Microscale immunoaffinity SPE and MEKC in fast determination of testosterone in male urine

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.