1. Results and discussion Technical innovations
GC/Q-TOF technical innovations allow trace analysis and screening of drug related substances
Peter Van Eenoo1, Michaël Polet1, Wim Van Gansbeke1, Remko Van Loon2, Ken Brady2
1DoCoLab Ghent University, 2Agilent Technologies
Introduction
androsterone
5α-androstan-3α,17β-diol
Screening methods for drug related substances in doping control have stringent analytical demands, as they require combining a number of critical prerequisites in one single method. The screening method needs to be able to detect qualitative compounds at very low concentrations and simultaneously quantify very high concentrations of endogenous steroids. On top of that, a lot of these target analytes are epimers and the method needs the fulfil the World Anti-Doping Agency (WADA) requirements on substance detection criteria and minimum required performance limits (MRPL). [1] Gas chromatography triple quadrupole mass spectrometry (GC/QQQ) is typically used for screening due to the high sensitivity and excellent linearity. [2] GC/QQQ also has a number of limitations such as the lack of retrospectivity, cumbersome addition of new compounds due to MRM and incompatibility with libraries. Screening with high resolution acquisition would solve these issues. However, up to now, in order to translate the current GC/QQQ screening methods into a full scan high resolution acquisition screening, GC high resolution instruments lacked sensitivity and had inadequate linearity. In liquid chromatography (LC), a similar switch from LC/QQQ screening to LC high resolution screening occurred over the past years. [3]
R2 = 0,999
R2 = 0,999
24 – 9600 ng/mL
2 – 800 ng/mL
Figure 2. Calibration curves for androsterone and 5α-androstan-3α,17β-diol on the GC/Q-TOF.
Table 1. Translation of the GC/QQQ screening method into an equivalent GC/Q-TOF screening method
GC/QQQ
GC/Q-TOF
0,5 mL urine
12 m HP-1MS
(250 µm I.D. x 0,25 µm df)
15 m HP ultra 1
(200 µm I.D. x 0,11 µm df)
2 µL injection from 60 µL
0,8 µL injection from 40 µL
14,8 min run
13,3 min run
Figure urine samples analysed on both the GC/QQQ and the GC/Q-TOF.
Results and discussion
Technical innovations
The current GC/QQQ screening method for doping control substances was successfully translated into an equivalent screening method on GC/Q-TOF for both the quantitative and qualitative compounds (> 250 compounds). The same calibration ranges were used on the GC/Q-TOF as on the GC/QQQQ (Figure 2). In addition, the GC/Q-TOF screening method is compliant with the WADA requirements and is capable of reaching the WADA MRPLs (Figure 3).
Owing to a number of technical innovations such as an extended flight tube (higher resolution) and the high efficiency low energy EI source, the GC/Q-TOF exhibits significant higher sensitivity and improved linearity. The 7250 GC/Q-TOF closes the gap with GC/QQQ, allowing the transfer of the current GC/QQQ screening method for drug related substances to a GC/Q-TOF screening method. The 7250 GC/Q-TOF is optimized for low energy EI experiments with adapted magnets, lenses and tuning. Low energy EI is a softer ionization with less fragmentation. As visualized in Figure 1, this results in a higher relative abundance of the diagnostic ions (i.e., high mass to charge ratio ions).
Taking into account that anti-doping samples can be stored and reanalysed for up to ten years, the retrospectivity and sensitivity offered by GC/Q-TOF opens the door to a cleaner sport.
calusterone metab.
5 ng/mL
fluoxymesterone tetrol
5 ng/mL
βMeT metab.
5 ng/mL
clenbuterol
0.2 ng/mL
low energy EI (17 eV)
conventional EI (70 eV) A B M+
αMeT metab.
2 ng/mL
Figure 4. Urine sample spiked at MRPL and analysed on the GC/Q-TOF in full scan high resolution aqcuisition (18 eV).
Conclusions
Switching to a high resolution full scan acquisition open screening method brings up a new chapter for GC screening and opens many new doors and possibilities for the future. There are no limitations on the amount of compounds that can be added to the method and the approach allows retrospectivity and compatibility with libraries.
Figure 1. Low energy EI (17 eV) and conventional EI (70 eV) spectrum of fluoxymesterone metabolite
Contact
References
Michaël Polet
DoCoLab, Ghent University
World Anti-Doping Agency, WADA technical document – TD2018MRPL
W. Van Gansbeke, M. Polet, F. Hooghe, C. Devos, P. Van Eenoo. Improved sensitivity by use of gas chromatography-positive chemical ionization triple quadrupole mass spectrometry for the analysis of drug related substances. J. Chromatogr. B 2015, 1001, 221.
A, Jimenez Giron, K. Deventer, K. Roels, P. Van Eenoo. Development and validation of an open screening method for diuretics, stimulants and selected compounds in human urine by UHPLC-HRMS for doping control. Analytica chimica acta 2012, 721, 137.