Determination of Eight Estrogens in Milk by UHPLC and the Agilent 6495 Triple Quadrupole Mass Spectrometer

Significance of the Topic

A rising concern in food safety and public health is the presence of natural and synthetic estrogens in dairy products. Since cows are often pregnant during milking, their milk contains elevated levels of female hormones. Chronic exposure, especially in infants and children, may disrupt normal development and is linked to hormone-sensitive cancers. Regulatory bodies worldwide set stringent maximum residue limits (MRLs) for estrogens in milk, driving the need for sensitive, robust analytical methods.

Study Objectives and Overview

This application note presents a rapid, precise workflow to quantify eight estrogens in milk using ultra-high performance liquid chromatography (UHPLC) coupled to an Agilent 6495 Triple Quadrupole Mass Spectrometer. The goals were to achieve detection limits well below 1 µg/kg without derivatization, minimize sample preparation, and ensure compliance with international regulations.

Methodology and Instrumentation

The method employs the QuEChERS extraction-partitioning technique followed by dispersive SPE cleanup. Ten-gram milk samples are fortified, extracted with acetonitrile and extraction salts, then subjected to C18 dispersive cleanup. The dried extracts are reconstituted in 50% methanol/water and injected.

Instrumentation Used

• Agilent 1290 Infinity UHPLC with Poroshell 120 Phenyl-Hexyl column (2.1×100 mm, 2.7 µm)
• Agilent 6495 Triple Quadrupole MS with JetStream ESI source operating in negative ion dynamic MRM mode
• Agilent MassHunter software for data acquisition and quantitation

Main Results and Discussion

The instrument detection limits (IDLs) in solvent ranged from 1.2 to 3.7 ppt, and the lower limits of quantitation (LLOQs) in milk were 0.1 µg/kg, far below the 1 µg/kg regulatory threshold. Calibration was linear over 0.1–10 µg/kg (R2 > 0.998). Recoveries for the eight estrogens spiked at 0.2, 1, and 5 µg/kg ranged from 83% to 106% with relative standard deviations below 10%. Matrix effects were minimal, and blank milk samples showed negligible interference at the LLOQ level.

Benefits and Practical Applications

• High sensitivity enables reliable quantitation of estrogens at sub-regulatory levels
• QuEChERS-based sample prep reduces solvent usage, labor, and waste
• No derivatization simplifies workflow and shortens turnaround time
• Applicable to routine QA/QC in dairy and infant formula testing

Future Trends and Potential Applications

Advances may include automated QuEChERS workstations, expanded multi-residue panels covering other endocrine disruptors, and integration with high-resolution mass spectrometry for non-targeted screening. Improved data processing algorithms and cloud-based platforms could further enhance throughput and data reproducibility.

Conclusion

The UHPLC-MS/MS method using Agilent 6495 Triple Quadrupole provides a fast, sensitive, and robust solution for estrogen analysis in milk. Its low detection limits, high recovery, and simplified sample preparation make it suitable for regulatory compliance and routine monitoring across the dairy industry.

References

1. Maruyama K, Oshima T, Ohyama K. Exposure to exogenous estrogen through intake of commercial milk produced from pregnant cows. Pediatrics Int. 2010;52(1):33–38.
2. Candyce H, et al. High- and Low-Fat Dairy Intake, Recurrence, and Mortality After Breast Cancer Diagnosis. J Natl Cancer Inst. 2013;105(22):1759–1760.
3. Qin LQ, et al. Estrogen: one of the risk factors in milk for prostate cancer. Med. Hypotheses. 2004;62(1):133–142.
4. GB/T 22992-2008. P.R. China: National Standard for Estrogen Residues in Milk.
5. Directive 96/23/EC. European Union Council Directive on monitoring of hormones in foodstuffs.
6. Stubbing G, Bigwood T. The development and validation of a multiclass LC-MS/MS procedure for determination of veterinary drug residues using QuEChERS. Anal Chim Acta. 2009;637:68–78.
7. Parra NP, Taylor L. Why Instrument Detection Limit (IDL) is a Better Metric for Determining the Sensitivity of Triple Quadrupole LC/MS Systems. Agilent Technol Tech Overview. 2014;5991-4089EN.