Method for detecting effect of PQQ on lactic acid by combining HPLC with UPLC-MS

Abstract

The invention relates to a method for detecting the effect of PQQ on lactic acid by combining HPLC with UPLC-MS and belongs to the technical field of bioanalysis. The method provided by the invention adopts an UPLC method for studying reaction between PQQ and lactic acid, and separation conditions are optimized, so that separation of the PQQ from multiple reaction products is achieved; and qualitative analysis is carried out on the reaction products by adopting UPLC-MS. The method provided by the invention firstly studies the reaction behavior of the PQQ and the lactic acid, three products are found in reaction liquid, and quasi-molecular ions [M+H]<+> are 403, 475 and 547 respectively. Esterification reaction is respectively carried out on the PQQ as well as monomolecular lactic acid, dimolecular lactic acid and trimolecular lactic acid, and products PQQLa1, PQQLa2 and PQQLa3 are obtained respectively. Peak areas of products obtained by different reaction times show that the PQQLa1 exists mainly, and products after esterification enter extracellular fluid by passing a blood brain barrier, so that the lactic acid content of the extracellular fluid is reduced. The method provided by the invention has extremely important research values on a neuroprotective mechanism of the PQQ, especially in the aspect of disease study related to senility.

Description

technical field [0001] The invention discloses a method for detecting the action of PQQ on lactic acid by combining HPLC with UPLC-MS, belonging to the technical field of bioanalysis. Background technique [0002] The energy supply in the central nervous system is mainly dependent on glucose in the peripheral blood. Astrocytes, the main component of the blood-brain barrier, take up glucose, produce lactic acid through glycolysis, and release it into the extracellular fluid, which reflects the living environment of nerve cells. Lactic acid, as a metabolite of glycolysis, has long been considered to have a damaging effect on nerve cells. Therefore, the role of lactic acid in the nervous system has been valued by scientists. It has been reported that the lactic acid content of cerebrospinal fluid in patients with acute brain trauma is elevated; the lactic acid content of cerebrospinal fluid in normal people is very low, and it has been reported that the lactic acid content in...

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Problems solved by technology

[0007] The determination of lactic acid content includes colorimetric method, EDTA titration method, HPLC analysis method, enzymatic method, and GC analysis method. The colorimetric method and EDTA method are simple and fast, but the sensitivity and precision are poor; the enzymatic method requires harsh experimental conditions and costs High; lactic acid is easy to decompose in gas chromatography, which affects its determination accuracy

The content of lactic acid in brain tissue is extremely low, and the ultraviolet absorption of lactic acid is extremely low. Most of the HPLC methods reported in the literature use 210nm detection, and there are many interference factors. The lowest detection concentration reported is 5 µM, which is too low for the detection sensitivity of biological samples of brain tissue.

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