Implementation of ultra-high performance supercritical fluid chromatography for screening and confirmation analysis

Code: 17A16CG 

The implementation project will review the capability of the proposed new technology (ultra-high performance supercritical fluid chromatography) within an anti-doping environment. The project will replicate the work done by Desfontaine, Novakova et al (2015/2016) and apply it into screening analysis for three months (circa 1500 samples). This will allow the methodology to be assessed with real samples which can be quitetdirty and more difficult to analyse. The method will also be considered as an orthogonal technology which could be ideally suited for use in confirmation methodology by anti-doping laboratories.

Main Findings: 

An Agilent 1260 Infinity II Hybrid SFC/UHPLC-MS system provided on loan by Agilent Technologies (Sydney, Australia) was used to review the capability of ultra-high performance supercritical fluid chromatography (SFC-MSMS) within a high throughput anti-doping environment by applying it to the screening of routine samples. The SFC-MSMS methodology was provided by Dr Bernhard Wüst, Agilent Technologies, Waldbronn, Germany and was developed by Parr et al (1) at the Institute of Pharmacy, Freie Universität Berlin. As part of this project the method was modified to include an additional 159 compounds with 10 compounds removed. The final modified method monitored 906 MRM transitions to screen for 400 compounds. This did not constitute the full suite of compounds currently monitored by the ASDTL UHPLC-HRMS (Orbitrap) screen however with further optimisation and rationalisation it may be possible to include additional compounds. The MS was operated in dynamic MRM mode with up to 281 concurrent transitions.
The current ASDTL automated sample preparation procedure with a minor modification was used to prepare extracts for SFC-MSMS analysis. Validation data was acquired for the 365 compounds in the laboratory's current multi-analyte standard mixes. A total of 306 compounds met the required validation performance criteria of detection at 50% MRPL (TD2019MRPL) in all ten samples with 310 compounds detected at 100% MRPL in all samples. Carry over was evident with 101 compounds visible in the third blank injected after injection of a sample spiked at 400% MRPL, with peak areas less than 1% of the 400% MRPL injection for most compounds. The SFC-MSMS system proved capable of screening for a large number of substances from multiple classes in a single analysis.
The scope of this project wasimpacted from on-going instrumnt issues that ultimately limited the size of the data set acquired during the routine sample analysis component of the project. Only 336 of the planned 1500 samples were analysed. This individual UHPSFC-MSMS system supplied for this project did not prove to be sufficiently robust during the period over which it was tested. SFC-MSMS however does offer an alternate selectivity and orthogonality to reverse phase chromatography which is complementary to existing methogologies and is therefore a potentialy useful orthogonal confirmatory technique. The volume of organic solvent consumed by the SFC-MSMS was significantly greater than that used by the UHPLC-HRMS making it less 'green' than anticipated.