Why does the solution of Trinder's reagent such as EHSPT and MAOS change color after storage?

Recently, some customers reported that the EHSPT (also called TOOS) solution prepared by myself was transparent, but it turned yellow or red after being left for two days. What is the reason? Can it still be used?

Reasons for discoloration of Trinder's reagent solution:

Chromogenic substrates such as EHSPT and MAOS are all new Trinder’s reagents, which are all derivatives of highly water-soluble sodium aniline, which will produce red or yellow substances when oxidized. Trinder's reagent powder is not easy to be oxidized. It needs to be sealed and refrigerated after preparation. If the solution is exposed to the air for a long time and the temperature is high, they may be slowly oxidized by the oxygen in the air. Discoloration of the solution occurred.

Trinder’s reagent for enzyme-linked immunoassay

Trinder's reagent detection principle:

Trinder's reagents such as EHSPT and MAOS can be oxidized by hydrogen peroxide (TOOS, MAOS, etc. need to be coupled with 4-AAP) in the presence of peroxidase to produce red quinone imine substances, which can be detected by measuring absorbance with a photometer The content of the test sample that can generate or oxidize hydrogen.

The new Trinder’s reagents are all reductive color-developing reagents. They are originally colorless. When they are catalyzed by peroxidase by oxygen, they can generate colored oxidation products. However, chromogen substrates such as EHSPT are relatively unstable in the solution. They are contaminated by oxidizing impurities or exposed to air for a long time, which will cause them to slowly oxidize, and the solution will change color. Therefore, when the chromogen substrate is formulated into a solution, it is generally recommended to prepare it for immediate use to prevent it from being contaminated or oxidized.

The advantages of Trinder's reagent compared to TMB:

When using TMB as a color reagent, the detection wavelength is 450nm maximum absorption wavelength and 405nm wavelength, and the absorption wavelength of bilirubin in the serum is 380~530nm, which will interfere with the detection results, and the color should be done under acidic conditions. Reaction limits its application. The UV absorption of the color reaction product of the new Trinder’s reagent is >540nm, MAOS and MADB are even as high as 630nm, and the reaction requires a wide pH range, and is less interfered by other substances in the serum. It can be used for detection items that require high precision values.

At present, in addition to chemiluminescence immunoassays, enzyme-linked immunoassays are more commonly used. The new Trinder's reagent is one of the important chromogenic substrates. Desheng is engaged in the R&D, production and sales of acridinium ester chemiluminescence reagents and new Trinder's reagents. All have extensive experience.