Method for determining contents of pentose, hexose, amino sugar and uronic acid in pneumonic polysaccharide vaccine hydrolysate

A determination method and hydrolyzate technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of simultaneous detection of multiple monosaccharides, influence on quantitative accuracy, long measurement time, etc., and achieve unique separation selectivity and The effect of anti-matrix interference ability, long-term stability, rapid equilibrium, good retention and separation selectivity

Active Publication Date: 2018-07-06
SHIMADZU (CHINA) CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have limitations: first, the measurement time is long (about 4 hours per sample); second, derivatization is required, and there is no separation, there are hidden dangers in the stability and accuracy of the method; third, the simultaneous determination of hexose , pentose, amino sugar and uronic acid, three methods are required to determine pentose, amino sugar and uronic acid respectively, and there is no hexose detection method, which is time-consuming and labor-intensive
HPLC-UV / Vis methods can provide higher separation selectivity than EP methods, but still require a responsible derivatization process, which is not conducive to rapid detection
HPLC-RID and HPLC-ELSD methods can realize the detection of monosaccharides without derivatization, but these two methods have poor selectivity and cannot realize the simultaneous detection of multiple monosaccharides in complex matrices (such as vaccines)
IC-ECD does not require derivatization and provides higher selectivity than HPLC-UV / Vis, HPLC-RID, and HPLC-ELSD, but the method usually requires a long separation time, and it is difficult to achieve simultaneous qualitative and quantitative, and complex matrices Affects quantitative accuracy

Method used

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  • Method for determining contents of pentose, hexose, amino sugar and uronic acid in pneumonic polysaccharide vaccine hydrolysate
  • Method for determining contents of pentose, hexose, amino sugar and uronic acid in pneumonic polysaccharide vaccine hydrolysate
  • Method for determining contents of pentose, hexose, amino sugar and uronic acid in pneumonic polysaccharide vaccine hydrolysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] 1. Experimental instruments and equipment:

[0072] High pressure binary pump, degasser, autosampler, column oven and triple quadrupole mass spectrometer.

[0073] 2. Experimental reagents:

[0074] Glucose, rhamnose, glucuronic acid, and rhamnose amino were purchased from Sigma, and the specifications were all 1 g; Ether glucose was synthesized in the laboratory, and the structure of the synthesized compound was confirmed by MS and literature data.

[0075] 3. Detection conditions:

[0076] Chromatographic column: stationary phase 1, R1 is silica gel;

[0077] Mobile phase: A-acetonitrile aqueous solution containing 10mmol / L ammonium formate (adjust pH to 4.3 with formic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 80%; B-10mmol / L ammonium formate (adjust pH to 4.3 with formic acid) aqueous solution ;

[0078] Specifically, the final concentration of ammonium formate in mobile phase A in aqueous acetonitrile is 10 mmol / L....

Embodiment 2

[0099] Detection of 2,4-diaminopentose and hexonic acid in polysaccharides from type 1 pneumonia

[0100] Type 1 pneumonia polysaccharides can be hydrolyzed to obtain 2,4-diaminopentose and hexonic acid. The detection conditions are as follows:

[0101] Stationary phase: with stationary phase described in embodiment 1;

[0102] Mobile phase: A-acetonitrile aqueous solution containing 10mmol / L ammonium formate (adjust pH to 6.5 with acetic acid), the volume concentration of acetonitrile in the acetonitrile aqueous solution is 80%; B-10mmol / L ammonium formate (adjust pH to 6.5 with acetic acid) aqueous solution ;

[0103] Gradient: 0-10min 80%A-20%A; LC-MS conditions: Ion source: ESI positive and negative ion scanning simultaneously, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230 °C, the temperature of the heating module is 400 °C, the drying gas flow rate is 10 L / min, and the interface voltage is 3 kV; other detection co...

Embodiment 3

[0105] Example 3: Detection of pentose, hexose and hexonic acid in type 2 pneumonia polysaccharide

[0106] Type 2 pneumonia polysaccharides can be hydrolyzed to obtain pentose sugar, hexose sugar and hexonic acid. The detection conditions are as follows:

[0107] Stationary phase: with stationary phase described in embodiment 1;

[0108] Mobile phase: A-contains 5mmol / L ammonium formate (pH 6.5) aqueous methanol solution, the volume concentration of methanol in the methanol aqueous solution is 80%; B-5mmol / L ammonium formate (pH 6.5) aqueous solution;

[0109] Gradient: 0-10min 75%A-20%A; LC-MS conditions: ion source: ESI negative ion, nebulizer flow rate 3L / min, heater flow rate 10L / min, interface temperature 200°C, DL temperature 230°C, heating The module temperature is 400° C., the drying gas flow rate is 10 L / min, and the interface voltage is 3 kV; other testing conditions are the same as those in Example 1.

[0110]The measured pentose, hexose and hexonic acid content...

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Abstract

The invention provides a method for determining contents of pentose, hexose, amino sugar and uronic acid in a pneumonic polysaccharide vaccine hydrolysate. The method is suitable for the quantitativedetection on a compound in the 23-valent pneumococcal polysaccharide vaccine hydrolysate. According to the method, a hydrophilic chromatography-zwitterion mixed mode stationary phase is mainly taken as a chromatographic column, and an acetonitrile, ammonium formate or (and) ammonium acetate aqueous solution is taken as an eluent, so that the pentose, the hexose, the uronic acid and the amino sugarremain on the stationary phase; a mass spectrometry is taken as a detector for MRM mode quantification. A new way is provided for quality evaluation on a polysaccharide pneumonia vaccine. The methodsolves the problems of long determining time, poor repeatability, easiness for interference and the like of a 'European pharmacopoeia' method as well as the problem of shortage of a hexose content determining method. The method is good in repeatability, strong in operability and easy for standardization and industrialization realization and has guidance significance in quality evaluation and batchrepeatability control on the polysaccharide pneumonia vaccine.

Description

technical field [0001] The invention specifically relates to a content determination method of pentose, hexose, amino sugar and uronic acid in the hydrolyzate of pneumonia polysaccharide vaccine. The invention belongs to the technical field of vaccine quality evaluation. Background technique [0002] Pneumonia is one of the important causes of death for the elderly, children and AIDS patients. The emergence of pneumonia polysaccharide vaccine has greatly reduced the incidence of such diseases. The main pneumonia polysaccharide vaccines currently on the market include 23-valent vaccines and 13-valent vaccines. Whether it is a 23-valent vaccine or a 13-valent vaccine, it is necessary to quantify the content of vaccine structural units in each valent state to evaluate the quality of the vaccine. Hexose sugar, pentose sugar, amino sugar and uronic acid are important structural units of pneumonia polysaccharide vaccine. "European Pharmacopoeia" adopts the method of derivatiza...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027
Inventor 龙珍李月琪王宇田李佳李长坤冀峰黄涛宏
Owner SHIMADZU (CHINA) CO LTD
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