Microwaves accelerate anti-doping analyses

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  • Published: Apr 26, 2010
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Microwaves accelerate anti-doping analyses

A record number of new psychoactive drugs was reported in 2009 in Europe to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol, according to the EMCDDA-Europol annual report which was published in April, 2010. The 24 new substances recorded represented the largest total for a single year and an increase of 11 over 2008.

The report states that "It can be anticipated that the concept of designer drugs ... will continue to change at an unprecedented speed. With rapid technological developments, for example cheap organic synthesis coupled with the increased use of the Internet for marketing and selling of new drugs, it may be expected that synthetic analogues of other major drug groups will appear."

This conclusion is supported within the sporting world, where some drug testing labs are trying to anticipate which drugs will be used next by the athletics cheats. They are actually developing methods for detecting new designer drugs before they have been found in real samples or added to the prohibited list maintained by the World Anti-Doping Agency (WADA).

Central to the performance of drug testing labs is the ability for high-throughput analysis, testing as many samples as possible within a given period. This is especially important during major sporting events such as the Olympic Games or the Football World Cup, when a frantic period of urine testing takes place.

Sample extraction is one area where there is generally scope for improving the timescale and it has attracted the attention of a team of Italian scientists working at the Antidoping Laboratory of the Italian Sports Medical Federation in Rome. Francesco Botre, who is also affiliated to the University La Sapienza, Monica Mazzarino, Sara Riggi and Xavier de la Torre took a look at microwave-assisted extraction (MAE) to see what effect it would have on urine processing times.

They reasoned that MAE in a closed system raises the temperature to the solvent boiling points quickly, due to the increased pressure, thereby reducing the overall extraction time. The closed system also prevents loss of volatiles or solvent by evaporation and allows lower volumes of extractant to be used.

The researchers tested the power of MAE for several classes of drugs of abuse and their metabolites, including glucocorticoids (16 compounds), diuretics (16), beta-blockers (17), narcotics (6), anabolic steroids (25), and beta2-agonists (8). The first three groups were extracted for analysis by GC/MS in selected ion monitoring mode and the last three groups by LC-tandem MS with electrospray ionisation and selected reaction monitoring mode.

Both analyses followed the accredited ISO17025 procedure. In both cases, the five solvents tested were ethyl acetate, diethyl ether, tert-butyl methyl ether (TBME), chloroform-TBME and dichloromethane-TBME.

Urine samples were adjusted to pH 7.4, spiked with internal standards and hydrolysed with beta-glucuronidase. For GC/MS, the solutions were adjusted to pH 9 and subjected to MAE at various powers, testing a range of organic solvents. The extracts were subjected to trimethylsilylation before analysis. For LC/MS, the hydrolysed urines were neither pH adjusted before MAE nor derivatised afterwards.

The optimum power for thermally labile compounds such as prednisolone and chlorthiazide was 600 W, generating a temperature of 70°C in the closed extraction cell. For the thermally stable drugs, the power was optimised at 1020 W, generating 150°C. Higher powers in both cases led to analyte losses due to thermal degradation.

The best solvent in both cases was ethyl acetate, although it introduced higher matrix effects and mass spectrometric ion suppression than the other solvents, especially for the androgenic anabolic steroids and glucocorticoids. In these cases, diethyl ether or TBME gave better recoveries and no interferences.

Nevertheless, recoveries of 100% were achieved in 30-60 seconds with detection limits of 1-10 ng/mL for anabolic steroids, 3-15 ng/mL for glucocorticoids, 2-100 ng/mL for diuretics and beta2-agonists and 30-50 ng/mL for beta-blockers and narcotics. These values are all below the minimum performance limits set by WADA.

By comparison, extraction with a mechanical shaker in an open system using the corresponding solvents was only 20% complete in 60-120 seconds and required up to 10 minutes for completion. For some of the drugs, shaking did not achieve full recovery.

So, the introduction of MAE can save up to 90% of the extraction time without diminishing extraction yields, while maintaining analytical performance. Botre recommended the procedure for drug anti-doping tests and suggested that it would also be applicable to clinical, forensic and toxicological analyses.

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



 Francesco Botré


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