Introduction
Mycotoxin contamination in corn is a critical food safety issue worldwide. Aflatoxins (B1, B2, G1, G2) and fumonisins (B1, B2) are among the most prevalent and toxic mycotoxins produced by Aspergillus and Fusarium species. Regulatory agencies such as the FDA and EU have set strict maximum limits (e.g., 20 ppb for aflatoxins in human food). Robust sample preparation using solid-phase extraction (SPE) is essential for accurate LC-MS/MS analysis.
Extraction with Acetonitrile-Water Mixtures
Corn samples (typically 5 g ground) are extracted with 20 mL of acetonitrile/water (80:20, v/v) containing 1% acetic acid. After homogenization and centrifugation, the supernatant is diluted with water to reduce the organic solvent content to ≤10% before SPE loading. This step ensures optimal retention of mycotoxins on the sorbent.
SPE Cartridge Selection for Toxin Purification
For multi-toxin analysis, mixed-mode reversed-phase/weak anion exchange (RPLC/WAX) cartridges such as Poseidon WAX SPE Cartridges are ideal. Aflatoxins are neutral and retained by reversed-phase interactions, while fumonisins (anionic) are retained by anion exchange. Alternatively, MAX SPE Cartridges (mixed-mode strong anion exchange) can be used if fumonisins are the primary target.
Conditioning and Loading Procedures
Condition the cartridge with 3 mL of methanol, followed by 3 mL of water. Load the diluted extract (pass through at 1–2 mL/min). After loading, wash with 3 mL of water to remove polar matrix components such as sugars and salts.
Washing Steps Removing Pigments and Fats
To eliminate chlorophyll, carotenoids, and lipids, wash with 3 mL of methanol/water (60:40, v/v) containing 1% formic acid. This step retains mycotoxins while eluting hydrophobic pigments. If necessary, a hexane wash (2 mL) can further remove nonpolar fats.
Elution Solvent Systems for Mycotoxins
Elute aflatoxins with 3 mL of methanol containing 0.1% formic acid. For fumonisins, use 3 mL of methanol/water (90:10, v/v) with 2% ammonia. Collect eluates separately or combine for multi-toxin analysis. Evaporate to dryness under nitrogen and reconstitute in 1 mL of mobile phase.
LC-MS/MS Detection and Quantification
Separation is achieved on a C18 column (2.1 × 100 mm, 1.7 μm) using a gradient of water/methanol (both with 0.1% formic acid and 5 mM ammonium formate). MRM transitions are monitored: aflatoxin B1 (m/z 313→285), B2 (315→287), G1 (329→243), G2 (331→245), fumonisin B1 (722→352), B2 (706→336). Quantify using matrix-matched calibration curves with internal standards (e.g., 13C-labeled analogs).
Method Validation and Regulatory Compliance
Validation parameters include linearity (R² > 0.99), recovery (70–120%), precision (RSD < 20%), and LOQ (≤ 0.5 ppb for aflatoxins). The method meets FDA and EU regulatory guidelines. For high-throughput analysis, consider 96-well SPE plates to process multiple samples simultaneously.
For detailed product specifications and to request sample cartridges, visit HLB SPE Cartridges (reversed-phase), MCX SPE Cartridges (mixed-mode cation exchange), and WCX SPE Cartridges (weak cation exchange) for alternative selectivity.
