A kind of high-efficiency liquid phase detection and content determination method of 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: 2020-03-24
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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of high-efficiency liquid phase detection and content determination method of ectoine
  • A kind of high-efficiency liquid phase detection and content determination method of ectoine
  • A kind of high-efficiency liquid phase detection and content determination method of ectoine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Selection of chromatographic conditions

[0053] 1 Selection of Column

[0054] Choose C18 and ADME two chromatographic columns. Tetrahydropyrimidine has almost no retention in traditional chromatographic columns dominated by C18 functional groups, with a retention time of about 3.011 min (pure water phase), and peaks together with the solvent peak. The accuracy is poor and the analysis and determination of the compound cannot be completed. The ADME chromatographic column with adamantyl group with a cage structure was selected to successfully make the tetrahydropyrimidine baseline separation, with good accuracy, method repeatability and precision, and it can be used for the determination of the compound.

[0055] 2 Selection of column temperature

[0056] The optimal temperature range for selecting ADME is 25-40 ℃, more preferably 30 ℃.

[0057] 3 Selection of detection wavelength

[0058] Take an appropriate amount of tetrahydropyrimidine standard, dissolve it in wate...

Embodiment 2

[0071] Example 2 Methodology establishment

[0072] 1 Establish a standard curve

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

[0074] Table 1 Linear regression equation and linear range of tetrahydropyrimidine

[0075]

[0076] 2 Precision test

[0077] Take the standard solution (concentration of 50.0 μg / mL), inject the sample according to the chromatographi...

Embodiment 3

[0092] Example 3 Determination of the content of tetrahydropyrimidine in the sample

[0093] 1 Test product

[0094] The test product is the extract of the bacterial fermentation cells of 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℃, 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 mobile phase ratio A:B is 5:95, and the injection volume is 5 μL.

[0097] 3 Preparation of related solutions and mobile phases

[0098] 3.1 Preparation of potassium dihydrogen phosphate aqueous solution: accurately measure 2.7218 g of potassium dihydrogen phosphate, add 900 ml of water, adjust the pH to 3.0 with a volume content of 50% glacial acetic acid aqueous solution, and t...

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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 present invention relates to the field of analytical chemistry, and more specifically to a high-performance liquid phase detection and content determination method of tetrahydropyrimidine. 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) is widely present in In most cells of moderately halophilic bacteria, cyclized amino acid derivatives with osmotic pressure compensation synthesized by certain 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 a wide range of application prospects in the food industry, enzyme technology production, genetic engineering, polymer production, biomedicine, and bioremediation of environment...

Claims

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Application Information

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Patent Type & Authority Patents(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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