57
Figure 3. Selectivity comparison of a single versus triple quadrupole system for fluoxymesterone-M2 at MRPL levels.
groups in order to reduce the polarity of the substance. Underivatised they would possibly stick in the glass vial or liner. The derivatisation step was fully automated using a Thermo Scientific Triplus RSH Autosampler. This enabled batch derivatisation in dedicated preparation cycles (a batch of 6 samples was derivatised prior to injection) or the use of a sequential procedure in which each sample was derivatised and injected. Prior to injection, the samples were transferred into Thermo Scientific Chromacol GOLD-Grade inert glass vials (Type 1 hydrolytic class glass, 29% free silanol groups on the surface) to minimise adsorption to the vial surface. [4]
GC-MS analysis
Following sample preparation, the samples were injected into the GC-MS system. The samples were separated using a Thermo Scientific TRACE 1310 GC system coupled with a Thermo Scientific TSQ 8000 triple quadrupole mass spectrometer. The triple quadrupole MS data were compared to an Q Exactive Orbitrap mass analyser to see, what benefits in resolution Orbitrap can provide. Table 1 shows the GC and GC-MS parameters used.
Results
GC-QqQ offers good selectivity for endogenous and exogenous steroids, as well as other relevant analytes of interest in urine. This technology is widely used and is well established in antidoping laboratories around the world. For the non-targeted
approach, results are shown on a GC- HRAM with 1 ppm mass accuracy across a broad linearity range, including good sensitivity, and linearity for a large number of compounds.
GC-QqQ enables doping control laboratories to perform screening, confirmation, and quantification of analytes in complex matrices in a single analysis without the need to change their current extraction and GC separation methods. The identification and quantitation of anabolic- androgenic steroids (AAS) is well studied using GC-QqQ technology. GC-QqQ offers significant benefits over LC-MS interfaced to atmospheric ionisation sources, which are limited by poor ionisation. Selected reaction monitoring (SRM) provides trace- level detection of the steroids of interest, separating the chemical noise of the matrix from the signal and assuring high sensitivity and selectivity even in very complex matrices, such as camel and horse urine.
GC coupled with single quadrupole MS in selected ion monitoring (SIM) mode has been widely used for anti- doping applications, and it is still used in laboratories for many applications. However, for steroids analysis, single quadrupole systems are limited in terms of selectivity and sensitivity [1].
Figure 3 shows the selectivity of single versus triple quadrupole systems for a real urine sample containing fluoxymesterone-M2. The results highlight that the single quadrupole system did not provide the necessary sensitivity and selectivity. Co-elution’s from the matrix make it difficult to determine the
identity of the analyte and also reduce the sensitivity of the measurement. SRM using the triple quadrupole system, on the other hand, provides both excellent sensitivity and selectivity.
The Rio de Janeiro Doping Control Laboratory (LBCD–LADATEC) analysed approximately 500 samples per day during the 2016 Summer Olympic Games. To meet these demanding sample throughput requirements, robust and reliable analytical workflows were essential. Table 2 shows the World Anti-Doping Agency Minimum Required Performance Levels (WADA/MRPL) and the LOD of banned substances and their metabolites.
Table 2. Limits of detection. Compound
Nandrolone
19-Norandrosterone (19-NA)
19-NE Clenbuterol
WADA MRPL (ng/mL)
5 2
2 0.2
3’-hydroxystanozolol 2 Etilefrine
Norfenefrine Methadone Zilpaterol
Ethylestrenol Oxycodone
Oxymorphone Nandrolone
Mesterolone P
100 100 50 5 5
50 50 5 5
LOD (ng/mL)
2.5 0.4
1
0.1 1
50 50 5 1 1 5
10
2.5 2.5
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