Abstract
Conventional methods for the determination of testosterone in body
fluids typically suffer from poor recovery, lack of specificity, complex
sample pretreatment, or the need for derivatization. Here, a simple, specific,
and fast analysis method for testosterone was developed, with a methodology
based on testosterone-specific immunoaffinity solid-phase extraction (IA-SPE)
and subsequent analysis by partial filling micellar electrokinetic
chromatography (PF-MEKC). An immunosorbent consisting of a recombinant
anti-testosterone Fab fragment covalently attached to activated Sepharose was
prepared. IA-SPE and PF-MEKC were set up in hyphenated and off-line
constructions, and the applicability of the two constructions in analysis of
testosterone in male urine was investigated. The results obtained with the
hyphenated construction proved to be only indicative of the presence of
testosterone. The off-line IA-SPE and PF-MEKC construction, however, was
successfully used in the determination of free testosterone in male urine
samples after enzymatic hydrolysis of the glucuronide conjugates. Except for
the hydrolysis reaction, no sample pretreatment was required. After
hydrolysis, the overall analysis time per sample was only 14 min. The off-line
IA-SPE PF-MEKC method proved to be robust, sensitive (limit of quantification
35 ?g/L), and specific, enabling separation of testosterone from four related
steroids. Thus, it provides attractive features when compared to traditional
methods for determination of testosterone in male urine.
Original language | English |
---|---|
Pages (from-to) | 3232-3241 |
Journal | Electrophoresis |
Volume | 28 |
Issue number | 18 |
DOIs | |
Publication status | Published - 2007 |
MoE publication type | A1 Journal article-refereed |
Keywords
- testosterone
- Fab fragment
- phage display
- immunoaffinity solid-phase extraction
- partial filling micellar electrokinetic chromatography
- immunoaffinity capillary electrophoresis