Extraction of Veterinary Growth Promoters from Animal Urine Using ISOLUTE® HYDRO DME+ Prior to UPLC-MS/MS Analysis

Significance of the Topic

Animal growth promoters such as steroids, resorcylic acid lactones and stilbenes are closely monitored due to regulatory and public health concerns. Reliable extraction from urine matrices is critical for sensitive and accurate screening in veterinary drug control and food safety.

Objectives and Study Overview

This application note presents a streamlined procedure for isolating 18 veterinary growth promoters from animal urine using Biotage ISOLUTE HYDRO DME+ sorbents, followed by UPLC–MS/MS detection.

Methodology and Instrumentation

• Sample hydrolysis: 75 µL urine diluted 1:1 with 200 mM ammonium acetate (pH 5.2) containing Helix pomatia glucuronidase; incubated at 60 °C for 2 h.
• Extraction: Load 150 µL hydrolyzed urine onto ISOLUTE HYDRO DME+ (400 mg column or 96-well plate), add 900 µL acetonitrile, mix and apply ~5 psi positive pressure.
• Post-elution: Evaporate at 40 °C and reconstitute in 200 µL 0.1% formic acid/50% methanol.
• UPLC conditions: Shimadzu Nexera UHPLC with ACE UltraCore Super C18 (2.5 µm, 100 × 2.1 mm); gradient of 0.5 mM ammonium acetate/acetic acid in water and methanol.
• MS/MS: Sciex TripleQuad 5500 operating in dual‐polarity ESI with optimized MRM transitions for each analyte.

Results and Discussion

Recoveries for all analytes exceeded 80% across bovine, equine, ovine and porcine urine, with relative standard deviations under 10%. Calibration curves over 0.4–10 ng/mL displayed r² values above 0.999, and limits of quantitation were 0.4 ng/mL with accuracies between 95% and 105%. Chromatograms in both positive and negative ion modes showed clear separation of all 18 targets.

Benefits and Practical Applications

• Rapid, high‐throughput workflow in both column and plate formats.
• Efficient matrix cleanup and enzyme removal for clean extracts.
• Applicability to routine residue monitoring, regulatory compliance and food safety analysis.

Future Trends and Opportunities

Further improvements in sorbent design and automation could boost throughput and sensitivity. Integration with high‐resolution mass spectrometry may enable broader multi‐residue screening. Sustainable solvent and waste management strategies will support green analytical chemistry initiatives.

Conclusion

The ISOLUTE HYDRO DME+ protocol delivers a robust, reproducible and sensitive method for extracting veterinary growth promoters from animal urine, achieving high recoveries, low LOQs and excellent linearity suitable for regulatory and research laboratories.

Reference

Biotage (2019) Application Note AN925: Extraction of Veterinary Growth Promoters from Animal Urine Using ISOLUTE HYDRO DME+ prior to UPLC–MS/MS Analysis.