IMPROVED PERFORMANCE OF MODERN MS-COMPATIBLE REVERSED-PHASE/ANION-EXCHANGE MIXED-MODE HPLC COLUMNS

Significance of the Topic

Polar acidic analytes such as nucleotides and small organic acids are difficult to retain on traditional reversed-phase (RP) columns without ion-pairing reagents, which impair mass spectrometry (MS) compatibility. Mixed-mode stationary phases combining RP and anion-exchange capabilities can address retention issues but often suffer from column bleed, poor MS signal stability, and metal interactions that degrade analyte recovery.

Objectives and Study Overview

This work evaluates the performance of the Atlantis PREMIER BEH C18 AX column, a modern mixed-mode reversed-phase/anion-exchange (RP/AX) stationary phase designed on a pH-stable bridged-ethyl hybrid (BEH) particle. Key aims include:

• Assessing retention of ionized acids at acidic mobile phase pH.
• Measuring matrix effects and MS signal stability compared to leading mixed-mode columns.
• Evaluating recovery of metal-sensitive analytes using MaxPeak HPS hardware.

Methodology and Used Instrumentation

Two LC-MS platforms were employed:

• ACQUITY UPLC I-Class with PDA detector for isocratic separations of a polar mixture (2.1 × 50 mm, 1.7 µm), mobile phase 10 mM ammonium formate pH 3.0 at 0.2 mL/min, UV detection at 254 nm.
• ACQUITY UPLC H-Class coupled to Xevo TQD MS for matrix-effect studies (2.1 × 150 mm, 1.8 µm), positive/negative ESI, formic acid or ammonium formate buffers, infusion of analytes post-column and post-union.

Main Results and Discussion

Retention Behavior:

• The BEH C18 AX column showed enhanced retention of negatively charged acids at pH 3 compared to conventional RP phases, while reducing retention of basic cations.

Matrix Effects:

• In both ESI modes, matrix suppression/enhancement was significantly lower on the Atlantis PREMIER BEH C18 AX than on two competing mixed-mode RP/AX columns, reflecting minimal bonded-phase hydrolysis and column bleed.

Metal-Sensitive Analyte Recovery:

• MaxPeak HPS column hardware delivered higher peak areas, improved peak shape, and more reproducible signals for nucleotides and other metal-interacting compounds versus stainless-steel hardware.

Benefits and Practical Applications

The Atlantis PREMIER BEH C18 AX column enables direct reversed-phase separations of polar anions without ion-pairing reagents, enhancing MS compatibility and sensitivity. Laboratories in bioanalysis, pharmaceutical QA/QC, and environmental testing can achieve reliable quantitation of nucleotides, organic acids, and other polar analytes with minimal method development.

Future Trends and Applications

Ongoing developments may include:

• Extending mixed-mode designs to cation-exchange/RP phases for basic compounds.
• Integrating high-throughput UPLC methods in automated bioanalysis.
• Applying advanced hardware coatings to further reduce metal-analyte interactions.

Conclusion

The Atlantis PREMIER BEH C18 AX column combines pH-stable BEH particles, controlled anion-exchange capacity, and MaxPeak HPS hardware to deliver superior retention of polar acids, low matrix effects, and enhanced recovery of metal-sensitive compounds. This platform simplifies LC-MS workflows and improves assay robustness.

Reference

1) Walter TH et al. A New Mixed-Mode Reversed-Phase/Anion-Exchange Stationary Phase Based on Hybrid Particles. Waters Application Note 720006742EN, 2020
2) Smith KM, Rainville P. Utilization of MaxPeak High Performance Surfaces and the Atlantis PREMIER BEH C18 AX Column to Increase Sensitivity of LC-MS Analysis. Waters Tech Brief 720006745EN, 2020