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Phospholipid detection method and application

A detection method and phospholipid technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of weak elution ability of phospholipids, large damage to chromatographic column hardware, difficult separation, etc., and achieve short separation time and short retention time , good separation effect

Pending Publication Date: 2019-07-12
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

Regarding the detection of phospholipids, normal phase or reversed phase liquid chromatography methods are generally used at home and abroad. Normal phase chromatography mostly uses silica gel chromatography columns, which generally only have a strong response to phosphatidylcholine and phosphatidylethanolamine, and other types of phospholipids. The removal ability is weak, and the equilibration time required for the detection is too long, so it is not suitable for rapid analysis of complex biofilm phospholipids
The reversed-phase chromatography is based on the difference in hydrophobicity caused by the difference in the length of the acyl chain of the phospholipid compound, so the various types of phospholipids are easily eluted within the same time period, resulting in overlapping peaks
In addition, it is also reported that mass spectrometry detects phospholipids on the detector, but its price is high, the maintenance cost is high, and the ionization of phospholipid molecules is inhibited, which affects the peak shape.
In contrast, the HPLC evaporative light detection method is simple to operate, high in accuracy, and low in cost. Related methods have been explored in existing studies, but there are still some problems that need to be improved. For example, n-hexane and iso-hexane are mostly used as mobile phases. Propanol, which will cause great damage to hardware such as chromatographic columns, liquid phase pumps, and split rings, will shorten their lifespan, and the reported detection methods can only detect a limited number of phospholipids such as phosphatidylcholine and phosphatidylethanolamine, or the equilibrium time Long, peak tailing, and difficult separation lead to inaccurate quantification. With the increase of phospholipid molecular species, the required separation time increases. Choosing the appropriate elution gradient and chromatographic parameters is very important for both separation effect and analysis efficiency

Method used

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  • Phospholipid detection method and application
  • Phospholipid detection method and application
  • Phospholipid detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Weigh 0.1 mg each of phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and glycerol phosphatidic acid, and dissolve in 1 mL of chloroform-methanol solution (2:1, v / v) to make 100 mg / L The single-standard solution was passed through a 0.45 μm membrane, and then detected by ultra-high performance liquid chromatography time-of-flight mass spectrometry (UPLC-Q-TOF-MS).

[0033] The chromatographic experimental conditions are CORTECS UPLC HILIC column (φ2.1mm×150mm, 1.6μm); mobile phase: A is acetonitrile, B is 10mM ammonium acetate aqueous solution (containing 0.1% formic acid, pH ≈ 3.65); elution program: 0 ~2min, 95%A, 5%B; 2~15min, 95%~70%A, 5%~30%B; 15~17min, 70%~60%A, 30%~40%B; 17~17.1 min, 60%~95%A, 40%~5%B; 17.1~20min, 95%A, 5%B; flow rate 0.3mL / min; injection volume: 1μL.

[0034] The mass spectrometry experiment conditions are electrospray positive and negative ion mode (ESI, scan range m / z 50~1500, scan time: 0.2s, capillary...

Embodiment 2

[0037] In view of the insufficient method in Example 1, this example adopts a high-performance liquid phase system (HPLC) and a HILIC chromatographic column with a wider diameter for phospholipid analysis.

[0038] To simplify the sample preparation process, weigh 0.1 mg each of phosphatidylglycerol, glycerol phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, and dissolve them in 1ml of chloroform-methanol solution (2:1, v / v) was formulated into a 100mg / L mixed standard solution, passed through a 0.45μm membrane, and then detected by high performance liquid chromatography. The detection condition is XBridge TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate is 1.0mL / min, the column temperature is 35°C, the injection volume is 10μL, the drift tube temperature is 50°C, and the gas pressure is 2.50bar.

[0039] Acetonitrile was mixed with ammonium acetate as mobile phase for elution. ...

Embodiment 3

[0041] Weigh 0.1 mg each of phosphatidylglycerol, glycerol phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, and dissolve them in 1 mL of chloroform-methanol solution (2:1, v / v) Prepare a 100 mg / L mixed standard solution, pass through a 0.45 μm membrane, and then enter high-performance liquid chromatography for detection. The detection condition is XBridge TM HILIC chromatographic column (φ4.6mm×250mm, 5μm), the flow rate is 1.0mL / min, the column temperature is 35°C, the injection volume is 10μL, the drift tube temperature is 50°C, and the gas pressure is 2.50bar.

[0042] Acetonitrile was mixed with ammonium acetate as mobile phase for elution. Start with acetonitrile: ammonium acetate = 95:5, do 2min equal concentration elution, then reduce the concentration of acetonitrile, reach acetonitrile:ammonium acetate = 90:10 in 8min, reach acetonitrile:ammonium acetate = 80:20 in 10min, 17min It reac...

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Abstract

The invention discloses a phospholipid detection method and application. A phospholipid detection method is eluted with acetonitrile and ammonium acetate, and a phospholipid detection eluent comprisesacetonitrile and ammonium acetate, wherein by volume percentage, the acetonitrile accounts for 50% to 95%, and the ammonium acetate accounts for 5% to 50%. An application of the phospholipid detection method is used for detection of phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidyl ethanolamine, glycerophosphatidic acid, sphingomyelin and lysophosphatidylcholine. The analysis method provided by the invention has the advantages of simple operation, large number, good peak shape and short retention time of phospholipids analyzed, convenience and speed, reliability andpracticality.

Description

technical field [0001] The invention belongs to the technical field of phospholipid detection, and in particular relates to a phospholipid detection method and application. Background technique [0002] Phospholipids are the main components of biological membranes. According to its polar acyl head group, phospholipids are divided into six categories: phosphatidyl cholines (Phosphatidyl cholines, PC), phosphatidylethanolamines (Phosphatidylethanolamines, PE), phosphatidyl serines (Phosphatidyl serines, PS), phosphatidyl glycerol (phosphatidyl Glycerol, PG), glycerol phosphatidic acid (phosphatidic acid, PA) and sphingolipid (SM), the Sn-2 acyl chain of phospholipids can be degraded into lysophospholipids, such as lyso-phosphatidylcholine (LPC ). The phospholipid bilayer is the basic structure of the cell membrane. People's understanding of the life function of phospholipids begins with the basic components of the biological membrane. Almost all the functions of cells at the...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06
Inventor 常明赵品贞陶冠军刘睿杰张涛王兴国金青哲
Owner JIANGNAN UNIV
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