High Sensitive Thiols Detection using a Gold Working Electrode

  • New – ECD with a gold working electrode
  • Highly sensitive with 1 fmol detection limit for GSH and 0.5 fmol for -SH compounds

Biological thiols such as cysteine (Cys) and glutathione (GSH) play a crucial role in redox status control. Homocysteine (Hcys), one of the main thiols, has a close relationship with the pathogenesis of arteriosclerosis. Looking through the literature, several analytical methods for measuring these thiols have been reported. One of the most common procedures is to use post or pre-column derivatization with UV-Vis or fluorescence detection. However, derivatization methods do not always satisfy the required sensitivity and one is forced to use a complicated protocol. One of the ways to avoid the derivatization procedure is to use electrochemical detection using an amalgam working electrode. However, this procedure demands intensive maintenance of the working electrode. One of the best and most realistic analytical procedures involves using electrochemical detection with a gold working electrode as we describe below.

We introduce a new concise analytical procedure for measurement of biological thiols using a gold working electrode. Thiols have a high affinity for gold working electrodes and this can result in pollution of the working electrode and can lead to less reproducible results. Our new pretreated gold working electrode is coated with a material to prevent such pollution from thiols and other compounds. Thus, this allows highly sensitive and reproducible thiol detection using a simple methodology. As far as injecting protein-precipitated samples, this electrode could be used for one year without any maintenance. Users do not need to perform any treatments such as generating an amalgam layer or applying pulse potentials in order to clean the gold working electrode.

The detection limit with this methodology is 1 fmol for GSH and 0.5 fmol for Cys at the conditions set when using Eicompak SC-3ODS column. Thiols (i.e. Cys, GSH, Glu-Cys, Cys-Gly and Hcys) as well as ascorbic acid can be analyzed using this procedure.

Chromatographic Condition

HPLC-ECDEicom HTEC-500
ColumnEicom SC-3ODS (3.0 ID x 100 mm)
M.P. flow rate400 μl/min
Applied potential+600 mV vs. Ag/AgCl
Working electrodeEicom WE-AU
Time constant filter1.5 sec
System Temperature25°C
Mobile Phase99% 0.1 M Sodium phosphate buffer (pH2.5), 1% MeOH and 50 mg/L Sodium Octanesulfonate (SOS), 5 mg/L EDTA-2Na. (In the situation where the target compound is only GSH, the SOS concentration should be 100 mg/L)

Sodium Octanesulfonate (SOS) increases the retention time of amine compounds but reduces it for other cations.

Figure 1 shows a typical chromatography from a human serum sample and standard solutions.

Sample Preparation

Human Blood Sampling

Healthy volunteer. Blood samples are collected from the volunteer’s arm and immediately mixed with EDTA (ethylenediaminetetraacetic acid). The samples are centrifuged within 2 hours of collection at 5,000G for 20 min. The plasma supernatant can then be stored for one day at -20˚C.

Rat Blood Sampling

Trunk blood (750 μl) from a male Sprague Dawley rat (~300 g) was mixed in EDTA tubes. Samples were then centrifuged at 5000G for 15 min. The plasma supernatant was then used in the eight-step protocol (detailed in the Application Manual, “Sample Preparation for Catecholamine Analysis in Plasma or Urine”).

Reference

Y. Hiraku, M. Murata and S. Kawanishi; Determination of intracellular glutathione and thiols by high performance liquid chromatography with a gold electrode at the femtomole level: comparison with a spectroscopic assay. Biochimica et Biophysica Acta. 1570 (2002) 47-52.


Fig. 1 – Hcys analysis in the human serum (black) and standard solution (grey)