HPLC (high performance liquid chromatography) detection method for content of polyamines in follicular granular cells of goose and application of HPLC detection method

Abstract

The invention discloses an HPLC (high performance liquid chromatography) detection method for content of polyamines in follicular granular cells of a goose and application of the HPLC detection method. The method provided by the invention comprises the following steps: (1), a solid-phase extraction method for polyamines in the cells: collecting the cells with 5% HClO4, adding a 1,6-hexamethylene diamine homogenate into the cells to abstract a supernatant fluid, adding benzoyl chloride into the supernatant fluid to carry out derivation, putting a derived fluid in a C18 solid-phase extraction column, filtering the column with ultra-pure water and methanol to remove impurities, afterwards, eluting the derived fluid with methanol again, and collecting the polyamines derived by the benzoyl chloride; and (2), a high performance liquid chromatography detection method for the polyamines in the cells: wherein a chromatographic separation column is a C18 chromatographic column with the sizes of 5 microns and 4.6*250mm; a volume ratio of mobile-phase methanol to the ultra-pure water is 66: 34; a flow velocity is 0.9mL / min; an ultraviolet detection wavelength is 229nm; a column temperature is 25 DEG C. According to the scheme, putrescine, spermidine and spermine in the follicular granular cells of the goose can be effectively separated, the content of the polyamines in the follicular granular cells of the goose can be rapidly and accurately detected, and the HPLC detection method has the characteristics of being simple and convenient to operate, short in detection time, low in cost, high in accuracy and good in repeatability.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and relates to a method for detecting the polyamine content of goose follicle granulosa cells by using high performance liquid chromatography (HPLC) and its application, which has the advantages of simple operation, short detection time and low cost , high accuracy and good repeatability. Background technique [0002] Polyamines (including putrescine, spermidine and spermine) are a class of low molecular aliphatic compounds with two or more amino groups. Polyamines can participate in the regulation of gene expression and translation, cell proliferation, cell signal transduction, cell membrane stability and other life activities, and can regulate the activity of certain cation channels. Studies have shown that when the concentration of intracellular polyamines decreases, cell growth slows down or even stops; and when intracellular polyamines are excessive, it can lead to excessi...

AI Technical Summary

Problems solved by technology

For example, if the amount of benzoyl chloride used reaches 10 μL, there will be many residual decomposition products and many miscellaneous peaks, and the peak time of impurities and putrescine will be close to each other, which will interfere with the peak shape of putrescine; A large number of double shoulders and interference peaks; ③The detection time is long, about 20 minutes; ④The detection peak type is continuous, the resolution is poor, and the effective separation interval between the peaks is small, and the peaks cannot be accurately separated; ⑤The standard and sample peaks The time variation is large, which interferes with the separation and identification of putrescine, spermidine and spermine; Some existing detection methods also need to combine mass spectrometers, which greatly increases the detection cost

In summary, the existing polyamine detection methods can only detect the polyamine content in tissues, but cannot effectively detect the polyamine content in cells; and the existing polyamine detection methods have certain deficiencies, mainly manifested in the need for expensive mass spectrometry The cost of polyamine detection is high, and it is not suitable for general application; the sample needs to be large; the pretreatment and purification of samples are insufficient, and there are many interfering impurities; organic reagents such as benzene and n-hexane are used, and the residue peaks interfere; the detection conditions are not optimized enough , insufficient separation

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