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Analytical Methods For Sports Drugs: Challenges and Approaches
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.ORCID iD: 0000-0002-2485-4276
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drugs used to enhance human performance in sport competitions are prohibited by the world anti-doping association (WADA). Biological samples from athletes are continuously tested for adverse analytical findings regarding the identity and/or quantity of the banned substances. The current thesis deals with the development of new analytical methods to determine the concentrations of certain drugs used by athletes and even by regular users for therapeutic purposes. The developed methods aim to analyze the contents of these drugs in the biological matrices; plasma, serum and saliva to provide a successful approach towards either doping detection or therapeutic monitoring. β-adrenergic blockers such as propranolol and metoprolol are used in sports to relief stress and as therapeutic agents in the treatment of hypertension. Both drugs are in chiral forms and available only as racemic mixtures. The different pharmacology of each enantiomer necessitates the monitoring of each enantiomer by stereoselective analytical technique such as chiral liquid chromatography for separation and mass spectrometry for selective detection. The Endogenous anabolic androgenic steroids (EAAS) on the other hand are only notoriously used in sports to increase muscle mass and strength. A method utilizing high-resolution mass spectrometry (HRMS) coupled to ultra-high performance liquid chromatography (UHPLC) was developed for the simultaneous determination of EAAS and their conjugated metabolites to provide a better insight into the steroidal module of the athlete biological passport (ABP). Moreover, the steroidal profile was assessed in serum using the proposed method after the administration of Growth hormone injection as an approach toward the implementation of a new endocrinological module based on steroids biomarkers to hormone doping.  Biological samples contain many components that may interfere with the analytical measurements. Therefore, sample preparation methods were developed using solid phase extraction (SPE) and miniaturized techniques such as microextraction by packed sorbents (MEPS) for the purification and pre-concentration of analytes prior to LC/MS analysis.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University , 2019. , p. 89
Keywords [en]
Sports Drugs, Doping in Sports, Steroids, LC-MS/MS, Chiral analysis, high-resolution mass spectrometry, Sample preparation, Biological samples, solid phase extraction
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-172566ISBN: 978-91-7797-835-0 (print)ISBN: 978-91-7797-836-7 (electronic)OAI: oai:DiVA.org:su-172566DiVA, id: diva2:1348253
Public defence
2019-10-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2019-09-25 Created: 2019-09-03 Last updated: 2019-09-13Bibliographically approved
List of papers
1. Online post-column solvent assisted and direct solvent-assisted electrospray ionization for chiral analysis of propranolol enantiomers in plasma samples
Open this publication in new window or tab >>Online post-column solvent assisted and direct solvent-assisted electrospray ionization for chiral analysis of propranolol enantiomers in plasma samples
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2015 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1418, p. 110-118Article in journal (Refereed) Published
Abstract [en]

An Online post-column solvent-assisted ionization (OPSAI) method was developed for enhancing the ionization of the beta-blocker propranolol utilizing normal phase LC-MS/MS. Solvent-assisted electrospray ionization (SAESI) was studied by the introduction of the assistant solvents A: 0.5% Formic acid in Isopropanolol, B: 0.5% Formic acid in lsopropanolol-Water (1:1), and C: 0.5% Formic acid in water into the electrospray ionization chamber using a spray needle. Analyte molecules can be directly ionized by the aid of the assistant solvent spray. Both methods were applied to the chiral separation of propranolol enantiomers using normal phase analysis on cellulose-based chiral column. Interestingly, both methods are easy to handle and offer a wide range of assistant solvents that can be used in order to gain the optimum ionization of the analyte molecules. The both methods considerably improved the analyte signal and the peak area greatly increased. The propranolol average signal-to-noise (S/N) ratio was enhanced from 26 +/- 1 and 42 +/- 1 to 2341 +/- 61 and 1725 +/- 29 for R-propranolol and S-propranolol, respectively, when the post-column solvent method (OPSAI) was used with isopropanol-assistant solvent (A). While in case of solvent-assisted electrospray ionization method (SAESI) signal was enhanced from 26 +/- 1 and 42 +/- 1 to 2223 +/- 72 and 2155 +/- 58 for R-propranolol and S-propranolol, respectively, with water as an assistant solvent. The limit of detection was 10 ng/mL and the method was linear in the range 50-2000 ng/mL. The NPLC-MS method was applied for the determination of propranolol enantiomers in human plasma after microextraction by packed C18 sorbent.

Keywords
Normal phase LC, Chiral separation, Propranolol Enantiomers, Electrospray ionization, Tandem mass spectrometry
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-123792 (URN)10.1016/j.chroma.2015.09.051 (DOI)000364268200012 ()26422307 (PubMedID)
Available from: 2015-12-15 Created: 2015-12-07 Last updated: 2019-09-04Bibliographically approved
2. Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry
Open this publication in new window or tab >>Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry
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2016 (English)In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 30, no 8, p. 1309-1317Article in journal (Refereed) Published
Abstract [en]

A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150x4.6mm, 5m) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2mL/min. The total chromatographic run time was 10min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.0872.09 for metoprolol and m/z 303.3154.3 for IS. The linearity range was 2.5-500ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories.

Keywords
metoprolol enantiomers, chiral chromatography, tandem mass spectrometry, therapeutic drug monitoring, plasma, saliva
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-133381 (URN)10.1002/bmc.3685 (DOI)000379971200020 ()26766521 (PubMedID)
Available from: 2016-09-09 Created: 2016-09-06 Last updated: 2019-09-04Bibliographically approved
3. Development and validation of a UHPLC-HRMS method for the simultaneous determination of the endogenous anabolic androgenic steroids in human serum
Open this publication in new window or tab >>Development and validation of a UHPLC-HRMS method for the simultaneous determination of the endogenous anabolic androgenic steroids in human serum
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Being performance enhancing hormones, endogenous anabolic androgenic steroids (EAAS) are banned from most competitive sports by the World Anti-doping Agency (WADA).. In anti-doping control laboratories, routine assays are mainly performed on urine samples of athletes in and out of competitions. Serum constitutes a promising alternative to urine as it is less subjected to manipulation or contamination issues that may influence the method sensitivity. The simultaneous determination of EAAS and their conjugated metabolites is very challenging due to their contradicting chemical behaviors at the ionization interface of the mass spectrometer. This may prejudice their detection or limit the method sensitivity. Herein, we have addressed these challenges and developed a new method for the simultaneous determination of unconjugated, sulphate and glucuronide EAAS (Androsterone, Etiocholanolone, testosterone, epitestosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione and 17a-hydroxyprogesterone) in human serum using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The use of mass spectrometric detection in full scan mode facilitated the study of the most versatile adducts for detection and quantitation. Using solid phase extraction as sample preparation protocol prior to analysis, the method limits of quantitation ranged from 0.006 to 7.904 ng/mL and the recoveries ranged from 70.2 to 96.5 %. The method calibration was performed in untreated serum representing realistic matrix composition with correlation coeffecients ranged from 0.9859 to 0.9988. Finally, the serum- levels of the investigated steroids were determined in 4 male and 1 female human subject to provide estimates of baseline levels based on individual values.

Keywords
Doping, Endogenous Anabolic androgenic steroids, Chromatography, UHPLC, Mass spectrometry, Solid phase extraction.
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-172564 (URN)
Available from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-04Bibliographically approved
4. Longitudinal studies of putative growth hormone (GH) biomarkers and hematological and steroidal parameters in relation to 2 weeks administration of human recombinant GH
Open this publication in new window or tab >>Longitudinal studies of putative growth hormone (GH) biomarkers and hematological and steroidal parameters in relation to 2 weeks administration of human recombinant GH
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Detection of low doses of recombinant growth hormone is a challenge in anti-doping testing. Future testing leads toward longitudinal monitoring of IGF-I and P-III-NP in an endocrine module. Additional biomarkers, for example vitamin D binding protein, alpha-HS-glycoprotein, fibronectin 1 and decoin have been identified in different omics studies. Here we have longitudinally studied the usefulness of these putative biomarkers in relation to two weeks administration of low doses of recombinant growth hormone in healthy male volunteers. Moreover, we studied the hematological parameters included in the athlete biological passport as well as serum concentration of dihydrotestosterone-sulfate. Alpha-HS-glycoprotein decreased 25 % up to three weeks after treatment period (p˂0.05), whereas fibronectin increased 20 % during the treatment (p˂0.05). The addition of these two biomarkers increased the likelihood to detect recombinant growth hormone intake based on individual calculated thresholds in some of the participants as compared to the GH2000 score. A minor 10 % increase (p=0.003) in reticulocytes percentage and OFF-score was observed, but the changes did not lead to any atypical profile based on individual passport thresholds. Vitamin D binding protein, decoin and dihydrotestosterone-sulfate were not affected by recombinant growth hormone. Dihydrotestosterone-sulfate was negatively correlated with the IGF-I biomarker at baseline (R=-0.50, p=0.003) and post dose (R=-0.59, p=0.01). In conclusion we have verified Alpha-HS-glycoprotein and fibronectin 1 as promising future biomarkers for detecting low doses of recombinant growth hormone and further studies of these proteins are encouraged.

Keywords
doping, growth hormone, IGF-I, fibronectin 1, alpha-HS-glycoprotein, DHT, athlete biological passport
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-172565 (URN)
Available from: 2019-09-03 Created: 2019-09-03 Last updated: 2019-09-04Bibliographically approved

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