peak of 287 is characterized by isotopic distribution which is highly specific for chlorine atom (Figure 3). The chlorine gives main isotopes of 35 and 37 which are stable isotopes and their natural molar fraction input is 0.7576 and 0.2424, respectively, according IUPAC Technical review for atomic weight of elements [15]. This leads to a conclusion, that the ion 287 still carries chlorine and cannot be produced after A-ring cleavage and loss of hydrogen chloride, as declared by authors. So, the conclusion of the authors goes in conflict with spectrum data.
The ion 251 (Figure 2 and Figure 3) cannot be the product of cleavage A-and B-ring at C2/C3 and C5/C10 atoms as well, because this ion is just a product of hydrogen chloride loss. It is clearly demonstrated after detailed investigation of this ion due to absence of chlorine-specific isotopic distribution. Thus, it means that ion 251 is chlorine-deficient ion with intact A-and B-rings, rather than destroyed rings as stated in manuscript.
The ion 185 cannot be the product of cleavage between C7/C8 as well, because these atoms (C7 and C8) are located in B-ring and the atom C8 is a stacked atom between B-and C-ring. However, by simple calculation it is clearly seen that ion 185 is a product of exactly A-ring cleavage. Even though, origination of this ion still debatable because ion with m/z =185 is mostly featured for 1-ene-3-keto steroids, however the authors declared that all novel metabolites are 3-OH function and no one has 3-oxo function. So, their conclusions are highly speculative.
The most true ion in these spectra of M2, M3 and M4 is ion 147, which is well characterized for 1-or 4-ene steroids, and the ion is displayed everywhere. This is very first ion which is related to exactly A-B-rings cleavage, but which has not been mentioned by authors. Probably, authors did not mentioned about this ion due to considering it as a product of TMS-group migration which is known phenomenon for steroids [16] with more than one TMS-groups and giving an   tabolite. But the precursor ions are not shown in the spectra provided for manuscript by some reasons (however, in the same manuscript spectra with existing precursor ion of "old" metabolite I is depicted in the Figure 1 of manuscript [2]). Notwithstanding, the ion 379 (after elimination of CH 2 O (TMS)) of M3 is also suitable to provide some kind of elemental composition. It is known that ions spectra can be used to calculate the number of carbon atoms in molecule.
For this purpose we need to know relative abundances of quasi-molecular ion and its first isotope ( Figure 6). Specific isotopes distribution due to chlorine atom and input of silicon atom isotopic distribution should be considered in this case. By this calculation we can estimate, that the number of carbon atoms is other hand, if one takes for calculations C = 26, which is obtained number from the presented spectrum, we will get the final number of DBE = 8, which is impossible in this structure even if we consider two double bonds in D-ring of the proposed structure (one originally existed and one derived after unsaturation). It is very questionable: either the structures are impossible, or spectra are not fully related to the proposed structures, but rather fitted to display the steroidal picture.
More details and conclusions can be obtained about the novel metabolites, their confidence and validity of the method. Despite a huge number of discrepancies and remarks found in the basic manuscript by Rodchenkov et al. [2], WADA-accredited laboratories use this method as highly confident and believable in spite of absence of control samples, post-administrative assay, lack of real standards, contradictions to previous and current research and many questionable aspects.

Concluding Remarks
Doping control as an analytical science always demonstrated a growing interest to accuracy, validity and precision. Actually the method is used by all WADAaccredited laboratories and has been recently employed for re-analyses of samples collected during 2008 and 2012 Olympic Games. Nobody of WADA-affiliated scientists has ever raised the question about validity of the used method; however lots of discrepancies are on the face of the presented material. The authors of "fresh-baked" questionable method know for sure that the scientific community (not only WADA-affiliated) should have been attracted to the discussion, but there was no discussion. There are many challenges of the discussed method that meet such problems as absence of negative controls, confirming absence of interference and side-effect from matrix and other substances, and absence of post-administration assay, confirming that the proposed structures derived from turinabol; therefore the conclusion of detection window for the novel metabolites is invalid.
It is assumed that synthetic standards of the proposed metabolites may enhance (or, in contrary, reduce) the postulated statements in the discussing paper.
Special attention should be attracted to inconsistence of the proposed structures with those found and validated by authors already after the publication of the presented method. Although ISL rule does not force laboratories to provide evidences by alternative approaches, but science usually requires confirming and re-confirming. In case of discovery of new substances, confirmation is strictly required instead of just believing. Eventually, huge discrepancies between presented data and its interpretation, and some wrong conclusions arise many questions about whether it has been done intentionally or not. Data analysis and additional data processing is highly desirable. If WADA accredited laboratories do have the internal data of method validity, including excretion study and high-resolution full fragmentation spectra, they have to highlight them for open access to provide method integrity. Otherwise, it seems that reputable organization employs mystification instead of science.
These issues are described in our critical review of the method for detection of long-term metabolites of turinabol, and altogether lead to the main conclusion of inability to apply the method in its current state; thus, the results cannot be absolutely believable and require a careful revision by wide scientific community.