阅读说明：本技术 一种HPLC-APCI/MS检测母乳中rac-OPL对映异构体的方法 (Method for detecting rac-OPL enantiomer in breast milk by HPLC-APCI/MS ) 是由 张兰威 陈玉洁 于 2019-03-06 设计创作，主要内容包括：本发明方法属于食品检测领域,涉及一种检测母乳中9种甘油三酯位置异构体(rac-OPO/rac-OOP,rac-PPO/rac-POP,rac-LaOO/rac-OLaO,和rac-OPL/rac-PLO/rac-POL)的方法。本发明主要包括以下步骤：1)母乳中甘油三酯的定性2)一维反相色谱靶向收集目标甘油三酯分子；3)二维银离子HPLC APCI/MS检测九种位置异构体；4)检测方法在母乳中的应用。本发明方法能够同时检测9种母乳中位置异构体,并且具有操作简单、分离度好,稳定性高等优点。(The invention belongs to the field of food detection, and relates to a method for detecting 9 triglyceride positional isomers (rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO, and rac-OPL/rac-PLO/rac-POL) in breast milk. The invention mainly comprises the following steps: 1) characterization of triglycerides in breast milk 2) targeted collection of target triglyceride molecules by one-dimensional reverse phase chromatography; 3) detecting nine position isomers by two-dimensional silver ion HPLC APCI/MS; 4) the use of the detection method in breast milk. The method can simultaneously detect the position isomers in 9 breast milks, and has the advantages of simple operation, good separation degree, high stability and the like.)

1. A method for detecting 1-oleic acid-2-palmitic acid-3-linoleic acid glyceride enantiomer in breast milk by HPLC-APCI/MS comprises the following steps: the method is characterized by being capable of effectively separating the enantiomers rac-OPL1 and rac-OPL2 of 1-oleic acid-2-palmitic acid-3-linoleic acid glyceride (rac-OPL); the method breaks through the bottleneck of triglyceride detection in breast milk, and is carried out according to the following steps:

1) lipid extraction: taking 2ml of breast milk, extracting total lipid by a Folch method, carrying out rotary evaporation on an extracting solution until the extracting solution is dry, dissolving the extracting solution by using normal hexane, and filtering the solution by using a 0.22 mu m organic phase filter membrane for later use; 2) one-dimensional chromatography: injecting a fat sample of 0.5 mg/ml into a liquid chromatography-triple quadrupole mass spectrometer connected with a C18 chromatographic column, wherein the injection volume is 5 mul, and determining the retention time of the definite OPL by using an ESI MS/MS technology, then injecting a fat sample of 10mg/ml into a liquid chromatography connected with a C18 chromatographic column, wherein the injection volume is 20 mul, performing targeted collection on target triglyceride molecules by using an automatic collector, drying the target triglyceride molecules by using nitrogen for standby, and re-dissolving the target triglyceride molecules to 50 mul by using normal hexane for standby, and 3) performing two-dimensional chromatography: the detection of the positional isomer of the target TAG,

the method comprises the following steps of (1) detecting OPL collected in one-dimensional chromatography by utilizing a chiral chromatographic column and matching with a high performance liquid chromatography-mass spectrometer with APCI-MS, wherein the mass spectrum conditions are as follows: APCI ion source, cation mode, fragmentor: 70V, scanning range: 350-1200; 4) the relative amounts of rac-OPL1 and rac-OPL2 were calculated from the peak areas of the rac-OPL1 and rac-OPL2 chromatographic peaks extracted from the total ion flow graph of breast milk samples.

2. The method of claim 1, wherein the separation of the enantiomer of rac-OPL is optimized by using two-dimensional chromatography and APCI/MS to effectively exclude interference from other triglyceride molecules in the sample.

3. The method of claims 1-2, wherein the chromatographic peaks of rac-OPL1 and rac-OPL2 extracted from the total ion flow graph of the breast milk sample are calculated for relative content, so that the accuracy of enantiomer quantification can be effectively improved.

4. The mass to charge ratio M/Z used to extract the chromatographic peaks of rac-OPL1 and rac-OPL2 from a total ion flow graph in a breast milk sample according to the methods of claims 1-3 was 857.5.

5. The method of claims 1-4, wherein the efficient targeted collection of OPL is performed using C18 reverse phase chromatography:

the column was Zorbax Eclipse Plus C18(5 μm,250 mm. times.4.6 mm, Agilent technologies, USA) and the liquid phase elution conditions are shown in Table 1.

Table 1C18 non-aqueous liquid chromatography mobile phase gradient elution conditions.

6. The method according to claims 1-5, characterized in that rac-OPL1 and rac-OPL2 can be efficiently separated using chiral chromatography with a Lux Cellulose-1(3 μm,250 mm x 4.6 mm, Phenomenex, Torrance, CA, U.S. A.) chiral chromatography column and liquid phase elution conditions: : 0-180min 90% A +10% B, A being n-hexane, B being n-hexane: isopropanol (99:1, v/v), flow rate 0.5 ml/min.

7. The method of claims 1-6, detecting two enantiomers of rac-OPL in breast milk, rac-OPL1 and rac-OPL2.

Technical Field

The invention belongs to the field of lipid detection, and relates to a method for detecting 1-oleic acid-2-palmitic acid-3-linoleic acid glyceride (rac-OPL) enantiomer in breast milk by using HPLC-APCI/MS.

Background

Triglycerides are the most predominant component of breast milk lipids, and account for approximately 97-98% of milk fat. The distribution of the positions of the fatty acids in the triglycerides determines their physiological function. The distribution of the fatty acids in the triglyceride in the mother milk is not random, for example, 70% or more of palmitic acid is in the Sn-2 position, while unsaturated fatty acids such as oleic acid and linoleic acid are distributed in the Sn-1 and 3 positions in many cases. The palmitic acid can be prevented from forming hard-to-absorb soap calcium with calcium ions when being distributed at the Sn-2 position, so that the absorption of the palmitic acid and the calcium ions is promoted. There is a great difference in the digestive absorption of fat in the infant intestine compared to adults. The enzymes that hydrolyze triglycerides primarily in the infant gut are gastric lipase, protein 2 lipase associated with the pancreas, and bile salt-stimulated hydrolysis lipase, rather than pancreatic triglyceride lipase in the adult gut. Wherein the gastric lipase hydrolyzes up to 60% of triglycerides in breast milk and the GL lipase has specificity for the Sn-3 position. Studies have shown that long-chain fatty acids hydrolyzed in the stomach by triglycerides activate pancreatic lipase activity in the infant's intestine, thereby promoting the digestive absorption of triglycerides in the intestine.

Oleic-palmitic-linoleic acid glyceride (OPL) is the most abundant triglyceride in chinese breast milk, with the main positional isomer rac-OPL (relative content higher than 85%). However, the detection of rac-OPL enantiomer has not been reported. This forms the bottleneck of deeper research on breast milk components and brings difficulties for the infant milk powder formula organization to deeply imitate breast milk. The establishment of the rac-OPL enantiomer detection method breaks through the bottleneck of breast milk triglyceride component detection, and the operation is simple, thereby being beneficial to component monitoring in the production process of the infant formula milk powder.

Disclosure of Invention

The purpose of the invention is as follows: provides a method for single-column two-dimensional liquid chromatography-APCI/MS of rac-OPL enantiomer in breast milk.

The technical problem is as follows: 1) since the detection of rac-OPL enantiomer in breast milk has not been reported. 2) The chiral chromatography technology is utilized to solve the problem of detecting rac-OPL enantiomer, and 3) the two-dimensional chromatography and the APCI/MS technology effectively eliminate the interference of TAGs except the target TAGs in the breast milk.

The technical scheme is as follows: the invention relates to a method for detecting rac-OPL enantiomer in breast milk. The method can eliminate interference of other triglyceride molecules in a complex sample, and can monitor the relative content of rac-OPL1 and rac-OPL2 enantiomers. The requirements of simple operation and easy quantification are met. The technical scheme adopted by the invention is as follows:

(1) accurate detection of rac-OPL enantiomer in breast milk

Sample pretreatment: extracting the total lipid of a sample by a Folch method, carrying out rotary evaporation on an extracting solution until the extracting solution is dry, then carrying out constant volume measurement by using normal hexane, and passing through a membrane for standby.Triglyceride one-dimensional reverse phase chromatographic separation process: HPLCC18 reverse phase chromatography for separating triglyceride molecules in sample according to different ECN (carbon atom equivalent), and primarily separating triglyceride molecules in sampleAnd (5) separating. The liquid phase gradient elution conditions are shown in table 1:

TABLE 1C18 gradient elution conditions for non-aqueous liquid chromatography mobile phase

Characterization of triglycerides. The structure of the triglyceride molecules was deduced using ionic fragments generated by ESI-MS/MS. And the retention time of the target triglyceride molecule (OPL) is well defined. The mass spectrometry conditions were as follows: ESI ion source, fragment: 70V, scanning range: 350-1200.Targeted collection of target triglyceride molecules. The fractions of the C18 reverse phase chromatogram were collected at a rate of 2 min/tube using an automatic collector, and the tubes with the corresponding retention times of the OPL triglyceride molecules were collected and blown dry with nitrogen for further use.

(2) The separation of rac-OPL is the establishment of the detection method of rac-OPL1 and rac-OPL2.

Preparing a standard solution: dissolving 25mg rac-OPL standard substance with n-hexane into a 25mL volumetric flask to prepare 1mg/mL standard stock solution, and passing through a membrane of 0.22 μm for later use.Optimizing the liquid phase condition of chiral chromatography. The chiral chromatographic column is matched with high performance liquid chromatography with an Evaporative Light Scattering Detector (ELSD) to detect the mixed standard substance of 1 mg/mL. The conditions for the evaporative light detector were: the drift tube temperature was constant at 40 deg.C, the sparge gas was nitrogen, and the pressure was 3.5 bar. An n-hexane-isopropanol system is adopted to optimize the flow rate, the mobile phase gradient and the chromatographic column temperature respectively, and the sample injection volume is 10 mu l. The liquid chromatography conditions of the final chiral chromatography were: 0-180min 90% A +10% B, the mobile phase is n-hexane, B mobileThe phases are n-hexane: isopropanol (99:1, v/v); the flow rate was 0.5 ml/min.Detection of chiral chromatographic HPLC-APCI/MS. The 1mg/L mixed standard was again tested using chiral chromatography in combination with an APCI/MS detector. The mass spectrum conditions are as follows: APCI ion source, fragment: 70V, scanning range: 350-1200.

The technical effect is achieved. The method can effectively separate the enantiomers of rac-OPL, namely rac-OPL1 and rac-OPL2. The bottleneck of fat detection in breast milk and infant formula milk powder is broken through, the target triglyceride component can be effectively and rapidly obtained by utilizing two-dimensional liquid chromatography and APCI/MS technology, the interference of other triglyceride molecules is eliminated, and the detection of enantiomers in breast milk and other complex samples is realized, as shown in figure 1, figure 2 and figure 3.

Description of the drawings: FIG. 1 is a graph of rac-OPL standard separated into rac-OPL1 and rac-OPL2 under elution conditions of the present invention, as analyzed by chiral chromatography HPLC-ELSD; FIG. 2 is a total ion flow graph of a sample of breast milk from an example analyzed by chiral HPLC APCI/MS under elution conditions of the present invention. FIG. 3 is a chromatogram of rac-OPL1 and rac-OPL2 extracted under elution conditions of the present invention from a sample of breast milk from an example analyzed by chiral HPLC APCI/MS.

Detailed description of the preferred embodiments. The present invention will be further illustrated with reference to the following specific embodiments. It should be understood that the following detailed description is illustrative of the invention only and is not intended to limit the scope of the invention.