LC-MS/MS Analysis of Glyphosate & Other Polar Contaminants in Food with an Ion Exchange/HILIC Column

Description

Glyphosate is a broad-spectrum herbicide widely used throughout the world. The International Agency for Research on Cancer classified glyphosate as a probable human carcinogen. The safety residual level of glyphosate in food is regulated in the USA and Europe as well as other regions. Direct analysis of underivatized glyphosate, however, can be challenging due to its minimum retention on a reversed-phase or hydrophilic interaction chromatography (HILIC) column and severe adsorption onto the stainless-steel flow path. On the other hand, glyphosate and other polar anionic contaminants tend to strongly retain on an anion-exchange column and require high salt mobile phase to elute, which is not friendly to mass spectrometric analysis. 

These challenges motivated the development of a hybrid ion-exchange/HILIC column that offers balanced retention of glyphosate as well as other polar contaminants. With the column, an LC-MS/MS method has been established for the detection of glyphosate, aminomethylphosphonic acid (AMPA), glufosinate, and 14 other polar contaminants in various food matrices in a single run. Additionally, the nonspecific binding of glyphosate and other chelating compounds onto stainless steel is minimized with a simple passivation solution.

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Transcript

Hello! This is Xiaoning Lu from Restek LC Solutions. Thank you very much for this opportunity to present our work on “LC-MS/MS Analysis of Glyphosate and Other Polar Contaminants in Food with a Novel Ion-Exchange/HILIC Column.”

Analysis of underivatized glyphosate, as well as other metabolites and analogs, can be challenging. On one hand, it has minimal retention on a reversed-phase or HILIC column. On the other hand, they tend to strongly retain onto an anion-exchange column and require high-salt mobile phase to elute, which is not friendly to mass spectrometric analysis. To make this more complicated, glyphosate and other metabolites severely adsorb on a stainless-steel flow path. To overcome these challenges, we have developed a novel hybrid ion-exchange/HILIC column, as well as a simple HPLC system passivation solution, and established an LC-MS/MS method for the analysis of glyphosate, AMPA, glufosinate—as well as 14 other polar contaminants in various food matrices in a single run.

The hybrid ion-exchange/HILIC column is built on a single ligand that is comprised of multiple functionalities including ion-exchange and polar chemical moieties. The hybrid ligand can be applied to the separation of a wide range of compounds through ion exchange, HILIC, or the mixed mode of ion exchange and HILIC. This novel column technology is patent pending.

One of the advantages of the single-ligand based stationary phase is its lot-to-lot reproducibility. As shown in Fig. 2, the variability (RSD) of the retention across the seven lots of column is <1.0% for both B1 and B3, the 1st and 2nd peak, and 2.4% for B9, the 3rd peak, respectively.

We also developed a simple solution to passivate the stainless-steel flow path of HPLC systems. Simply inject a full loop of the passivation solution and let it flow through the system at a flow rate 0.4 mL/min and divert your flow to waste so it does not enter your detector. Do this a couple of times, and your system is passivated.

Now, let’s look at the results. Figure 3 shows the LC-MS/MS separation of glyphosate, AMPA, and glufosinate on a short hybrid ion-exchange/HILIC column (the dimensions are 2.1 x 30 mm, 2.7 µm superficially porous particle). As you can see, we have reduced the cycle time to a mere 8 minutes with excellent retention, resolution, and peak shape. Note the fast equilibration time is only 1.5 minutes. You don’t typically see that when HILIC is involved.

Polar pesticides are challenging and often require several different columns and conditions to analyze. As you can see, we are retaining 17 of these compounds in one short 13-minute run. No complex buffers. And note this separation was performed on a short, 3 cm column!

Figure 5 illustrates the analysis of glyphosate, AMPA, and glufosinate in various food matrices, including strawberry, spinach, tea, and cereals. The samples were prepared according to simple QuPPe methods, basically an acidified methanol extraction. As can been seen in Figure 5, glyphosate, AMPA, and glufosinate are detected with clean baseline and minimal interference.

Figure 6 shows the robustness of the method where the analyte retention barely moves over 500 injections. The data also demonstrate a guard column protects and extends the column lifetime beyond 500 injections.

In summary, we have developed a novel hybrid ion-exchange/HILIC column as well as a simple HPLC system passivation solution for the analysis of glyphosate and other polar contaminants in foods.

Thanks again for your interest in my work. I hope it’s helpful to you. Should you have any questions or suggestions, please see the link below.