RESEARCH PAPER SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control Maria Kristina Parr 1 & Bernhard Wuest 2 & Edgar Naegele 2 & Jan F. Joseph 1 & Maxi Wenzel 1 & Alexander H. Schmidt 1,3 & Mijo Stanic 3 & Xavier de la Torre 4 & Francesco Botrè 4,5 Received: 12 March 2016 /Revised: 11 July 2016 /Accepted: 18 July 2016 /Published online: 23 August 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolu- tion capabilities for highly polar analytes as they interact in- sufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π–π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO 2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Keywords Sympathomimetic drugs . Beta-blockers . Polar stimulants . Doping control . Sulfoconjugates . Dilute-and-inject Introduction During the past few years high-performance liquid chroma- tography coupled to tandem mass spectrometry (HPLC-MS/ MS) gained importance for the detection of various classes of doping-relevant substances [1–5]. In contrast to GC-MS it allows for separation of analytes with different functional properties without derivatization. However some analytes are still challenging as HPLC-MS/MS shows limited resolu- tion capabilities and highly polar analytes interact insufficient- ly on the conventional analytical columns. Thus, the HPLC analysis of polar stimulants and their metabolites is in partic- ular dissatisfactory [6]. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. First reports on SFC, at that time called high pressure or dense chromatography, date back to the 1960s [7, 8]. Initially hampered by technical realization, the applicability of SFC for analytical chromatography increased with the re- cent accessibility of new commercially available instruments over the last 5 years [9–11]. SFC offers separation by the help Parts of this work were presented at the 53rd TIAFT meeting 2015 in Florence, Italy. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9805-4) contains supplementary material, which is available to authorized users. * Maria Kristina Parr maria.parr@fu-berlin.de 1 Institute of Pharmacy, Freie Universität Berlin, Koenigin-Luise-Str. 2-4, 14195 Berlin, Germany 2 Agilent Technologies, 5301 Stevens Creek Boulevard, Santa Clara, CA 95051, USA 3 Chromicent GmbH, Rudower Chaussee 29, 12489 Berlin, Germany 4 Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197 Rome, Italy 5 Department of Experimental Medicine, ‘Sapienza’ University of Rome, Viale Regina Elena 324, 00161 Rome, Italy Anal Bioanal Chem (2016) 408:6789–6797 DOI 10.1007/s00216-016-9805-4