Rapid, Simple, and Effective Clean-up of Bovine Liver Samples Prior to UPLC-MS/MS Multiresidue Veterinary Drugs Analysis

Significance of the topic

Monitoring veterinary drug residues in edible tissues is essential to ensure food safety and regulatory compliance. Beef liver presents analytical challenges due to its high content of fats and phospholipids, which can impair chromatographic performance, shorten column life, and cause ion suppression in mass spectrometry. A streamlined cleanup strategy that effectively removes these interferences while preserving a broad range of analytes is therefore of high practical importance.

Objectives and study overview

This study aimed to develop and evaluate a rapid, simple, and efficient sample cleanup protocol for bovine liver extracts, compatible with UPLC-MS/MS multiresidue analysis. The goal was to maximize removal of fats and phospholipids using a single-step solid-phase extraction (SPE) pass-through method, while maintaining high recoveries and method sensitivity for over 60 veterinary drug residues.

Methodology and used instrumentation

Sample Preparation:

• 2 g of homogenized bovine liver spiked with standards.
• Extraction with 10 mL of 0.2% formic acid in 85:15 acetonitrile:water.
• Centrifugation to precipitate proteins and collect supernatant.
• Pass-through cleanup on an Oasis PRiME HLB Cartridge (6 cc, 200 mg) without conditioning.
• Discard initial 0.6 mL, collect next 1 mL, dilute 200 µL of eluate with ammonium formate buffer (pH 4.5).

Instrumentation:

• UPLC: ACQUITY UPLC I-Class System with Fixed-Loop Sample Manager.
• Column: ACQUITY UPLC CSH C18, 1.7 µm, 2.1×100 mm, 30 °C.
• Mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in 50:50 acetonitrile:methanol (B) with linear gradient.
• MS: Xevo TQ-XS triple quadrupole with positive ESI, source at 150 °C, desolvation at 400 °C, MRM transitions optimized for each analyte.
• Data acquisition: MassLynx v4.1.

Main results and discussion

Recovery and matrix effects were evaluated at low (10 ng/g) and high (100 ng/g) levels (n=6). Overall recoveries averaged above 70% for most compounds, though more polar classes (e.g., tetracyclines) showed somewhat lower yields. The pass-through SPE removed over 95% of phospholipids and more than 90% of hexane-extractable fats, significantly reducing matrix interferences. Matrix effects averaged around 40%. Post-extraction SPE losses were minimal for most drugs, with slightly reduced recoveries observed for ivermectin, monensin, moxidectin, and novobiocin.

Benefits and practical applications

• Rapid, one-step cleanup compatible with high throughput screening.
• Elimination of conditioning and extensive solvent use.
• Significant reduction in matrix interferences improves instrument uptime and data quality.
• No additional filtration required before UPLC-MS/MS.

Future trends and potential applications

Further developments may include adaptation to other tissue types, automation in 96-well formats, expansion of analyte panels, and integration with high-resolution MS workflows. Advances in sorbent chemistries could enhance retention of very polar or lipophilic drugs, broadening the method’s applicability.

Conclusion

The streamlined sample preparation workflow using Oasis PRiME HLB pass-through cartridges delivers rapid, effective cleanup of bovine liver extracts, removing the majority of fats and phospholipids while maintaining satisfactory recoveries for a wide spectrum of veterinary drug residues. This approach supports reliable, high-throughput UPLC-MS/MS analysis in food safety and quality control laboratories.

References

• Young M.S., Tran K.V., Oasis PRiME HLB Cartridge for Effective Clean-up of Meat Extracts Prior to Multi-Residue Veterinary Drug UPLC-MS Analysis, Waters Application Brief, 2015.
• Lehotay S., High-Throughput Screening Analysis by UHPLC-MS/MS of >60 Veterinary Drugs in Animal Tissues, 125th AOAC Annual Meeting Presentation 2303, 2011.