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A method for analyzing endotoxin lipid a by liquid chromatography-mass spectrometry

A liquid mass spectrometry and endotoxin technology, applied in the field of analytical chemistry mass spectrometry identification, can solve the problems of different degrees of phosphorylation, achieve complete hydrolysis, improve the detection limit, and improve the effect of extraction

Active Publication Date: 2020-09-29
WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, different substrate systems lead to differences in the degree of phosphorylation detected

Method used

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  • A method for analyzing endotoxin lipid a by liquid chromatography-mass spectrometry
  • A method for analyzing endotoxin lipid a by liquid chromatography-mass spectrometry
  • A method for analyzing endotoxin lipid a by liquid chromatography-mass spectrometry

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preparation example Construction

[0032] Preparation of ammonium salt of EDTA: 700 mg of EDTA was dissolved in 1 ml of 30% ammonia water, freeze-dried and stored, and all reagents were of analytical grade.

[0033] It is found through research that the present invention is suitable for analyzing the cell membrane lipid A of Gram-negative bacteria or a small amount of cells. Specifically, the analysis strain in the embodiment is Campylobacter jejuni C. jejuni strains are examples and are not intended to limit the scope of the present invention.

Embodiment 1

[0035] A method for analyzing lipooligosaccharide lipid A by liquid chromatography-mass chromatography (HPLC-MS), comprising the steps of:

[0036] S1. Strain treatment (Campylobacter jejuni C. jejuni Strains 81176 and 11168): Suspend the dried bacterial cells evenly in the aqueous solution, control the cell concentration to 1 mg / mL, freeze at -40°C or -44°C or at -40°C or -44°C or -46°C or -48°C or -50°C) freeze-dried storage;

[0037] S2. Extract endotoxin lipid A: hydrolyze the freeze-dried cells, centrifuge the mixture after cooling at 0 or 5 or 8 or 12 or 15 or 21 or 25°C, add a volume ratio to the washed precipitate A 4:4:1 mixed solution of chloroform, methanol, and ammonium acetate was centrifuged, the supernatant was collected, and dried with nitrogen;

[0038] S3. HPLC-MS analysis:

[0039] Chromatographic conditions: amide column inner diameter 0.3mm, length 10cm; mobile phase A: 10 mM ammonium acetate in water; mobile phase B: 96% tetrahydrofuran solution conta...

Embodiment 2

[0048] Hydrolysis condition optimization

[0049] After step S1 was completed, 50 μg of freeze-dried cells were divided into 3 groups, suspended in 150 μL of 1% acetic acid, 150 μL of 50 mM pH4.0 ammonium citrate (NH 4 Cit) and 150 μL of 50 mM pH5.5 ammonium citrate, the suspension was heated to 100 °C, and the hydrolysis reaction continued for 1 hour under the same conditions. After the hydrolyzate was cooled, centrifuged, and washed, chloroform-methanol-water with a volume ratio of 4:4:1 was added to extract lipid A and the MALDI-TOF spectrum was obtained according to the MALDI-TOF / TOF analysis method described, as shown in figure 2 shown.

[0050] The target structure ions m / z 1921.4, m / z 1798.4 and m / z 1879.4 can be detected in the three hydrolysis methods, compared with figure 2 In (A) there are fewer impurity ions, and the proportion of target ions is relatively high, so 1% acetic acid can hydrolyze lipooligosaccharide LOS more thoroughly, which is the preferred hydr...

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Abstract

The invention discloses a method for analysis of endotoxin lipoid A by HPLC-MS (high performance liquid chromatography mass spectrometry). The method includes the steps of: S1. strain treatment; S2. extraction of endotoxin lipoid A: subjecting freeze-dried cells to hydrolysis reaction, after cooling, centrifuging the mixture, adding a mixed solution of chloroform, methanol and ammonium acetate ina volume ratio of 4:4:1 into a cleaned precipitate, carrying out centrifugation, then collecting the supernate, and performing blow-drying with nitrogen; S3. HPLC-MS analysis: acquiring a fragment ionspectrum by an enhanced product ion scanning mode to determine the structural information of lipoid A. The invention establishes a method for analysis of endotoxin lipoid A by HPLC-MS, on-line separation and identification are carried out to determine the structural information of lipoid A; 1% acetic acid is employed for hydrolysis, and the hydrolysis is thorough; moreover, 1M concentration ammonium acetate is added to enhance the extraction effect of lipoid A. The method provided by the invention can detect the structure of lipoid A with a molecular weight of 2002.2 and 2126.4 that cannot bedetected by MALDI-TOF, and can be widely applicable to lipid A analysis of different Gram-negative bacteria.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry mass spectrometry identification, and more specifically relates to a method for analyzing endotoxin lipid A by high performance liquid chromatography-mass spectrometry (HPLC-MS). Background technique [0002] Campylobacter jejuni (C. jejuni ) is a major pathogenic bacterium of zoonotic and infectious diarrhea worldwide. It is a typical Gram-negative bacterium. Its infection rate is generally on the rise around the world. Cases caused by Campylobacter jejuni were listed as common foodborne diseases in 1980. In developed countries such as Europe and the United States, such as the United States and New Zealand, foodborne bacterial infections caused by C. jejuni have surpassed Salmonella and Shigella to a certain extent. Poultry animals are the main hosts of C. jejuni, as are unprocessed dairy products and untreated water sources. Acute gastroenteritis is C .jejuni Typical symptoms o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062G01N2030/067
Inventor 胡锐杨运煌蒋滨刘买利
Owner WUHAN INST OF PHYSICS & MATHEMATICS CHINESE ACADEMY OF SCI
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