LC-MS quantitative screening method for 18 anabolic steroids in oral fluid using MS2 spectra data collected with Q Exactive Orbitrap mass spectrometer

Importance of the Topic

A reliable and sensitive method for quantifying anabolic steroids in oral fluid addresses critical needs in anti-doping, clinical toxicology, and forensic investigations. Oral fluid sampling is minimally invasive and reflects recent drug intake, making it valuable for rapid screening of androgenic-anabolic steroids in athletes and patients.

Study Objectives and Overview

This work aimed to develop and validate a high-resolution LC-MS/MS screening protocol for 18 anabolic steroids in human oral fluid. The approach combined liquid–liquid extraction with ultra-high-resolution MS2 spectra acquisition on a Q Exactive Orbitrap instrument to achieve low detection limits and robust confirmation of compounds.

Methodology and Instrumentation

Sample Preparation:

• 200 μL of preserved oral fluid spiked with 13C₃-testosterone internal standard.
• Liquid–liquid extraction using methyl tert-butyl ether, cold precipitation, evaporation, and reconstitution in 50% methanol.

Chromatography:

• Thermo Acquity C18 column (100×3 mm, 2.6 μm).
• Gradient elution over 15 minutes with aqueous formic acid, methanolic formic acid, and acetonitrile/isopropanol/acetone mixture.

Mass Spectrometry:

• Thermo Scientific Q Exactive Orbitrap with APCI source, resolution 35,000.
• Targeted MS2 acquisition (t-MS2) using inclusion list, optimized collision energies, and time windows.
• Quantification based on two most abundant fragments per analyte and ion ratio confirmation following EU guidelines.

Main Results and Discussion

Limit of quantification (LOQ) was 1 ng/mL for 17 steroids, and 6 ng/mL for 6β-hydroxyfluoxymesterone. Upper linear limits ranged from 60 to 1500 ng/mL depending on signal intensity. Calibration curves showed R² values above 0.98 for all targets. Intra- and inter-assay precision remained within acceptable limits (<20% CV) across four QC levels (2, 15, 90, 450 ng/mL). Matrix effects were negligible, with recoveries between 78.5% and 118% in spiked oral fluid. Method specificity was confirmed by high-accuracy MS2 spectra and consistent ion ratios. The protocol successfully detected all analytes in both LLE and protein precipitation processed positive samples from a collaborator laboratory.

Benefits and Practical Applications

This quantitative screening method offers:

• High specificity and confidence in steroid identification via full MS2 spectra.
• Low detection limits suitable for recent intake monitoring.
• Compatibility with routine lab workflows and various sample preparation techniques.
• Applicability in anti-doping controls, clinical toxicology, and workplace drug testing.

Future Trends and Opportunities

Advances may include integration of automated sample preparation platforms, expansion to additional steroid metabolites, and coupling with high-throughput data analysis tools such as machine learning for rapid result interpretation. Ultra-high-resolution mass spectrometers could further enhance multiplexed screening capabilities and reduce analysis time.

Conclusion

A robust LC-MS/MS method using Q Exactive Orbitrap technology was established for quantitative screening of 18 anabolic steroids in oral fluid, demonstrating high sensitivity, precision, and reliability for anti-doping and toxicological applications.

Reference

1. Draft SANCO 1805/2000 Rev.1 [Revised Commission Decision 93/256 of 14 April 1993] laying down performance criteria for analytical methods to be used for certain substances and residues thereof in live animals and animal products according to Council Directive 96/23/EC.