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.
Abstract Bachelor Project FBT 2018-2019: OPTIMIZATION OF SAMPLE PREPARATION FOR THE CONFIRMATION OF DOPING IN URINE USING GC-MS
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:
- Methyl tert-buthyl ether (MTBE)
The different pHs used are:
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 2016-2017: Direct 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.
9052 Zwijnaarde (Gent)
09/331 32 90
Prof. Peter Van Eenoo
Pieter Van Renterghem