Extraction of a Comprehensive Steroid Panel from Horse Hair Using ISOLUTE® SLE+ Prior to LC/MS-MS Analysis

Importance of the Topic

The precise quantitation of multiple steroid hormones in horse hair is vital for doping control and veterinary diagnostics, offering a non-invasive matrix that reflects long-term exposure. Supported liquid extraction (SLE) simplifies sample cleanup and enhances throughput in bioanalytical workflows.

Objectives and Study Overview

This study demonstrates an efficient protocol for extracting a comprehensive panel of 18 steroid analytes from equine hair using ISOLUTE SLE+ plates, followed by UHPLC-MS/MS analysis. Performance metrics include recovery, repeatability, sensitivity, and linearity across a wide concentration range.

Methodology and Instrumentation

Sample Preparation:

• 20 mg hair samples pulverized in Biotage Lysera tubes with stainless steel beads.
• Two-step micropulverization with 0.1% NH4OH in IPA and 50 pg/mg deuterated internal standards.
• Extraction on 400 µL ISOLUTE SLE+ plates; elution with dichloromethane.
• Evaporation using Biotage SPE Dry 96; reconstitution in 50:50 aqueous ammonium fluoride–methanol.

Instrumentation:

• Shimadzu Nexera X2 UHPLC with ACE C18 column (100 x 2.1 mm, 1.7 µm).
• Shimadzu 8060 triple quadrupole MS with ESI, operated in both positive and negative ion modes using 0.2 mM NH4F and methanol as mobile phases.

Main Results and Discussion

Recoveries exceeded 80% for most analytes with RSDs below 5% (n=7). LLOQs ranged from 0.5 to 25 pg/mg, with several steroids detected at or below 0.5 pg/mg. Calibration curves spanning 0.5–500 pg/mg yielded r² values above 0.999. Chromatographic conditions produced clear separation of isobaric steroids.

Benefits and Practical Applications

The SLE+ workflow avoids emulsion formation, reduces solvent consumption, and supports high-throughput 96-well processing. This method is directly applicable to routine doping control, residue monitoring, and research in equine health and related animal matrices.

Future Trends and Opportunities

Emerging directions include novel sorbent technologies for SLE, full automation of sample preparation, miniaturization for lower sample inputs, and application to other keratinous matrices (e.g., hoof or fur). Integration with ambient ionization techniques may further accelerate analysis.

Conclusion

This protocol delivers a robust, sensitive, and reproducible approach for comprehensive steroid profiling in horse hair, ideally suited for routine bioanalytical laboratories engaged in veterinary drug monitoring and sports anti-doping programs.

References

Biotage Application Note AN921 (2019)