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Method for detecting monosaccharide and preparation method for derivative reagent

A technology of derivatizing reagents and monosaccharides, which is applied in the field of medicine, can solve problems such as unsatisfactory separation results of monosaccharides, waste of biological samples, and large sample consumption, and achieve the effects of extremely small size, easy operation, and high detection sensitivity

Active Publication Date: 2014-02-12
OCEAN UNIV OF CHINA
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Problems solved by technology

1-Naphthalene-3-methyl-5-pyrazolone (NMP) is also a commonly used derivatization reagent. For example, Sun Zhiwei et al. used NMP to determine the composition of monosaccharides in rapeseed pollen polysaccharides (Sun Zhiwei, Liu Lingjun, Hu Baojun, etc. Preparation of 1-(2-naphthyl)-3-methyl-5-pyrazolone derivative reagent and its application in the determination of sugar compounds by high performance liquid chromatography-mass spectrometry[J]. Chromatography, 2008(02 ): 200-205), its reaction mechanism is the same as that of PMP, and both can react with monosaccharides to generate sugar-double derivatization reagent products with UV absorption, but the above two derivatization reagents have high detection limits and large sample consumption. Mass spectrometry detection can only qualitatively sample, not quantitative and other disadvantages
Using ordinary derivatization reagents combined with LC-MS to quantify monosaccharides requires a series of standard curves, which is not only cumbersome, but also a waste of precious biological samples, and the separation results of monosaccharides are not ideal.

Method used

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  • Method for detecting monosaccharide and preparation method for derivative reagent
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  • Method for detecting monosaccharide and preparation method for derivative reagent

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Preparation of D3NMP

[0030] Weigh 5g of naphthalenehydrazine hydrochloride, put it in 60mL of water, heat it to 100°C to dissolve it completely, adjust it to 8-9 with 20% NaOH solution, obtain a solid after suction filtration, wash with water until the pH is 7, and suction filtration After vacuum drying, 3.5 g of naphthalenehydrazine was obtained.

[0031] Add 2g of naphthalenehydrazine into 20mL of absolute ethanol, stir in a water bath at 55°C until it dissolves, add 1.37g of deuterated ethyl acetoacetate dropwise, and react for 2h after the dropwise addition, then raise the temperature to 80°C and reflux for 7h, and the reaction The solution was concentrated under reduced pressure to 1 / 3 of the original volume, and recrystallized three times with 10 mL of methanol to obtain 0.9 g of D3NMP.

[0032] The calculated yield of D3NMP is 33.3%

Embodiment 2

[0033] Example 2 Liquid Phase Analysis of D3NMP Derivatives

[0034] Make D3NMP into a 0.5mol / L ethanol solution, and mix glucose, glucuronic acid, glucosamine, mannose, xylose, galactose, and fucose (0.05mol / L) at 70°C in an alkaline medium of ammonia water Response 90mm. After the reaction was completed, it was extracted with chloroform three times, centrifuged, and the supernatant was taken with a final volume of 25uL for LC-MS analysis. Liquid phase conditions: liquid phase column 0.3×250mm SB-C18 column (5um, Agilent), flow rate 1mL / min, mobile phase A is acetonitrile, mobile phase B is 0.01mol / L ammonium acetate solution, and the sample volume is 2uL; Phase conditions: mobile phase 29%A15min, 29%A30min linearly increased to 33%A, 33%A15min linearly increased to 35%A, 29%A10min, 245nm detection. see results figure 1 .

[0035] The results showed that the seven monosaccharides derived from D3NMP were well separated.

Embodiment 3

[0036] Example 3 NMP, D3NMP derived LC-MS analysis

[0037] Make NMP / D3NMP into a 0.5mol / L ethanol solution, react NMP with a known amount of glucose, glucuronic acid, and glucosamine (0.01mol / L) in a total of 10uL in an alkaline medium of ammonia water, D3NMP and the glucose to be tested, Glucuronic acid and glucosamine react at 70°C for 90 minutes. After the reaction was completed, extract with chloroform three times, centrifuge, take the supernatant, the final volume is 50uL, mix the known and unknown samples in equal volumes, and perform LC-MS analysis. Liquid phase conditions: liquid phase column 0.3×250mm SB-C18 column (5um, Agilent), flow rate 15uL / min, mobile phase A is acetonitrile, mobile phase B is 0.01mol / L ammonium acetate solution, and the sample volume is 0.02uL; Mobile phase conditions: mobile phase 27%A15min, 27%A30min linearly increased to 31%A, 31%A15min linearly increased to 34%A, 27%A10min, 245nm detection. Mass spectrometry was detected in negative ion ...

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Abstract

The invention belongs to the field of medicines and relates to a method for detecting monosaccharide and a preparation method for a derivative reagent. The method for detecting the monosaccharide comprises the steps of (1) deriving the monosaccharide through the derivative reagent; (2) performing liquid chromatography-mass spectrum (LC-MS) analysis on a derived product, wherein the derivative reagent comprises 1-naphthalene-3-perylene methyl-5-pyrazolone (d3nmp) and 1-naphthalene-3-methyl-5-pyrazolone (NMP). The detection method disclosed by the invention can be used for qualifying and quantifying trace samples, is high in detection sensitivity and easy to operate and can be used for performing relative quantification on two unknown samples at the same time; the signal-to-noise ratio of a mass spectrum is low, and the polarity of a flow phase required for separation is low, so that the service life of a column is greatly prolonged. The sensitivity of the method disclosed by the invention is 4-5 times higher than that of the conventional 1-naphthalene-3-methyl-5-pyrazolone (NMP) deriving method; furthermore, the monosaccharide can be better separated by the method disclosed by the invention than the conventional NMP deriving method, and the phenomenon that chromatographic peaks of the monosaccharide are superposed is avoided.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a method for detecting monosaccharides and a preparation method for derivatization reagents. Background technique [0002] Currently, there are many challenges in the field of polysaccharides. The study of polysaccharides is not limited to the analysis of a large number of samples, but also more and more studies on samples of biological origin. Due to the small amount of polysaccharides in samples from biological sources (such as blood, cells, and tissues), the monosaccharides have strong polarity, similar structures, and lack of optical activity, so it is difficult to separate and identify them by conventional methods. Sugar has no absorption in the ultraviolet region, and the traditional differential refractometric detection method has low sensitivity and is not conducive to gradient elution. In order to improve its separation selectivity and detection sensitivity, people often use deri...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06C07D231/22
Inventor 张丽娟韩章润曾洋洋兰莹邱培菊
Owner OCEAN UNIV OF CHINA
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