Analysis of Trenbolone at low levels in animal tissues using the EVOQ GC-TQ

Significance of the Topic

Trenbolone esters are potent anabolic steroids banned in food producing animals in the European Union due to potential endocrine and carcinogenic risks. Sensitive monitoring of such residues in meat is essential to ensure consumer safety and regulatory compliance under EU directives.

Objectives and Overview of the Study

This application note describes the development and validation of a gas chromatography–tandem mass spectrometry method using the EVOQ GC-TQ system to detect and confirm 17α- and 17β-trenbolone in animal tissues at sub-ppb levels. The method meets the identification criteria of Commission Decision 2002/657/EC, employing two selective reaction monitoring transitions per analyte for unequivocal confirmation.

Methodology and Instrumentation

Sample Preparation

• 15 g of fresh muscle tissue freeze-dried and ground to powder.
• Fortification with deuterated 17β-trenbolone-d3 internal standard at 1.0 ng/g.
• Liquid–solid extraction with methanol and acetate buffer, followed by centrifugation.
• Enzymatic hydrolysis with Helix pomatia extract at 50°C for 15 hours.
• SPE cleanup on Envi-ChromP and SiOH cartridges with hexane, diethyl ether, and ethyl acetate solvents.
• Derivatization of dry residue using MSTFA with iodine catalysis.

Instrumentation

• Gas Chromatograph: Bruker 436 GC equipped with a DB-5MS column (30 m × 0.25 mm × 0.25 µm), helium carrier at 1.0 mL/min.
• Mass Spectrometer: EVOQ GC-TQ triple quadrupole MS, electron impact ionization at 70 eV, source at 250°C, transfer line at 310°C.
• Acquisition Mode: Selected Reaction Monitoring with unit resolution on both Q1 and Q3 quadrupoles; argon collision gas at 2.0 mTorr.

Main Results and Discussion

Chromatographic separation achieved a resolution greater than one between the α and β epimers of trenbolone. Two SRM transitions were optimized for each epimer, with one common quantifier and one specific qualifier transition. The method demonstrated limits of quantification of 0.1 ng/g in meat. Recovery was approximately 10 percent, owing to analyte distribution across fractions in a multi-residue workflow. Analysis of spiked equine muscle samples showed clean ion chromatograms without matrix interferences, and unknown incurred samples were declared compliant under EU criteria.

Benefits and Practical Applications of the Method

• High sensitivity allows sub-ppb screening and confirmation in complex matrices.
• Specific SRM transitions ensure unambiguous identification of trenbolone epimers.
• Compliance with EU regulatory standards for banned growth promoters.
• Adaptable multi-residue workflow enables simultaneous monitoring of multiple steroid classes.

Future Trends and Opportunities

Advancements may include integration of high-resolution mass spectrometry for broader screening, automation of sample preparation to increase throughput, miniaturized extraction techniques to reduce solvent consumption, and data-driven approaches using machine learning for enhanced pattern recognition and quantification.

Conclusion

The GC-MS/MS method using the EVOQ GC-TQ provides a robust, sensitive, and compliant approach for trace-level analysis of trenbolone in animal tissues. It supports food safety laboratories in enforcing bans on anabolic steroids and protecting consumer health.

References

• Maume F, Le Bizec B, Marchand P, Montrade M, André F. Analyst 1998;123:2645–2648.
• Marchand P, Le Bizec B, Gade S, Monteau F, André F. J Chromatogr A 2000;867:219–231.
• EU Commission Decision 2002/657/EC, Implementing Directive 96/23/EC.
• Council Directive 96/23/EC of 29 April 1996 on monitoring substances in live animals and products.