Automated Determination of Steroids in Urine of Food-Producing Animals

Significance of the Topic

The illegal use of steroids as growth promoters in food-producing animals is strictly prohibited in the European Union and requires monitoring under national residue control plans. Analytical challenges such as matrix interferences in urine demand efficient sample cleanup to ensure accurate LC–MS/MS or HPLC–HRMS measurements. High sample throughput in official laboratories highlights the need for automated methods that maintain performance while reducing hands-on time.

Objectives and Study Overview

This application note describes development and validation of an automated dual solid phase extraction workflow for the determination of ten synthetic and endogenous steroids in animal urine. The method aims to meet EU decision 2002/657/EC requirements for detection limits and recoveries, while enabling efficient processing of large sample numbers.

Methodology and Instrumentation

Sample Pretreatment:

• Centrifugation to remove particulates.
• Enzymatic hydrolysis with glucuronidase/arylsulfatase in sodium acetate buffer (pH 5.2), overnight incubation at 37 °C.

Solid Phase Extraction:

• Dual SPE cleanup using CHROMABOND® HR-X (200 mg) for initial removal of matrix components, followed by CHROMABOND® NH2 (500 mg) for final purification.
• Manual protocol adapted to LCTech FREESTYLE SPE robotic system for automation of conditioning, loading, washing and elution steps.

Reconstitution and Analysis:

• Drying of extracts under nitrogen, reconstitution in 0.1% formic acid acetonitrile/water (10/90).
• Chromatographic separation on a Phenomenex Luna C18(2) column (150 × 2.0 mm, 3 µm) with gradient elution over 20 min at 0.2 mL/min and 30 °C.
• Detection by HPLC–MS/MS on an Agilent 1290 Infinity/6495 system with positive ESI and MRM transitions for each analyte and deuterated internal standards.

Instrumentation Details:

• LCTech FREESTYLE SPE system with dual column racks, solvent delivery, plungers, and software wizards for automated SPE.
• Agilent 6495 Triple Quadrupole with ESI source (gas temperature 200 °C, sheath gas 380 °C, capillary voltage 3000 V).

Main Results and Discussion

Recovery studies at concentrations below 1 µg/kg demonstrated compliance with EU requirements, with recoveries within –50% to +20% deviation from target. Reproducibility testing on ten urine samples processed manually versus automated SPE showed relative standard deviations of 16–21% for automated cleanup compared to 22–39% manually. Automated processing yielded slightly improved precision and consistent peak area mean values.

Benefits and Practical Applications of the Method

• High throughput sample cleanup reduces labor and time per sample.
• Improved reproducibility and method robustness through reduced operator variability.
• Flexibility to adapt to other analytes by modifying SPE protocols via software wizards.
• Compliance with regulatory limits for steroid residues ensures reliable enforcement in food safety monitoring.

Future Trends and Potential Applications

• Integration with high-resolution mass spectrometry for expanded analyte panels and enhanced confirmation.
• Miniaturization of SPE formats to reduce solvent consumption and waste.
• Incorporation of real-time monitoring and data analytics for workflow optimization.
• Extension to other complex biological matrices such as serum or tissues.

Conclusion

Automated dual SPE on the FREESTYLE platform delivers equivalent or improved recovery and reproducibility compared to manual protocols, supporting high-throughput steroid screening in animal urine. The method meets stringent EU requirements and provides a scalable solution for official control laboratories.

References

• Confirmatory Method for the Determination of Steroids in Bovine Urine by LC–MS/MS; Bundesamt für Verbraucherschutz und Lebensmittelsicherheit; 2011.
• Polemans S., De Wasch K., Noppe H., et al.; Endogenous occurrence of some anabolic steroids in swine matrices; Food Additives and Contaminants; 2005; 22(9): 808–815.