Analysis of Chlorpromazine in Milk and Chicken Egg Extracts using Triple Quadrupole LC/MS/MS

Importance of the Topic

Chlorpromazine, a veterinary tranquilizer banned in food-producing animals by Japanese regulations and the Positive List system, poses a food safety risk if residues remain in milk or eggs. Sensitive and reliable testing methods are essential to ensure compliance and protect public health.

Objectives and Overview of the Study

This study evaluates a newly developed triple quadrupole LC–MS/MS test method for trace determination of chlorpromazine in milk and chicken egg extracts, following the Pharmaceutical Affairs and Food Sanitation Council’s draft guidelines. The goal is to achieve lower detection limits and simplified sample handling compared to existing protocols.

Methodology and Instrumentation Used

Sample pretreatment and cleanup:

• Weigh 10 g of milk or egg; extract twice with acetone and dilute to 100 mL.
• Load 10 mL of extract onto a sulfonate-modified methacrylate copolymer SPE mini-column.
• Elute, concentrate to ~1 mL at 40 °C, and adjust to 5 mL with 0.1% formic acid/acetonitrile (3:2 v/v).

Instrumentation details:

• LC–MS/MS platform: Shimadzu LCMS-8050 with electrospray ionization (positive mode).
• Column: Shim-pack HR-ODS (150 mm × 2.1 mm i.d., 3 μm).
• Mobile phase: 0.1% formic acid in water/acetonitrile (72:28 v/v), 0.20 mL/min flow, 40 °C column temperature.
• Injection volume: 2 μL; ESI probe voltage: +1.0 kV; DL temp: 250 °C; block heater: 350 °C; interface: 300 °C.
• Gas flows: nebulizing 2 L/min; drying 5 L/min; heating 15 L/min.
• MRM transitions: m/z 319.10 → 86.15 (quantifier), 321.10 → 58.10 (qualifier).

Main Results and Discussion

Calibration over 10–1 000 ng/L yielded excellent linearity (R2 > 0.9998). The method’s quantitative limit reached 10 ng/L with a 2 µL injection—half the LOD of conventional protocols. Blank milk and egg extracts showed no detectable chlorpromazine, and minor interferences in milk were below one-fifth of the LLOQ. Spike-and-recovery assays at 0.0001 mg/kg (equivalent to 20 ng/L) demonstrated recoveries of 103% (milk, RSD 5.7%) and 102% (egg, RSD 2.5%), confirming accuracy and precision.

Benefits and Practical Applications of the Method

• Enhanced sensitivity down to 10 ng/L with minimal sample volume.
• Reduced solvent usage and streamlined pretreatment steps.
• High selectivity through MRM mode, minimizing matrix effects.
• Applicable to routine regulatory screening of milk, eggs, and other food matrices.

Future Trends and Applications

This targeted LC–MS/MS strategy may be adapted for other prohibited veterinary drugs and complex food samples. Automation of SPE and integration with high-throughput LC systems will improve laboratory efficiency. Combining targeted MRM with high-resolution mass spectrometry and non-targeted screening can expand monitoring capabilities for emerging contaminants.

Conclusion

The developed triple quadrupole LC–MS/MS method provides a robust, sensitive, and accurate tool for monitoring chlorpromazine residues in milk and eggs, meeting strict food safety and regulatory requirements.