Quantification of dimethylsulfoxide (DMSO) in equine plasma and urine using HILIC-MS/MS.

This study developed and validated a simple HILIC‑MS/MS method to measure dimethylsulfoxide (DMSO) in horse plasma and urine around international doping-control thresholds. It offers a faster, solvent-friendly alternative to older gas chromatography approaches and worked on real samples that exceeded regulatory limits.

What the researchers studied and why it matters

• DMSO is a highly polar solvent with analgesic and anti-inflammatory effects; because it can alter performance or mask pain, it is prohibited in horse racing.
• DMSO also occurs naturally in the environment, so racing authorities set threshold concentrations to distinguish incidental exposure from prohibited use: 1 µg/mL in plasma and 15 µg/mL in urine.
• The goal was to create and validate a robust, routine laboratory method to quantify DMSO in equine plasma and urine that is accurate around these thresholds and practical for high-throughput doping control.

Analytical challenge and rationale for the chosen technique

• DMSO’s high polarity makes it poorly retained on conventional reversed-phase LC and historically pushed labs to use gas chromatography (GC).
• GC methods typically require laborious liquid–liquid extraction to move DMSO from water-rich biofluids into an organic injection solvent, increasing time and risk of loss or contamination.
• Hydrophilic interaction liquid chromatography (HILIC) retains polar analytes effectively, enabling direct handling of aqueous samples after simple preparation.
• Coupling HILIC to tandem mass spectrometry (MS/MS) provides the selectivity and sensitivity needed to quantify DMSO near low µg/mL thresholds in complex matrices like plasma and urine.

Sample preparation and chromatographic conditions

• Sample preparation was intentionally minimal:
  • Plasma: protein precipitation to remove major interferents.
  • Urine: dilution to bring matrix salts and DMSO into a range compatible with HILIC-MS/MS.
• Chromatography used an Acquity BEH HILIC column, suitable for retaining small polar molecules such as DMSO.
• The mobile phase was a gradient of acetonitrile and an ammonium acetate buffer, a standard HILIC system that balances retention, peak shape, and MS compatibility.

Quantitation strategy and internal standardization

• Quantitation relied on stable-isotope dilution with hexadeuterated DMSO (d6‑DMSO) as the internal standard.
• The isotopically labeled internal standard co-extracts and co-elutes with native DMSO, correcting for:
  • Variations in sample preparation and injection volume,
  • Ionization suppression/enhancement from matrix constituents, and
  • Instrumental drift over time.

Method validation design

• Validation covered the concentration ranges relevant to regulatory thresholds in both matrices.
• Assessed performance characteristics included:
  • Selectivity: absence of interfering signals at the DMSO retention time/transition.
  • Carry-over: negligible analyte residue after high-concentration injections.
  • Linearity: proportional response across the calibration range around 1 µg/mL (plasma) and 15 µg/mL (urine).
  • Precision: repeatability and intermediate precision within predefined acceptance limits.
  • Accuracy: agreement of measured versus nominal concentrations using quality controls.
  • Stability: DMSO stability under typical storage and analytical conditions (e.g., bench-top, autosampler).
  • Inter-individual matrix variation: consistent performance across plasma and urine from different horses.
• All criteria were met; the methods were deemed fit for purpose for doping-control testing.

Applications to real-world samples

• The validated methods were applied to actual equine doping-control specimens.
• Measured DMSO concentrations in some samples exceeded the international thresholds, demonstrating the method’s practical utility for regulatory enforcement.

Advantages compared with traditional GC approaches

• Simplified workflow: precipitation/dilution replaces time-consuming extraction into GC-compatible solvents.
• Improved throughput: fewer manual steps and HILIC-MS/MS’s rapid analysis shorten turnaround times.
• Enhanced selectivity and robustness: MS/MS with isotope dilution mitigates matrix effects common in biological samples.
• Better fit to polar analytes: HILIC provides reliable retention and peak shape for highly water-soluble compounds like DMSO.

Practical considerations and potential limitations

• Environmental ubiquity of DMSO requires vigilant contamination control (e.g., avoiding DMSO-containing lab reagents and plastics).
• Carry-over must remain tightly managed due to high-solubility analytes being run near regulatory decision limits.
• Laboratories should verify matrix effects across diverse horse populations and diets, as done here via inter-individual matrix checks.

Implications for equine anti-doping and surveillance

• This HILIC‑MS/MS method enables reliable differentiation between environmental background and pharmacological use by targeting established thresholds.
• The streamlined protocol supports routine, high-volume testing, improving responsiveness in racing oversight.
• The approach is broadly transferable to other small, polar veterinary analytes where GC extraction burdens testing capacity.

Key takeaways

• A validated, isotope-dilution HILIC‑MS/MS method accurately quantifies DMSO in equine plasma (around 1 µg/mL) and urine (around 15 µg/mL) with minimal sample prep.
• Performance met stringent criteria for selectivity, carry-over, linearity, precision, accuracy, stability, and cross-subject matrix effects.
• Real-world application confirmed its suitability for enforcing DMSO thresholds in horse racing.