Chemoinformatics-based classification of prohibited substances employed for doping in sport

Representative molecules from 10 classes of prohibited substances were taken from the World Anti-Doping Agency (WADA) list, augmented by molecules from corresponding activity classes found in the MDDR database. Together with some explicitly allowed compounds, these formed a set of 5245 molecules. Five types of fingerprints were calculated for these substances. The random forest classification method was used to predict membership of each prohibited class on the basis of each type of fingerprint, using 5-fold cross-validation. We also used a k-nearest neighbors (kNN) approach, which worked well for the smallest values of k. The most successful classifiers are based on Unity 2D fingerprints and give very similar Matthews correlation coefficients of 0.836 (kNN) and 0.829 (random forest). The kNN classifiers tend to give a higher recall of positives at the expense of lower precision. A na?ve Bayesian classifier, however, lies much further toward the extreme of high recall and low precision. Our results suggest that it will be possible to produce a reliable and quantitative assignment of membership or otherwise of each class of prohibited substances. This should aid the fight against the use of bioactive novel compounds as doping agents, while also protecting athletes against unjust disqualification.     

Comprehensive plasma‐screening for known and unknown substances in doping controls

Occasionally, doping analysis has been recognized as a competitive challenge between cheating sportsmen and the analytical capabilities of testing laboratories. Both have made immense progress during the last decades, but obviously the athletes have the questionable benefit of frequently being able to switch to new, unknown and untested compounds to enhance their performance. Thus, as analytical counteraction and for effective drug testing, a complementary approach to classical targeted methods is required in order to implement a comprehensive screening procedure for known and unknown xenobiotics. The present study provides a new analytical strategy to circumvent the targeted character of classical doping controls without losing the required sensitivity and specificity. Using 50 µL of plasma only, the method potentially identifies illicit drugs in low ng/mL concentrations. Plasma provides the biological fluid with the circulating, unmodified xenobiotics; thus the identification of unknown compounds is facilitated. After a simple protein precipitation, liquid chromatographic separation and subsequent detection by means of high resolution/high accuracy orbitrap mass spectrometry, the procedure enables the determination of numerous compounds from different classes prohibited by the World Anti‐Doping Agency (WADA). A new hyphenated mass spectrometry technology was employed without precursor ion selection for higher collision energy dissociation (HCD) fragmentation experiments. Thus the mass spectra contained all the desired information to identify unknown substances retrospectively. The method was validated for 32 selected model compounds for qualitative purposes considering the parameters specificity, selectivity, limit of detection (<0.1–10 ng/mL), precision (9–28%), robustness, linearity, ion suppression and recovery (80–112%). In addition to the identification of unknown compounds, the plasma samples were simultaneously screened for known prohibited targets. Copyright © 2010 John Wiley & Sons, Ltd.     

An improved gas chromatography screening method for doping substances using triple quadrupole mass spectrometry, with an emphasis on quality assurance

A GC‐QqQ‐MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, β‐blockers, β‐2‐agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti‐doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro‐organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α‐androstane‐3,17‐dione and 5β‐androstane‐3,17‐dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass‐to‐charge ratios and the obtained ranges were compared with the World Anti‐Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast screening of anabolic steroids and other banned doping substances in human urine by gas chromatography/tandem mass spectrometry

A fast and sensitive method for the comprehensive screening of anabolic agents and other banned doping substances using gas chromatography/tandem mass spectrometry (GC/MS/MS) with an external ionization ion trap mass spectrometer is presented. The method takes advantage of the resolving power of MS/MS to eliminate background interferences, thus speeding up the chromatographic analysis. For each compound, different fragmentation reactions were studied and their collision energies optimized to obtain the best sensitivity in terms of their signal-to-noise ratio (S/N). A dramatic reduction in overall analysis time was achieved compared with other common approaches. More than 50 substances could finally be monitored in less than 7.4 min with detection limits (S/N >3) lower than 0.5 ng ml(-1) for most of the compounds with special sensitivity requirements according to the International Olympic Committee (IOC). A validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes. Good intermediate precision, below 25% for most of the compounds, and robustness were observed. The optimized method was successfully applied to analyse more than 100 real human urine samples with optimum sensitivity and specificity rates.     

Forensic differentiation of biogenic organic compounds from petroleum hydrocarbons in biogenic and petrogenic compounds cross-contaminated soils and sediments

“Total petroleum hydrocarbons” (TPHs) or “petroleum hydrocarbons” (PHCs) are one of the most widespread soil pollutants in Canada, North America, and worldwide. Clean-up of PHC-contaminated soils and sediments costs the Canadian economy hundreds of million of dollars annually. Much of this activity is driven by the need to meet regulated levels of PHC in soil. These PHC values are legally required to be assessed using standard methods. The method most commonly used in Canada, specified by the Canadian Council of Ministers of the Environment (CCME), measures the total hydrocarbon concentrations in a soil by carbon range (Fraction 1: C₆–C₁₀; Fraction 2: C₁₀–C₁₆, Fraction 3: C₁₆–C₃₄: and Fraction 4: C₃₄+). Using the CCME method, all of the materials extractible by a mixture of 1:1 hexane:acetone are considered to be petroleum hydrocarbon contaminants. Many hydrocarbon compounds and other extractible materials in soil, however, may originate from non-petroleum sources. Biogenic organic compounds (BOCs) is a general term used to describe a mixture of organic compounds, including alkanes, sterols and sterones, fatty acids and fatty alcohols, and waxes and wax esters, biosynthesized by living organisms. BOCs are also produced during the early stages of diagenesis in recent aquatic sediments. BOC sources could include vascular plants, algae, bacteria and animals. Plants and algae produce BOCs as protective wax coating that are released back into the sediment at the end of their life cycle. BOCs are natural components of thriving plant communities. Many solvent-extraction methods for assessing soil hydrocarbons, however, such as the CCME method, do not differentiate PHCs from BOCs. The naturally occurring organics present in soils and wet sediments can be easily misidentified and quantified as regulated PHCs during analysis using such methods. In some cases, biogenic interferences can exceed regulatory levels, resulting in remediation of petroleum impacts that are not actually present. Consequently, reliance on these methods can trigger unnecessary and costly remediation, while also wasting valuable landfill space. Therefore, it is critically important to develop new protocols to characterize and differentiate PHCs and BOCs in contaminated sediments. In this study, a new reliable gas chromatography–mass spectrometry (GC–MS) method, in combination with a derivatization technique, for characterization of various biogenic compounds (including biogenic alkanes, sterols, fatty acids and fatty alcohols) and PHCs in the same sample has been developed. A multi-criteria approach has been developed to positively identify the presence of biogenic compounds in soil and sediment samples. More than thirty sediment samples were collected from city stormwater management (SWM) ponds and wetlands across Canada. In these wet sediment samples, abundant biogenic n-alkanes, thirteen biogenic sterols, nineteen fatty carboxylic acids, and fourteen fatty alcohols in a wide carbon range have been positively identified. Both PHCs and BOCs in these samples were quantitatively determined. The quantitation data will be used for assessment of the contamination sites and toxicity risks associated with the CCME Fraction 3 hydrocarbons.     

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.     

Electrochemical doping of polyaniline with the tetracyanoquinodimethane anion

A new complex of aniline (An) with tetracyanoquinodimethane (TCQM) An(TCQM)₄ has been obtained and studied. The redox cycling of polyaniline (PAni-sulfo) in a neutral electrolyte containing Cl^– and ClO₄^? anions led to a replacement of the sulfate anion by the Cl^– and ClO₄^? anions. The TCQM anion can also replace the sulfate anions in polyaniline (PAni) during the cycling of the latter in a neutral electrolyte containing TCQM anions. The introduction of TCQM in PAni creates conditions for generating nanocrystalline regions inside the polymer.     

Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone

Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4%, respectively, for the LC/IT-MS method and lower than 8.4 and 4.4%, respectively, for the LC/TOF-MS method. Accuracy (bias percentage) was less than 9.7% for both methods. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Gibberellins and related substances

Conclusions The principles of the reduction of 2-keto-derivatives of gibberellic acid by complex metal hydrides were studied, and the synthesis of the epimer of gibberellic acids and its ester was carried out.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1805–1809, August, 1969.The authors would like to thank V. I. Zaretskii and I. B. Papernaya for taking the mass spectra and discussing the results and A. V. Kessenikh for the NMR spectra.     

Gibberellins and related substances

Chemistry of Natural Compounds - 1. A study of the gibberellin-like substances of the leaves of tobaccoN. tabacum has shown that the latter contains gibberellin A3 and possibly A6 in the free...     

Bioavailability of silicon from food and food supplements

The bioavailability of silicon from three different silicon sources was studied. A diet rich in silicon, a tablet containing a dry extract of horsetail and a solution of silicon in a choline-glycerol matrix were compared. Blood and urine of one healthy test person were sampled to monitor the silicon uptake. The silicon content of blood and serum samples was determined by graphite furnace atomic absorption spectrometry. Strongly diverging results were obtained for the three different silicon sources. Neither an increase in urinary silicon excretion nor in serum silicon content was observed when feeding the silicon rich diet. Urinary silicon excretion did significantly (P < 0.05) rise during supplementation with tablets containing dry extract of horsetail. Intake of a solution of silicon in a choline-glycerol matrix resulted in a significantly (P < 0.05) increased urinary silicon excretion and serum silicon content. From these results it can be concluded that speciation (chemical form, matrix) strongly influences the bioavailability of silicon. Received: 3 August 1998 / Revised: 25 September 1998 / Accepted: 30 September 1998     

Gibberellins and related substances

Conclusions 1. A study of the gibberellin-like substances of the leaves of tobaccoN. tabacum has shown that the latter contains gibberellin A₃ and possibly A₆ in the free state.The amount of GA₃ in the leaves of this plant is approximately 0.2–2 µg/kg.2. In addition, tobacco leaves contain neutral gibberellin-like substances in the bound form which are liberated on hydrolysis.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 4, pp. 296–304, 1969