Quantitative Analysis of QACs in Milk Using a QuEChERs Sample Preparation Approach

Importance of the topic

Quaternary ammonium compounds (QACs) such as benzalkonium chlorides (BACs) and didecyldimethylammonium chloride (DDAC) are widely used disinfectants in dairy farming and processing. Their residual presence in raw milk can affect product safety, interfere with fermentation (starter cultures), and raise regulatory or consumer-safety concerns. Rapid, sensitive and routine-capable analytical methods are therefore necessary to monitor QACs at low ng/mL (µg/kg) concentrations and support surveillance and compliance testing.

Objectives and study overview

This application study presents a practical LC–MS/MS workflow for simultaneous quantification of nine common QAC homologues in milk using a QuEChERS-based sample preparation. The goal was to demonstrate a fast, robust method with limits of quantification (LOQs) suitable for routine monitoring and below typical general residue limits applied in food safety frameworks.

Methodology and sample preparation

Sample preparation employed a standard QuEChERS extraction (RESTEK Q‑Sep QuEChERS Extraction Packets, En Method) adapted for milk. Key steps:

• 10 g blank milk spiked to prepare calibration/QC levels.
• QuEChERS extraction as primary cleanup.
• Freezer precipitation cleanup: raw extract frozen for 2 hours, supernatant decanted to remove precipitates.
• Matrix-matched calibration prepared by diluting spiked extract with blank extract; QC samples prepared from different milk types (whole, organic, alpine).

Used instrumentation

The method was implemented on Shimadzu instrumentation:

• Nexera X3 UHPLC coupled to an LCMS-8045RX triple-quadrupole mass spectrometer.
• Analytical column: Shim-pack Velox SP‑C18 (100 mm × 2.1 mm, 2.7 µm).
• Delay (solvent) column to reduce background QAC contamination: Shim-pack GIST HP C18‑AQ (30 mm × 3 mm, 3 µm) placed between mixer and autosampler.
• Mobile phases: 5 mM ammonium formate + 0.01% formic acid in water (A) and methanol (B).
• Injection volume 1 µL; column oven ~40 °C; autosampler cooled to 15 °C.
• MS conditions: electrospray ionization (positive mode), interface and heat-block temperatures elevated to assist desolvation, collision-induced dissociation (CID) used for MRM transitions; optimized MRM pairs (quantifier + qualifier) for each QAC homologue were used.

Analytical conditions and run time

Analysis used scheduled MRM acquisition with at least two transitions per analyte. Total chromatographic runtime was approximately 12 minutes per injection. Use of a delay column minimized contribution of ubiquitous QAC background from solvents or plumbing, improving specificity for sample-derived residues.

Main results and discussion

Key performance characteristics observed:

• Calibration range: 2.5–100 ng/mL (matrix-matched), with weighted linear regression (1/conc) and R² > 0.999 for all analytes.
• Limit of quantification (LOQ): set at 2.5 ng/mL (2.5 µg/kg) for individual QACs, driven by persistent low-level background of some congeners (DDAC, BAC‑C12, BAC‑C14).
• Method precision and accuracy: QC samples at 7.5, 30 and 80 ng/mL prepared in three milk types showed mean accuracies generally in the 80–96% range and relative standard deviations typically below ~7% (most values lower), indicating acceptable trueness and repeatability for routine monitoring.
• Sensitivity is well below the commonly applied general residue guidance of 0.01 mg/kg (10 ng/mL), supporting surveillance needs.

Chromatograms and calibration plots demonstrated good peak separation and linear detector response. The freezer precipitation cleanup step provided practical removal of matrix interferences while keeping sample preparation fast and compatible with laboratory throughput.

Benefits and practical applications

The described workflow offers several practical advantages for food testing laboratories:

• High sensitivity and selectivity for a panel of C8–C18 BAC homologues and DDAC homologues using a single 12‑minute LC–MS/MS run.
• Minimal and rapid sample preparation (QuEChERS + freeze‑out) compatible with routine throughput and multi-sample campaigns.
• Matrix-matched calibration and the use of a delay column mitigate matrix effects and ubiquitous background contamination, improving reliability for low‑level monitoring.
• Method performance meets surveillance needs where regulatory maximum residue limits are absent or broadly defined, enabling detection well below common guidance thresholds.

Future trends and potential applications

Potential directions to extend or enhance the method include:

• Incorporation of isotopically labelled internal standards to further improve quantification accuracy and correct for matrix effects.
• Automation of QuEChERS and sample handling to increase throughput in high‑volume labs.
• Method extension to processed dairy products (cheese, yogurt) and other food matrices with appropriate validation.
• Lowering LOQs through alternative cleanup strategies or more sensitive instrumentation where tighter regulatory limits are required.
• Development of multi-class workflows combining QACs with other antimicrobial residues or surfactants to streamline routine monitoring panels.
• Harmonization of regulatory limits across jurisdictions to standardize surveillance and compliance testing.

Conclusions

This application demonstrates a practical LC–MS/MS approach coupling QuEChERS extraction with a Shimadzu Nexera X3 / LCMS‑8045RX system for reliable quantification of nine QAC homologues in milk. The method achieves LOQs of 2.5 ng/mL, linear response across 2.5–100 ng/mL with R² > 0.999, and acceptable QC recoveries and precision across milk types, making it suitable for routine monitoring and surveillance of QAC residues in dairy matrices.

References

1. EU Reference Laboratory for Pesticides Requiring Single Residue Methods, CVUA Stuttgart. Analysis of BACs and DDAC in Milk using QuEChERS method and LC‑MS/MS, Version 2.