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DoCoLab (DopingControleLaboratorium)

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Abstract Bachelor Project FBT 2019-2020: Validation of three GC-MS/MS confirmation methods for the detection of doping products

The world of doping is everchanging. This leads to doping laboratories having to constantly adapt to this everchanging environment. For de last couple of years DoCoLab used a single GC-MS/MS based confirmation method for all the doping products that are detected using GC-QTOF. Due to recent demands stated by the World Anti-Doping Agency (WADA), DoCoLab has decided to make three new confirmation methods derived from the one they have been using.

These three new methods consist of one specifically for small stimulants, one for endogenous steroids and one big one for all exogenous compounds detected using GC-QTOF. Of course, these new methods can’t be put into practice instantly since DoCoLab is an accredited lab and the careers of athletes are on the line. So, in order to assure that the methods work as intended, they must be validated.

The process of these validations is described in this thesis. The parameters used for the validation of these methods are imposed by WADA. They differ depending on if the method is quantitative or qualitative. During the course of this thesis two qualitative methods and one quantitative method will be validated.

The first method to be validated is the method for small stimulants, since DoCoLab needs this method to be able to report small stimulants at a lower concentration. This method is a qualitative one, so the validation is based on peak recognition. Peak recognition is based on retention time and ion-ratios. This method is completely validated for the minimum required proficiency level (MRPL). All that remains to be done is the reanalysis of some analytes that didn’t make the cut on the ½ MRPL in the initial analysis. This due to a problem that probably occurred during the injection of a sample.

The second method is a quantitative method for the confirmation of endogenous steroids. This method is validated with parameters such as linearity, repeatability… Sadly due to the worldwide outbreak of Corona virus the validation of this method and the final method were done from home. This method is completely validated and ready to be used for the quantification of endogenous steroids. All parameters were validated and there are no problems in the method.

The method for exogenous compounds is validated in a similar fashion to the method for stimulants. Because of Corona virus, there was no longer time to generate the data needed for the third method. As an alternative, the data used in the validation of this method is data from an earlier validation. A very large portion of the compounds in this method have been validated. Some remain unvalidated due to poor detector response or due to a shift in the retention time.



Doping control happens in two stages: a screening stage and a confirmation stage. Today only one pH and solvent are used to extract doping products. Also, some products extract better in a basic environment and some in a more acidic environment. Because doping control makes use of many low concentrations, an as high as possible extraction recovery is needed. To get this highest recovery a different extraction solvent and pH could be needed. The focus of this study is to find the best solvent and pH for each component that is prohibited in sports for analyses with the gas chromatography-mass spectrometry. These components are extracted with a liquid-liquid extraction, derivatized and analyzed with the time of flight gas chromatography-mass spectrometry.

The different solvents used are:

  • Ethylacetate
  • Pentane
  • Methyl tert-buthyl ether (MTBE)

The different pHs used are:

  • 5,2
  • 7
  • 9,6
  • 14

These combination test showed the following solvent and pH give the best results. These results are generalized and there are a few exceptions.

  • Anabolic agents: MTBE and pH 9,5
  • Beta-2 Agonist: MTBE and pH 9,5
  • Hormone and metabolic modulators: A lot of variation for each compound
  • Diuretics: MTBE and pH vary a lot
  • Stimulants: Ethyl acetate or MTBE and a pH 9,5 or 14
  • Narcotics: MTBE and pH 9,5
  • Others: MTBE and pH 9,5

Not all prohibited substances were tested in this short period or showed a good result and need to be retested. In total 62 of the 77 tested products are optimized, although a lot more products need to be investigated.

Abstract bachelorproef 2016-2017Direct detection of testosterone esters in sports, Development of an ultra-sensitive GC/MSMS method for the detection of testosterone esters in blood plasma

The goal of this study was to develop an ultra-sensitive method for detecting T esters in blood plasma. The reason for the development is, that the detection of an intact ester of testosterone could lead towards unequivocal proof of misuse through administration of the exogenous testosterone.

In this study, an ultra-sensitive detection method was created on GC/MSMS including a custom sample preparation protocol. In our study protocol a liquid-liquid extraction (LLE) was used for the sample clean-up. Furthermore the use of a HPLC with a fraction collector was used to exclude the samples of cholesterol.

After the method development, it was validated according to the in-house validation protocol of DoCoLab.

The method was then tested on blood samples of 12 volunteers. These were collected in a Nebido® study. In this study volunteers were injected with a single dose of 1g of Nebido®, a drug containing T undecanoate. After the administration, blood samples were collected over a time period of 3 months.

Through this method it was possible to detect T undecanoate between 32 and 86 days. This is a similar result as the available methods in up-to-date literature. In the future the interference of cholesterol could be eliminated which would result in clean and better compound response. Still the method can detect 8 other T esters, so it could be used as direct method which in the future could result in a standalone detection method or as a confirmation test. As a confirmation test it could replace the more expensive analysis with GC/C/IRMS, which would lower the cost and expensive for the analyst and the client.

Samenvatting eindwerk 2013-2014: In vivo uPA+/+-SCID chimeric mice and in vitro human liver microsomes as model for the study of methyldienolone and methyltrienolone metabolism
Doping related substances are often used in sports because of their performance-enhancing effects. Furthermore, new products, especially developed to circumvent doping control, are continuously brought on the market. Therefore it is necessary to optimize the screening methods in order to detect these products. In this case, the metabolism of two anabolic androgenic steroids is investigated, for which the metabolism was previously not described, namely methyldienolone and methyltrienolone. Because the steroids are intensively metabolized after administration, the parent compounds have limited diagnostic value and the metabolites need to be identified. These metabolites allow a longer detection of the abuse than the actual steroid itself. Therefore it is important that the metabolism is known.
The study of the metabolism is performed by in vivo and in vitro techniques. The in vitro technique uses human liver microsomes. In vivo the uPA+/+-SCID mouse model transplanted with human hepatocytes is used. The excretion urine of these chimeric mice is analyzed to detect the formed human metabolites. Prior to this, non-chimeric mice are used to determine a sufficient dose and to verify the excretion study.
An indication of the human metabolism is confirmed by obtaining the same metabolites in the human liver microsomes as in the chimeric mouse model. Through analysis on GC-MS a metabolite of methyldienolone was detected in both models, and this was also confirmed through detection on LC-MS2. In the analysis of methyltrienolone, which can only be detected through LC-MS2, a metabolite was detected that was obtained by the human metabolism. As metabolic reaction, hydroxylations of methyldienolone and methyltrienolone take place. The metabolism is known and in further research it’s important that the structure of the metabolites is determined, so that methyldienolone and methyltrienolone can be identified more specifically in the urine and could be taken up in the routine screening to detect their presence.
Samenvatting eindwerk 2012-2013: Onderzoek naar de metabolisatie van mentabolan via in vitro humane levermicrosomen en in vivo uPA+/+-SCID chimere muizen
Doping is een stof of methode die door sporters wordt gebruikt om beter te kunnen presteren, maar die totaal in strijd is met de moraal van de sport. Elk jaar worden er nieuwe producten ontwikkeld en op de markt gebracht die mogelijks als doping kunnen gebruikt worden. Dit zorgt ervoor dat er steeds optimalisatie en onderzoek moet gebeuren naar mogelijke detectiemethoden. Er zijn excretiestudies nodig om te bepalen tot welke componenten de steroïden gemetaboliseerd worden in de lever en vervolgens geëxtraheerd worden uit de urine. Omdat het gebruik van sommige steroïden ernstige gezondheidsrisico's kunnen opleveren, is het ethisch gezien niet toegestaan om onderzoek te voeren via humane excretiestudies. Daarom worden de excretiestudies uitgevoerd via in vitro humane lever microsomen (HLM) en het via in vivo uPA+/+-SCID chimere muismodel. HLM worden gerekend tot de meest populaire in vitro techniek. Als in vivo techniek is het uPA+/+-SCID chimere muismodel een goed alternatief voor de humane excretiestudies. Deze muizen beschikken over een gehumaniseerde lever door de regeneratie met humane hepatocyten in aanwezigheid van leverinsufficiëntie. Daarnaast zijn de muizen immunodefficiënt (SCID) waardoor de getransplanteerde humane hepatocyten niet afgestoten worden.
In dit onderzoek werd de metabolisatie van het steroïde 7a-methyl-19-norandrostenedione of mentabolan onderzocht. Dit is een product die tot de androgene anabole steroïden (AAS) behoort en als doping aanwezig is in het milieu van het bodybuilden. Het is een 19-nor steroïde en het prohormoon van 7a-methyl-19-nortestosteron of MENT.
Met het muismodel werd er via een vergelijking van de 24u pre- en postadministratie urinestalen van zowel chimere als niet-chimere muizen geprobeerd een profiel op te stellen van de verschillende metabolieten. De steroïde en mogelijke metabolieten werden geëxtraheerd uit de muisurine met behulp van vloeistof/vloeistof extractie na enzymatische hydrolyse met β-glucuronidase. De analyse gebeurde op GC-MS na derivatisatie met trimethylsilyl en op LC-MS². Er werden verschillende dosissen getest voor het uitvoeren van de steroïde administratie studie met als hoogste dosis 1mg.
Met de HLM werd  een profiel opgesteld van de verschillende metabolieten via een vergelijking tussen mentabolan en de negatieve controle. De HLM werden geïncubeerd op 37°C in een milieu waar o.a. een NADPH regeneratie systeem en een fosfaatbuffer (pH 7,4) in aanwezig zijn. De negatieve controle en mentabolan verschillen in de aanwezigheid van het steroïde mentabolan. De steroïde werd op een gelijkaardige manier geëxtraheerd als de extractie uit de muisurine.
Bij de GC-MS analyse van de 24u pre- en postadministratie urinestalen na het toedienen van de hoogste dosis (1mg) werd er geen verschil gedetecteerd tussen de 24u pre- en postadministratie urinestalen. Bij de LC-MS² analyse van dezelfde urinestalen werden er 4 metabolieten vastgesteld, met als moleculaire ionen m/z 302 en 304 die ontstonden door een monohydroxylatie en het moleculair ion m/z 320 dat ontstond door dihydroxylatie. Het verschil in detectie is te wijten aan de hogere selectiviteit van de LC-MS² analyse.
Via de GC-MS analyse van de HLM werden er 7 metabolieten vastgesteld die ontstonden via hydroxylatie of reductie van mentabolan. Bij de LC-MS² van de HLM werden er 6 metabolieten vastgesteld met als moleculaire ionen m/z 302, 304 en 318 die ontstonden door mono- en dihydroxylaties. Vermits mentabolan de voorloper is van MENT, is de tweede metaboliet die via GC-MS gedetecteerd werd met belangrijkste ionen m/z 417 en 432 mogelijks MENT. Door het ontbreken van een referentie van MENT kan dit echter niet bevestigd worden.
Aan de hand van het massaspectrum en de verschillende retentietijden gebeurde enkel de detectie van de verschillende metabolieten. In het vervolgonderzoek zullen de metabolieten geïdentificeerd worden door het bepalen van hun structuur.


Technologiepark 30
9052 Zwijnaarde (Gent)
09/331 32 90


Traineeship supervisor
Prof. Peter Van Eenoo
Traineeship supervisor
Leen Lootens
Traineeship supervisor
Pieter Van Renterghem
Fiona Hooghe
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