Analysis of Chloramphenicol in Shrimp and Chicken Egg Extracts Using Triple Quadrupole LC/MS/MS

Importance of the Topic

The antibiotic chloramphenicol is widely used in veterinary medicine due to its broad antimicrobial spectrum, but regulatory bodies have banned its residues in food because of potential genotoxic and carcinogenic effects. In vivo hydrolysis of chloramphenicol glucuronide conjugates back to the parent compound further complicates monitoring. Reliable, sensitive analysis methods are essential to ensure food safety and regulatory compliance.

Study Objectives and Overview

This study describes the quantitative determination of chloramphenicol and its glucuronide conjugates in shrimp and chicken eggs following the 2017 Japanese Ministry of Health, Labour and Welfare test method revision. The aim was to validate a triple quadrupole LC/MS/MS protocol that meets the zero-tolerance requirement under the positive list system.

Methodology and Instrumentation

Sample Pretreatment:

• Homogenize 10 g shrimp or egg sample with methanol and centrifuge; adjust to 100 mL.
• Evaporate 4 mL of supernatant; hydrolyze with β-glucuronidase in phosphate buffer.
• Extract with ethyl acetate (3×), centrifuge, and purify on a divinylbenzene–N-vinylpyrrolidone copolymer column.

LC/MS/MS Conditions:

• Column: Shim-pack™ HR-ODS (150 × 2.1 mm, 3 µm).
• Mobile Phase: 10 mmol/L ammonium acetate : acetonitrile (70 : 30 v/v).
• Flow Rate: 0.35 mL/min; Column Temp: 40 °C; Injection: 5 µL.
• Probe Voltage: –1.0 kV (ESI negative); DL: 300 °C; Block Heater: 500 °C; Interface: 400 °C.
• MRM Transitions: chloramphenicol m/z 321.10>152.25 (Quantifier), 321.10>257.05 (Qualifier); glucuronide m/z 496.90>193.05, 496.90>113.00.
• Instrument: Shimadzu LCMS-8050 triple quadrupole MS.

Main Results and Discussion

• Calibration: 0.1–2 µg/L for chloramphenicol; 0.5–10 µg/L for conjugate; R2>0.999.
• Detection Limit: 0.005 mg/kg in produce; Quantitative Limit: 0.1 µg/L.
• Spike Recovery: shrimp 87.9% (RSD 7.6%), eggs 95.5% (RSD 13.5%).
• No target compounds detected in unspiked shrimp or eggs; complete hydrolysis confirmed by absence of conjugate and detection of chloramphenicol after conjugate addition.

Benefits and Practical Applications

The validated method enables high-throughput, accurate quantification of chloramphenicol residues at sub-µg/L levels, supporting regulatory surveillance and quality control in food safety laboratories. Robust clean-up and hydrolysis steps ensure reliable detection of both free and conjugated forms.

Future Trends and Potential Applications

• Integration of high-resolution mass spectrometry for wider screening of banned compounds.
• Automation of sample preparation to improve throughput and reproducibility.
• Development of portable MS platforms for on-site monitoring.
• Expansion to multi-residue workflows covering diverse veterinary drugs.

Conclusion

The described LC/MS/MS protocol fulfills zero-tolerance requirements for chloramphenicol and its glucuronide conjugates in shrimp and eggs, achieving reliable sensitivity (0.1 µg/L), excellent linearity, and acceptable recoveries. It represents a robust tool for food safety testing and regulatory compliance.

Used Instrumentation

• Shim-pack™ HR-ODS Column (150 × 2.1 mm, 3 µm).
• Shimadzu LCMS-8050 Triple Quadrupole Mass Spectrometer.

References

1. Ministry of Health and Welfare Notification No. 370, 1959.
2. Ministry of Health, Labour and Welfare Notification No. 499, 2005.
3. Ministry of Health, Labour and Welfare Notification No. 49, 2017.
4. Shimadzu Corporation. Application Note C199: Analysis of Chloramphenicol in Shrimp and Chicken Egg Extracts Using Triple Quadrupole LC/MS/MS, First Edition Nov. 2019.