Development and Validation of Sensitive, Fast and Simple LC-MS/MS Method to Investigate the Association between Adrenocortical Steroidogenesis and the High Intensity Exercise in Elite Athletes.

The very high intensity of exercise accompanied by mental stress triggers adaptive mechanisms associated with adrenocortical steroidogenesis. However, the association between adrenocortical steroidogenesis and the high intensity of exercise in elite athletes is poorly studied. A significant obstacle to solving this complex task is the wide range (4-5 orders) of steroid concentrations in serum and limitations related to the amount of biological samples taken from professional athletes. To solve this task, we have developed and validated a non-trivial approach for targeted serum metabolic profiling based on the use of LC-MS/MS with dual-polarity electrospray ionization. The developed method based on the proposed approach allows for the quantitative determination of 14 stress resistance biomarkers in elite athletes using a small amount of specimen within 8.5 min.

Determination of Blood Plasma Aminothiols Using Derivatization-enhanced Capillary Transient Isotachophoresis.

A sensitive capillary electrophoresis method was developed for the determination of aminothiol (cysteine, homocysteine, and glutathione) total levels in human blood plasma. Analytes were derivatized with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)) after reduction with dithiothreitol. Liquid-liquid extraction was applied to purify the samples and concentrate the analytes. Total analysis time was 7.5 min using a silica capillary (50 µm i.d.; effective separation length 23.5 cm). Electrophoretic separation was performed using 50 mM citric acid with 20 mM triethanolamine (pH 3) containing 2% Ficoll 400. Detection limit was 0.8 µM for glutathione and 0.3 µM for both cysteine and homocysteine. Accuracy was 94 - 107%, repeatability and reproducibility were ca. 2.7 - 3.5 and 2.5 - 6.5%, respectively.

Application of wavelet analysis to detect dysfunction in cerebral blood flow autoregulation during experimental hyperhomocysteinaemia.

The purpose of the present study was to investigate the use of laser Doppler flowmetry (LDF) signals coupled with spectral wavelet analysis to detect endothelial link dysfunction in the autoregulation of cerebral blood flow in the setting of hyperhomocysteinaemia (HHcy). Fifty-one rats were assigned to three groups (intact, control, and HHcy) according to the results of biochemical assays of homocysteine level in blood plasma. LDF signals on the rat brain were recorded by LAKK-02 device to measure the microcirculatory blood flow. The laser operating wavelength and output power density were1064 nm and 0.051 W/mm2, respectively. A Morlet mother wavelet transform was applied to the measured 8-min LDF signals, and periodic oscillations with five frequency intervals were identified (0.01-0.04 Hz, 0.04-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz, and 2-5 Hz) corresponding to endothelial, neurogenic, myogenic, respiratory, and cardiac origins, respectively. In initial state, the amplitude of the oscillations decreased by 38% (P < 0.05) in the endothelial range in HHcy rats than in control rats. Cerebral autoregulation was challenged by hemorrhagic hypotension. The lower limit of autoregulation raised in a rat model of chronic HHcy (71.5 ± 0.7 mmHg in HHcy vs. 62.3 ± 0.5 mmHg in control). The data obtained indicate that the laser Doppler method and wavelet analysis may be successfully applied to detect the dysfunction of the endothelial link in cerebral vessel tone and to reveal the pathological shift of lower limit of autoregulation.

High-temperature high-performance liquid chromatography on a porous graphitized carbon column coupled to an Orbitrap mass spectrometer with atmospheric pressure photoionization for screening exogenous anabolic steroids in human urine.

RATIONALE: The presence in a urinary matrix of a large number of endogenous steroids and corticosteroids with similar structures can hamper the detection of specific exogenous steroids using liquid chromatography/mass spectrometry (LC/MS) with reversed-phase columns. Therefore, the development of LC/MS methods using alternative columns is of great interest. Porous graphitized carbon is a unique stationary phase for high-performance liquid chromatography (HPLC), with properties differing from traditional silica-based and polymeric stationary phases. METHODS: The new method involves enzymatic hydrolysis, liquid-liquid extraction, and determination by high-temperature HPLC/Orbitrap mass spectrometry (HTLC/Orbitrap MS) with atmospheric pressure photoionization (APPI). To achieve APPI of doping substances, the mobile phase consisted of 0.1% CF3COOH (A) and a mixture of acetonitrile/2-propanol (25:75 v/v), containing 0.1% CF3COOH (B), which was used as an effective proton source. RESULTS: A screening method for the detection of 57 exogenous steroids has been developed. The method was validated by spiking 10 different blank urine samples at different concentration levels. Validation parameters included limit of detection (LOD), selectivity, ion suppression, extraction recovery, and repeatability. All studied compounds had an LOD lower than the minimum required performance level. Of the 57 steroids studied, 55 showed recovery better than 70%. For all of the analytes, the relative retention times proved to be stable between days, with relative standard deviations (RSDs) smaller than 0.3%. In addition, the interday RSDs of the peak area ratios ranged between 0.7% and 14.5%. CONCLUSIONS: The proposed method matches the basic requirements of all methods used to analyze drugs or metabolites in an antidoping laboratory, i.e., sensitivity, selectivity, and specificity. The acquisition of full-scan mass spectra with accurate masses can be a valuable tool in the retrospective evaluation of analyzed samples for anabolic steroids recently added to the prohibited list.

Detection of S-adenosylhomocysteine and methylation index in blood by capillary electrophoresis.

This work proposes an approach to the direct analysis of S-adenosylhomocysteine (SAH) and the methylation index in blood using CE with UV detection (CE-UV). After application of meglumine postinjection, we achieved SAH in-capillary preconcentration in the HClO4 extracts of erythrocytes, which improved the detection limit (S/N = 3) of SAH up to 3 fmol or 180 nmol/L at the injection volume of 50 nL, taking into account the sample dilution rate. CE-UV was carried out in 30 mM glycine and 45 mmol/L HCl (pH ~1.8) at 17 kV in a capillary 48 cm in length and 50 µm id. Accuracy of the technique was 101% and reproducibility was about 12%.