Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques: Dissertation

Lotta K. Amundsen

Research output: ThesisDissertationCollection of Articles

Abstract

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC-UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC-UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC-ESI-MS. We discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC-UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Sirén, Heli, Supervisor, External person
Award date23 May 2008
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-7085-0
Electronic ISBNs978-951-38-7086-7
Publication statusPublished - 2008
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Electromigration
Testosterone
Chromatography
Steroids
Spectrophotometry
Electrospray ionization
Mass spectrometry
Capillary electrophoresis
Immunoglobulin Fab Fragments
Body fluids
Cyclodextrins
Bovine Serum Albumin
Surface-Active Agents
Electrolytes
Ionization
Doping (additives)
Association reactions
Ligands

Keywords

  • capillary electromigration techniques
  • capillary electrophoresis
  • micellar electrokinetic chromatography
  • electrospray ionization mass spectrometry
  • immunoaffinity
  • solid-phase extraction
  • Fab fragment
  • testosterone
  • epitestosterone
  • androgen
  • anabolic steroid
  • albumin

Cite this

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title = "Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques: Dissertation",
abstract = "Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC-UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC-UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC-ESI-MS. We discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC-UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.",
keywords = "capillary electromigration techniques, capillary electrophoresis, micellar electrokinetic chromatography, electrospray ionization mass spectrometry, immunoaffinity, solid-phase extraction, Fab fragment, testosterone, epitestosterone, androgen, anabolic steroid, albumin",
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year = "2008",
language = "English",
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publisher = "VTT Technical Research Centre of Finland",
number = "675",
address = "Finland",
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}

Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques : Dissertation. / Amundsen, Lotta K.

Espoo : VTT Technical Research Centre of Finland, 2008. 114 p.

Research output: ThesisDissertationCollection of Articles

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AB - Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC-UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC-UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC-ESI-MS. We discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC-UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

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KW - solid-phase extraction

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KW - epitestosterone

KW - androgen

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KW - albumin

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