UPLC™ Separation of Fifteen Bisphenols Using a Waters Acquity™ Biphenyl RP Column with MaxPeak™ Premier Technology and UV Detection

Significance of the topic

The bisphenol family includes numerous structural analogs of bisphenol A (BPA) used in plastics, coatings and thermal papers. Several bisphenols display endocrine-disrupting properties and are therefore monitored in food, consumer goods and environmental matrices. Analytical challenges arise from the strong structural similarity between many bisphenols, which can lead to co-elution and ambiguous detection. Robust, high‑throughput chromatographic methods that maximize selectivity and allow orthogonal detection (UV and MS) are therefore valuable for routine monitoring and method development.

Objectives and overview of the study

This application note evaluated reversed‑phase UPLC separations of 15 bisphenol compounds to identify a stationary phase that provides baseline resolution in a short analysis time. Three BEH‑based bonded phases packed in inert MaxPeak Premier hardware were compared: C18, phenyl, and biphenyl. The aim was to demonstrate whether enhanced π–π interactions provided by phenyl or biphenyl ligands improve selectivity sufficiently to resolve all analytes within an eight‑minute run using a simple methanol/water mobile phase and UV detection at 220 nm, with MS used for peak identification.

Methodology and used instrumentation

The separation was performed on an ACQUITY I‑Class UPLC system (HPS flowpath) with a tunable UV detector set to 220 nm. For compound identification a Xevo TQ‑S micro triple quadrupole mass spectrometer operated in negative ESI mode (scan range 50–500 m/z) was used. Columns evaluated:

• BEH C18, 2.1 × 50 mm, 1.7 µm (MaxPeak Premier inert hardware)
• BEH Phenyl, 2.1 × 50 mm, 1.7 µm (MaxPeak Premier inert hardware)
• BEH Biphenyl, 2.1 × 50 mm, 1.7 µm (MaxPeak Premier inert hardware)

Key chromatographic conditions:

• Mobile phases: A = water; B = methanol (no additives)
• Gradient: linear methanol ramp from 50% to 90% B over 6.5 minutes (representative short UPLC gradient)
• Flow rate: 0.45 mL/min
• Column temperature: 25 °C
• Injection volume: 1.0 µL
• Sample: mixture of 15 bisphenols at 0.1 mg/mL prepared in 75:25 water:methanol, injected in triplicate

Main results and discussion

All three columns were evaluated under identical chromatographic conditions. Peak identities were confirmed by MS. Observed outcomes:

• BEH C18: produced multiple co‑elutions (three sets of overlapping peaks) and failed to fully resolve the 15 bisphenols.
• BEH Phenyl: improved separation relative to C18 but still showed at least one pair of partially co‑eluting peaks.
• BEH Biphenyl: achieved baseline separation of all 15 bisphenols within the short gradient (approx. eight minutes) without additional method modification.

Discussion points:

• The increased π–π interaction surface of the biphenyl ligand enhances secondary retention mechanisms for aromatic analytes beyond hydrophobic interactions alone, increasing selectivity for closely related bisphenols.
• Chromatographic resolution on the biphenyl phase enabled reliable UV detection at 220 nm as a standalone or complementary approach to MS, which is advantageous for labs with limited MS access or when MS quantitation is complicated by matrix interferences.
• Use of inert MaxPeak Premier flowpath hardware reduced surface adsorption and contributed to consistent peak shapes for potentially active bisphenols.

Key benefits and practical applications

The described biphenyl UPLC method offers several practical advantages:

• Fast, baseline separation of 15 structurally similar bisphenols in a single short run, increasing sample throughput for monitoring programs.
• Improved selectivity reduces reliance on complex MS/MS transitions to distinguish co‑eluting isomers, simplifying data processing and lowering risk of interference.
• Compatibility with UV detection provides a cost‑effective alternative for screening assays while preserving the option to confirm identities by MS.
• Inert hardware minimizes adsorption losses for reactive phenolic compounds, improving quantitative robustness.

Limitations and practical considerations

Important caveats for implementation:

• The method was demonstrated with neat standard mixtures at relatively high concentrations (0.1 mg/mL). Real sample workflows will require appropriate extraction, cleanup and concentration steps to reach relevant regulatory detection limits.
• UV detection at 220 nm is non‑selective; chromatographic resolution is therefore critical to avoid false positives from co‑eluting matrix components.
• No mobile phase additives or pH modulation were used; matrices or target analytes with different ionization states may require buffer or pH adjustments for optimal retention and MS sensitivity.

Future trends and opportunities for use

Potential developments and extensions of this work include:

• Integration with tandem MS quantitation (MRM) or high‑resolution MS for lower limits of detection and higher specificity in complex matrices.
• Optimization for trace‑level analysis using sample preparation (SPE, QuEChERS) and dilution/reconstitution strategies tailored for food and environmental samples.
• Exploration of modified gradients, column lengths or particle sizes to balance throughput and resolution for isomeric bisphenols in more challenging matrices.
• Screening additional stationary‑phase chemistries and mixed‑mode phases to address novel bisphenol analogs or metabolites.
• Adoption of greener mobile phases and method miniaturization to reduce solvent consumption while maintaining performance.

Conclusion

This study demonstrates that a BEH Biphenyl stationary phase in inert MaxPeak Premier hardware provides superior chromatographic selectivity for a panel of 15 bisphenols compared with BEH C18 and BEH Phenyl phases. Baseline separation within an approximately eight‑minute UPLC run enables reliable UV monitoring and reduces dependence on selective MS transitions. The approach supports higher throughput screening and robust quantitation strategies for bisphenol surveillance in food and environmental testing, while further method tuning and sample preparation will be required for trace‑level, matrix‑rich analyses.

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