Efficient and Traceable Aflatoxin Immunoaffinity Sample Cleanup with Extraction+™ Connected Device

Significance of the topic

The reliable detection and quantification of aflatoxins in agricultural commodities is critical for food safety and regulatory compliance. Aflatoxins B1, B2, G1 and G2 are potent, heat-stable mycotoxins produced by Aspergillus species that can persist through processing, bioaccumulate in fatty tissues and present carcinogenic risks. Immunoaffinity (IA) cleanup is a widely accepted selective sample preparation approach (AOAC 991.31) for aflatoxin isolation prior to chromatographic analysis. Automating IA workflows can reduce operator variability, increase throughput, and provide digital traceability required in modern high-throughput testing laboratories.

Objectives and study overview

This application note evaluated an automated immunoaffinity cleanup workflow for aflatoxins using Waters VICAM AflaTest 1 cc IA cartridges combined with the Extraction+ Connected Device vacuum manifold, Pipette+ automated pipetting (easy pipetting mode) and OneLab software. The goals were to (1) reproduce AOAC 991.31 performance for corn across 0.1–100 ng/g, (2) optimize vacuum pressure gradients to maintain controlled flow rates (≤1 drop/s) across challenging matrices, (3) reduce hands-on time and increase throughput for up to 24 samples, and (4) demonstrate end-to-end traceability via LIMS and Empower integration.

Methodology

Sample preparation followed AOAC 991.31 with specific implementation details summarized here:

• Sample matrix: ground corn (Trilogy reference material), 25 g aliquots spiked with aflatoxins B1, B2, G1 and G2.
• Extraction: 25 g corn + 5 g NaCl and 125 mL 70% methanol, blended 2 minutes, gravity filtered. 15 mL filtrate diluted with 30 mL water and filtered again.
• Immunoaffinity cleanup: 15 mL of twice-filtered sample loaded onto a 1 cc VICAM AflaTest IA cartridge, washed twice with 10 mL water and eluted with 1.0 mL methanol. Eluate diluted 1:1 with water prior to injection.
• Automation: Loading, washing and elution steps were executed on the Extraction+ Connected Device using programmable vacuum gradients to control flow rate and prevent sorbent drying or clogging. Pipette+ assisted pipetting (easy mode) was used to reduce manual handling.
• Chromatography: ACQUITY UPLC H-Class PLUS with ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 µm). Isocratic mobile phase methanol/water 45/55 v/v, flow 0.350 mL/min, column 40 °C, injection 5 µL, 6 min run time.
• Detection: UHPLC fluorescence detector (Excitation 360 nm, Emission 440 nm) with Empower CDS for data processing.

Instrumentation used

• Extraction+ Connected Device vacuum manifold (programmable vacuum gradients, up to 1000 mbar absolute)
• Pipette+ Automated Pipetting (easy pipetting mode; compatible with connected mechanical pipettes)
• VICAM AflaTest 1 cc immunoaffinity cartridges
• ACQUITY UPLC H-Class PLUS System with ACQUITY UPLC HSS T3 column
• ACQUITY UHPLC FLR Detector
• OneLab Software for automation scripting and device control
• Empower Chromatography Data System and LIMS integration for chain-of-custody and result transfer

Main results and discussion

Key analytical performance and workflow outcomes reported in the study:

• Linearity: Calibration across 0.1–100 ng/g exhibited excellent linearity for all four aflatoxins (R² ≥ 0.995).
• Accuracy and precision: Mean recoveries ranged 95–110% with repeatability RSDs <6% across concentrations; overall reproducibility was <10% RSD.
• Sensitivity: The workflow enabled reliable detection and quantification down to 0.1 ng/g in the tested corn matrix using direct UHPLC-FLR without derivatization.
• Flow control and robustness: Programmable vacuum gradients preserved the target flow rate (≤1 drop/s), prevented sorbent drying and minimized clogging when processing viscous or particulate-rich samples. Only a fraction (~20%) of the vacuum device’s maximum capacity was required to process challenging matrices.
• Throughput and hands-on time: The automated-assisted workflow processed 24 samples with reduced operator involvement. The app note reports manual processing of 24 samples required approximately 90 minutes versus ~60 minutes with assisted pipetting, reducing hands-on time by ~30 minutes for that batch; broader claims of up to 50% processing-time reduction are presented as a benefit for high-throughput settings.
• Traceability: OneLab integration with LIMS and Empower enabled automated sample lists, chain-of-custody tracking, and seamless transfer of sample metadata and results, reducing manual entry errors and improving auditability.

Practical benefits and applications

The automated IA workflow offers several practical advantages for routine aflatoxin testing laboratories:

• High-throughput capacity: concurrent processing of up to 24 cartridges in a compact footprint.
• Consistent IA performance: Maintains AOAC-level selectivity and quantitative performance for aflatoxins B1, B2, G1 and G2.
• Reduced operator variability: Programmable pressure profiles and automated pipetting lower inter-operator differences.
• Robust handling of difficult matrices: gradient vacuum control mitigates clogging and uneven flow typical for particulate or viscous extracts.
• End-to-end digital traceability: LIMS → OneLab → Empower integration supports audit trails and reduces data transfer errors.

Future trends and applications

Potential developments and broader applications stemming from this workflow include:

• Extension to other mycotoxin panels and matrices: leveraging IA cartridges or multiplexed cartridges for simultaneous cleanup of multiple toxins.
• Hybrid workflows coupling automated IA cleanup with LC-MS/MS detection to increase specificity, enable multi-class quantitation and meet diverse regulatory requirements.
• Scaled automation and robotic integration for fully walkaway sample preparation in high-throughput contract laboratories and food safety hubs.
• Software-enhanced quality control: integration of decision-support tools, automated QC checks and predictive maintenance of vacuum manifolds to further reduce failures.
• Data interoperability and cloud-enabled audit trails to support remote review and regulatory reporting.

Conclusion

This application note demonstrates that immunoaffinity cleanup using VICAM AflaTest cartridges can be successfully automated with the Extraction+ Connected Device and Pipette+ workflows while retaining AOAC 991.31 performance for aflatoxins in corn. The optimized vacuum gradients maintain controlled flow and minimize matrix-related issues, delivering excellent recoveries, linearity and precision across 0.1–100 ng/g. Integration with OneLab, LIMS and Empower provides traceable, lower-touch workflows suitable for laboratories requiring higher throughput, reproducibility and digital auditability.

Reference

1. AOAC INTERNATIONAL. Aflatoxins in Corn, Raw Peanuts, and Peanut Butter, Immunoaffinity Column (AflaTest) Method 991.31, 2005.
2. Sun, T.; Dreolin, N.; Hird, S.; Collette, N. Analysis of Aflatoxins in Corn and Peanuts Using Immunoaffinity Chromatography and the Arc HPLC System. Waters Application Note, 2023.
3. Benvenuti, M. E.; Di Gioia, A. Rapid Analysis of Aflatoxins without Derivatization Using Ultra Performance Liquid Chromatography and Fluorescence Detection. Waters Application Note, 2019.