Extraction of Multiple Mycotoxins From Nuts Using ISOLUTE® Myco prior to LC-MS/MS Analysis

Significance of the topic

Mycotoxins are harmful secondary metabolites produced by fungi that can contaminate nuts, such as brazil nuts and peanuts. Due to their toxicity and strict regulatory limits in food, accurate and reliable analytical methods are essential for ensuring public health and meeting legislative requirements.

Study objectives and overview

This application note presents a solid phase extraction (SPE) procedure using ISOLUTE Myco columns followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to extract and quantify multiple mycotoxin classes—aflatoxins B1, B2, G1, G2 and ochratoxin A—from nut matrices. The goal is to achieve high recoveries, low limits of quantification and reproducible results in line with European regulations.

Methodology and instrumentation

Sample preparation:

• Grind 50 g nut sample; store at 2–8 °C.
• Extract 5 g ground sample with 20 mL acetonitrile:water (80:20) by shaking for 30 min; centrifuge at 3000 g for 10 min.
• Take 4 mL supernatant, dilute to 32 mL with water; centrifuge again.

SPE protocol (flow rate 1 mL/min):

• Condition column with 2 mL acetonitrile; equilibrate with 2 mL 10 mM ammonium acetate.
• Load 3 mL sample extract; wash with 3 mL 10 mM ammonium acetate and 3 mL 90:10 ammonium acetate:acetonitrile.
• Dry column; elute sequentially with 2 mL 0.1% formic acid in acetonitrile and 2 mL 0.1% formic acid in methanol.
• Evaporate eluates under air or nitrogen; reconstitute in 1 mL 0.1% acetic acid in 20% acetonitrile:methanol; filter through 0.2 µm PTFE.

LC-MS/MS conditions:

• UHPLC on Kinetex XB-C18 column (50×2.1 mm, 2.6 µm) with 10 min gradient (20–100% organic) at 0.45 mL/min.
• Triple quadrupole MS in positive ESI MRM mode using optimized transitions for each target analyte.

Main results and discussion

The method delivered excellent linearity (r²>0.993) over 0.1–10 ng/mL, with actual LOQs of 1–3 µg/kg in both nuts. Repeatability (%RSDr) was below 7% and recoveries ranged from 90 to 113%, meeting EU repeatability and recovery criteria. Representative retention times were 3.3–4.1 min for aflatoxins and 6.1 min for ochratoxin A.

Benefits and practical applications

This workflow offers a single SPE product for simultaneous cleanup of multiple mycotoxin classes in diverse nut samples. The streamlined protocol reduces sample preparation time, ensures robust performance and complies with regulatory requirements, making it ideal for routine food safety testing and quality control laboratories.

Future trends and potential applications

Future developments may include automated SPE platforms, on-line coupling with UHPLC-MS/MS for higher throughput, application to additional food matrices and integration with high-resolution mass spectrometry for expanded mycotoxin screening.

Conclusion

The ISOLUTE Myco SPE method combined with LC-MS/MS provides a reliable, efficient and sensitive approach for the simultaneous extraction and quantification of key mycotoxins in nut matrices. The protocol meets stringent regulatory criteria and supports high-confidence food safety monitoring.

Used instrumentation

• ISOLUTE Myco 60 mg/3 mL SPE columns
• Shimadzu Nexera UHPLC system with Kinetex XB-C18 column
• AB Sciex Triple Quad 5500 mass spectrometer (ESI positive MRM)
• SPE drying with TurboVap® LV or equivalent evaporator