58 August / September 2019
Table 3. GC-HRAM data. Compounds
Clenbuterol (SIM)
18-norepimetendiol (SIM)
Epimetendiol
RT (min) XIC m/z 4.08 5.75
9.17
19-norandrosteron 8.86 Noretiocholanolon THMT-5α THMT-5β
*Stanozolol (3’OH+4bOH)
9.82
12.10 12.24 18.77
R2
335.06897 253.19508
358.26864 405.26396 405.26396 270.23459 270.23459 254.12661
Bromantane-6OH and Fluoxymesterone M2 (17,17-dimethyl-18-nor) were not analysed in this experiment, Testosterone-D3 is used as an internal standard.
GC-HRAM enables a simultaneous quantitative and qualitative analysis of exogenous and endogenous steroids. Orbitrap MS provides excellent sensitivity and high mass accuracy in the SRM workflow, and exceptional linear range is demonstrated, especially for endogenous steroids. Spectral fidelity is maintained across the required concentration range including mass accuracy. Additional benefits of the non-targeted acquisition workflow include the ability to perform retrospective data analysis and post-acquisition data processing, enabling future investigations to be made on existing samples, should new compounds of interest be identified.
The electron energy also plays an important role in the determination of steroids. Reducing the energy from 50 to 30 eV resulted in a 2.5–10-fold decrease in LODs. Reducing the electron energy further did not lead to additional sensitivity improvements.
0.9959 0.9978
0.9991 0.9991 0.9988 0.9990 0.9990 0.9912
Range (ng/mL) 0.1–0.5 0.05–5
0.1–5
0.05–5 0.05–5 0.1–5 0.1–5 0.1–5
The GC Orbitrap delivered optimal results for the steroids at an electron energy of 30 eV. Table 3 shows the linearity and LOD for the exogenous steroids, while Figure 5 highlights the benefits of using an exact mass GC for endogenous steroids.
The high mass accuracy allows lower detection limits utilising the 5th decimal after the comma in the exact mass, to detect apexing coeluting substances and to get rid of disrupting background m/z. This is not possible using QqQ, since those are nominal m/z instruments.
Conclusion
This study highlights the exceptional results generated from this complete GC-MS workflow. Several factors were important in delivering these results, including the choice of sample preparation method and glassware [4].
The GC-QqQ system showed the expected results in an anti-doping laboratory under routine conditions for screening,
confirmation and quantification in a single analysis workflow. The SRM approach delivered trace-level detection of the analytes of interest as required by WADA/ MRPL methods.
Using HRAM Orbitrap technology-enabled simultaneous qualitative and quantitative analysis of exogenous and endogenous steroids. The high selectivity of the exact mass measurements could also be used to determine new substances. Additionally, the wide linear range and the potential for non-targeted analysis makes GC-HRAM a valuable complementary method to GC- QqQ for anti-doping applications.
Acknowledgments
We would like to thank Sporthochschchule Köln, Landschaftsverband Rheinland and UFRJ Instituto de Chimica for their support with this study.
References
1. ICRAV 2018, Dubai UAE, Friday 9th of March, Workflow solution for Steroids and Anti-Doping Analysis in Urine with GC-MS/ MS and GC HRAM
2. European Pharmacopoeia 7, 01/2008, Chapter 3.2., Glass Containers for Pharmaceutical Use
3. Thermo Fisher Scientific Webinar, 25.7.2019, Fundamentals of Solid Phase Extraction
4. White Paper 21833, Thermo Fisher Scientific, Lennartz et al,
Why is high glass quality so important when you want to detect low concentrations of analytes?
Figure 5. Coeluting compounds at RT=13.70 min.
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