Methods for the determination of residual veterinary drugs in Raw Milk using LCMS-8050

Importance of Topic

Veterinary drug residues in raw milk pose potential health risks, especially for vulnerable populations such as infants and children. Ensuring compliance with stringent food safety regulations (e.g., FSSAI, EU) requires rapid, sensitive, and reliable multiresidue analytical methods. Advances in ultra-fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) enable efficient monitoring of a broad spectrum of veterinary drugs at trace levels, supporting high-throughput testing in quality control and research laboratories.

Objectives and Study Overview

This study aimed to develop and validate four high-throughput multiresidue methods (Methods I–IV) on a Shimadzu LCMS-8050 system coupled with a Nexera X3 auto-sampler. Collectively, the methods cover 69 veterinary drug compounds with diverse chemical properties. Key objectives included achieving regulatory compliance, minimizing analytical runtime, and ensuring robust performance at low limits of quantification (LOQs).

Methodology and Instrumentation

Used Instrumentation

• Liquid Chromatograph–Mass Spectrometer: Shimadzu LCMS-8050
• Auto-sampler: Nexera X3 with SIL-40C
• Column: Shim-pack Velox C18 (3.0 mm i.d. × 100 mm, 2.7 µm)
• Software: LabSolutions Insight for data processing and validation

Sample Preparation

Extraction relied on modified QuEChERS protocols and solid-phase extraction (SPE) across four methods:

• Method I & II: Acetonitrile-based protein precipitation, salt partitioning, C18 dispersive SPE, evaporation, and reconstitution in mobile phase.
• Method III: Trichloroacetic acid deproteinization, HLB cartridge cleanup, methanol elution, and dilution.
• Method IV: Dilution with ion-pairing reagent and filtration prior to injection.

Analytical Conditions

Each method employed a 17-minute gradient run at 0.3 mL/min, 40 °C column temperature, and 10 µL injection volume. Mobile phases consisted of aqueous formic acid or oxalic acid buffers and methanol, with an electrospray ionization source in positive mode and multiple reaction monitoring (MRM) transitions (one target, two qualifiers).

Main Results and Discussion

Validation followed SANTE guidelines for specificity, linearity, recovery, and precision:

• Linearity: Calibration curves (weighted 1/C2) demonstrated R2 values ≥ 0.989 for representative compounds.
• LOQs: Achieved 2 µg/kg for 40 compounds, 5 µg/kg for 14 compounds, 10 µg/kg for 11 compounds, and 20 µg/kg for 5 compounds.
• Recovery: Mean recoveries for 48 compounds were within 70–120%; 20 compounds showed recoveries of 35–70%. All recoveries met reproducibility criteria (≤ 20 % RSD).
• Precision: Repeatability (RSDr) and within-lab reproducibility (RSDR) at LOQ and 2× LOQ levels were ≤ 20 % for all analytes.

Benefits and Practical Applications

The developed methods offer:

• High sensitivity and compliance with FSSAI and EU regulatory limits.
• Rapid analysis with a 17-minute runtime per injection, facilitating high throughput.
• Versatility across chemically diverse veterinary drugs using unified instrumentation and software packages.

Future Trends and Applications

Emerging trends include integration of automated sample preparation platforms, expansion to isotopically labeled internal standards for enhanced accuracy, and incorporation of high-resolution mass spectrometry for untargeted screening. The methods can be adapted for other food matrices and extended surveillance of emerging veterinary pharmaceuticals.

Conclusion

The four multiresidue LC–MS/MS methods validated on the Shimadzu LCMS-8050 demonstrate robust performance for quantifying 69 veterinary drugs in raw milk. Achieving low LOQs, high recoveries, and precise quantitation supports regulatory compliance and routine quality control in dairy analysis.

Reference

1. M. Anastassiades, S.J. Lehotay, D. Štajnbaher, F.J. Schenck, "Fast and Easy Multiresidue Method Employing acetonitrile Extraction/Partitioning and Dispersive Solid-Phase Extraction for the Determination of Pesticide Residues in Produce," J. AOAC Int., 86, 412–431 (2003).
2. European Commission, "Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues and Analysis in Food and Feed," SANTE/12682/2019.