LC-MS Quantitative Screening Method for 18 Anabolic Steroids in Oral Fluid Using MS2 Spectra Data Collected with Q Exactive Orbitrap Mass Spectrometer

Significance of the Topic

Monitoring anabolic steroids in oral fluid offers a non-invasive matrix for timely detection of performance-enhancing compounds. The use of high-resolution MS2 data enhances specificity and reduces false positives, which is critical for anti-doping control and clinical toxicology applications.

Objectives and Study Overview

This work aimed to develop and validate a quantitative screening method capable of detecting 18 anabolic steroids in human oral fluid. The protocol covers sample preparation, chromatographic separation, MS2 acquisition, and data processing under EU performance guidelines.

Methodology

Oral fluid samples (200 µL) were spiked with a 13C3-labeled testosterone internal standard and subjected to liquid–liquid extraction with MTBE. Extracts were evaporated, reconstituted in 50% methanol, and injected (30 µL) onto a Thermo Acquacore C18 column. A 15-minute gradient across water/formic acid, methanol/formic acid, and acetonitrile/isopropanol/acetone mobile phases achieved chromatographic separation. Targeted MS2 spectra were acquired on a Q Exactive Orbitrap using APCI ionization; two diagnostic fragment ions per analyte were monitored, and ion ratios were evaluated against EU criteria for confirmation.

Used Instrumentation

• Thermo Scientific Q Exactive Orbitrap mass spectrometer with APCI source
• Thermo Acquacore C18 (100×3 mm, 2.6 µm) column
• Mobile phase A: 0.2% formic acid in water; B: 0.1% formic acid in methanol; C: ACN/IPA/acetone (45/45/10 v/v/v)

Main Results and Discussion

The method achieved LLOQs of 1 ng/mL for all analytes except 6β-hydroxyfluoxymesterone (6 ng/mL). Calibration curves exhibited linear ranges up to 1500 ng/mL (R2 > 0.98). Intra- and inter-assay precision CVs were below 20% at the LOQ and below 15% at higher QC levels. Matrix effects were minimal, with recoveries between 78.5% and 118% in spiked oral fluid. High-resolution MS2 spectra provided clear fragment patterns and reliable ion ratio confirmation, supporting robust identification.

Benefits and Practical Applications

The protocol supports rapid, high-confidence screening for anti-doping controls, forensic investigations, and clinical monitoring. Oral fluid sampling simplifies collection logistics and improves user compliance compared to blood or urine.

Future Trends and Opportunities

Advances may include automated sample handling, integration of multiplexed drug panels, and application of machine learning algorithms for spectral interpretation. Expansion to additional biological matrices and real-time data processing could further enhance monitoring capabilities.

Conclusion

A robust LC-MS2 screening method was established for 18 anabolic steroids in oral fluid, combining sensitive quantification, minimal matrix interference, and confirmatory ion ratio criteria. This approach is suitable for diverse analytical environments requiring reliable performance.

References

1. Draft SANCO 1805/2000 Rev.1: Performance criteria for analytical methods for residues in live animals and animal products (Council Directive 96/23/EC).