Column Selection Study for Analysis of Multiple Mycotoxins by LC-MS/MS

By: Emily R. Barrey1, Olga I. Shimelis1, and Christine Dumas2, 1Div. of Sigma-Aldrich, Bellefonte, PA 16823 USA, 2Sigma-Aldrich, St.Galen, Switzerland

Introduction

Importance of Mycotoxin Analysis

  • Mycotoxins exist in food as a result of fungal infection of crops.
  • Mycotoxins remain in the food upon heating and processing.
  • Complex food matrices and desired low detection limits provide challenges for analysis of mycotoxins.
  • In recent years, significant advances in the analytical techniques have been applied to detection of mycotoxins.

Trends in Recent Analytical Methods for Mycotoxins

  • Simplified extraction.
       – Involves modified QuEChERS cleanups.
  • Multi-class and multi-component assays, single method for multiple mycotoxins.
  • LC/MS and LC-MS/MS detection in place of LC/UV and LC/FL detection.

MS Conditions for Mycotoxin Analysis

MS Conditions for Mycotoxin Analysis

 

Selectivity

  • Interactions that contribute to retention and selectivity.
       – Dispersive or hydrophobic interactions.
       – Polar interactions (hydrogen bonding and other dipole interactions).
       – Ionic interactions.
  • Dispersive interactions are commonly considered most important in reversed- phase chromatography.
  • Polar and ionic interactions, however, are responsible for a significant amount of retention and selectivity.

Experimental

Mycotoxin Standard (50 ng/mL) on Shell-Type C18

Mycotoxin Standard (50 ng/mL) on Shell-Type C18

 

Mycotoxin Standard (50 ng/mL) on Sub-2 µm C18

Mycotoxin Standard (50 ng/mL) on Sub-2 µm C18

 

Discussion on Fused-Core®versus Porous Sub-2 μm
 

  Solid-Core Asymmetry Porous Asymmetry
3-Acetyl DON 1.55 0.836
15-Acetyl DON 3.10 1.81
Fumonisin B1 2.48 1.26
Fumonisin B2
0.836 0.461
  • Slight difference in elution orders wereobt ained with the two different C18 phases.
  • Separation of isobaric Fumonisin B2 and Fumonisin B3 was achieved on both C18 phases.
  • Separation of the isobaric acetyl-DON analytes were not achieved on either 18 column.
  • Similar analyte peak asymmetries were observed on the sub-2 µm porous and solid-core columns. Some analyte peak shapes were improved on the sub 2 µm porous column.

Phenyl Bonded Phase

  • Phenyl is a Lewis base or electron donor; π-π interaction can occur with solutes that are deficient in electrons (Lewis acids).
  • Due to the rigid nature of the aromatic ring, solute shape can dictate selectivity (how closely solutes can approach the ring).

Phenyl Bonded Phase

 

Mycotoxin Standard (50 ng/mL) on Shell-type Phenyl-Hexyl

Mycotoxin Standard (50 ng/mL) on Shell-type Phenyl-Hexyl

 

Mycotoxin Standard (50 ng/mL) on Shell-type Biphenyl

Mycotoxin Standard (50 ng/mL) on Shell-type Biphenyl

 

Acetyl-DON Separations (50 ng/mL)

Acetyl-DON Separations (50 ng/mL)

 

Retention Comparison Between C18 and Phenyl-Hexyl Stationary Phases for Mycotoxins

Retention Comparison Between C18 and Phenyl-Hexyl Stationary Phases for Mycotoxins

 

Discussion on HPLC Analysis

  • Elution orders varied slightly between the three different phases.
  • The Biphenyl phase allowed for greater retention of the mycotoxins, but less peak separation for the later eluting compounds. Better resolution (in comparison to C18) was obtained for the isobaric acetyl-DON peaks but not baseline separation.
  • The Phenyl-Hexyl column offered the best separation for the majority of the peaks including the isobaric acetyl-DON compounds.

Conclusion

  • The Phenyl-Hexyl Fused-Core technology improved the separation of the 15 mycotoxins compared to C18. This will enable chromatographic analysis of multiple mycotoxins in a single assay.
  • This work will be expanded to evaluate whether an improved separation will result in lower matrix effects (better separation from matrix components).

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC.
Titan is a trademark of Sigma-Aldrich Co. LLC.
Fused-Core is a registered trademark of Advanced Separations Technology, Inc.

Materials