Prohibited Substances in Racehorse Urine: Validated SPE Method with Clean Screen® DAU

The Application

Horse racing is a multi-million pound industry with zero tolerance for doping. The International Federation of Horseracing Authorities (IFHA) maintains a list of prohibited substances, and antidoping labs need methods that screen broadly across drug classes from a single extraction. That means one SPE cartridge handling anesthetics, analgesics, sedatives, opioids, beta-blockers, stimulants, anti-inflammatories, and more, all from one of the dirtiest biological matrices in routine testing.

A Turkish antidoping laboratory validated exactly that.

The Method

63 prohibited substances extracted from racehorse urine and analysed by LC-MS/MS, validated to 2021/808/EC guidelines.

Product used: Clean Screen® DAU, 500 mg / 3 mL (P/N CSDAU503)

Sample Preparation

1. Mix 14 mL urine with internal standard, 1 M phosphate buffer (pH 5.8), ammonium sulfate, protease, and β-glucuronidase
2. Incubate 1.5 hours at 55°C (hydrolysis)
3. Transfer 7 mL, centrifuge, dilute with phosphate buffer and water
4. Adjust to pH 7 with 10% NaOH

SPE Protocol (Automated, Gilson Aspect GX-274)

1. Condition with water, methanol, and phosphate buffer (0.1 M, pH 6)
2. Load sample
3. Wash with 1 M acetic acid (dry), then methanol (dry)
4. Elute with ethyl acetate:aqueous ammonia (97:3 v/v)
5. Evaporate and reconstitute for LC-MS/MS

The Clogging Problem (and Two Solutions)

Horse urine is viscous and protein-rich. During development, cartridge clogging became an issue. The researchers solved it by adding protease during hydrolysis and precipitating proteins with ammonium sulfate.

The alternative: UCT's XtrackT® DAU columns. Large-particle silica (80–200 µm) delivers better, more consistent flow through dirty samples without requiring additional sample pre-treatment. If your lab processes high volumes of equine urine, XtrackT® eliminates the clogging problem at the sorbent level.

Performance

• Screening limits and low LODs achieved for all 63 substances
• Matrix effects: -17.88% to +19.7% (effective matrix removal confirmed)
• No interferences detected in selectivity/specificity testing
• Full compliance with 2021/808/EC validation criteria

Why Chromatography Direct

The home of chromatography consumables. We stock both Clean Screen® DAU and XtrackT® DAU for equine antidoping work. High-quality consumables at unrivalled prices, making your lab budget go further.

XtrackT® DAU has previously been validated for 89 banned or controlled racing industry drugs in horse urine. Whether you need standard or large-particle SPE, we carry the range and we price it for labs running high-throughput antidoping screening. Specialists, not generalists.

Reference

Kabil, E.; Göktaş, E. F. Development and Validation of Confirmatory Quantitative and Screening Methods for 63 Prohibited Substances in Horse Urine Using LC-MS/MS. Chromatographia 2025, 88, 151–168. DOI: 10.1007/s10337-024-04384-2