Structure-activity relationship for peptídic growth hormone secretagogues.

Growth hormone releasing peptides (GHRPs) could be widely used by cheating athletes because they produce growth hormone (GH) secretion, so may generate an ergogenic effect in the body. Knowledge of the essential amino acids needed in GHRP structure for interaction with the target biological receptor GHSR1a, the absorption through different administration routes, and the maintenance of pharmacological activity of potential biotransformation products may help in the fight against their abuse in sport. Several GHRPs and truncated analogues with the common core Ala-Trp-(D-Phe)-Lys have been studied with a radio-competitive assay for the GHSR1a receptor against the radioactive natural ligand ghrelin. Relevant chemical modifications influencing the activity for positions 1, 2, 3, and 7 based on the structure aa-aa-aa-Ala-Trp-(D-Phe)-Lys have been obtained. To test in vivo the applicability of the activities observed, the receptor assay activity in samples from excretion studies performed after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin was confirmed. Overall results obtained allow to infer structure-activity information for those GHRPs and to detect GHSR1a binding (intact GHRPs plus active metabolites) in excreted urines. Copyright © 2016 John Wiley & Sons, Ltd.

'Wrong-way-round ionization' and screening for doping substances in human urine by high-performance liquid chromatography/orbitrap mass spectrometry.

To free analytical resources for new classes of doping substances, such as banned proteins, maximization of the number of compounds that can be determined with high sensitivity in a single run is highly urgent. This study demonstrates an application of 'wrong-way-round ionization' for the simultaneous detection of multiple classes of doping substances without the need to switch the polarity. A screening method for the detection of 137 compounds from various classes of prohibited substances (stimulants, diuretics, ß(2)-agonists, ß-blockers, antiestrogens, glucocorticosteroids and anabolic agents) has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography/orbitrap mass spectrometry with wrong-way-round ionization. Up to 64% of compounds had a 10-fold lower limit of detection (LOD) than the minimum required performance limit. To compare the efficiency of conventional ionization relative to wrong-way-round ionization of doping substances in + ESI, a fortified blank urine sample at the minimum required performance limit was analyzed using two ESI approaches. All compounds were detected with markedly better S/N in a high-pH mobile phase, with the exception of acetazolamide (minimal change in S/N, < 20%).The method was validated by spiking 10 different blank urine samples at five different concentrations. Validation parameters included the LOD, selectivity, ion suppression, extraction recovery and repeatability.

[Profile of sex steroids in healthy volunteers' urine during experiment in isolated object].

The quantitative determination a number of endogenous steroids and their metabolites in urine of healthy volunteers by means of gas chromatography - mass spectrometry was performed. The dynamic of steroid profile of healthy individuals as well as possible ranges of several endogenous steroid parameters have been investigated. Samples were obtained during 105-days experiment with 6 volunteers in isolated on ground modules where were modeling the main life conditions which could influence the steroid profile: meal volume and composition, water consumption, motion activity, air composition and temperature, rate sleep - wakefulness and emotional tension. The parameters of urine steroid profile of healthy volunteer which were affected by life conditions in isolated object were revealed. The parameters of individual and group variability of steroid profile and its dependence from definite experiment conditions - change of salt consumption periods, autonomy of vital activity were detected.

High sensitive analysis of steroids in doping control using gas chromatography/time-of-flight mass-spectrometry.

The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric (GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with fast spectral acquisition rate) was used. This method is based on the formation of the silyl derivatives of steroids; exchange of the reagent mixture (pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample volume injection of this solution based on sorption concentration of the respective derivatives from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire analytes solvent-free concentrate transfer into the injector of the gas chromatograph. Detection limits for 100 µl sample solution volume were 0.5-2 pg/µl (depending on the component). Application of TOF-MS model 'TruTOF' (Leco, St Joseph, MO, USA) coupled with gas chromatograph and ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass spectra and resolving coeluted peaks. Due to use of the proposed method (10 µl sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in the urine extract in comparison with the injection of 1 µl of the same sample solution.

[The use of the deuterated internal standard for morphine quantitation for the purpose of doping control by gas chromatography with mass-selective detection].

The objective of this study was to demonstrate the possibility to use deuterated compounds as internal standards for the quantitative analysis of morphine by gas chromatography with mass-selective detection for the purpose of doping control. The paper is focused on the problems associated with the use of deuterated morphine-D3 as the internal standard. Quantitative characteristics of the calibration dependence thus documented are presented along with uncertainty values obtained in the measurements with the use of deuterated morphine-D6. An approach to the assessment of method bias associated with the application of morphine-D6 as the deuterated internal standard is described.

[Effect of mexidol on 6beta/free hydroxycortisol ratio. Possibility of CYP3A4 activation].

The influence of mexidol (2-ethyl-6-methyl-3-oxypyridine) after a single peroral administration on the levels of 6beta-hydroxycortisol (6beta-OHC) and free cortisol (FC) in human urine has been evaluated. The 6beta-OHC/FC ratio is increased (approximately 2.96 +/- 0.76 times) against the basal 6beta-OHC/FC ratio during the first 24 hour after drug administration. Data analysis on the second and third day after mexidol administration did not show evident changes in 6beta-OHC/FC ratios. It is suggested that CYP3A4 activation after mexidol administration occurred only during active drug biotransformation and excretion and ceased after full excretion from the human body.

[Experimental pharmacokinetics of the new neurotensine-derived antipsychotic drug dilept].

The metabolism and pharmacokinetics of a new neurotensine-derived dipeptide drug dilept (N-caproyl-L-prolyltyrosine methyl ester) and its tentative metabolites after intravenous and peroral administration of the parent drug and its tabletized form in rats have been studied by HPLC-ESI(-)-MS/MS method. It is established that unchanged dilept (detected in the blood plasma for no less than 30 min) as well as N-caproyl-L-proline and N-caproyl-L-prolyl-L-tyrosine (both detected in the blood over more than 4 h) are the major metabolites in the bloodstream upon peroral administration of the drug. The proposed structures of metabolites were confirmed by countersynthesis. Dilept and N-caproyl-L-prolyl-L-tyrosine penetrate through the blood - brain barrier. The drug is rapidly absorbed, distributed, and metabolized in the rat organism. Peroral administration of dilept in rats in the form of tablets (at a dose of 200 mg/kg) resulted in a significant increase in intestinal absorption, as evidenced by a 22% improvement in the bioavailability, whereas dilept alone showed an absolute bioavailability of less than 1%.

[HPLC-MS determination of 2-ethyl-6-methyl-3-oxypyridine].

An HPLC-ESI-MS method has been developed for determining 2-ethyl-6-methyl-3-oxypyridine (EMO) in human urine upon peroral administration of this substance in form ofmexidol. Various sample preparation (extraction) procedures were tested and compared for evaluating the recovery and matrix effect. Solid-phase extraction procedure followed by derivation with dansyl chloride is proposed as a method of choice. The recovery of analyte was 48.1 +/- 3.4%, and the matrix effect was 99.4 +/- 4.1%. The MS and MS/MS spectra of EMO and its dansyl derivatives are presented and interpreted. The analyses were performed using a mass spectrometer of the ion trap type with electrospray ionization at atmospheric pressure, operating in the regime of positive ion detection.

Introduction of HPLC/orbitrap mass spectrometry as screening method for doping control.

A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta(2) agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70%. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17%, respectively. The relative standard deviation (RSD) of the intraday precision (n = 6) ranged from RSD = 16-22%, whereas the interday precision (n = 18), ranged from RSD = 17-26%, depending on the solute concentration investigated.

[Gas chromatography/tandem mass-spectrometry assay for trace amounts of 3'-hydroxystanozolol].

3'-Hydroxystanosolol detection in biological fluids at pg levels by gas chromatography/tandem mass spectrometry is described. Gas chromatography/high resolution mass spectrometry results can be confirmed with gas chromatography/tandem mass-spectrometry.

[Doping in sports].

Drugs used by athletes for the improvement of results are described and classified with respect to chemical structure and pharmacological action. The main groups of drugs treated as doping are considered and the WADA requirements to prohibited preparations are formulated. The main effects produced by drugs on the athletes and animals (race horses, fight dogs, etc ) are described and the measures of therapy against side effects are outlined.

Characterization of prednisone, prednisolone and their metabolites by gas chromatography-mass spectrometry.

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography-mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.

Determination of methylprednisolone metabolites in human urine by gas chromatography-mass spectrometry.

Methylprednisolone and its metabolites were studied as their methoxyamine-trimethylsilyl derivatives by means of capillary gas chromatography-mass spectrometry. The expected unchanged drug and 11-keto and 20-hydroxy metabolites were found in human urine. The typical metabolites are 6,7-dehydro analogues of the above-mentioned compounds. Characteristic gas chromatographic profiles of urine steroids were obtained. Retention indices and m/z values are presented for methylprednisolone and its main metabolites.