Detection methods for autologous blood doping.

The use of blood doping is forbidden by the World Anti-Doping Agency. Several practices, such as blood transfusions are used to increase oxygen delivery to muscles and all of them are highly pursued. In this regard, the development of accurate methodologies for detecting these prohibited practices is one of the current aims of the anti-doping control laboratories. Flow cytometry methods are able to detect allogeneic blood transfusions but there is no official methodology available to detect autologous blood transfusions. This paper reviews protocols, including the Athlete Biological Passport, that use indirect markers to detect misuse of blood transfusions, especially autologous blood transfusions. The methods of total haemoglobin mass measurements and the detection of metabolites of blood bags plasticizers in urine are reviewed. The latter seems to be an important step forward because it is a fast screening method and it is based on urine, a fluid widely available for doping control. Other innovative approaches to blood transfusion detection are also mentioned. A combination of the reported methodologies and the implementation of the Athlete Biological Passport is becoming a promising approach.

Identification of free and conjugated metabolites of mesocarb in human urine by LC-MS/MS.

The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases, the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was carried out using a C(18) column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-, di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time. Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries of 100.3 +/- 0.8 and 105.9 +/- 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL(-1) were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng mL(-1), and relative standard deviations were lower than 15% in all cases (n = 4).

Ultraperformance liquid chromatography tandem mass spectrometric method for direct quantification of salbutamol in urine samples in doping control.

A fast and reliable quantitative method for salbutamol using direct analysis of the urine sample by ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) has been developed. Urine samples were spiked with salbutamol-d6 (internal standard), and, then, they were diluted with ultrapure water (1:1, v/v). Aliquots of 1 microl of the mixture were directly analyzed by UPLC/MS/MS. The chromatographic separation was performed in a UPLC BEH C18 (100 mm x 2.1 mm, 1.7 microm) column with a mobile phase contained 0.01% formic acid in ultrapure water (v/v) and 0.01% formic acid in acetonitrile (v/v), using gradient elution at 0.6 ml/min. The temperature of the column was set to 45 degrees C. The total run time was 3.2 min. Electrospray ionization in positive ion mode was used under multiple reaction monitoring (MRM) at different collision energies. Nitrogen and argon were used as desolvation and collision gas, respectively. The method was shown to be linear from 200 to 5000 ng/ml (r2>0.99). The limit of quantitation was estimated in 200 ng/ml. Intra-assay precision and accuracies, evaluated by using quality control samples containing 550 and 1100 ng/ml salbutamol, were always better than 8.4%. The intermediate precision was estimated to be in the range of 5.6-8.9%. The method was shown to be reliable when applying to routine samples, and the short analysis time resulting from a simple sample preparation and a fast instrumental analysis makes it of great interest for antidoping control purposes.

Ethanol metabolism in Drosophila melanogaster.

A quantitative study of the transformation of ethanol into acetaldehyde shows that, in Drosophila melanogaster, the mitochondrial ethanol oxidizing system is not very active but that the part played by catalase appears more important than expected. For a strain without alcoholdehydrogenase, ethanol is highly toxic. The presence of acetaldehyde in the culture medium is toxic for all the strains studied. But, since even a strain without any aldehydeoxidase lives normally, the metabolic production of acetaldehyde does not seem dangerous.