Application Notebook - Solutions for Food Safety

Significance of Concern

Rapid, reliable screening for veterinary drug residues in foodstuffs is essential to protect public health. Quinolone antimicrobials in meat (chicken, pork, fishery products) are strictly regulated, with maximum residue limits (MRLs) as low as 0.01 mg/kg in the EU, Japan, and many other regions. Traditional microbiological methods and time-consuming multi-step sample preparations limit laboratory throughput and data quality. There is a pressing need for streamlined, high-sensitivity instrumental screening methods that deliver both quantitative and qualitative confidence and reduce the chances of false positives or negatives.

Objectives and Study Overview

• Develop a rapid, multi-residue LC-UV/FLD screening method for twelve common quinolone antibiotics at MRL levels in meat products.
• Implement a simplified pretreatment workflow suitable for high-fat matrices such as chicken, pork, and shrimp.
• Combine i-Series UHPLC with a highly sensitive RF-20AXS fluorescence detector and PDA detection to achieve low-level quantitation and built-in UV library spectral confirmation.
• Evaluate method performance: sensitivity, repeatability, linearity, and qualitative confirmation via UV similarity matching.

Methodology and Instrumentation

• Pretreatment -- simple QuEChERS-style acetonitrile extraction followed by hexane partitioning to remove lipids, evaporation, and redissolution in 80:20 acetonitrile:water (sample load equivalent to 0.025 mg/L for 0.01 mg/kg MRL).
• Chromatography -- Shim-pack FC-ODS reversed-phase column (150 × 4.6 mm, 3 μm) at 40 °C; mobile phases (20 mM phosphate + 0.1 M perchlorate buffer and organic modifier acetonitrile:methanol 90:10); 0–100% organic gradient over 7.5 min; 5 μL injection; 1 mL/min flow.
• Detection -- dual detection on i-Series: PDA at 280 nm for initial library-based identification, and RF-20AXS fluorescence at Ex/Em = 290/495 nm for second-generation quinolones (e.g. ciprofloxacin, ofloxacin) and 325/365 nm for older compounds (e.g. oxolinic acid, nalidixic acid).
• Data processing -- LabSolutions Insight software performs automatic peak integration, concentration calculations against calibration standards, and qualitative confirmation by UV spectral similarity matching to an onboard library.

Key Results and Discussion

• Limits of quantitation achieved at 0.025 mg/L (equivalent to 0.01 mg/kg MRL) with signal-to-noise ratios >20 for all twelve quinolones.
• Excellent repeatability: RSD < 3.0% for peak areas across six consecutive injections in chicken, pork, and shrimp matrices.
• Calibration curves linear across 0.01–0.25 mg/L (1–25% above MRL) with R² > 0.999 for all compounds.
• Fluorescence detection provided high sensitivity and selectivity, while simultaneous PDA UV spectra allowed library-based qualitative confirmation, e.g. piromidic acid matched with >90% UV similarity score.
• Screening report templates automatically flag samples above MRL, enabling rapid pass/fail assessment without manual calculation.

Benefits and Practical Applications

• Single-run analysis (14 min cycle time) simultaneously screens all target quinolones in high-fat matrices, replacing multi-step microbiological assays.
• Simple, robust sample pretreatment reduces labor and consumable costs, while minimizing the potential for operator error.
• Combined quantitative and qualitative confirmation in one system enhances data confidence, reducing false positives/negatives and accelerating decision-making in quality control.
• Automated report generation and built-in UV library matching greatly improve laboratory throughput and traceability of analytical results.

Future Trends and Potential Uses

• Extension of the screening panel to include sulfonamides, antifolates, and additional veterinary drug classes for comprehensive residue surveillance.
• Integration with high-resolution mass spectrometry for non-targeted screening and retrospective data analysis of emerging contaminants.
• Adoption of on-line sample preparation (e.g. online SPE or SFE) to further streamline workflows and reduce sample handling.
• Application in regulatory testing, feed safety, and global import/export compliance programs requiring high-throughput, high-sensitivity screening.

Conclusions

A rapid, high-sensitivity LC-UV/FLD screening method for twelve quinolone antimicrobials at regulatory levels in meat matrices was developed using the i-Series UHPLC and RF-20AXS fluorescence detector. The simplified acetonitrile/hexane pretreatment effectively removed lipids, allowing direct injection without laborious solid-phase extraction. The method achieved LOQs at 0.01 mg/kg, excellent repeatability (RSD < 3%), and linearity (R² > 0.999) across the targeted range. The combined PDA library matching and fluorescence detection approach provides both quantitative and qualitative assurance, making this system ideal for routine residue screening to support compliance with MRL regulations.

References

1) Japanese Ministry of Health, Labour and Welfare, Positive List System for Agricultural Chemical Residuals, May 2006. 2) Mikanagi, K. et al., Simultaneous Analysis Method for Veterinary Drugs by HPLC (Livestock and Marine Products), Jpn. J. Antibiotics, 2003. 3) Japanese Pharmacopoeia 17th ed., Veterinary Drug Residue Analytical Methods.