Streamlined stationary phase selection facilitated by a “sample-plug retention test” in supercritical fluid extraction-supercritical fluid chromatography-mass spectroscopy (SFE-SFC-MS) method development for on-line extraction of anabolic agents

Abstract

A novel method development (MD) approach was established to aid column selection for online supercritical fluid extraction-supercritical fluid chromatography-mass spectrometry (SFE-SFC-MS). An adapted stationary phase (SP) selection procedure was used to expand traditional SFC-MS screening by inclusion of an “Analyte-Plug Retention Test.” This test enabled an early evaluation of “trapping” capability for a plug of targeted analytes on the SP. Using an SFE-simulation method, the on-line trapping of an SFE-based extract-plug was mimicked by trapping an SFC-based injection-plug under instrument conditions similar to an actual on-line extraction. Thirteen SPs were screened using the adapted MD procedure as part of the SFC-separation optimization in the development of an on-line SFE-SFC-MS extraction method for anabolic agents (AAS) in anti-doping testing. A cyano SP was chosen for further development, based on chromatographic separation potential and high plug-retention potential for targeted analytes. The chosen column was used in “proof-of-concept” on-line extractions using a generic SFE-SFC-MS method. Successful extract-plug trapping was demonstrated. Retention times and peak areas (4–20%RSD) were reproducible for targeted AAS. Successful first round SFE-SFC-MS SP selection was demonstrated to circumvent tedious re-optimization in future development stages and streamlining of the hyphenated SFE-SFC-MS MD process.

Graphical Abstract

Keywords:

• Anabolic androgenic steroids
• extraction simulation
• hyphenated method development
• on-line extraction
• SFC column scouting

Acknowledgments

The authors acknowledge Shimadzu Scientific Instruments, Inc. for instrumentation and research support for this work. We would also like to acknowledge the support from Restek Corporation, in the form of chromatography columns and stationary phases used during this work. We offer this study in honor of and in remembrance to Professor Harold McNair, a wonderful teacher, researcher, mentor, friend, and human being.

Disclosure statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: YF, WH, and MN are employed by Shimadzu Corporation or Shimadzu Scientific Instruments, Inc., the manufacturer of the instrumentation utilized in this work.

Additional information

Funding

Further support is acknowledged from the National Science Foundation [CHE-2108767].