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 SPE (IA-SPE) and subsequent analysis by partial filling MEKC (PF-MEKC). An immunosorbent consisting of a recombinant antitestosterone 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 and PF-MEKC method proved to be robust, sensitive (LOQ 35 mug/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.     

Comparison of partial filling MEKC analyses of steroids with use of ESI-MS and UV spectrophotometry

Until now, partial filling micellar EKC-ESI-MS (PF-MEKC-ESI-MS) has seldom been applied for human biomolecules. In this study, determination of three electrically neutral endogenous steroid hormones, namely androstenedione, testosterone, and epitestosterone, by PF-MEKC-ESI-MS was investigated. Since ESI-MS and ESI-MS/MS behaviors of testosterone and epitestosterone proved to be nearly identical, efficient separation of the two compounds was required to obtain reliable identification. The chemical conditions as well as the instrumental parameters affecting the PF-MEKC-ESI-MS analysis were researched. In optimal conditions, ESI-MS showed excellent selectivity, and all the steroids could be identified using SIM. LODs were 0.75-5 microg/mL. The results obtained by PF-MEKC-ESI-MS were compared with those obtained by corresponding PF-MEKC-UV. PF-MEKC-UV provided better resolution, repeatability, and more than ten-fold higher sensitivity, in terms of LODs, than PF-MEKC-ESI-MS. The reasons for this were carefully examined. In comparison with PF-MEKC-UV, PF-MEKC-ESI-MS suffered from greater band broadening owing to the sheath-liquid interface. Resolution was also decreased owing to the elevated capillary temperature. Finally, we discovered that in the analysis of electrically neutral compounds, in-capillary sample concentration by micellar sweeping could be more efficiently utilized in PF-MEKC-UV than in PF-MEKC-ESI-MS.     

Radioactive contamination of groundwater at the Andreeva Bay Shore Technical Base,Northwest Russia

The Andreeva Bay Shore Technical Base is one of the largest, most contaminated nuclear legacy sites in Northwest Russia. Radioactive contamination at the site stems from servicing and maintenance activites for Russian Northern Fleet nuclear submarine. Studies of groundwater contamination have been conducted using measurements taken at different boreholes around the site. Results indicate that groundwater contamination has occurred in some areas of the Andreeva Bay facility. These areas are primarily located near Building 5 where accidental releases occurred in 1982 during storage of spent nuclear fuel (SNF) and near the liquid and solid radioactive waste (LRW and SRW) storage facilities which have been infiltrated by precipitation waters.     

Partial filling micellar electrokinetic chromatography analysis of androgens and testosterone derivatives using two sequential pseudostationary phases

Separation of anabolic and androgenic steroids by micellar electrokinetic chromatography (MEKC) has been little studied. Simultaneous separation of the endogenous alpha-epimers testosterone and epitestosterone has not been achieved with any electroseparation technique. Here, a partial filling micellar electrokinetic chromatographic (PF-MEKC) method is described for the analysis of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone). The resolution efficiency of single-isomer sulphated gamma-cyclodextrins and the surfactants sodium dodecyl sulphate and sodium taurocholate was exploited. The method is based on the sequential introduction of short plugs of two different pseudostationary phases into the capillary. The separation was completed in less than 10 min. The method can be used in quantitative analysis. Linear correlation was obtained between concentration and peak area of 0.996 or better. The repeatability (RSD) of the compound peak areas ranged from 3.6% (methyltestosterone) to 6.2% (androstenedione). Limits of detection were between 73 microg/L (testosterone) and 160 microg/L (fluoxymesterone). As a demonstration of the method, androstenedione, testosterone and epitestosterone were determined in a spiked urine sample.     

Assessing toxicity and mobilisation of impregnation salts at a contaminated site

Severe soil contamination is often encountered at wood-impregnation plants due to spills, dripping and deposition of sludge associated with dissolved salts of copper, chromium and arsenic (CCA). Soil samples from a CCA-plant in southern Norway were analysed via a factorial extraction design to investigate mobilisation of contaminated soils. Various concentrations of organic acids, sea-salts, and pH showed that contaminants were not stable, and could be mobilised to the aqueous phase. To further investigate mobilisation of impregnation salts, soil solution collectors were installed at various depths at the site. Concentrations varied considerably. Hydrological changes revealed elevated levels of dissolved salts, which agree with the factorial experiment. Soil chemical processes (not total solid-phase concentrations) dominated the mobilisation and subsequent leaching. Soil solutions were tested for changes in toxicity by chemical analysis and degree of inhibition of luminescence in Vibrio fisheri (Microtox). Changes in toxicity corresponded to changes in soil solution.     

Asymptotic analysis of steady solutions of the KdVB equation with application to resonant sloshing

The forced Korteweg-de Vries equation with Burgers’ damping (fKdVB) on a periodic domain, which arises as a model for water waves in a shallow tank with forcing near resonance, is considered. A method for construction of asymptotic solutions is presented, valid in cases where dispersion and damping are small. Through variation of a detuning parameter, families of resonant solutions are obtained providing detailed insight into the resonant response character of the system and allowing for direct comparison with the experimental results of Chester and Bones (1968).     

Simultaneous entrainment of oil and water droplets in high Reynolds number gas turbulence in horizontal pipe flow

We develop a 1D cross sectional concentration profile model for oil and water droplets that coexist in the turbulent gas phase (of Re ∼ 10⁶) in near horizontal stratified pipe flows. Entrainment of the oil and water mixture from a liquid film near the bottom of the pipe into the gas is modeled based on earlier single-fluid entrainment correlations. A Gamma distribution for the droplet sizes based on the breakup of liquid filaments, is adopted. An explicit algebraic–exponential formula for the total concentration profile for either phase can then be derived.