Sensitive Quantitation of Cereulide in Infant Formula Following ISO 18465:2017

Significance of the topic

Infant formula is consumed by one of the most vulnerable population groups, and contamination with cereulide — a heat-stable, lipophilic emetic toxin produced by Bacillus cereus — poses acute health risks. Because cereulide is odorless and tasteless and resists common food processing conditions, highly sensitive and robust analytical methods are essential for routine surveillance, regulatory compliance, and rapid response to contamination incidents. This study demonstrates a streamlined LC–MS/MS workflow that attains quantitation sensitivity well below current EFSA and EU guidance values, enabling earlier detection and mitigating recall risks.

Objectives and study overview

• Implement and validate a quantitative workflow for cereulide in infant formula that follows ISO 18465:2017 guidance.
• Demonstrate a simple, cost-effective sample preparation compatible with high-throughput testing.
• Establish method performance characteristics: limit of quantification (LOQ), linearity, recovery, repeatability, matrix effects, and applicability to naturally contaminated material.

Methodology and sample preparation

The procedure uses a minimal extraction and cleanup approach optimized for the lipophilic cereulide molecule: weigh 2.5 g of powdered infant formula, spike if required, add 30 mL acetonitrile, shake for 60 minutes (horizontal shaker) and incubate 30 minutes at room temperature, centrifuge 10 minutes at low temperature, and filter the supernatant through a 0.22 µm PTFE filter prior to LC–MS/MS analysis. Matrix-matched calibration standards were prepared in extracted blank matrix across seven concentrations from 0.0021 to 0.84 µg/L. Extraction recovery experiments were performed by spiking pre- and post-extraction at low, mid, and high levels to quantify procedural losses. Matrix effects were evaluated by comparing post-extraction spiked matrix to neat solvent standards.

Used Instrumentation

• UHPLC: Agilent 1290 Infinity III (High-Speed Pump, Multisampler, Multicolumn Thermostat).
• Analytical column: Agilent Altura ZORBAX Eclipse Plus C18, 2.1 × 50 mm, 1.8 µm, with a ZORBAX RRHD guard column.
• Mass spectrometer: Agilent 6495D Triple Quadrupole with Jet Stream (AJS) ion source operating in positive electrospray MRM mode.
• Consumables: 0.22 µm PTFE Captiva Econofilter, standard lab vortexer and centrifuge.

Key LC conditions: 40 °C column temp, multisampler at 4 °C, flow 0.4 mL/min, mobile phases of 10 mM ammonium formate + 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B), 5 µL injection. A divert valve protects the MS from early-eluting matrix components.

MRM acquisition used the predominant ammonium adduct precursor (m/z ~1170.7) with multiple product ions (including a quantifier transition to m/z 172.1) to provide both quantitation and ion-ratio confirmation for increased specificity. Automated MRM optimization and MassHunter software were employed for acquisition and quantitative processing.

Main results and discussion

• Limit of quantification (LOQ): 0.0021 µg/L in liquid equivalent (0.025 µg/kg sample equivalent), determined at S/N ≥ 6 per ISO 18465:2017; observed S/N > 12.9 (n = 6).
• Linearity: Seven-point matrix-matched calibration from 0.0021 to 0.84 µg/L yielded excellent linearity (R2 > 0.9995), exceeding ISO performance criteria.
• Extraction recovery: Consistently >80% across low (0.0042 µg/L), mid (0.0167 µg/L), and high (0.167 µg/L) spike levels (82.9%, 82.7%, 82.3% respectively) with %RSD ≤ 2.21%.
• Repeatability: At the LOQ (0.0021 µg/L), %RSD for peak area/concentration was 3.06% across six replicates, demonstrating high precision at trace levels.
• Matrix effects: Measured at 0.0167 µg/L, matrix effect was ~108% (≤2.51% RSD), indicating minimal ion suppression/enhancement; matrix-matched calibration compensates residual effects.
• Run time and throughput: Efficient chromatographic conditions with cereulide eluting at ~5.6–5.8 min and a short total cycle enable high-throughput routine analysis; the diverter program minimizes carryover into the MS.
• Real sample application: A naturally contaminated infant formula sample was quantified at 0.0036 µg/L (0.043 µg/kg), illustrating the method’s ability to detect low-level, authentic contamination well below regulatory action thresholds.

The combination of an optimized reversed-phase column, gradient into a highly organic phase (>90%), elevated desolvation temperatures, and targeted MRM transitions enhanced sensitivity for the lipophilic cereulide molecule. Using matrix-matched calibrants and a simple acetonitrile-based extraction yielded a balance between analytical performance and operational simplicity.

Benefits and practical applications

• Regulatory compliance: LOQ substantially below EFSA/EU guidance enables confident screening and compliance verification for infant formula.
• Routine surveillance: The streamlined sample prep and short analysis time support high sample throughput in quality control and public health laboratories.
• Cost-effectiveness: Minimal solvent and consumable needs, plus limited cleanup steps, reduce per-sample cost and complexity.
• Outbreak investigation and root-cause analysis: High sensitivity and specificity allow trace detection and confirmation in contaminated lots and environmental samples.

Future trends and potential applications

• Broader matrix scope: Adaptation and validation for other high-starch or ready-to-eat foods (rice, pasta) and processed infant products would expand surveillance coverage.
• Isotope-labeled internal standards: Incorporation of labeled cereulide analogs would further improve accuracy and compensate for residual matrix variability.
• High-resolution MS adjuncts: Complementary HRMS screening could enhance untargeted surveillance and identification of related depsipeptides or novel analogs.
• Automation of sample prep: Robotics or on-line extraction approaches could further increase throughput while reducing manual variability.
• Integration with risk-based monitoring: Combining highly sensitive analytics with targeted sampling strategies will optimize resource allocation for food safety programs.

Conclusion

A validated, ISO 18465:2017-aligned LC–MS/MS workflow using a simple acetonitrile extraction and Agilent 1290/6495D instrumentation achieves trace-level quantitation of cereulide in infant formula with LOQ = 0.0021 µg/L, high recovery (>80%), excellent precision, and negligible matrix interference. The approach supports routine, high-throughput monitoring and early detection of contamination events, providing practical utility for regulatory testing, manufacturer quality control, and public health surveillance.

Reference

1. European Food Safety Authority (EFSA). EFSA Provides Rapid Risk Assessment on Cereulide in Infant Formula; EFSA: Parma, Italy, February 2, 2026.
2. European Commission, Directorate-General for Health and Food Safety. Result of the EFSA Rapid Risk Assessment (RRA) on Acute Reference Dose (ARfD) of Cereulide in Infants and Information on Acute Consumption of Infant Formulae; European Commission: Brussels, Belgium, 2026.
3. European Commission. Commission Implementing Regulation (EU) 2026/459 of 24 February 2026 Amending Implementing Regulation (EU) 2019/1793 as Regards the Temporary Increase of Official Controls and Emergency Measures; Official Journal of the European Union, 2026.