Electrochemical investigations of corticosteroid isomers—testosterone and epitestosterone and their simultaneous determination in human urine (original) (raw)
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TheScientificWorldJournal, 2012
The validated micellar electrokinetic chromatography (MEKC) was proposed for the determination of five steroid hormones in human urine samples. That technique allowed for the separation and quantification of cortisol, cortisone, corticosterone, testosterone, and epitestosterone and was sensitive enough to detect low concentrations of these searched steroids in urine samples at the range of 2-300 ng/mL. The proposed MEKC technique with solid-phase extraction (SPE) procedure was simple, rapid, and has been successfully applied as a routine procedure to analyze steroids in human urine samples. The MEKC method offered a potential in clinical routine practice because of the short analysis time (8 min), low costs, and simultaneous analysis of five endogenous hormones. Due to its simplicity, speed, accuracy, and high recovery, the proposed method could offer a tool to determine steroid hormones as potential biomarkers in biomedical investigations, what was additionally revealed with health...
Quantification of testosterone and epitestosterone in human urine by capillary liquid chromatography
Journal of Microcolumn Separations, 2000
A capillary-liquid chromatography LC method was developed for the quantification of the endogenous steroids testosterone and epitestosterone in human urine. One milliliter of urine was used for the overall method. Free testosterone was first separated by liquid᎐liquid extraction with n-pentane at pH 7. Glucuronides of testosterone and epitestosterone were enzymatically hydrolyzed and the free compounds were extracted with n-pentane at pH 11. A capillary Ž . column switching system with a low back pressure precolumn PC was used for Ž . fast loading of large sample volumes 20 L . Chromatographic separation was Ž . carried out on a 15 cm = 300 m inner diameter i.d. column, packed with 3 m Hypersil BDS-C at a flow rate of 4 Lrmin with isocratic elution and UV 18 Ž . absorbance detection 240 nm . Limit of detection for free testosterone was established at 0.5 ngrmL. Limits of detection were established at 1.5 and 3.2 ngrmL for testosterone and epitestosterone, respectively, after being hydrolysed from their glucuronides. Good reproducibility and robustness were observed Ž . through the entire calibration range up to 250 ngrmL . ᮊ
Talanta, 2011
A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination of testosterone (T) and epitestosterone (E) in human urine samples has been developed and elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis using 36% hydrochloric acid for determination of total level of T has been applied. Unconjugated (free) form of the both androgens were determined without hydrolysis steps, what makes novelty of the method, because simplifies the proposed procedure. In turn, the measurements of urinary free T and E provided the diagnostic information for excess adrenal production of steroids. The proposed LC assay was evaluated by analyzing a series of urine samples containing T, E and methyltestosterone (MT) as internal standard at the range of concentration 2-300 ng −1 mL of both analyzed hormones. The proposed method was fully validated for specificity, linearity, limits of detection and quantitation, precision and trueness according to the current requirements concerning analytical methods. Interestingly, the developed LC method allows to obtain a sensitive enhancement with respect to UV detection with the quantitation limit for T and E equaled 2 ng mL −1 . The method was selective and reliable for identity and enable to detect changes of endogenous levels of T and E in urine independently of fluctuations characteristic for both analyzed endogenous hormone level in plasma. .pl (L. Konieczna). androgen deficiency in clinical conditions . The normal amounts of total endogenous T and epitestosterone (E) practically measured in healthy male in urine are in the range 30-60 ng mL −1 [11]. T and E and their ratio T/E is stable in males, what was well established . Since 1983, T was forbidden in sports by the International Olympic Committee (IOC). The detection of illicit use of T is currently carried out measuring the ratio between the concentration of T and its isomer E. A ratio of their concentrations (T/E ratio) higher than 4 is considered as potentially indicative of T administration. On the other hand, because the T/E ratio can be artificially modified by the administration of E, a urinary concentration of epitestosterone above 200 ng mL −1 has been established as indicative of its misuse as a masking agent . The World Anti-Doping Agency (WADA) indicated that if the T/E ratio was equal or above 4, or concentration of E higher than 200 ng mL −1 , a confirmation procedure to prove doping would be necessary .
Biosensors and Bioelectronics, 2010
A disposable electrochemical immunosensor using screen-printed carbon electrodes (SPCEs) and protein A-functionalized magnetic beads (MBs) was developed for the determination of testosterone. Antitestosterone was immobilized onto MBs and a direct competitive immunoassay involving testosterone labeled with peroxidase (HRP) was performed. The resulting conjugate was trapped on the SPCE with a small magnet. Testosterone determination was carried out by amperometry at −0.2 V upon H 2 O 2 additions using hydroquinone (HQ) as the redox mediator. The experimental variables involved in the immunosensor response to testosterone were evaluated. Under the optimized conditions, a calibration plot for testosterone was obtained with a linear range between 5.0 × 10 −3 and 50 ng/mL (r = 0.995). The detection limit was 1.7 pg/mL and the EC 50 was 0.25 ± 0.04 ng/mL. These characteristics are notably better than those achieved with other reported immunosensors. Furthermore, anti-testosterone/MBs conjugates were shown to be stable for at least 25 days. A good selectivity was also found against other steroid hormones. The usefulness of the immunosensor was demonstrated by analyzing human serum spiked with 1 and 10 ng/mL testosterone.
Journal of Solid State Electrochemistry, 2013
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Electroanalysis, 1995
The electroanalytical behavior of testosterone thiosemicarbazone (TT) has been investigated by linear sweep and differential pulse voltammetry with and without adsorptive preconcentration on a hanging mercury drop electrode (HMDE). The adsorptive stripping response has been evaluated with respect to preconcentration time and potential, drop size, scan rate, and other variables. Measurement of a selected reduction wave enables the determination of TT in the μg L−1 range, with a detection limit of 100 ng L−1 (10 min preconcentration time). The determination can also be carried out in a urine/aqueous NaClO4 (2/8) medium. The immunological interaction of TT with the testosterone specific antibody can be electrochemically monitored via the decrease in the reduction wave, as described previously. This property is discussed as a tool for the determination of testosterone and its specific antibody by competitive immunoassay with amperometric detection in real matrices such as urine.