Quantitative analysis of anabolic steroids in control samples from food-producing animals using a column-switching LC-HESI-MS/MS assay

Importance of the Topic

Monitoring anabolic steroid residues in food-producing animals is essential for ensuring consumer safety and enforcing regulatory limits within the European Union. This approach helps prevent health risks associated with hormone residues in meat products.

Objectives and Study Overview

The study aimed to develop a rapid, sensitive, and automated online-SPE column-switching LC-HESI-MS/MS assay for simultaneous quantification of eight anabolic steroids in bovine urine. Key goals included minimizing sample preparation time and achieving detection limits compliant with EU screening requirements.

Methodology and Instrumentation

• Sample preparation: Centrifugation, enzymatic hydrolysis with β-glucuronidase, dilution and filtration of urine samples.
• Online SPE: EXP Trap HALO C8 cartridge coupled via a 6-port switching valve to a Chromolith Fast Gradient RP-18e monolithic column.
• Chromatography: Mobile phases of 0.2 mM ammonium fluoride in water (A) and methanol (B), flow rate 0.4 mL/min, 40 °C, 20 min cycle time.
• Mass spectrometry: Shimadzu LCMS-8050 triple quadrupole with heated ESI source in positive and negative ion modes; MRM transitions optimized using LabSolutions software.

Main Results and Discussion

• Limits of quantification below 0.25 pg/µL for all analytes, suitable for EU screening thresholds.
• Calibration linearity (0.25–6 pg/µL) with R2 > 0.998 and 1/x weighting.
• Intraday precision: RSD < 4.6 % at 2 pg/µL and < 3.6 % at 6 pg/µL.
• Matrix effects ranging up to 90 % suppression for dienestrol, mitigated by deuterated internal standards.
• Injection strategy: Three discrete 5 µL injections onto the trap column improved low-level sensitivity compared to a single large injection.

Practical Benefits and Applications

• Automated online SPE reduces hands-on preparation and solvent use versus traditional offline SPE.
• High sample throughput with 20 min analysis time supports routine monitoring in food safety labs.
• Trace-level detection ensures compliance with EU directives governing hormone use in livestock.

Future Trends and Applications

• Coupling to high-resolution mass spectrometry for expanded screening of metabolites and unknown residues.
• Further miniaturization and multiplexing of sample preparation to boost throughput and reduce costs.
• Exploration of novel stationary phases and mobile phase additives to enhance selectivity and minimize matrix effects.
• Application of similar online-SPE-LC-MS/MS workflows to other classes of veterinary drugs and contaminants.

Conclusion

The developed column-switching LC-HESI-MS/MS method delivers sensitive, precise, and efficient quantification of anabolic steroids in bovine urine. It streamlines workflow, meets regulatory requirements, and is well-suited for routine food safety testing.

Instrumental Setup

• UHPLC system: Shimadzu Nexera with six-port switching valve.
• Trap cartridge: EXP Trap HALO C8 (1 × 5 mm).
• Analytical column: Chromolith Fast Gradient RP-18e (50 × 2 mm).
• Mass spectrometer: Shimadzu LCMS-8050 triple quadrupole with heated electrospray ionization source.
• Software: LabSolutions for data acquisition and MRM optimization.