Solid Phase Extraction and SFC-MS/MS Method for Analysis of Aatoxins M1, M2, B1, B2, G1 and G2 in Milk Powders

Importance of the Topic

Aflatoxins B1, B2, G1 and G2 are toxic secondary metabolites produced by Aspergillus species, and their hydroxylated forms M1 and M2 can appear in milk following animal exposure.
The presence of these compounds in milk powders poses significant public health concerns, as B- and G-series are classified as carcinogens and M1 is potentially carcinogenic.
Regulatory bodies such as the European Union strictly limit aflatoxin M1 in milk to below 0.050 µg/kg, driving the need for highly sensitive and reliable analytical approaches.

Aims and Study Overview

This study aimed to develop and validate a novel analytical workflow combining solid phase extraction (SPE) with supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) for simultaneous quantitation of six aflatoxins (B1, B2, G1, G2, M1 and M2) in milk powder matrices.
The goal was to achieve low limits of quantitation, high throughput, and minimal environmental impact through the use of CO₂ as a mobile phase.

Použitá instrumentace

• Shimadzu Nexera UC supercritical fluid chromatography system
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer with ESI source
• Shim-pack UC-X Sil column (250 mm × 2.1 mm I.D., 3 µm)
• Supel™ Tox Alfazea SPE cartridges and Q-Sep extraction salts

Methodology

Sample Preparation:

• Five grams of milk powder were extracted with 20 mL of water/acetonitrile (1:1 v/v) containing 0.1% formic acid and Q-Sep salts, vortexed, centrifuged, and the supernatant applied to conditioned Supel™ Tox Alfazea SPE cartridges.
• After washing with acetonitrile, eluates were filtered (0.22 µm PTFE) and, for higher-level spikes, concentrated under nitrogen.

SFC-MS/MS Conditions:

• Mobile phases: supercritical CO₂ (A) and methanol modifiers with ammonium formate or formic acid (B, C).
• Gradient elution over 7 minutes at 1.0 mL/min (CO₂) and 0.4 mL/min make-up flow, column temperature 40 °C.
• ESI positive-ion mode, MRM transitions optimized for quantifier and qualifier ions, source temperatures between 250–400 °C, nitrogen and argon gases for nebulization and collision.

Main Results and Discussion

Chromatographic separation of the six aflatoxins was achieved within 7 minutes, eluting in order of increasing polarity (B1/B2 first, followed by G1/G2, then M1/M2).
Calibration curves were linear (r² > 0.999) across relevant concentration ranges, with LODs of 7.6–22.4 ng/kg and LOQs of 22.8–68.0 ng/kg.
Repeatability was acceptable (%RSD ≤ 14.6% at low spikes, ≤ 6.0% at higher spikes).
Recovery studies (n = 3) at multiple levels yielded 70–135% recovery and revealed matrix effects ranging from modest suppression (82–129%) for B/G toxins to higher effects (136–174%) for M1/M2, indicating co-extracted interferences after SPE concentration.

Benefits and Practical Applications

• High sensitivity and specificity allow compliance with strict regulatory limits in dairy testing.
• Rapid analysis and high throughput support routine QA/QC workflows.
• Supercritical CO₂ reduces organic solvent consumption and environmental impact.

Future Trends and Potential Uses

Further developments may include automation of SPE steps, expansion to other food matrices, and integration with high-resolution mass spectrometry for enhanced screening of mycotoxin analogues.
Miniaturized SFC systems and green chemistry approaches could improve sustainability and lower operational costs in routine laboratories.

Conclusion

The combined SPE and SFC-MS/MS method provides a robust, sensitive, and environmentally friendly solution for simultaneous quantitation of aflatoxins B1, B2, G1, G2, M1 and M2 in milk powders, meeting regulatory requirements and supporting food safety monitoring.

Reference

• Benkerroum N. International Dairy Journal. 2016;62:63–75.
• Commission Regulation (EC) No 1881/2006. Official Journal of the European Union, 2006.
• Sartori A.C.A.V., Mattos J.S. de, Moraes M.H.P. de, Nóbrega A.W. da. Food Analytical Methods. 2015;8:2321–2330.