A generic approach for simultaneous detection and quantification of pesticides, veterinary drugs, and aflatoxin M1 in milk using LC-MS/MS

Importance of the Topic

Milk is a highly consumed food product and a potential carrier of chemical contaminants. Residues of pesticides, veterinary drugs, and mycotoxins such as aflatoxin M1 pose risks to human health and dairy industry economics. Regulatory bodies including the EU and FSSAI have established stringent maximum residue limits (MRLs) and minimum required performance limits (MRPLs) to protect consumers. A unified, high-throughput analytical method is essential to ensure milk safety while reducing analysis time and costs.

Objectives and Study Overview

The primary objective was to develop and validate a generic LC-MS/MS workflow capable of simultaneously extracting, detecting, and quantifying multi-class pesticides (192 compounds), veterinary medicines/drugs (54 compounds), and aflatoxin M1 in milk. The method was evaluated against SANTE 12682/2019, EU Directive 2002/657/EC, and EC Regulation 1881/2006 guidelines.

Methodology and Instrumentation

Sample Preparation and Clean-Up
The Veterinary Drugs Explorer (VDX) QuEChERS-based protocol was applied to 4 g of milk. Samples were spiked, then extracted with 1% acetic acid in acetonitrile. After addition of ammonium oxalate/EDTA and sodium sulfate, centrifugation was followed by C18 dispersive SPE. The extract was evaporated under nitrogen and reconstituted in water/methanol/acetonitrile (70:15:15).
Instrumentation Used

• UHPLC: Thermo Scientific Vanquish Flex Binary UHPLC system with Accucore aQ column (100 × 2.1 mm, 2.6 µm).
• MS/MS: Thermo Scientific TSQ Quantis triple quadrupole with HESI II source and fast polarity switching in timed-SRM mode.
• Data Processing: Thermo Scientific TraceFinder software v4.1, using automatic flagging for retention time, ion ratios, linearity, recovery, and precision.

Main Results and Discussion

The method achieved quantitation limits of 0.005 mg/kg for most pesticides, 0.25–25 µg/kg for veterinary drugs, and 0.025 µg/kg for aflatoxin M1. Procedural standard calibration provided 70–120% recovery and <15% RSD (n = 6) for >94% of analytes. Fast polarity switching enabled monitoring of 650 SRM transitions in one 17 min run. Auto-optimized dwell times (2–46 ms) maintained >12 data points per peak. Repeatability over 100 injections showed <15% area RSD and <0.05 min retention time drift, demonstrating robustness and high throughput.

Advantages and Practical Applications

• Simultaneous multi-class analysis reduces turnaround time and cost by up to 50% compared to separate assays.
• Compliance with EU and FSSAI MRLs ensures regulatory acceptance for routine milk testing.
• High sensitivity and precision support quality control in dairy, feed, and environmental monitoring.

Future Trends and Potential Applications

Continued advances may include integration of high-resolution mass spectrometry for non-target screening, full automation of sample preparation, and expansion to other food and environmental matrices. Enhanced software algorithms and machine learning could further streamline data review and reporting.

Conclusion

The generic LC-MS/MS method combining a modified QuEChERS extraction with advanced UHPLC-MS/MS instrumentation offers a powerful solution for simultaneous detection of pesticides, veterinary drugs, and aflatoxin M1 in milk. It meets stringent regulatory criteria, delivers robust performance, and increases laboratory throughput.

Reference

1. National Dairy Development Board. India milk production statistics 2018–2019.
2. Bedi JS, Gill JPS, Kaur P, Aulakh RS. Pesticide residues in milk and feedstuffs in Punjab (India). J Anim Feed Sci. 2018;27:18–25.
3. Akhtar S, Ahad K. Pesticides residue in milk and milk products: mini review. Pak J Anal Environ Chem. 2017;18(1):37–45.
4. Food Safety and Standards Authority of India. Food Safety and Standards (Contaminants, Toxins and Residues) Regulations, 2011.
5. European Commission. EU Pesticides Database. Public access.
6. Thermo Fisher Scientific. VetDrugs Explorer workflow. Application Brief 65118; 2020.
7. European Commission. SANTE/12682/2019: Analytical quality control and method validation for pesticide residues.
8. European Commission. Commission Decision 2002/657/EC: performance of analytical methods.
9. European Commission. Regulation (EC) No 1881/2006: maximum levels of contaminants in foodstuffs.