Highly Efficient LC-MS/MS Analysis of Multiple Mycotoxins Utilizing Biphenyl Column Selectivity with Inert Column Technology

Importance of the Topic

Mycotoxins are toxic secondary metabolites produced by fungi and pose significant risks to food safety and public health worldwide. Routine monitoring of multiple mycotoxins in agricultural products is required to meet regulatory limits, ensure quality control, and prevent economic losses. However, the chemical diversity of over 300 known mycotoxins demands robust, high-throughput, and sensitive analytical methods capable of simultaneous multi-analyte detection.

Objectives and Overview of the Study

This study compares coated “Inert” column hardware technology against traditional stainless steel flowpath for LC-MS/MS analysis of 34 structurally diverse mycotoxins. Goals include:

• Assessing sensitivity and peak shape improvements using Raptor Inert Biphenyl columns.
• Eliminating high pH requirements that reduce silica column lifetime.
• Evaluating reproducibility and ease of use with minimal chemical passivation.

Methodology and Instrumentation

A side-by-side comparison employed two Restek 100×2.1 mm, 2.7 µm Biphenyl columns: one standard stainless steel and one with an inert glass-like coating applied via Chemical Vapor Deposition. Key parameters:

• LC System: High-performance liquid chromatography with Raptor Biphenyl stationary phase.
• Mass Spectrometer: Triple quadrupole MS operating in positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode.
• Mobile Phases: A: water + 0.05% formic acid; B: methanol + 0.05% formic acid.
• Gradient: 75% A initially, ramp to 100% B over 9 min, then re-equilibrate.
• Column Temperature: 60 °C; Flow Rate: 0.4 mL/min; Injection Volume: 5 µL; Analyte Concentration: 10 ng/mL.

Results and Discussion

Comparison of area and peak height ratios (inert vs stainless steel) revealed:

• Improved Sensitivity for 33 of 34 mycotoxins (97%). Signal area and height enhancements reached up to 10-fold.
• Fumonisin B1–B3 showed 3.8–6.5× area and 6.0–10.1× height increases, with sharper peak shapes and reduced tailing.
• Aflatoxin B1 exhibited a modest decrease (< 0.9×) in both metrics but remained quantitatively reliable.
• Coated hardware suppressed non-specific adsorption/binding (NSA/NSB) interactions, enhancing reproducibility across multiple injections without extensive chemical passivation.

Benefits and Practical Applications

• Single-run multi-mycotoxin analysis combining Alternaria toxins, ergot alkaloids, fumonisins, trichothecenes, and aflatoxins under acidic conditions.
• Extended column lifetime by avoiding high pH conditions and mitigating metal surface interactions.
• Enhanced sensitivity lowers detection limits, improving compliance testing for regulatory laboratories.
• Reduced need for laborious column conditioning and chemical passivation accelerates sample throughput.

Future Trends and Opportunities

Advancements may include:

• Expansion of inert coating technologies to other stationary phases for broader compound classes.
• Integration with micro- and nano-flow LC to further reduce solvent consumption and enhance sensitivity.
• Automated real-time monitoring and AI-driven method optimization for rapid multi-toxin screening in complex food matrices.
• Development of universal sample preparation protocols compatible with coated hardware to streamline workflows.

Conclusion

The inertly coated Biphenyl column under acidic LC-MS/MS conditions offers a robust solution for high-throughput multi-mycotoxin analysis. This technology delivers substantially improved sensitivity, peak shape, and reproducibility while extending column life and simplifying routine laboratory operations.