ECD Innovations

Improved Technology for the Direct Analysis of Biologically Relevant Thiols, Disulfides, and Related Compounds
Thiols, disulfides, nitrosothiols, and related compounds play many critical roles in the body. Glutathione (GSH) is a crucial antioxidant that is involved in many aspects of cellular protection. Furthermore, maintenance of a high GSH/GSSG ratio (i.e., reductive environment) is essential for normal cellular functioning – a change in this ratio is associated with numerous diseases. Nitrosothiols play a key role in signal transduction, and, along with nitric oxide, help regulate the cardiovascular system.

There is a substantial body of literature showing that HPLC-electrochemical detection (ECD), with its inherent selectivity and sensitivity, is the preferred technique for the direct measurement of thiols, disulfides and related compounds – notoriously hard-to-measure analytes. Thiols and their reaction products are reactive and quickly foul the working electrode leading to rapid loss of sensitivity. Moreover, measurement of disulfides requires a very high potential often close to the oxidation potential of the mobile phase, leading to increased noise, baseline instability, and shortened electrode life. Therefore, traditional ECD systems are often unstable, difficult to use, and require extensive routine maintenance in order to keep operating.

No more. ESA Biosciences has developed the first stable, sensitive, and robust analytical system that enables routine measurement of thiols, disulfides, thioethers, and other sulfur-containing compounds – without the cumbersome maintenance routines required of traditional ECD techniques. The new system contains a specially developed electrochemical cell featuring a unique Boron-Doped Diamond (BDD) working electrode. This new inert electrode provides excellent sensitivity and durability, while minimizing or eliminating the problems associated with other carbon-based working electrode materials. Now researchers studying thiols, disulfides, nitrosothiols, and thioethers will finally be able to answer many of the important questions that have eluded them for years.

Simplifying the Analysis of Difficult Compounds
The BDD working electrode offers many advantages over other carbon-based materials:

It is inert. No more loss of signal due to electrode fouling. No more routine maintenance.
It can be used at high potentials (often >1400 mV). Accurately analyze previously hard-to-measure analytes – without high background currents, noise, and chromatographic instability.
Measure analytes requiring a high oxidation potential without mobile phase interference! The upper potential used with current carbon-based working electrodes is limited by the oxidation of water in the mobile phase. The potential at which water oxidizes on the BDD is shifted much more positive relative to oxidation of analytes.
Unique chip design. Easy to replace, when required.

Chromatograms showing the resolution of numerous thiols, disulfides, and thioethers in under 40 mins, for standards (10 ng, each on column), plasma, and standard-spiked plasma samples.

Typical chromatograms for liver tissue homogenates showing standards, sample, and sample spiked with GSNO (10 ng). Insets show chromatograms at higher sensitivity, for clarity.

Looking beyond thiols and disulfides...
Use the BDD for other compounds that show poor electrochemical response or require high oxidation potentials. The BDD electrode takes ECD into new areas and extends its usefulness to other analytes....