Skip to main content
SpringerLink
Log in

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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 resolution capabilities for highly polar analytes as they interact insufficiently 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 CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced.

SFC-MS/MS vs. LC-MS/MS

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Botre F. New and old challenges of sports drug testing. J Mass Spectrom. 2008;43(7):903–7. doi:10.1002/jms.1455.

    Article  CAS  Google Scholar 

  2. Reddy IM, Beotra A, Jain S, Ahi S. A simple and rapid ESI-LC-MS/MS method for simultaneous screening of doping agents in urine samples. Indian J Pharm. 2009;41(2):80–6. doi:10.4103/0253-7613.51347.

    Article  CAS  Google Scholar 

  3. Georgakopoulos C, Saugy M, Giraud S, Robinson N, Alsayrafi M. Analytical progresses of the International Olympic Committee and World Anti-Doping Agency Olympic laboratories. Bioanalysis. 2012;4(13):1549–63. doi:10.4155/bio.12.148.

    Article  CAS  Google Scholar 

  4. Gosetti F, Mazzucco E, Gennaro MC, Marengo E. Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review. J Chromatogr B. 2013;927:22–36. doi:10.1016/j.jchromb.2012.12.003.

    Article  CAS  Google Scholar 

  5. Thevis M, Thomas A, Pop V, Schanzer W. Ultrahigh pressure liquid chromatography-(tandem) mass spectrometry in human sports drug testing: possibilities and limitations. J Chromatogr A. 2013;1292:38–50. doi:10.1016/j.chroma.2012.12.048.

    Article  CAS  Google Scholar 

  6. Mazzarino M, Fiacco I, de la Torre X, Botre F. Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry. J Chromatogr A. 2011;1218(11):8156–67.

    Article  CAS  Google Scholar 

  7. Klesper K, Chorwin AH, Turner DA. High pressure gas chromatography above critical temperatures. J Org Chem. 1962;27:700–1.

    Article  CAS  Google Scholar 

  8. Giddings CG, Myers MN, King JW. Dense gas chromatography at pressures to 2000 atmospheres. J Chromatogr Sci. 1969;7:276–83.

    Article  CAS  Google Scholar 

  9. Berger TA, Berger BK. Minimizing UV noise in supercritical fluid chromatography. I. Improving back pressure regulator pressure noise. J Chromatogr A. 2011;1218(16):2320–6. doi:10.1016/j.chroma.2011.02.030.

    Article  CAS  Google Scholar 

  10. Grand-Guillaume Perrenoud A, Veuthey JL, Guillarme D. Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds. J Chromatogr A. 2012;1266:158–67. doi:10.1016/j.chroma.2012.10.005.

    Article  CAS  Google Scholar 

  11. Grand-Guillaume Perrenoud A, Boccard J, Veuthey JL, Guillarme D. Analysis of basic compounds by supercritical fluid chromatography: attempts to improve peak shape and maintain mass spectrometry compatibility. J Chromatogr A. 2012;1262:205–13. doi:10.1016/j.chroma.2012.08.091.

    Article  CAS  Google Scholar 

  12. Bartle KD. Theory and principles of supercritical fluid chromatography. In: Smith RM, editor. Supercritical fluid chromatography. Loughborough: Royal Society of Chemistry; 1988. p. 1–28.

    Google Scholar 

  13. Langley GJ. Supercritical fluid chromatography (SFC). 2012. http://www.southampton.ac.uk/~gjl/Research/sfc.htm. Accessed 20 Jan 2014.

  14. King JW, Hill HH, Lee ML. Analytical supercritical fluid chormatography and extraction. In: Rossiter BW, Baetzold RC, editors. Physical methods of chemistry series, vol X. 2nd ed. Hoboken: Wiley; 1993.

    Google Scholar 

  15. Weller HN, Ebinger K, Bullock W, Edinger KJ, Hermsmeier MA, Hoffman SL, et al. Orthogonality of SFC versus HPLC for small molecule library separation. J Comb Chem. 2010;12(6):877–82.

    Article  CAS  Google Scholar 

  16. Parr MK, Bokland MH, Liebetrau F, Schmidt AH, Schänzer W, Sterk SS. Enantiomeric separation of clenbuterol as analytical strategy to distinguish abuse from meat contamination. In: Schänzer W, Geyer H, Gotzmann A, Mareck U, editors. Recent advances in doping analysis (21). Cologne: Sportverlag Strauß; 2013. p. 26.

    Google Scholar 

  17. Doue M, Dervilly-Pinel G, Pouponneau K, Monteau F, Le Bizec B. Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry. Anal Bioanal Chem. 2015;407(15):4473–84. doi:10.1007/s00216-015-8573-x.

    Article  CAS  Google Scholar 

  18. Novakova L, Grand-Guillaume Perrenoud A, Nicoli R, Saugy M, Veuthey JL, Guillarme D. Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions. Anal Chim Acta. 2015;853:637–46. doi:10.1016/j.aca.2014.10.004.

    Article  CAS  Google Scholar 

  19. Novakova L, Perrenoud AG, Francois I, West C, Lesellier E, Guillarme D. Modern analytical supercritical fluid chromatography using columns packed with sub-2 mum particles: a tutorial. Anal Chim Acta. 2014;824:18–35. doi:10.1016/j.aca.2014.03.034.

    Article  CAS  Google Scholar 

  20. Novakova L, Rentsch M, Grand-Guillaume Perrenoud A, Nicoli R, Saugy M, Veuthey JL, et al. Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: analysis of biological samples. Anal Chim Acta. 2015;853:647–59. doi:10.1016/j.aca.2014.10.007.

    Article  CAS  Google Scholar 

  21. Görgens C, Guddat S, Orlovius AK, Sigmund G, Thomas A, Thevis M, et al. “Dilute-and-inject” multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing. Anal Bioanal Chem. 2015;407(18):5365–79. doi:10.1007/s00216-015-8699-x.

    Article  Google Scholar 

  22. Oatis Jr JE, Russell MP, Knapp DR, Walle T. Ring-hydroxylated propranolol: synthesis and beta-receptor antagonist and vasodilating activities of the seven isomers. J Med Chem. 1981;24(3):309–14. doi:10.1021/jm00135a014.

    Article  CAS  Google Scholar 

  23. Parr MK, Orlovius A, Guddat S, Gütschow M, Thevis M, Schänzer W. Sulfoconjugates of heavy volatile nitrogen containing doping substances for improved LC-MS/MS screening. In: Schänzer W, Geyer H, Gotzmann A, Mareck U, editors. Recent advances in doping analysis (15). Köln: Sport und Buch Strauß; 2007. p. 97–102.

    Google Scholar 

  24. Orlovius AK, Guddat S, Parr MK, Kohler M, Gutschow M, Thevis M, et al. Terbutaline sulfoconjugate: characterization and urinary excretion monitored by LC/ESI-MS/MS. Drug Test Anal. 2009;1(11-12):568–75.

    Article  CAS  Google Scholar 

  25. Orlovius A, Guddat S, Parr M, Koch A, Gütschow M, Thevis M, et al. Identification and monitoring of octopamine sulfoconjugate in urine by LC/(ESI)-MS/MS. In: Schänzer W, Geyer H, Gotzmann A, Mareck U, editors. Recent advances in doping analysis (19). Cologne: Sportverlag Strauß; 2011. p. 34–43.

  26. Mazzarino M, de la Torre X, Botre F. A screening method for the simultaneous detection of glucocorticoids, diuretics, stimulants, anti-oestrogens, beta-adrenergic drugs and anabolic steroids in human urine by LC-ESI-MS/MS. Anal Bioanal Chem. 2008;392(4):681–98. doi:10.1007/s00216-008-2292-5.

    Article  CAS  Google Scholar 

  27. World Anti-Doping Agency. Mimimum criteria for chromatographic-mass spectrometric confirmation of the identity of analytes for doping control purposes - TD2015IDCR. World Anti-Doping Agency. 2015. https://www.wada-ama.org/en/resources/science-medicine/td2015-idcr. Accessed 11 Oct 2015.

  28. World Anti-Doping Agency. Mimimum required performance levels for detection and identification of non-threshold substances - TD2015MRPL. World Anti-Doping Agency. 2015. https://www.wada-ama.org/en/resources/science-medicine/td2015-mrpl. Accessed 11 Oct 2015.

Download references

Acknowledgments

The World Anti-Doping Agency is acknowledged for their financial support (research grant 14A03KP). The authors thank Mr. Felix Bredendiek for technical assistance.

Author information

Authors and Affiliations

  1. Institute of Pharmacy, Freie Universität Berlin, Koenigin-Luise-Str. 2-4, 14195, Berlin, Germany

    Maria Kristina Parr, Jan F. Joseph, Maxi Wenzel & Alexander H. Schmidt

  2. Agilent Technologies, 5301 Stevens Creek Boulevard, Santa Clara, CA, 95051, USA

    Bernhard Wuest & Edgar Naegele

  3. Chromicent GmbH, Rudower Chaussee 29, 12489, Berlin, Germany

    Alexander H. Schmidt & Mijo Stanic

  4. Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197, Rome, Italy

    Xavier de la Torre & Francesco Botrè

  5. Department of Experimental Medicine, ‘Sapienza’ University of Rome, Viale Regina Elena 324, 00161, Rome, Italy

    Francesco Botrè

Authors
  1. Maria Kristina Parr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bernhard Wuest
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Edgar Naegele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jan F. Joseph
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maxi Wenzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexander H. Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mijo Stanic
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xavier de la Torre
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Francesco Botrè
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Kristina Parr.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 36.1 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Parr, M.K., Wuest, B., Naegele, E. et al. SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control. Anal Bioanal Chem 408, 6789–6797 (2016). https://doi.org/10.1007/s00216-016-9805-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9805-4

Keywords

Search

Navigation