CN109001360A - A kind of method of high-throughput detection phospholipid in lipid content - Google Patents

Abstract

The invention relates to a method for high-throughput detection of phospholipid content in oils and fats. The method uses ultra-high performance liquid chromatography separation and electrospray ion source high-resolution tandem time-of-flight mass spectrometry positive and negative ion scanning modes to characterize and analyze the phospholipid components in the sample. Quantitative, which belongs to the field of bioanalytical chemistry. In the method, the sample to be tested is pretreated, extracted with a reaction solvent and an extractant, and detected by liquid chromatography-mass spectrometry. The method is simple in operation, high in selectivity, and can quickly and accurately determine phospholipids in oil.

Description

A method for high-throughput detection of phospholipid content in oil

technical field

The invention relates to a method for high-throughput detection of phospholipid content in oil, belonging to the field of biochemical analysis and detection.

Background technique

Oil is mainly composed of triglycerides, which is an important part of people's food. It can not only improve the taste and flavor of food, but also provide calories and nutrients for the human body. The content of phospholipids in oils is quite different due to different raw materials. The phospholipid components affect the nutritional value of oils to a large extent, and also have a certain impact on the quality of oils. Studies have shown that the quality of phospholipids in oils will affect the color and flavor of oils, and can promote the photooxidation of oils, which in turn affects the storage properties of oils. Therefore, the detection and separation of phospholipid components in oil is of great significance for revealing its mechanism of action in oil quality changes and for precise control of oil products during processing and storage.

The types and structures of phospholipid components are complicated, and its analysis methods have also experienced the development stage from thin-layer chromatography to high-performance liquid chromatography to liquid chromatography-mass spectrometry. Due to the limitation of technical level and other factors, up to now, the phospholipid components in oils Most of the detection is still at the stage of relative quantification, so it is impossible to accurately know the specific content of phospholipids in oil.

Contents of the invention

In order to solve the limitations of the prior art, the present invention provides a method for high-throughput detection of phospholipid content in oil, which is efficient and accurate.

Technical solutions

The invention adopts ultra-high performance liquid phase (UPLC) separation and tandem time-of-flight mass spectrometry (Triple-TOF-MS/MS) to simultaneously measure the content of phospholipid components in oil. Among them, the ultra-high performance liquid chromatography uses methanol-water solution containing formic acid as the mobile phase, and the AB6600Triple TOF mass spectrometer can perform primary and secondary mass spectrometry data acquisition based on the IDA function under the control of the control software (Analyst TF 1.7, AB Sciex). Internal standard method for qualitative and quantitative analysis of phospholipid components. The specific plan is as follows:

A method for high-throughput detection of phospholipid content in oils, comprising the steps of:

(1) Preparation of oil sample test solution

Take 10 μL oil sample, add it to 390 μL water, then add 9 μL mixed internal standard solution and 951 μL extraction solution, then vortex, ultrasonically extract and centrifuge, take the supernatant, spin the supernatant dry, then add 100 μL complex solution dissolved to obtain the oil sample solution to be tested;

The mixed internal standard solution consists of 10ppm d15:0/18:1 phosphatidylethanolamine internal standard solution, 10ppmd18:1 hemolytic phosphatidylcholine internal standard solution and 100ppm d15:0/18:1/15:0 glycerol MTBE/methanol internal standard solution composition of triester;

Described extraction solution is the methyl tert-butyl ether/methanol solution that volume ratio is 5:1;

(2) Detection

Use UPLC-Triple-TOF-MS/MS to measure the sample to be tested, and determine whether the sample contains the target phospholipid component by comparing the retention time/first-order mass spectrum and second-order mass spectrum information in the sample, and then calculate it according to the peak area normalization method The relative mass percentage of each component;

The UPLC detection was carried out on an ultra-high performance liquid chromatograph with an injection volume of 6 μL. The mobile phase included phase A and phase B. Phase A was a mixed solution of ammonium acetate in a water/acetonitrile mixed solvent, and ammonium acetate was mixed in the mixed solution. The concentration in the solution is 10mM; phase B is a mixed solution of ammonium acetate in acetonitrile/isopropanol mixed solvent, and the concentration of ammonium acetate in the mixed solution is 10mM; the flow rate of the mobile phase is 0.3mL/min, and the column temperature is 50 ℃;

MS/MS detection is carried out on a mass spectrometer, and the conditions for positive ion detection mass spectrometry: ion source spray voltage is 5500V, ion source temperature is 550°C, cluster dissociation voltage is 100V, the pressure of ion source gas 1 and gas 2 are both 60psi, and the air curtain pressure The intensity is 30psi, the scanning range is 200~1000u; the negative ion detection mass spectrometry conditions: the ion source spray voltage is -4500V, the ion source temperature is 400°C, the declustering voltage is 100V, the pressure of the ion source gas 1 and 2 is 60psi, the air curtain The air pressure is 35psi, and the scanning range is 500~1000u.

Further, in step (1), in the mixed internal standard solution, 10ppm of d15:0/18:1 phosphatidylethanolamine internal standard solution, 10ppm of d18:1 hemolytic phosphatidylcholine internal standard solution and 100ppm of d15 :0/18:1/15:0 The volume ratio of the methyl tert-butyl ether/methanol internal standard solution of triglycerides is 1:1:1.

Further, in step (1), the vortex shaking time is 60s.

Further, in step (1), the centrifugation speed is 3000rpm, and the centrifugation time is 15-20min.

Further, in step (1), the complex solution is a dichloromethane/methanol mixture with a volume ratio of 1:1.

Further, in step (2), in the water/acetonitrile mixed solvent, the volume ratio of water/acetonitrile is 2:3.

Further, in step (2), in the acetonitrile/isopropanol mixed solvent, the volume ratio of acetonitrile/isopropanol is 1:9.

Further, in step (2), during the UPLC process, the gradient elution program is: at 0.01min, the volume ratio of phase A/B is 60/40, gradually change the gradient to 12min, become 0/100, and maintain until At 13.5min, the gradient was further changed to 60/40 at 13.7min and maintained at 18.0min.

The beneficial effects of the present invention are: the present invention pretreats the oil sample to be tested, then extracts it, and then performs ultra-high performance liquid chromatography-mass spectrometry detection. The method is simple in operation, high in accuracy and good in selectivity, can rapidly detect and perform qualitative and quantitative determination of phospholipid components in oil, and has high detection sensitivity.

Description of drawings

Fig. 1 is that rapeseed oil sample phospholipid detects positive ion mode TIC chromatogram in embodiment 1;

Fig. 2 is that rapeseed oil sample phospholipid detects negative ion mode TIC chromatogram in embodiment 1;

Fig. 3 is the QC detection result in embodiment 1.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. In the following examples, the ultra-high performance liquid chromatograph model used for UPLC detection is 1290UPLC, Agilent, and the chromatographic column is Kinetex C18 (2.1×100 mm, 1.7 μm; Phenomen, USA); the mass spectrometer used for MS/MS detection is AB 6600 Triple TOF mass spectrometer, but not limited thereto.

Example 1

Detection of phospholipid components in rapeseed oil samples

A method for high-throughput detection of phospholipid content in oils, comprising the steps of:

(1) Preparation of oil sample test solution

Take 10 μL rapeseed oil sample, add it to 390 μL water, then add 9 μL mixed internal standard solution and 951 μL extract solution, then vortex and shake for 60 s, ultrasonically extract for 10 min and centrifuge (centrifugation speed is 3000 rpm, centrifugation time is 15 min), take the supernatant solution, repeated 3 times, the supernatants were combined and spin-dried, and then 100 μL reconstitution solution was added to redissolve to obtain the oil sample test solution;

The mixed internal standard solution consists of 3 μL 10 ppm d15:0/18:1 phosphatidylethanolamine internal standard solution, 3 μL 10 ppm d18:1 hemolytic phosphatidylcholine internal standard solution and 3 μL 100 ppm d15:0/18:1/15: 0 triglyceride MTBE/methanol internal standard composition;

Described extraction solution is the methyl tert-butyl ether/methanol solution that volume ratio is 5:1;

The complex solution is a dichloromethane/methanol mixture with a volume ratio of 1:1.

(2) Detection

Use UPLC-Triple-TOF-MS/MS to measure the sample to be tested, and determine whether the sample contains the target phospholipid component by comparing the retention time/first-order mass spectrum and second-order mass spectrum information in the sample, and then calculate it according to the peak area normalization method The relative mass percentage content of each component; do a quality control (QC) every 5 samples to ensure the detection accuracy;

The UPLC test was carried out on an ultra-high performance liquid chromatograph with an injection volume of 6 μL, and the mobile phase included phase A and phase B, wherein phase A was formed in a mixed solvent of ammonium acetate in water/acetonitrile (2:3, V/V) The mixed solution, the concentration of ammonium acetate in the mixed solution is 10mM; Phase B is the mixed solution formed by ammonium acetate in acetonitrile/isopropanol (1:9, V/V) mixed solvent, the ammonium acetate in the mixed solution The concentration is 10mM; the flow rate of the mobile phase is 0.3mL/min, and the column temperature is 50°C;

During the UPLC process, the gradient elution program is: at 0.01min, the volume ratio of phase A/phase B is 60/40, gradually change the gradient to 12min, become 0/100, and maintain it to 13.5min, further change the gradient to 13.7 min changed to 60/40 and maintained to 18.0min;

The MS/MS test was carried out on a mass spectrometer, and the conditions of positive ion detection mass spectrometry were as follows: ion source spray voltage (IS) was 5500V, ion source temperature (TEM) was 550°C, declustering voltage (DP) was 100V, ion source gas 1 and 2, the air pressure is 60psi, the air curtain air pressure is 30psi, and the scanning range is 200-1000u.

Negative ion detection mass spectrometry conditions: ion source spray voltage (IS) is -4500V, ion source temperature (TEM) is 400°C, cluster dissociation voltage (DP) is 100V, the pressure of ion source gas 1 and 2 is 60psi, the air curtain pressure is It is 35psi, and the scanning range is 500～1000u.

Fig. 1 is the positive ion mode TIC chromatogram of the rapeseed oil sample phospholipid detection in embodiment 1; Fig. 2 is the negative ion mode TIC chromatogram of the rapeseed oil sample phospholipid detection in embodiment 1.

Use ProteoWizard software to convert the original mass spectrum into mzXML format, and then use XCMS to do retention time correction, peak identification, peak extraction, peak integration, peak alignment, etc., minfrac is set to 0.5, cutoff is set to 0.6, and this laboratory is based on XCMS Software, self-written R program package and self-built secondary database of phospholipids were used to identify phospholipids, and then the absolute content of phospholipids in the sample was calculated according to the added IS concentration and its peak area, the peak area of each phospholipid substance, and RF data into the corresponding formula.

After testing, a total of 32 kinds of phospholipids were detected in rapeseed oil, including 4 phosphatidylcholines, 14 phosphatidylethanolamines (PE), 11 phosphatidic acids, and 3 phosphatidylglycerols.

The QC test results are shown in Figure 3. It can be seen from Figure 3 that the peaks of the three groups of quality control basically overlap, indicating that the instrument test results under this method are very stable, ensuring the accuracy of the experiment.

Claims (8)

1. a kind of method of high-throughput detection phospholipid in lipid content, which comprises the steps of:

(1) oil sample prepare liquid is prepared

10 μ L oil samples are taken, are added in 390 μ L water, 9 μ L mixing inner mark solutions and 951 μ L extracting solutions are then added, then Be vortexed concussion, is centrifuged after ultrasonic extraction, takes supernatant, supernatant is spin-dried for, and 100 μ L are then added and redissolve liquid redissolution, obtain oil Rouge sample prepare liquid；

The d15:0/18:1 phosphatidyl-ethanolamine internal standard solution that inner mark solution is mixed by 10ppm, 10ppmd18:1 hemolytic phosphorus The methyl tertiary butyl ether(MTBE) of the d15:0/18:1/15:0 triglycerides of phosphatidylcholine internal standard solution and 100ppm/methanol internal standard solution composition；

The extracting solution is methyl tertiary butyl ether(MTBE)/methanol solution that volume ratio is 5:1；

(2) qualitative detection

Sample to be tested is measured using UPLC-Triple-TOF-MS/MS, passes through retention time/first mass spectrometric and two in contrast sample Whether grade Information in Mass Spectra determines in sample containing target phospholipid ingredient, the phase of each ingredient is then acquired by areas of peak normalization method To mass percentage；

UPLC detection carries out in Ultra Performance Liquid Chromatography instrument, and sample volume is 6 μ L, and mobile phase includes A phase and B phase, and wherein A phase is The mixed solution that ammonium acetate is formed in water/acetonitrile in the mixed solvent, concentration of the ammonium acetate in mixed solution are 10mM；B phase is The mixed solution that ammonium acetate is formed in acetonitrile/isopropyl alcohol mixed solvent, concentration of the ammonium acetate in mixed solution are 10mM；Stream The flow velocity of dynamic phase is 0.3mL/min, and column temperature is 50 DEG C；

MS/MS detection carries out on mass spectrograph, positive ion detection Mass Spectrometry Conditions: ion source spray voltage is 5500V, ion source temperature Degree is 550 DEG C, and solution cluster voltage is 100V, and the air pressure of ion source gas 1 and 2 is 60psi, and gas curtain air pressure is 30psi by force, scanning Range is 200~1000u；Anionic textiles Mass Spectrometry Conditions: ion source spray voltage is -4500V, and ion source temperature is 400 DEG C, Solution cluster voltage is 100V, and the air pressure of ion source gas 1 and 2 is 60psi, and gas curtain air pressure is 35psi, scanning range 500 by force ~1000u.

2. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that described in step (1) It mixes in inner mark solution, the d18:1 hemolytic phosphatidyl gallbladder of the d15:0/18:1 phosphatidyl-ethanolamine internal standard solution of 10ppm, 10ppm The methyl tertiary butyl ether(MTBE) of the d15:0/18:1/15:0 triglycerides of alkali internal standard solution and 100ppm/methanol internal standard solution volume ratio is 1:1:1。

3. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that in step (1), be vortexed The concussion time is 60s.

4. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that in step (1), centrifugation Revolving speed is 3000rpm, centrifugation time 15-20min.

5. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that described in step (1) Redissolve the methylene chloride/methanol mixed liquor that liquid is volume ratio 1:1.

6. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that in step (2), water/ Acetonitrile in the mixed solvent, water/acetonitrile volume ratio are 2:3.

7. the method for high-throughput detection phospholipid in lipid content as described in claim 1, which is characterized in that in step (2), second In nitrile/isopropyl alcohol mixed solvent, acetonitrile/isopropanol volume ratio is 1:9.

8. the method for high-throughput detection phospholipid in lipid content as described in any one of claim 1 to 7, which is characterized in that step (2) in, during UPLC, gradient elution program are as follows: when 0.01min, A phase/B phase volume ratio is 60/40, gradually changes gradient To 12min, become 0/100, and maintain to arrive 13.5min, further changes gradient to 13.7min and become 60/40, and maintain to arrive 18.0min。