Expanding doping detection: Screening for carbohydrate-based plasma volume expanders

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  • Published: Jan 15, 2012
  • Author: Steve Down
  • Channels: Base Peak
thumbnail image: Expanding doping detection: Screening for carbohydrate-based plasma volume expanders

Indirect drugs of abuse

The prohibited list of substances maintained by the World Anti-Doping Agency (WADA) contains hundreds of entries which are banned for athletes. Some substances are prohibited at all times like anabolic steroids, and others during competition only, such as stimulants.

The list also contains compounds which do not act directly on an athlete to improve performance, like masking agents. For instance, plasma volume expanders fall into this category even though they have legitimate medical applications such as increasing the volume of blood during acute blood loss after accidents or surgery, and replacing blood when patients refuse to accept transfusions.

In the illicit world of sports doping, volume expanders are taken to prevent dehydration and to disguise the use of recombinant erythropoietin by controlling the haemocrit or haemoglobin values. They consist of solutions of mannitol, dextran or hydroxyethyl starch and WADA-accredited labs use GC/MS or LC-tandem-MS methods for their detection.

A team of Italian scientists from the Antidoping Laboratory at the Italian Sports Medicine Federation noted that these methods require toxic reagents such as pyridine and involve lengthy pretreatment steps. In their place, they have devised a quicker method which is still based on mass spectrometry, to take advantage of the excellent specificity it gives.

Francesco Botre, who is also affiliated to the Sapienza University of Rome, worked with Monica Mazzarino, Ilaria Fiacco and Xavier de la Torre and published details of their novel procedure in Drug Testing and Analysis.

Carbohydrate markers of doping with volume expanders

Urine samples from 300 athletes provided during competition and 300 out of competition were collected from a range of sports such as cycling, basketball, football, swimming, rugby and volleyball. They had all tested negative in screening procedures and were analysed along with 30 urines from healthy volunteers who had not taken any medication.

All samples were treated with the enzyme alpha-amylase from human saliva to break down the polysaccharides present into smaller saccharides such as glucose, hydroxyethylated glucose, maltose, isomaltose or hydroxyethylated maltose. Preliminary studies with urines collected during excretion studies of pharmaceutical formulations containing dextran or hydroxyethyl starch had confirmed the identities of the breakdown products.

The reaction mixture was spiked with carbon-13-labelled derivatives of mannitol and glucose to act as internal standards and reconstituted in aqueous acetonitrile for direct analysis by LC/MS with electrospray ionisation. The saccharides were separated on an amino column, optimising the mobile phase and column temperature for the best peak shape, sensitivity and selectivity.

The mass spectra recorded in positive mode displayed few diagnostic ions but the negative-ion spectra were far more informative. Under tandem mass spectrometry conditions, unique combinations of selected reaction monitoring transitions and retention times were selected for each of the breakdown products and standards to enable quantitation.

Good analytical performance was achieved with limits of detection and quantification in the range 30-100 µg/mL and the method was validated in accordance with the official ISO 1725 criteria and the WADA requirements for accredited drug testing labs.

Hydroxyethylated maltose and isomaltose were selected as specific markers of the presence of hydroxyethyl starch and dextran, respectively, since they are absent from control urine. Other hydrolysis products such as maltose, glucose and sucrose are present in blank urine at levels similar to those in the doping control, so are less specific.

The picture was less clear for mannitol because it was detected in all urines tested, including the 30 control urines. So, this carbohydrate originates from other sources, with fruit, food and drug additives the most likely candidates. However, the data were used to recommend threshold urinary concentrations which would be indicative of the abuse of volume expanders by athletes.

Mannitol levels in the 600 doping samples ranged from 50-1200 µg/mL, with the majority (more than 70%) at 200-500 µg/mL. These levels are about 20 times lower than those reported following intravenous administration of mannitol, so can be regarded as a threshold figure.

However, the researchers cautioned against accepting this figure just yet because "the number of urine samples examined in this study is not sufficient for a statistical evaluation." In addition, more work should be carried out "to establish a way to distinguish between the permitted oral and the prohibited intravenous administration."

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

The plasma volume expanders mannitol, dextran and hydroxyethyl starch, which are on the WADA prohibited list as masking agents, have been determined in urine by a mass spectrometric method. The procedure has been validated to WADA requirements for accredited laboratories

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