Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.     

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis‐stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO‐mimetic peptides is under development from which peginesatide (also referred to as Hematide?), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase‐III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO‐mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL). Copyright © 2011 John Wiley & Sons, Ltd.     

Measurement uncertainty in sports drug testing

This paper discusses some of the contentious issues raised by van der Veen (Accred Qual Assur 8:334, 2003[1]), and Van Eenoo and Delbeke (Accred Qual Assur 8:477, 2003 [2]) concerned with the evaluation of the uncertainty of measurement and compliance decisions based on regulatory limits. Also, the criticisms of accreditation by van der Veen are considered and some general remarks about the strengths and limitations of accreditation provided. It is concluded that a position between those adopted by van der Veen and by Van Eenoo and Delbeke would be more appropriate.     

New and old challenges of sports drug testing

This brief note gives a general overview on the activity of the antidoping laboratories accredited by the World Anti-Doping Agency (WADA), outlining the evolution, over the last four decades, of the analytical methods and techniques in the detection of prohibited substances and methods. Special emphasis is given to the future trends of the fight against doping in sports, as seen from the perspective of a laboratory scientist, in the wider context of fair play, health protection, and perception of the activity of the antidoping laboratories by the general public.     

Development and validation of a mass spectrometric detection method of peginesatide in dried blood spots for sports drug testing

As recently reported, dried blood spot (DBS) analysis is an advantageous technique for doping control purposes due to the minimal invasive sample collection, the simple and economic manner, as well as the low susceptibility to manipulation. Its general applicability to the sports drug testing arena has been shown for analytes of various substance classes, all of which comprise exclusively low molecular mass compounds. The aim of the present study was to investigate whether the technique of DBS analysis is applicable also to (pegylated) peptides with relevance for doping controls. As target analyte, peginesatide (Omontys, Hematide), a recently approved pegylated erythropoietin-mimetic peptide of approximately 45 kDa, tested for the treatment of anaemia in patients with renal failure, was chosen, which has been prohibited in elite sports due to its assumed endurance enhancing effects. Therefore, a detection method for peginesatide employing DBS was developed based on extraction, proteolytic digestion and cation-exchange purification followed by liquid chromatography-tandem mass spectrometry analysis. Eventually, the assay was validated for qualitative purposes and proved to be specific, sensitive (limit of detection, 10 ng/mL) and precise (relative standard deviations below 18%), demonstrating the general suitability of DBS analysis in sports drug testing also for (pegylated) peptides.     

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.     

Synthesis and characterization of thiochromone S,S-dioxides as new photolabile protecting groups

3-Arylthiochromone derivatives were synthesized as new photolabile protecting groups, in which the photoreactivity was switchable based on oxidation of the sulfur atom (sulfide and sulfone); the protected substrates , released the corresponding alcohols, amines or carbonxylic acids almost quantitatively under UV-light in neutral condition and the photoproduct showed high fluorescence intensity.     

Synthesis and characterization of new nano-particles as blue ceramic pigment

The synthesis of a new nano-blue ceramic pigment CoxMg1-xAl2O4 (0 < or = x < or = 0.1) using the combination between co-precipitation and combustion synthesis (CS) method. The structure of pigments is assigned based on thermogravimetric and differential thermogravimetric analysis (TG-DTGA), X-ray diffractions (XRD), and UV-vis spectroscopy. Also, diffuse reflectance spectroscopy (DRS) using CIE L*a*b* parameter measurement method, infrared spectroscopy (IR) and transmission electron microscopy (TEM) is used. The result revealed that the nano-size particle pigment was obtained in range 24-35 nm by using 3-methyl-pyrozole-5-one (3MP5O) as a fuel at 400 degrees C in open furnace. Also, results show the varying colors and particles' size as a result of different calcination temperatures from 500 to 1200 degrees C for 2h.     

Amphiphilic hydrogel nanoparticles. Preparation, characterization, and preliminary assessment as new colloidal drug carriers

Inverse emulsion photopolymerization of acrylated poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol) and poly(ethylene glycol) was successfully employed to prepare stable, cross-linked, amphiphilic nanoparticles. Even at low emulsifier concentrations (2%) and high water-to-hexane weight ratios (35/65), the stability of the inverse emulsion allowed for the formation of well-defined colloidal material. Inverse emulsion characteristics and polymerization conditions could be controlled to vary the size of the nanoparticles between 50 and 500 nm. The presence of hydrophobic nanodomains within these otherwise hydrophilic nanoparticles was verified by using pyrene as a microenvironmentally sensitive probe. The hydrophobic poly(propylene glycol)-rich domains appear to be suitable for incorporation of hydrophobic drugs, encapsulating Doxorubicin up to 9.8% (w/w). We believe that the complex nano-architecture of these materials makes them a potentially interesting colloidal drug delivery carrier system and that the method should be useful for a number of amphiphilic macromolecular precursors.     

Synthesis, characterization, and biological evaluation of new oxime-phosphazenes

Hexachlorocylotriphosphazene (1) was reacted with 4-hydroxy-3-methoxybenzaldehyde to give hexakis[(4-formyl-2-methoxy)phenoxy]cyclotriphosphazene (2). Hexakis[(4-(hydroxyimino)2-methoxy)phenoxy]cyclotriphosphazene (3) was synthesized by reaction of 2 with hydroxlamine hydrochloride in pyridine. Compound 3 was reacted with benzyl chloride, acetyl chloride, allyl bromide, benzoyl chloride, propanoyl chloride, 4-methoxybenzoyl chloride, 2-chlorobenzoyl chloride, chloroacetyl chloride, methyl iodide, and thiophene-2-carbonyl chloride. From these reactions, full or partially substituted compounds were obtained, usually in high yields. Pure or defined products could not be obtained from reaction of 3 with methacryloyl chloride and O-acetylsalicyloyl chloride. The structures of the compounds were determined by elemental analysis, and IR, ¹H, ¹³C, and ³¹P NMR spectroscopy. The synthesized compounds were screened for in-vitro antimicrobial activity against two Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis), two gram-negative bacteria (Escherichia coli and Klebsiella pneumonia), and fungal strains (Aspergillus niger, and Candida albicans) by the agar well diffusion method. Few compounds had significant activity against both Gram-positive and Gram-negative bacteria. None of the compounds had antifungal activity except compounds 7 and 9, which had moderate activity.     

Synthesis and characterization of dicyanovinyl-substituted thienylpyrroles as new nonlinear optical chromophores

[structure: see text] New dicyanovinyl-substituted 1-(alkyl)aryl-2-(2'-thienyl)pyrroles 2 were synthesized and characterized. The solvatochromic behavior of the synthesized compounds was investigated. All the derivatives showed reversible oxidation and reduction on the CV time scale. The hyperpolarizabilities (beta) of compounds 2 were measured using hyper-Rayleigh scattering. The results are among the highest beta values reported for donor-acceptor-substituted thienylpyrroles.     

Rapid determination of urinary di(2-ethylhexyl) phthalate metabolites based on liquid chromatography/tandem mass spectrometry as a marker for blood transfusion in sports drug testing

Methods of blood doping such as autologous and homologous blood transfusion are one of the main challenging doping practices in competitive sport. Whereas homologous blood transfusion is detectable via minor blood antigens, the detection of autologous blood transfusion is still not feasible. A promising approach to indicate homologous or autologous blood transfusion is the quantification of increased urinary levels of di(2-ethylhexyl) phthalate (DEHP) metabolites found after blood transfusion. The commonly used plasticizer for flexible PVC products, such as blood bags, is DEHP which is known to diffuse into the stored blood. Therefore, a straight forward, rapid and reliable assay is presented for the quantification of the main metabolites mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethylhexyl) phthalate that can easily be implemented into existing multi-target methods used for sports drug testing. Quantification of the DEHP metabolites was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography/tandem mass spectrometry. The method was fully validated for quantitative purposes considering the parameters specificity, linearity (1-250 ng/mL), inter- (2.4%-4.3%) and intra-day precision (0.7%-6.1%), accuracy (85%-105%), limit of detection (0.2-0.3 ng/mL), limit of quantification (1 ng/mL), stability and ion suppression effects. Urinary DEHP metabolites were measured in a control group without special exposure to DEHP (n?=?100), in hospitalized patients receiving blood transfusion (n?=?10), and in athletes (n?=?468) being subject of routine doping controls. The investigation demonstrates that significantly increased levels of secondary DEHP metabolites were found in urine samples of transfused patients, strongly indicating blood transfusion.     

Synthesis,characterization and electrochemical properties of new phthalocyanines

The chemical synthesis and characterization with spectroscopic and electrochemical properties of [bis(4-fluorophenyl)-methoxy]-substituted metallo-phthalocyanines were reported for the first time. The new phthalocyanines have been characterized by elemental analysis, UV-Vis, FT-IR, and mass spectroscopies. The aggregation behavior of the phthalocyanine compounds was investigated in different solvents and concentrations. It is found that the fluoro substituents of peripherally Co and Cu complexes are examined and induce a shift to the redox processes toward the negative potentials and formed more reversible processes. Metal-based reduction and oxidation reactions were obtained for the Co(II) complex, whereas Cu(II) complexes exhibited Pc-ring-based electron-transfer reactions. The voltammetric measurements supported the proposed structure of the complexes.     

Synthesis, characterization, and application of cationic water-soluble oligofluorenes in DNA-hybridization detection

A simple and efficient approach was developed for the synthesis of a series of cationic water-soluble oligofluorenes up to a chain length of a heptamer. Bromoalkyl-substituted fluorenyl boronic esters as the key intermediates were synthesized by using a modified Miyaura reaction. With an increasing number of repeat units (trimer to hexamer), the size-specific oligomers have shown redshifts in both the absorption and emission maxima. The emission maximum reaches the limit for the hexamer in both water and buffer solution. The quantum yields of the oligomers decreased with increased oligomer size in water. Both fluorescence quenching of the oligomers by 9,10-anthraquinone-2,6-disulfonate and the fluorescence resonance energy transfer experiments with the oligomers as the donor and fluorescein (Fl)-labeled double-stranded DNA (dsDNA-Fl) as the acceptor revealed the chain-length-dependent behavior. The Stern-Volmer quenching constant increased with the molecular size, whereas the highest donor-sensitized Fl emission was observed for the hexamer. These size-specific oligomers also served as a model to study the structure-property relationships for cationic polyfluorenes.     

Synthesis,characterization and effects of arm number on properties of amphiphilic polyurethanes as drug delivery carriers

Amphiphilic polyurethanes based on methoxy poly(ethylene glycol) (mPEG) and poly(?-caprolactone) diol (PCL) with different arm numbers such as two, three and four were successfully synthesized. Their structures were confirmed by Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (¹H NMR) and gel permeation chromatography (GPC). The effects of arm number on properties of amphiphilic polyurethanes were studied. Pyrene fluorescence probe technique and dynamic light scattering (DLS) analyses showed that the CMC value and the micellar size of the resultant amphiphilic polyurethanes decreased and the micellar stability against dilution enhanced with increasing the arm number of polyurethane. Using indometacin (IMC) as a model drug, the results indicated that the drug loading capacity and in vitro drug sustained release effect of polyurethane with four arms were better than those of polyurethanes with two and three arms.