Efficient Sub-1 Minute Analysis of Selected Biomarker Catecholamines by Core-Shell Hydrophilic Interaction Liquid Chromatography (HILIC) with Nanomolar Detection at a Boron-Doped Diamond (BDD) Electrode

At a Glance

Metadata	Details
Publication Date	2021-08-18
Journal	Separations
Authors	Majidah Alsaeedi, Huda A. Al‐Ghamdi, Phyllis E. Hayes, Anna Hogan, Jeremy D. Glennon
Institutions	University College Cork
Citations	2
Analysis	Full AI Review Included

Executive Summary

This study details the development of an ultra-rapid and highly sensitive method for analyzing critical catecholamine (CA) biomarkers (Dopamine, Epinephrine, Norepinephrine) using core-shell Hydrophilic Interaction Liquid Chromatography (HILIC) coupled with Boron-Doped Diamond (BDD) electrochemical detection (ECD).

Ultra-Rapid Separation: Achieved simultaneous separation of all three polar CAs in less than 60 seconds using a Poro-shell Z-HILIC column (2.7 µm core-shell particles).
High Efficiency: The core-shell column provided superior performance, demonstrating an efficiency of up to 49,040 N/m for Dopamine, nearly double that of fully porous alternatives.
Nanomolar Sensitivity: BDD electrochemical detection provided enhanced sensitivity, achieving Limits of Detection (LODs) of 40 nM (DA) and 50 nM (EPI, NE), significantly lower than traditional UV detection.
Robust Detection Material: The BDD electrode was selected for its wide potential window, low background current, and high resistance to fouling, enabling reliable detection in complex biological samples.
Method Validation: The method demonstrated excellent linearity (R² > 0.99) and high precision (Intra-day RSD < 0.42%).
Real Sample Applicability: Successfully applied to the analysis of CAs in human urine samples following Phenylboronic Acid (PBA) Solid Phase Extraction (SPE) pretreatment, with recovery rates ranging from 90% to 94%.

Technical Specifications

Parameter	Value	Unit	Context
Separation Time	<1 (60)	minute (s)	Simultaneous analysis of DA, EPI, NE
Column Type	Poro-shell Z-HILIC	N/A	Core-shell zwitterionic stationary phase
Column Dimensions	2.1 x 50	mm	Optimal column size
Particle Size (d_p)	2.7	µm	Core-shell particle diameter
Optimal Flow Rate	1.5	mL/min	Achieved backpressure: 360 bar
Mobile Phase Composition	85:15	ACN:10 mM Ammonium Formate	pH 3.0
Working Electrode Material	Boron-Doped Diamond (BDD)	N/A	High stability, low fouling
Optimal Detection Potential	+1.3	V	Amperometric ECD vs. Ag/AgCl
LOD (Dopamine, DA)	40	nM	Signal-to-Noise (S/N) = 3
LOD (Epinephrine, EPI)	50	nM	Signal-to-Noise (S/N) = 3
LOD (Norepinephrine, NE)	50	nM	Signal-to-Noise (S/N) = 3
Efficiency (DA)	49,040	N/m	Poro-shell Z-HILIC column
Intra-day Precision (RSD)	0.35-0.42	%	Retention time repeatability
Sample Recovery (Urine)	90-94	%	Following PBA SPE pretreatment

Key Methodologies

Column Selection and Optimization:
- Three zwitterionic HILIC columns were tested (Poro-shell Z-HILIC core-shell, Fully Porous Z-cHILIC, Fully Porous Z-HILIC).
- The Poro-shell Z-HILIC (2.7 µm) was selected due to its superior efficiency (up to 49,040 N/m) and ability to achieve rapid separation.
Chromatographic Conditions:
- Mobile phase was optimized to 85% ACN in 10 mM ammonium formate buffer (pH 3.0). High ACN concentration enhances HILIC partitioning of polar analytes.
- Flow rate was maximized at 1.5 mL/min to achieve the sub-1 minute separation time while maintaining acceptable backpressure (360 bar).
Electrochemical Detection (ECD):
- A bare BDD electrode was used as the working electrode in a three-electrode configuration (Ag/AgCl reference, Pt counter).
- The optimal oxidation potential for simultaneous detection of all three CAs was determined to be +1.3 V via hydrodynamic voltammograms.
Sample Pretreatment (SPE):
- Phenylboronic Acid (PBA) Solid Phase Extraction (SPE) was employed for selective extraction of CAs from urine samples.
- The elution solvent (80:20 ACN:H₂O with 1% formic acid) was compatible with the subsequent HILIC separation.

Commercial Applications

The combination of rapid HILIC separation and highly sensitive BDD detection is valuable for high-throughput analysis in fields dealing with polar, electroactive biomarkers and complex matrices.

Clinical Diagnostics and Screening:
- Traumatic Brain Injury (TBI): Rapid, high-throughput screening of plasma or urine for elevated DA, EPI, and NE levels, serving as prognostic biomarkers.
- Neurodegenerative Disease Monitoring: Early identification and monitoring of Parkinson’s disease (PD) and Alzheimer’s disease (AD) through peripheral CA analysis.
- Oncology: Screening for CA overproduction associated with tumors like pheochromocytomas and paragangliomas.
BDD Electrode Technology:
- High-Fouling Sample Analysis: Utilizing the BDD electrode’s resistance to fouling for reliable analysis of complex biological fluids (urine, blood, CSF) and environmental samples.
- Sensitive Trace Analysis: Applications requiring nanomolar or picomolar detection limits for electroactive compounds, leveraging the BDD’s wide potential window and low noise.
High-Throughput Analytical Labs:
- Core-Shell Chromatography: Implementation of core-shell HILIC columns for rapid analysis of polar metabolites, reducing solvent consumption and increasing sample throughput in pharmaceutical and metabolomics laboratories.

View Original Abstract

A rapid, sensitive method for the separation of catecholamine biomarkers (CAs), of importance in traumatic brain injury (TBI) and in Parkinson’s disease (PD), has been successfully developed using hydrophilic interaction liquid chromatography (HILIC). Dopamine (DA), epinephrine (EPI), and norepinephrine (NE) are known to be three to fivefold elevated above normal in traumatic brain injury (TBI) patients. HILIC facilitates the rapid and efficient separation of these polar biomarkers, which can be poorly retained by reversed-phase liquid chromatography (RPLC), while electrochemical detection (ECD) at the boron-doped diamond (BDD) electrode provides enhanced nanomolar detection. Three HILIC columns were compared, namely the superficially porous (core-shell) Z-HILIC column and the Z-cHILIC and Z-HILIC fully porous columns. The core-shell Z-HILIC showed the highest efficiency with a rapid separation within 60 s. The HILIC method utilizing the core-shell Z-HILIC column was initially optimized for the simultaneous analysis of DA, EPI, and NE using UV detection. The advantages of using the BDD electrode over UV detection were explored, and the improved limits of detection (LODs, S/N = 3) measured were 40, 50, and 50 nM for DA, EPI, and NE, respectively. Method validation is reported in terms of the linearity, repeatability, reproducibility, and LODs. Furthermore, the proposed method was successfully applied to the real sample analysis of urinary CAs following phenylboronic acid (PBA) solid phase extraction (SPE) pretreatment.

Tech Support

Original Source

DOI: https://doi.org/10.3390/separations8080124

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