Last Month's Most Accessed Feature: Screening for 200 illegal sports drugs: The power of high-resolution mass spectrometry

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  • Published: Jan 8, 2015
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Mass spectrometry for drug testing

A single LC/MS method using high-resolution time-of-flight mass spectrometry has been developed to measure 200 illegal sports drugs in urine down to the WADA minimum required performance levels.

Sports drug testing authorities are having to cope with an increasing number of drugs in the WADA prohibited list as more substances are banned for athletes. This makes it more difficult for the testing labs as the screening process is continually being expanded to include them. There are a number of GC/MS and LC/MS methods that have been approved for testing but neither technique alone can cover all of the illegal agents.

More recently, high-resolution mass spectrometry has been applied to this growing problem. It brings the advantage that the entire data set can be recorded to allow for the retrospective analysis of data for compounds that were recently added to the prohibited list. Several of the proposed screening methods employ a time-of-flight mass spectrometer and can detect dozens of different sports drugs.

Now, a team of Spanish scientists from the University of Jaén has developed an LC/MS method with a TOF instrument that can measure 200 sports drugs of different classes in urine. They combined it with a single-stage SPE procedure that works satisfactorily for them all. Antonio Molina-Díaz, Juan C. Domínguez-Romero, Juan F. García-Reyes and Felipe J. Lara-Ortega described how the procedure can also be extended to metabolites of the target drugs and to other drugs with similar structures.

Fragmentation without preselection

The set of 200 drugs included steroids, glucocorticosteroids, cannabinoids, stimulants, analgesics, β-blockers, diuretics, antidiabetics, narcotics, opiates and anti-inflammatories. For method development, they were added to human urine that was extracted by SPE using a mixed mode cartridge packed with material that operated in ion exchange and nonpolar adsorption modes.

Even with the large number of targeted drugs, the dual action cartridge provided recoveries for the majority of the drugs of 70-120%, which fulfils the WADA minimum required performance levels. The few drugs with poor recoveries were highly hydrophobic and poorly retained on the column.

The extracts were analysed by LC/MS using two different instrument setups, both with electrospray ionisation in positive- or negative-ion mode. In the first, in-source collision-induced dissociation was initiated to produce the fragments. In contrast, a dedicated collision cell was in place in the second instrument and the performances of the two were compared.

The main criteria for confirming the presence of each drug are the retention times and the presence of at least two high-resolution ions with the expected abundances. With in-source CID, 75% of the compounds were unambiguously identified but that was exceeded by the second method, Here, all ions were fragmented without precursor ion isolation, providing comprehensive identification of the compounds, including those that were "missed" by the first method.

One key advantage of subjecting all of the ions in the collision cell to CID is that a full data set for all ions is recorded, paving the way for retrospective searching in the future. This could not be accomplished by fragmenting selected ions.

Metabolites and more

The researchers used the same principles to look for other compounds, such as other compounds from the same class, like methamphetamines. A database containing diagnostic ions for a particular class of compounds was created then the extracted ion chromatograms (EICs) for the ions were produced.

The principle was illustrated by searching for the ion of m/z 163.0754, which is characteristic of methylenedioxyamphetamine derivatives. In a test run, the three related amphetamines MDA, MDEA and MDMA were all detected and their presence confirmed.

Similarly, the technique can detect derivatives or metabolites of the 200 target drugs by calculating the accurate masses of the potential compounds and searching for them in the data set. It was illustrated by searching for metabolites of the diuretic drug bumetanide in rat urine following intraperitoneal administration, six of which were found. They were produced by mono- and dihydroxylation, oxidation and dealkylation of the parent drug.

It is the high-resolution capabilities of the mass spectrometer and the fragmentation of all the ions without preselection that makes this method so powerful. It should prove useful for drug testing facilities as it covers many different compound classes and complies with the WADA reporting limits.

Related Links

Talanta 2015, 134, 74-88: "Screening and confirmation capabilities of liquid chromatography-time-of-flight mass spectrometry for the determination of 200 multiclass sport drugs in urine"

Article by Steve Down

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

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