Determination of Testosterone Esters in Serum by Liquid Chromatography – Tandem Mass Spectrometry (LC-MS-MS)
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Department of Physics, Chemistry and Biology Final Thesis Determination of testosterone esters in serum by liquid chromatography – tandem mass spectrometry (LC-MS-MS) Erica Törnvall Final Thesis performed at National Board of Forensic Medicine 2010-06-03 LITH-IFM-EX--10/2263--SE Department of Physics, Chemistry and Biology Linköping University 581 83 Linköping, Sweden 1 Department of Physics, Chemistry and Biology Determination of testosterone esters in serum by liquid chromatography – tandem mass spectrometry (LC-MS-MS) Erica Törnvall Final Thesis performed at National Board of Forensic Medicine 2010-06-03 Supervisors Yvonne Lood Martin Josefsson Examiner Roger Sävenhed 2 Avdelning, institution Datum Division, Department Date 2010-06-03 Chemistry Department of Physics, Chemistry and Biology Linköping University Språk Rapporttyp ISBN Language Report category Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-EX--10/2263--SE Engelska/English Examensarbete _________________________________________________________________ C-uppsats D-uppsats Serietitel och serienummer ISSN ________________ Övrig rapport Title of series, numbering ______________________________ _____________ URL för elektronisk version Titel Title Determination of testosterone esters in serum by liquid chromatography – tandem mass spectrometry (LC-MS-MS) Författare Author Erica Törnvall Sammanfattning Abstract Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum. The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible. Nyckelord Keyword LC-MS-MS, MRM, testosterone, testosterone esters 3 Abstract Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum. The technique used to separate and identify the testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible. 4 Abbreviations MeOH Methanol ACN Acetonitrile LC Liquid chromatography MS Mass spectrometry TIC Total ion chromatogram AAS Anabolic androgenic steroids MRM Multiple reaction monitoring CID Collision induced dissociation C18 Octadecyl Rt Retention time ESI Electrospray ionization WADA World Anti-Doping Agency T/E Testosterone glucuronide/Epitestosterone glucuronide TA Testosterone acetate TB Testosterone benzoate TC Testosterone cypionate TD Testosterone decanoate TE Testosterone enanthate TP Testosterone propionate TPh Testosterone phenylpropionate TI Testosterone isocaproate TU Testosterone undecanoate 5 Table of contents Abstract Abbreviations 1. Introduction 8 1.1. Anabolic Androgenic Steroids 8 1.2. Testosterone and Testosterone Esters 8 1.3. Methods of Analysis 9 2. Methodology 10 2.1. Liquid Chromatography 10 2.2. Tandem Quadrupole Mass Spectrometry 11 2.3. Multiple Reaction Monitoring 11 2.4. Aim 12 3. Experimental 13 3.1. Chemicals and Reagents 13 3.2. Solutions 13 3.3. Instrumentation 13 3.4. Sample Preparation 14 3.4.1. Infusion Study 14 3.4.2. Mixed Standards 14 3.4.3. Sample Preparation by LLE 14 3.5. Optimization 14 3.5.1. MS-Specificity 14 3.5.2. Chromatographic Selectivity 15 3.5.3. Linearity and Sensitivity 15 4. Results and Discussion 16 4.1. Tandem-MS Detection 16 4.2. Gradient Profile 17 4.3. Mobile Phase Composition 20 4.4. Stationary Phase 22 4.5. Final Gradient Profile 24 4.6. Matrix Test 26 4.7. Linearity 30 4.8. Final Method 32 5. Conclusion 34 6. Acknowledgement 35 References 36 6 Appendix A. Names and Structures of Testosterone Compounds 37 Appendix B. Transitions in the MS-method Tested with Reference Solutions 38 Appendix C. Transitions Based on the Serum Analysis 42 Appendix D. Linearity Study 45 D.1. Chromatograms from the Linearity Study 45 D.2. Linearity Evaluated by Using Transition 97 47 Appendix E. Transitions Used in the Final Study 49 E.1. Final Transition Method 49 E.2. Tests of Final Transitions 50 7 1. Introduction 1.1. Anabolic Androgenic Steroids Anabolic androgenic steroids (AAS) are testosterone and its derivates. All AAS have both anabolic properties such as increased muscle hypertrophy and androgenic such as masculinisation [2]. Strength training is widely used to increase performance in sports with high physical demands. The use of drugs to further enhance the performance happens via forbidden substances, methods and manipulations. AAS are wide spread among athletes and the youth today in gyms. The effects of these drugs on physical performance are documented [1]. AAS increase the muscle hypertrophy induced by strength training further for athletes involved in doping. The number of nuclei per muscle fibre increases [2]. Those who have withdrawn from anabolic steroid usage and training for several years still have a remaining high number of myonuclei [1]. AAS are used in medical purpose as substitution treatment for men with no natural production of testosterone. Testosterone is predominantly administrated as intramuscular injection but is also available as gel and plaster. In forensic investigation AAS are involved in violent behaviour, depression and criminality and could cause more serious harm such as sudden cardiac death, damaged liver function and disturbances in the lipid metabolism [1, 2]. In Sweden it is restricted by law the use of synthetic AAS, testosterone and its derivates, growth hormone and chemical substances increasing production and secretion of testosterone and its derivates or growth hormone [3]. 1.2. Testosterone and Testosterone Esters Testosterone is produced in the Leydig cells in the testicles and even in females by the ovaries in small quantities. Testosterone is naturally secreted to urine [4]. Men produce 6-10 mg testosterone daily of which approximately 1% is excreted in urine [1]. Because of the short half-life of only one hour exogenous intake of pure testosterone do not have any effect, since only 2% of oral intake reaches the muscles. To slow down the metabolism and receive better effect the testosterone molecule has been modified at its 17-position. This modification