UHPLC-MS/MS Analysis of Polar Pesticides on the Supel™ Carbon LC Column

Importance of the Topic

Polar pesticides such as glyphosate, chlorate, fosetyl-Al, ethephon and related metabolites pose significant analytical challenges due to their high polarity and poor retention on conventional reversed-phase columns. Accurate monitoring of these compounds at trace levels is essential for ensuring food safety, environmental protection and compliance with regulatory limits.

Objectives and Overview of the Study

This application note explores the capabilities of a porous graphitic carbon column (Supel™ Carbon LC) coupled with UHPLC-MS/MS for the separation and quantitation of a broad range of polar pesticides and their metabolites. Key goals include:

• Evaluating chromatographic retention and peak shape of glyphosate and its analogs.
• Demonstrating rapid isocratic and gradient separations of chlorate, perchlorate and other anionic species.
• Assessing resolution of fosetyl-aluminum and its primary metabolites HEPA and methylphosphinico propionic acid.
• Separating bialaphos, ethephon and phosphonic acid with good sensitivity and reproducibility.

Methodology

A single Supel™ Carbon LC column (5 cm × 3.0 mm I.D., 2.7 μm) was used for all analyses. The general chromatographic conditions consisted of:

• Mobile Phase A: 20 mM ammonium bicarbonate in water, adjusted to pH 9.0 with ammonium hydroxide.
• Mobile Phase B: Acetonitrile.
• Flow Rate: 0.3 mL/min; Column Temperature: 40 °C.
• Injection volumes ranged from 5 to 8 µL, depending on the analyte group.

MS detection was performed in negative electrospray ionization mode (ESI-) on a triple quadrupole mass spectrometer. Optimized source settings included a 4000 V spray voltage, 325 °C interface temperature and nitrogen as curtain and auxiliary gases. Multiple reaction monitoring (MRM) transitions were selected for each analyte to ensure selectivity and sensitivity.

Used Instrumentation

• UHPLC system equipped with a Supel™ Carbon LC PGC column (5 cm × 3.0 mm, 2.7 µm).
• Triple quadrupole mass spectrometer with ESI in negative mode.
• Mobile phase delivery and gradient control modules.

Main Results and Discussion

The Supel™ Carbon LC column provided strong retention and sharp peak shapes for all target analytes without the need for derivatization or special buffers beyond ammonium bicarbonate. Highlights include:

• Glyphosate and related analogs (AMPA, glufosinate, acetyl-n-glufosinate) were baseline-resolved in under 7 minutes with a simple gradient from 0 % to 100 % acetonitrile.
• Chlorate and perchlorate ions eluted isocratically in a three-minute window with excellent symmetry, eliminating the need for dedicated ion chromatography equipment.
• Fosetyl-Al and its metabolites HEPA and 3-(methylphosphinico) propionic acid were separated in under 6 minutes, enabling accurate quantitation of both parent and degradation products.
• Bialaphos, ethephon and phosphonic acid displayed clear resolution in a seven-minute gradient method, facilitating simultaneous monitoring of precursor, active pesticide and degradation products.

Benefits and Practical Applications

The PGC stationary phase combines reversed-phase, hydrophilic and ionic retention mechanisms, allowing a single column to handle multiple classes of polar pesticides. Key benefits include:

• Reduced analysis time and solvent consumption through fast gradients and isocratic runs.
• Elimination of derivatization or specialized sample preparation for highly polar analytes.
• Compatibility with routine UHPLC-MS/MS workflows in food safety, environmental and agricultural testing laboratories.

Future Trends and Opportunities

As regulatory agencies continue to lower allowable limits for polar pesticide residues, analytical methods must adapt to deliver higher sensitivity and throughput. Potential developments include:

• Integration with automated sample preparation techniques for high-throughput screening.
• Expansion to multi-class pesticide panels by exploiting the broad retention characteristics of PGC columns.
• Coupling with high-resolution mass spectrometry for non-targeted metabolite profiling and emerging contaminant surveillance.

Conclusion

The Supel™ Carbon LC column paired with UHPLC-MS/MS offers a robust, versatile solution for the separation and quantitation of a diverse set of polar pesticides and their metabolites. By streamlining analysis times and minimizing specialized reagents or instrumentation, this approach supports modern regulatory and quality-control laboratories in achieving reliable, sensitive detection of challenging analytes.