Multi-class veterinary drugs analyses of QuEChERS extracts using an automated online μSPE cleanup coupled to LC-MS/MS

Significance of the Topic

The control of veterinary drug residues in food products is essential to protect public health and comply with global maximum residue limits (MRLs). Efficient sample cleanup and accurate quantification are critical to prevent false positives or negatives, minimize instrument contamination, and ensure reliable long-term operation of LC-MS/MS systems.

Objectives and Study Overview

This work aimed to evaluate a fully automated, online micro–solid phase extraction (µSPE) cleanup workflow coupled to LC-MS/MS for the rapid screening and quantitation of 103 veterinary drug residues in bovine muscle, liver, and kidney tissues. Manual dispersive SPE (dSPE) cleanup was compared with two automated µSPE sorbents (CEC18 and HyperSep Retain-PEP) to assess recovery, precision, matrix effects, linearity, and robustness.

Methodology and Instrumentation

Sample Preparation and Cleanup

• Tissues (5 g) homogenized, extracted by QuEChERS (acetonitrile plus salts).
• Manual cleanup: 500 mg C18 dSPE, vortex, centrifuge.
• Automated cleanup: TriPlus RSH µSPE autosampler with two miniaturized cartridges (15 mg CEC18 or 10 mg HRP), controlled syringe flow to condition, load, wash, and elute directly into the LC injection loop.

LC-MS/MS Analysis

• UHPLC: Vanquish Flex with Accucore VDX column (100 × 2.1 mm, 2.6 µm), gradient of 0.05% formic acid in water (A) and 1:1 MeOH:ACN with 0.05% FA and 5% water (B).
• Injection: Solvent sandwich technique (20 µL mobile phase A–2 µL sample in ACN–20 µL mobile phase A, plus air gaps) to improve early-eluting peak shape.
• Mass spectrometry: TSQ Altis triple quadrupole with HESI source, positive/negative modes, scheduled SRM, cycle time 0.35 s.

Key Results and Discussion

Recovery and Precision

• Automated CEC18 µSPE achieved 30–140% recovery with ≤20% RSD for >95% of compounds at 50 ng/g in all tissues.
• HyperSep PEP sorbent yielded poor retention for certain analytes (e.g., rafoxanide, lasalocid).
• Precision: Five replicates at 5 and 50 ng/g showed ≤20% RSD and recoveries within 70–120% for >90% of compounds.

Matrix Effects and Linearity

• Most analytes exhibited low to moderate matrix effects (<50%) across tissues and cleanup methods.
• Excellent calibration linearity (R2 > 0.99) from 1–100 ng/g using procedural matrix-extracted standards or automated on-board calibration.

Robustness and Throughput

• Over 100 consecutive injections with automated cleanup and sandwich injection maintained <±20% response drift without maintenance, including a 24 h pause.
• Automated workflows reduced sample-prep labor by 4–5 h per 50 samples and decreased solvent and reagent usage.

Benefits and Practical Applications

Automated online µSPE cleanup streamlines multi-residue veterinary drug analysis in food matrices by minimizing manual errors, improving throughput, and extending instrument uptime. The method supports routine compliance testing in veterinary diagnostic laboratories, QA/QC environments, and food safety monitoring.

Future Trends and Applications

Continued development of sorbent chemistries and further miniaturization of SPE devices will expand automated cleanup to new compound classes and complex matrices. Integration with high-resolution mass spectrometry and smart data workflows will enhance screening flexibility and enable real-time quality control.

Conclusion

The automated online µSPE-LC-MS/MS workflow using CEC18 cartridges offers a robust, precise, and high-throughput solution for multi-class veterinary drug residue analysis in animal tissues. It delivers consistent recoveries, controlled matrix effects, and prolonged instrument performance while reducing labor and consumable costs.

Instrumentation Used

• CTC Analytics TriPlus RSH µSPE autosampler (PAL3-RTC based)
• Thermo Scientific Vanquish Flex UHPLC system
• Thermo Scientific TSQ Altis triple quadrupole mass spectrometer
• Accucore VDX column, 100 × 2.1 mm, 2.6 µm

References

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3. AOAC SMPR 2018.010 Standard Method Performance Requirements for Veterinary Drug Residue Analysis, 2018.
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