Determination of testosterone thiosemicarbazone and study of its immunological reactions in urine by adsorptive stripping voltammetry (original) (raw)
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Analytica Chimica Acta, 2010
Voltammetric investigation of two corticoid isomers-testosterone and epitestosterone has been carried out at bare and single-wall carbon nanotubes (SWNT)-modified edge plane pyrolytic graphite electrode (EPPGE). Square wave voltammetry (OSWV) has been used for the simultaneous determination of isomeric steroids. The reduction of the two isomers occurred in a pH dependent, 2e, 2H + process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5-1000 nM for both the steroids with the limit of detection 2.8 × 10 −9 and 4.1 × 10 −9 M for testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results obtained from the proposed voltammetric method were also compared with HPLC analysis and found to be similar.
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.
Canadian Journal of Chemistry, 2004
Flutamide is a nonsteriodal anti-androgen drug, which is commonly used in the treatment of advanced prostate cancer. Based on the reduction of the nitro organic moiety of the drug molecule in acetate buffer of pH 5 at the hanging mercury drop electrode, three adsorptive cathodic stripping voltammetric procedures were optimized for determination of flutamide in bulk, tablets, and human serum applying linear-sweep, differential-pulse, and square-wave waveforms. The achieved limits of detection of the bulk drug were 1.9 × 107, 8.7 × 108, and 9.7 × 109 mol L1 by using the optimized differential-pulse, linear-sweep, and square-wave adsorptive stripping voltammetric procedures, respectively. Repeatability of the results was studied for 1 × 106 mol L1 of the drug and the recoveries obtained were 98.51 ± 1.56% (LSV), 98.89 ± 0.87% (DPV), and 99.21 ± 1.03% (SWV). The proposed procedures were successfully applied to the determination of the drug in pharmaceutical formulation (Eulexin® t...
Thiosemicarbazide; Adsorptive stripping Voltammetry; Real sample. KEYWORDS ABSTRACT In the present work, an adsorptive stripping voltammetric method using a hanging mercury drop electrode (HMDE) was described in order to determine the ultra trace of thiosemicarbazide in different real samples. The method is based on accumulation of thiosemicarbazide on mercury electrode. The potential was scanned to the negative direction and the differential pulse stripping voltammograms were recorded. The instrumental and chemical parameters were optimized. The optimized conditions were obtained in pH of 10.0, accumulation potential of 0.00 mV, accumulation time of 60 s, scan rate of 80 mV/s and pulse height of 50 mV. The relationship between the peak current versus concentration was linear over the range of 0.50-100.00 ng/ml. The limits of detection were 0.03 ng/ml and the relative standard deviation at 5.00 and 50.00 ng/ml of thiosemicarbazide ion were obtained 2.1 and 1.7%, respectively (n = 8).
Unique potentiometric detection systems for HPLC determination of some steroids in human urine
Journal of Separation Science, 2009
Isocratic HPLC with potentiometric detection is used for the determination of some 17-ketosteroids (17-KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17-KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p-chlorophenyl) borate containing PVC-based rubber phase membranes. The neutral 17-KS compounds were derivatized with Girard's reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17-KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion-sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC-based, rubber phase membrane. The three 17-KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time-consuming cleanup and preconcentration step.
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 .
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
Determination of selected drugs in human urine by differential pulse voltammetry technique
Bioelectrochemistry, 2008
A new, simple and selective differential pulse voltammetry (DPV) method for the simultaneous determination of selected drugs in model solutions and spiked human urine samples with prior extraction was developed and validated. The objects of analysis were paracetamol, furosemide, dipyrone, cefazolin and dexamethasone belonging to four different therapeutic groups (antibiotics, analgesic, demulcent and diuretic). Analytical methods for the preparation of urine samples for voltammetric analysis (liquid-liquid extraction-LLE and solid-phase extraction-SPE) were worked out and optimized. Hanging mercury drop electrode (HMDE) and graphite electrode were used as working electrodes. Reference electrode was Ag|AgCl| KCl (sat.) , whereas auxiliary electrodeplatinum electrode. The optimal conditions for quantitative determination were obtained in a Britton-Robinson (BR) buffer at pH 2.4. Quantification was performed by means of calibration curve and standard addition methods. The calibration curves of analysed drugs are linear within the range of concentration
Analysis of Pharmaceuticals and Biological Fluids Using Modern Electroanalytical Techniques
Critical Reviews in Analytical Chemistry, 2003
A review of the principles and application of modern electroanalytical techniques, namely, cyclic voltammetry, linear sweep voltammetry, differential pulse voltammetry, differential pulse polarography, square wave voltammetry, square wave polarography, stripping voltammetric, and stripping polarographic techniques, is presented. The use and advantages of these techniques at different electrodes are discussed. The analytical applications of these techniques to pharmaceutical compounds in dosage forms and biological media are also discussed. Various selected studies on these subjects since 1995 are reviewed.