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

Importance of the Topic

Monitoring steroid residues in milk is critical due to potential health risks associated with long-term exposure to exogenous hormones. Estrogens and progesterone naturally increase during dairy cow lactation, especially in late pregnancy, raising concerns about dietary intake of these bioactive compounds and their links to hormone-related disorders.

Objectives and Study Overview

This application note describes the development of a highly sensitive LC-MS/MS method on the Shimadzu LCMS-8050 platform for the quantitative determination of testosterone, progesterone, and estradiol at sub-parts-per-trillion levels in cow milk. The study aims to demonstrate method repeatability, specificity, and low detection limits in a complex food matrix.

Methodology and Instrumentation

Sample Preparation

• QuEChERS extraction: 10 mL milk was mixed with 10 mL acetonitrile and CEN QuEChERS salts, followed by centrifugation and collection of the organic layer.
• d-SPE cleanup: 1 mL extract was treated with MgSO₄, PSA and C₁₈ sorbents, centrifuged, and supernatant taken for analysis.

Used Instrumentation

• LCMS-8050 triple quadrupole MS with heated electrospray ionization probe, UF scanning (30,000 Da/s) and UF polarity switching (5 ms).
• Nexera UHPLC system with Shim-pack XR-ODS column (75 × 2.0 mm, 2.2 µm).

Chromatographic Conditions

• Mobile phase A: 0.1 % formic acid in water; B: 0.1 % formic acid in acetonitrile.
• Gradient from 70 % A to 10 % A over 3.5 min, hold, then return to initial conditions.
• Flow rate: 0.4 mL/min; column temperature: 40 °C; injection volume: 20 µL.

MS Conditions

• Positive ESI; nebulizing, drying, and heating gas flows optimized.
• Interface temperature: 350 °C; DL temp: 250 °C; heat block: 500 °C.
• MRM transitions: testosterone 289.40→109.20, progesterone 315.10→97.15, estradiol 255.10→159.20.

Main Results and Discussion

Specificity

• No interfering peaks observed in blank milk matrices at analyte retention times.

Linearity

• Testosterone and estradiol: 0.05–10 ppb; progesterone: 0.25–50 ppb with r² > 0.999.

Accuracy and Precision

• Measured accuracies ranged from 91.9 % to 118 % across calibration levels, meeting acceptance criteria.
• Carry-over effects were negligible due to the fast autosampler and optimized washing cycles.

Recovery

• Spiking experiments at low, mid, and high levels yielded recoveries within acceptable ranges, demonstrating efficient extraction and cleanup.

Benefits and Practical Applications

This protocol offers rapid, user-friendly sample preparation suitable for small and large dairy operations. The high sensitivity and specificity of the LCMS-8050 enable reliable monitoring of steroid contaminants in milk, supporting regulatory compliance and consumer safety assessments.

Future Trends and Opportunities

Advancements may include:

• Automation of QuEChERS workflows to further increase throughput.
• Integration with high-resolution mass spectrometry for broader screening of unknown bioactive contaminants.
• Application to other food matrices and environmental samples.

Conclusion

The combination of QuEChERS extraction and Shimadzu’s LCMS-8050 delivers a robust, sensitive method for quantifying low-level steroids in milk. This approach ensures high throughput, reproducibility, and compliance with stringent food safety standards.

References

1. Maruyama K., Oshima T., Ohyama K., Pediatric International, 52 (2010), 32–38.
2. Farlow D.W., Xu X., Veenstra T.D., J. Chromatogr. B 877 (2009), 1327–1334.
3. Ganmaa D., Sato A., Medical Hypotheses 65 (2005), 1028–1037.
4. Noppe H., Le Bizec B., Verheyden K., De Brabander H.F., Analytica Chimica Acta 611 (2008), 116.