High performance liquid chromatography (HPLC), also known as "high pressure liquid chromatography", "modern column chromatography", etc., is an important branch of chromatography. It has the characteristics of high pressure, high speed, high sensitivity and a wide range of applications. The application of important separation analysis technology in the fields of medicine, industry, agronomy, commodity inspection and legal inspection. In recent years, high performance liquid chromatography has been widely used in the separation and determination of pharmaceutical ingredients, functional ingredients of health foods, nutritional supplements, vitamins, and proteins. Luo Ning et al. used high performance liquid chromatography to simultaneously detect DTT and DTTox in drugs. DTT, dithiothreitol, is a small molecule organic reducing agent. One of the uses of DTT is as a reducing agent and deprotecting agent for sulfhydryl DNA. In DNA extraction experiments, DTT is often added to break the disulfide bonds between proteins, so that the DNA is fully freed and the efficiency of DNA extraction is improved.
DTT is also often used for the reduction of disulfide bonds in proteins, and can be used to prevent intramolecular or intermolecular disulfide bonds formed between cysteines in proteins. DTTox is the oxidation state of DTT, a six-membered ring structure containing disulfide bonds, and has high reducibility. Dithiothreitol (DTT) is also used mainly in clinical and experimental fields. Adding DTT in the production process of some drugs can protect the structure of the target protein to maintain drug activity. However, DTT has strong reducibility and may destroy protein structure in the body, causing enzyme inactivation and other phenomena. DTTox will change the structure of creatine kinase, and even completely inactivate creatine kinase when it reaches a certain concentration. Therefore, it is necessary to detect DTT and DTTox in drugs.
At present, it has been reported to use dithiothreitol spectrophotometer to determine the content of cobalt ion, but DTT is very easy to be oxidized, and the sensitivity of the titration method is not high, so cobalt ion can not be used to back titrate DTT. DTTox also has no effective detection method. The use of high performance liquid chromatography to detect DTT and DTTox in pharmaceuticals has the advantages of simple operation, good resolution, high sensitivity, good repeatability, and high recovery rate. It is an effective detection method.