High-performance liquid detection and content determination method for ectoine

An tetrahydropyrimidine and high performance liquid phase technology, applied in the field of analytical chemistry, can solve the problems of high analysis cost, poor accuracy and long time, and achieve the effects of improving detection efficiency, reducing cost and high reproducibility

Active Publication Date: 2018-06-05
青海出入境检验检疫局检验检疫综合技术中心 +1
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  • Abstract
  • Description
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Problems solved by technology

[0003] About the HPLC analysis method and content determination of ectoine, existing reports in the prior art adopt C18 reverse-phase chromatographic column to measure its content, but ectoine is not retained in C18 reverse-phase chromatographic column, and it goes out together with the solvent peak, and the accuracy is high. poor
Also reported (He Bing, Tao Ping, Chen Ailian, Shi Chaoou. Determination of ectoine and hydroxyl ectoine in moderately halophilic bacteria by hydrophilic interaction chromatography. Analytical Laboratory. Volume 35, Issue 1.93 -96) Use a hydrophilic chromatographic column Merck-SeQuant ZIC-HILIC (150 × 4.6mm, 5 μm), mobile phase acetonitrile: water (0.3% NH4H2PO4, pH 5) = 80: 20 (V / V) ;Flow rate: 1 mL / min; Detection wavelength: 210 nm; Column temperature: Analysis at 30 ℃, the retention time of ectoine is more than 12 minutes, the time is long, and the analysis cost is high
In addition, patent CN105669560A also discloses a method for determining the purity of ectoine, in which UltiMate 3000 Thermo Scientific high-performance liquid chromatography is used to measure ectoine, and the chromatographic column is a TSK-GEL C18 chromatographic column with a column temperature of 30°C and a mobile phase It is 2% acetonitrile, the flow rate is 1mL / min, and the ultraviolet detection wavelength is 210nm. In this method, although the ectoine peak time is fast, the ectoine chromatographic peak overlaps with the solvent peak, and the accuracy is poor.

Method used

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  • High-performance liquid detection and content determination method for ectoine
  • High-performance liquid detection and content determination method for ectoine
  • High-performance liquid detection and content determination method for ectoine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The selection of embodiment 1 chromatographic conditions

[0053] 1 Selection of chromatographic column

[0054] Choose C18, ADME two chromatographic columns. Ectrahydropyrimidine has almost no retention in the traditional chromatographic column mainly composed of C18 functional groups, and the retention time is about 3.011min (pure water phase). The ADME chromatographic column with the adamantane group in the cage structure was selected, and the ectoine baseline was successfully separated with good accuracy, method repeatability and precision, and it can be used for the content determination of this compound.

[0055] 2 Selection of column temperature

[0056] The optimum temperature range for ADME is 25-40°C, more preferably 30°C.

[0057] 3 Selection of detection wavelength

[0058] Take an appropriate amount of ectoine standard substance, add water to dissolve and dilute to make solutions with concentrations of 2 mg / ml, 2 mg / ml and 0.02 mg / ml respectively. The ...

Embodiment 2

[0071] Embodiment 2 Methodological establishment

[0072] 1 to establish a standard curve

[0073] Accurately weigh 10.0 mg of ectoine standard substance, put it in a 10 mL brown measuring bottle, add water to dissolve it and adjust the volume to the mark to prepare a standard solution with a concentration of 1.0 mg / mL, and dilute it with water successively to obtain a series of Standard solutions of different concentrations. According to the chromatographic conditions, 5 μL was injected for determination, and the peak area y was linearly regressed with the standard concentration x (μg / mL). The standard curve thus drawn is figure 2 As shown, it shows that ectoine has a good linear relationship in the range of 0.5-100 μg / mL, and the results are shown in Table 1.

[0074] The linear regression equation, linear range of table 1 ectoine

[0075]

[0076] 2 Precision test

[0077] Take the standard solution (concentration: 50.0 μg / mL), inject samples according to the chrom...

Embodiment 3

[0092] The content determination of ectoine in the sample of embodiment 3

[0093] 1 test article

[0094] The test product is the extract of bacteria fermented in Qinghai Lake, provided by Dr. Zhu Derui, School of Medicine, Qinghai University.

[0095] 2 Chromatographic conditions

[0096] Chromatographic column Shiseido CAPCELL PAK ADME (4.6×250mm, 5μm, Shiseido, Japan), flow rate 1.0mL / min, column temperature 30°C, detection wavelength 204nm, mobile phase A (methanol), mobile phase B (20 mM / L diphosphate Potassium hydrogen aqueous solution, phosphoric acid to adjust the pH to 3.0), the ratio of mobile phase A:B is 5:95, and the injection volume is 5 μL.

[0097] 3 Preparation of relevant solutions and mobile phases

[0098] 3.1 Preparation of potassium dihydrogen phosphate aqueous solution: Precisely measure 2.7218 g of potassium dihydrogen phosphate, add 900m1 of water, adjust the pH to 3.0 with 50% glacial acetic acid aqueous solution by volume, and then add water to m...

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Abstract

The invention provides a high-performance liquid detection and content determination method for ectoine. Chromatographic conditions are as follows: the chromatographic column adopts an adamantine-bonded chromatographic column; the detector adopts a DAD detector; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is methanol; the mobile phase B is an aqueous buffersolution of monopotassium phosphate; and the elution mode adopts isocratic elution. By utilizing the chromatographic conditions, a test solution and a standard solution are injected into a liquid chromatograph, the chromatogram is recorded, the peak area is calculated according to an external standard method, and the content of ectoine in the sample can be accurately determined. The method disclosed by the invention is short in detection time, excellent in separation effect, high in precision and accuracy and low in cost and has an important application value in aspects of strain screening and related quantitative researches during ectoine production.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a method for high-efficiency liquid phase detection and content determination of ectoine. Background technique [0002] Tetrahydropyrimidine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid; 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid; ectoine) widely exists in In the cells of most moderately halophilic bacteria, there are cyclic amino acid derivatives with osmotic pressure compensation synthesized by some microorganisms in response to environmental osmotic pressure stress. Tetrahydropyrimidine plays a very important role in protecting cell membranes, intracellular enzymes and proteins, and can be used as cosmetic moisturizers and enzyme protein stabilizers. It has wide application prospects in the fields of food industry, enzyme technology production, genetic engineering, polymer production, biomedicine, and environmental pollution bioremediation. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
CPCG01N30/06G01N30/88G01N2030/065G01N2030/884
Inventor 苏姗姗李占强张荣魏玉海林童姚志敏赵光跃凤晓博戴达勇
Owner 青海出入境检验检疫局检验检疫综合技术中心
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