The lack of observed intoxications may justify the absence of legal actions in the EU against phenethylamine designer drugs-of-abuse of the 2C-series. However, this may also be explained by either the inability of toxicologists to detect the abuse of substances of the 2C-series or the unawareness of the phenomenon of these drugs. Therefore, EU authorities should promote the availability of relevant standards, validated assays and scientific knowledge regarding these drugs.
Dextran and methacrylated dextrans (dex-MA) were degraded with dextranase, and the formed degradation products were characterized by electrospray mass spectrometry. It was shown that the main degradation product was isomaltose for both dextran and dex-MA. In degraded dex-MA, the main methacrylated product was isomaltotriose. The relative contribution of oligosaccharides with a higher molecular weight (up to isomaltohexaose) and of multiply methacrylated oligosaccharides increased with the degree of substitution of dex-MA. Enzyme kinetics with a three-substrate model showed that the Michaelis−Menten constant for the monomethacrylated substrate was smaller than for the unsubstituted substrate, whereas the Michaelis−Menten constant for multiply methacrylated susbtrates was higher. This indicates a favorable interaction of one methacrylate group with a hydrophobic binding subsite in the enzyme. The maximum degradation rate, however, was substantially lower for the substituted substrates than for the native substrate. From these results, it is concluded that the enzyme hydrolyzes a glycosidic bond between a methacrylated glucopyranose residue and an unsubstituted one in the dex-MA chain. This hypothesis is further supported with electrospray mass spectrometry because of both the presence of an ion formed by fragmentation at the nonreducing end of an oligosaccharide and the absence of oligosaccharides in which the number of methacrylates equals or exceeds the number of glucopyranose residues.
The enzymatic degradation of intramolecularly polymerized methacrylated dextran [dex-poly(MA)] and hydrogels based on methacrylated dextran was studied. Dex-poly(MA) was synthesized as a soluble model compound to investigate the enzymatic degradation of dextran hydrogels. After degradation of hydrogels and dex-poly(MA) by dextranase, the formed products were analyzed by gel permeation chromatography (GPC) and electrospray mass spectrometry (ES-MS). GPC analysis revealed that besides low-molecular-weight fractions (180−1100 Da) a soluble high-molecular-weight fraction (9−94 kDa) also was formed for both dextran hydrogels and dex-poly(MA). The high-molecular-weight products in degraded dextran hydrogels and dex-poly(MA) increased both in relative amount and in molecular weight with increasing degree of methacrylate substitution. This fraction most likely consisted of oligosaccharides bound to polymerized methacrylate groups. ES-MS showed that the low-molecular-weight fractions consisted of glucose, isomaltose and some larger unsubstituted oligosaccharides. The same products were formed after enzymatic degradation of native dextran. The enzymatic degradation of dex-poly(MA) as well as dextran hydrogels was described with kinetic models. Combination of enzyme kinetics, GPC and ES-MS lead to the conclusion that the enzymatic degradation can be divided in two processes. First, long unsubstituted chains with a length of probably about 18 or more glucopyranose residues are hydrolyzed at a similar rate and extent as native dextran. Second, the enzyme binds to unsubstituted chain segments of 6 to about 18 glucopyranose residues, which are hydrolyzed slower. Shorter unsubstituted chain segments are not enzymatically degraded.
The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.
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