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

Technical Analysis: BDD for Sub-1 Minute Catecholamine Biomarker Detection

This documentation analyzes the research detailing the rapid and sensitive analysis of Catecholamines (CAs) using core-shell Hydrophilic Interaction Liquid Chromatography (HILIC) coupled with Electrochemical Detection (ECD) utilizing a Boron-Doped Diamond (BDD) electrode. This application highlights the critical role of high-performance BDD material in advanced bioanalytical separation science.

Executive Summary

Application Focus: Development of a rapid and highly sensitive method for the simultaneous analysis of key Catecholamine biomarkers (Dopamine, Epinephrine, Norepinephrine) relevant to Traumatic Brain Injury (TBI) and Parkinson’s Disease (PD).
Core Technology: The method successfully couples high-efficiency core-shell HILIC separation with highly sensitive Electrochemical Detection (ECD) using a Boron-Doped Diamond (BDD) electrode.
Speed and Efficiency: Achieved complete separation of the three biomarkers in less than 60 seconds, demonstrating superior performance compared to traditional fully porous HILIC columns.
Nanomolar Sensitivity: The BDD electrode enabled enhanced nanomolar detection limits (LODs) of 40 nM for DA and 50 nM for EPI/NE, significantly lower than those achieved using conventional UV detection.
BDD Advantage: The BDD electrode was selected for its wide potential window, low background capacitive current, high sensitivity, and robust resistance to fouling, which are essential for complex biological sample analysis.
Validation: The method demonstrated excellent linearity (R² > 0.99) and high precision (RSD% < 0.65% inter-day) and was successfully applied to the analysis of Catecholamines in human urine samples following solid phase extraction (SPE).

Technical Specifications

The following hard data points were extracted from the optimized LC-BDD method:

Parameter	Value	Unit	Context
Working Electrode Material	Boron-Doped Diamond (BDD)	N/A	Used in a thin layer flow cell (0.7 µL volume)
BDD Electrode Diameter	8	mm	Working electrode dimension
Optimal Oxidation Potential	+1.3	V	Applied potential for amperometric detection (vs. Pd/H₂)
Limit of Detection (DA)	40	nM	HPLC-ECD (Signal/Noise = 3)
Limit of Detection (EPI/NE)	50	nM	HPLC-ECD (Signal/Noise = 3)
Separation Time	< 60	seconds	Achieved using core-shell Z-HILIC column
Column Type	Poro-shell Z-HILIC	N/A	Zwitterionic stationary phase
Column Dimensions	2.1 x 50, 2.7	mm, µm	Length, I.D., Particle Size
Mobile Phase Composition	85:15	ACN: Buffer (v/v)	10 mM Ammonium Formate (pH 3)
Optimal Flow Rate	1.5	mL/min	Resulting in 360 bar backpressure
Column Efficiency (DA)	49,040	N/m	Poro-Shell Z-HILIC column
Linearity Range (CAs)	0.1 - 25	µM	Correlation Coefficient (R²) > 0.99

Key Methodologies

The rapid and sensitive LC-BDD method relied on the precise control of chromatographic and electrochemical parameters:

Electrode Configuration: A three-electrode system was employed, featuring the BDD as the working electrode, a HyREF (Pd/H₂) reference electrode, and a carbon-loaded PTFE counter electrode within an Antec Flexcell thin layer flow cell.
Chromatographic Mode: Isocratic separation was performed using Hydrophilic Interaction Liquid Chromatography (HILIC), which is optimized for highly polar analytes like Catecholamines.
Stationary Phase Selection: Three zwitterionic HILIC columns were compared. The core-shell Z-HILIC column (2.7 µm particle size) was selected for its superior efficiency (nearly twofold increase) and ability to achieve separation in under 1 minute.
Mobile Phase Optimization: The optimal mobile phase consisted of 85% Acetonitrile (ACN) and 15% 10 mM ammonium formate buffer, carefully maintained at pH 3 to prevent peak broadening and tailing associated with higher pH values.
Flow Rate: The flow rate was optimized to 1.5 mL/min to achieve the sub-1 minute separation time, resulting in a system backpressure of 360 bar.
Electrochemical Detection (ECD): Amperometric detection was performed at a fixed oxidative potential of +1.3 V, maximizing the oxidation current for DA, EPI, and NE.
Sample Pretreatment: Real urine samples required pre-treatment using Phenylboronic Acid (PBA) Solid Phase Extraction (SPE) to isolate the CAs prior to injection.

6CCVD Solutions & Capabilities

The successful implementation of this high-speed, high-sensitivity LC-ECD method is directly dependent on the quality and customization of the BDD working electrode. 6CCVD is uniquely positioned to supply the necessary materials and fabrication services to replicate and advance this research.

Applicable Materials

To replicate the nanomolar detection achieved in this study, researchers require high-quality, electrochemically optimized BDD material.

Research Requirement	6CCVD Material Solution	Technical Advantage
High Sensitivity/Low Noise	Boron-Doped Diamond (BDD) Plates	MPCVD BDD offers the widest potential window and lowest background current for superior signal-to-noise ratio in ECD.
Electrode Robustness	Polycrystalline Diamond (PCD) Substrates	BDD grown on PCD substrates provides high mechanical strength and exceptional resistance to fouling, crucial for long-term analysis of complex biological matrices (urine, plasma).
Specific Doping	Custom BDD Doping Levels	We offer precise control over boron doping concentration to optimize conductivity and electrochemical activity for specific analyte oxidation potentials (e.g., +1.3 V for Catecholamines).

Customization Potential

The paper utilized a specific 8 mm diameter BDD working electrode integrated into a thin-layer flow cell. 6CCVD provides the necessary fabrication services to meet these precise engineering demands:

Custom Dimensions: 6CCVD can supply BDD plates and wafers up to 125 mm in diameter (PCD) and up to 10 mm thick (Substrates). We provide precise laser cutting and shaping services to produce custom electrode geometries, such as the 8 mm diameter discs required for the thin-layer flow cell used in this research.
Surface Finish: To ensure optimal flow characteristics and minimize non-specific adsorption in the flow cell, 6CCVD offers ultra-smooth polishing services: Ra < 1 nm for SCD and Ra < 5 nm for inch-size PCD/BDD.
Metalization Services: While the paper used a bare BDD surface, future integration often requires robust electrical contacts. 6CCVD offers in-house custom metalization stacks (e.g., Ti/Pt/Au, W/Au, Cu) tailored for electrochemical applications and bonding processes.

Engineering Support

The successful development of this rapid LC-BDD method required careful optimization of pH, potential, and column chemistry.

Expert Consultation: 6CCVD’s in-house PhD team can assist researchers with material selection, surface termination, and electrochemical optimization for similar LC-ECD biomarker detection projects, including the analysis of neurotransmitters, phenolic compounds, and other electroactive species.
Global Supply Chain: We ensure reliable, global shipping (DDU default, DDP available) of custom BDD electrodes and diamond materials, supporting time-sensitive research worldwide.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

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

References

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