Determination of 58 Glucocorticoids in Milk

Significance of the Topic

This study addresses the critical need to monitor glucocorticoid residues in milk, a key requirement for food safety and regulatory compliance worldwide. Glucocorticoids are widely used in veterinary medicine, but their residues can pose health risks and must remain below established maximum residue limits (MRLs). Reliable multiresidue methods enable laboratories to screen large numbers of samples efficiently and ensure consumer protection.

Objectives and Study Overview

The main goal was to develop and validate a streamlined, high-throughput method for simultaneous determination of 58 glucocorticoids in cow and goat milk. This approach combines a modified QuEChERS extraction with Agilent Captiva EMR–Lipid HF cartridges for enhanced matrix cleanup, followed by quantitation using LC/MS/MS. Validation focused on limits of quantitation (LOQs), recovery, precision, and compliance with Chinese national standards (GB 31650-2019).

Methodology

Sample preparation begins by weighing 5 g of milk and extracting with acetonitrile and QuEChERS salts. After centrifugation, an aliquot of the supernatant is diluted with water and loaded onto a Captiva EMR–Lipid HF cartridge for gravity-driven lipid removal. The eluent is dried under nitrogen, reconstituted in acetonitrile/water, and filtered prior to analysis. Chromatographic separation uses an Agilent InfinityLab Poroshell 120 EC-C18 column with a gradient of water containing 0.1% formic acid and 1 mmol/L ammonium fluoride (mobile phase A) and acetonitrile (mobile phase B). Detection is performed on an Agilent 6470B triple quadrupole LC/MS in positive ESI dMRM mode.

Instrumentation Used

• Agilent 1290 Infinity II high-performance liquid chromatograph
• Agilent 6470B triple quadrupole mass spectrometer
• Agilent InfinityLab Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 µm)
• Captiva EMR–Lipid HF cartridges (6 mL format)
• Bond Elut QuEChERS extraction kit for veterinary drugs
• PTFE syringe filters (0.2 µm) and ceramic homogenizers

Main Results and Discussion

• Gravity elution on Captiva EMR–Lipid HF cartridges was completed within 15–25 minutes for most complex matrices, a 20–50% time reduction compared to current and competing cartridges.
• Matrix removal evaluated by residue weight and GC/MS background screening showed equivalent or improved cleanup performance.
• Target recoveries for all 58 analytes ranged from 90% to 109% with RSDs of 1.2%–14.0%, meeting regulatory requirements.
• Introducing 1 mmol/L ammonium fluoride in mobile phase A enhanced ionization, providing significantly higher signal intensity for the glucocorticoids.
• Compared to traditional dispersive SPE cleanup, the EMR–Lipid HF approach delivered higher recoveries, especially for hydrophobic compounds.
• Method validation achieved LOQs of 0.2 µg/kg for 47 compounds and 2 µg/kg for 11 compounds, with linear calibration (R² > 0.99) over relevant ranges.

Benefits and Practical Applications

This protocol reduces solvent usage and processing time while maintaining robust lipid removal and broad analyte coverage. It suits routine screening and confirmatory analyses in quality control and regulatory laboratories. Compliance with national MRLs for betamethasone and dexamethasone in milk is demonstrated, supporting food safety monitoring programs.

Future Trends and Potential Applications

Ongoing developments may include automation of the EMR–Lipid HF cleanup for higher throughput, expansion of analyte panels to other steroid classes, and integration with high-resolution mass spectrometry for non-targeted screening. Green chemistry approaches may further reduce solvent consumption and environmental impact.

Conclusion

The presented method offers a simplified, efficient, and sensitive workflow for simultaneous quantitation of 58 glucocorticoids in milk. By leveraging Captiva EMR–Lipid HF cartridges and optimized LC/MS/MS conditions, laboratories can achieve rapid sample cleanup, reliable quantitation, and compliance with regulatory standards.

References

1. FAOLEX Database: National Food Safety Standard GB 31650–2019. FAOLEX, Food and Agriculture Organization of the United Nations.
2. China Ministry of Agriculture Announcement No. 1031-2-2008: Determination of Multiresidue Glucocorticoid Drugs in Animal-Origin Foods by LC/MS/MS.