Lorenz Göschl

Avhandlingen er tilgjengelig her!
The doctoral thesis.

Prøveforelesningen over oppgitt emne starter kl. 1015 samme sted:
"Mass spectrometric methods for anti-doping analysis of peptides and proteins"
Direktelenke prøveforelesning/ The trail lecture will be streamed here.

Disputasen starter 12.15
Direktelenke Disputas/ The defense will be streamed here. 

Opptak av disputasen vil være tilgjengelig i et døgn. 
A recording of the disputation will be available for 24 hours.

Since the mid-1970s, anti-doping measures have been an essential part of professional sports worldwide. The World Anti-Doping Agency, or WADA for short, is responsible for this. The drug class of Anabolic Androgenic Steroids (AAS) has always been one of the most commonly used illicit substances. AAS compounds allow athletes to grow muscles faster and recover better. Therefore, AAS are especially popular for weight training and many other sports.
In the field of anti-doping analysis, analyses are mainly performed from urine, but to a lesser extent also from blood. The implementation, research, and further development of such analytical methods are part of analytical chemistry. The conventional method for analysing AAS is gas chromatography combined with mass spectrometry (GC-MS). In this method, a complex mixture of substances is separated using a gas flow and an analytical separation column, and the individual substances are then measured using mass spectrometry (MS). With MS, the individual molecules can be distinguished by their weight respectively their mass.
However, before this method can be used, the samples must be prepared in a complex way. For this purpose, the desired substances are extracted from the sample and chemically prepared for the subsequent measurement. As a result, this approach is very time and resource-consuming, although it is very sensitive and selective. An alternative procedure to this method is the so-called liquid chromatography coupled with MS (LC-MS). Here, instead of gas, a liquid is used to separate the mixture of substances. The sample preparation is much less complex than with GC-MS and, in some cases, can even be omitted entirely. However, there has been little research and development on the analysis of AAS with LC-MS. Therefore, the main goal of this work was to improve the detection of steroids in a time- and resource-efficient manner by shifting the analysis from GC-MS to LC-MS.

When analysing substances in urine, usually, not the substances themselves are detected but a metabolic product. These so-called metabolites are chemically slightly modified versions of the original substance. Metabolites that can be detected over a very long period of time are of particular interest. These are referred to as “long-term metabolites”. This work focuses on analysing so-called phase-II metabolites, as these are easily detectable by LC-MS. A particular form of phase-II metabolites are the so-called glucuronide conjugates, to which special attention was paid in this work.

In the first part of this thesis, we present a novel, convenient analytical method developed to analyse steroid phase-II metabolites in which sample preparation is reduced to a minimum. For this purpose, we developed a simple but powerful online solid-phase extraction method coupled with LC-MS. Two comprehensive validation studies demonstrated the analytical performance of this method. Initially developed for rapid confirmation analysis, this method also showed high potential for identifying and characterizing novel steroid phase-II metabolites.

newly developed method. Both substances are among the most commonly used steroids in professional sports and are of great interest to anti-doping research. In routine analysis, both substances are conventionally measured using the GC-MS method described above.

For stanozolol, we achieved for the first time to unequivocally confirm the existence of four different phase-II glucuronide conjugates using newly synthesized high-quality reference standards. Furthermore, using urine samples from excretion studies, we generated elimination curves for all four metabolites showing elimination windows of up to 28 days. The long excretion time makes this metabolite very interesting for the long-term detection of stanozolol. With these results and the newly developed method, we significantly contributed to the efficient and reliable detection of stanozolol abuse in professional sports.

In the case of dehydrochloromethyltestosterone, we identified two different glucuronide conjugates of the important long-term metabolite M3 for the first time. Due to the lack of reference standards, we used a combination of different analytical approaches to characterize and elucidate the chemical structure of these conjugates. Since these metabolites are well suited for LC-MS analysis, we present for the first time a way to analyse metabolite M3 without requiring a laborious GC-MS approach. These findings can save time in the future and significantly reduce the use of expensive and ecologically hazardous substances.

In conclusion, this work provides valuable contributions to the field of anti-doping analysis on two different levels. On the one hand, an important step was taken at the methodological level towards an efficient and resource-efficient analysis of AAS. On the other hand, this work has provided important new insights into the nature of phase-II metabolism of anabolic androgenic steroids.

Main Supervisor
Professor Guro Forsdahl, Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway.

Co-Supervisor
Dr.Günter Gmeiner, Doping Control Laboratory, Seibersdorf Laboratories, Austria.

Evaluation Committee
Dr. Tiia Kuuranne, LAD- Laboratoire d'Analyse du Dopage, University Center of Legal Medicine, Lausanne, Switzerland - 1. opponent.
Professor Stig Pedersen-Bjergaard, Department of Pharmacy, UiO - 2. opponent.
Associate Professor Kjell H. Halvorsen, Department of Pharmacy, Faculty of Health Sciences, The Arctic University of Norway – Third member and chair of the evaluation committee.

Chair of the Defense: Professor Natasa Skalko-Basnet, Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway