High Sensitive Thiols Detection using a Gold Working Electrode
- New – ECD with a gold working electrode
- Highly sensitive to 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 a 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 way 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 the measurement of biological thiols using a gold working electrode. Thiols have a high affinity for gold working electrodes and this can cause pollution of the working electrode and can lead to less reproducible results. Our new pre-treated 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 method. As far as injecting protein-precipitated samples, this electrode could be used for one year with no 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 method is 1 fmol for GSH and 0.5 fmol for Cys at the conditions set when using the 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.
|Column||Eicom SC-3ODS (3.0 ID x 100 mm)|
|M.P. flow rate||400 μl/min|
|Applied potential||+600 mV vs. Ag/AgCl|
|Working electrode||Eicom WE-AU|
|Time constant filter||1.5 sec|
|Mobile Phase||99% 0.1 M Sodium phosphate buffer (pH2.5), 1% MeOH and 50 mg/L Sodium Octane sulfonate (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 Octane sulfonate (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.
Human Blood Sampling
Healthy volunteer. Blood samples are collected from the volunteer’s arm and immediately mixed with EDTA (ethylenediaminetetraacetate 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”).
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)