Low level quantitation of steroids in milk using LC/MS/MS

Significance of the Topic

The presence of natural and synthetic steroid hormones in cow milk has emerged as a critical food safety and public health issue. Dairy cows produce milk throughout pregnancy when circulating estrogen and progesterone levels increase. Chronic dietary exposure to these compounds may contribute to hormone-related cancers and disrupt endocrine balance in consumers. Reliable detection at trace levels is essential for regulatory compliance and risk assessment.

Objectives and Study Overview

This study aimed to establish a highly sensitive, high-throughput LC/MS/MS method for quantifying testosterone, progesterone, and estradiol in whole milk. The approach focused on achieving parts-per-trillion detection limits and excellent reproducibility in a complex matrix using the Shimadzu LCMS-8050 triple quadrupole system.

Methodology

Sample preparation employed a QuEChERS protocol:

• 10 mL whole milk extracted with 10 mL acetonitrile and a standardized QuEChERS salt pouch.
• Dispersive SPE cleanup using magnesium sulfate, PSA, and C18 sorbents.
• Supernatant collected after centrifugation for direct LC/MS/MS injection.

Instrumentation

Chromatography and mass spectrometry were performed on a Shimadzu Nexera UHPLC coupled to the LCMS-8050:

• Column: Shim-pack XR-ODS (75 × 2.0 mm, 2.2 μm).
• Mobile phase: 0.1% formic acid in water (A) and acetonitrile (B) with gradient elution at 0.4 mL/min and 40 °C.
• Interface: heated electrospray ionization in positive mode with rapid polarity switching.
• Optimized MRM transitions: testosterone 289.4→109.2; progesterone 315.1→97.2; estradiol 255.1→159.2.

Main Results and Discussion

The method demonstrated:

• High specificity with no detectable interferences in blank samples.
• Linear calibration ranges of 0.05–10 ppb for testosterone and estradiol, 0.25–50 ppb for progesterone (r² > 0.999).
• Accuracy spanning 92–118% across all levels.
• Recoveries within acceptance criteria for low (0.1–0.5 ppb), medium, and high (10–50 ppb) spikes.
• Minimal carryover enabled by the fast autosampler and optimized flow paths.

Benefits and Practical Applications

This workflow offers a robust, cost-effective solution for routine monitoring of steroid residues in dairy products. The simple QuEChERS-based preparation and ultrahigh-sensitivity triple quadrupole analysis enable both small laboratories and large producers to ensure compliance with safety standards and support quality control programs.

Future Trends and Opportunities

Emerging developments may include automated sample preparation, expanded multi-residue panels for veterinary drugs, integration with high-resolution MS for non-targeted screening, and portable sensors for in-field testing. Advances in ionization sources and data processing will further enhance sensitivity and throughput.

Conclusion

The validated LCMS-8050 method achieves low-ppt quantitation of key steroid hormones in milk with excellent linearity, accuracy, and recovery. Its combination of streamlined sample handling and ultrahigh sensitivity positions it as a valuable tool for food safety surveillance and regulatory compliance.

References

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