阅读说明：本技术 一种同时检测母乳中九种重要位置异构体的方法 (Method for simultaneously detecting nine important positional isomers in breast milk ) 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 simultaneously detecting nine important positional isomers in breast milk comprises the following steps: the method is characterized in that the method not only can simultaneously detect nine position isomers to eliminate the interference of other triglyceride molecules, but also can solve the problem of chromatographic peak overlapping of rac-OPO and rac-OLaO, and breaks through the bottleneck of triglyceride detection in breast milk, and the method 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: targeted collection of targets PPO, OPO, LaOO and OPL;

injecting fat sample 0.5 mg/ml into liquid chromatography-triple quadrupole connected with C18 chromatographic column

In a spectrum combination instrument, the sample injection volume is 5 mu l, and the retention time of a definite target is determined by using an ESI MS/MS technology; then injecting 10mg/ml fat sample into a liquid chromatogram connected with a C18 chromatographic column, wherein the sample injection volume is 20 mul, performing targeted collection on target triglyceride molecules by using an automatic collector, and drying by using nitrogen for later use;

(3) two-dimensional chromatography: detecting positional isomers of the target triglyceride;

one-dimensional chromatography is performed by using silver ion chromatographic column in combination with high performance liquid chromatography-mass spectrometry (HPLC-MS) instrument with APCI-MS

Further detecting positional isomers of the triglyceride molecules collected in (a); the mass spectrum conditions are as follows: APCI ion source, cation mode, fragmentor: 70V, scanning range: 350-1200;

(4) the relative content of positional isomers was calculated from the peak areas of the rac-OPL1 and rac-OPL2 chromatographic peaks extracted from the total ion flow graph of the breast milk sample.

2. The method of claim 1, wherein interference of other triglyceride molecules in the sample is effectively eliminated by using two-dimensional chromatography and APCI/MS technology, so that nine target position isomers can be effectively separated in the silver ion chromatographic column.

3. The method of claims 1-2, characterized in that the overlapping problem of rac-OPO and rac-LaOO chromatographic peaks is effectively solved by APCI/MS extraction chromatographic peak technique.

4. The method of claims 1-3, 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.

5. The method according to claims 1-4, characterized in that it enables efficient separation of rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO, and rac-OPL/rac-PLO/rac-POL using silver ion chromatography on a Varian ChromSphere 5 Lipids (5 μm,250 mm x 4.6 mm, Agilent Technologies, USA): the liquid phase elution conditions were: 0-20 min 70% A +30% B, 20-21min 70% A +30% -57% A +43% B, 21-45min 57% A +43% B, wherein A is n-hexane, B is n-hexane: isopropanol (99:2, v/v), and the flow rate is 0.5 ml/min.

6. The method of claims 1-5, wherein nine positional isomers of rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO, and rac-OPL/rac-PLO/rac-POL are detected in breast milk.

Technical Field

The invention belongs to the field of lipid detection, and relates to a method for simultaneously detecting nine important positional isomers in breast milk.

Background

Maternal milk fat is not only able to provide 50% of the energy and essential fatty acids to infants but also different lipid structures have different physiological functions. Triglycerides are the most predominant component of breast milk lipids, and account for approximately 97-98% of milk fat. The specific location distribution of fatty acids in breast milk triglycerides combined with the specific digestive absorption conditions in infants results in a specific digestive absorption of fat in infants. Such as saturated fatty acid, especially palmitic acid can avoid the palmitic acid and calcium ions to form soap calcium when the palmitic acid is distributed at the Sn-2 position, thereby promoting the absorption of the palmitic acid and the calcium ions. Breast milk contains dozens or even hundreds of triglycerides and a large number of isomers exist. And only one isomer, such as 1, 3-oleic acid-2-palmitic acid (rac-OPO) and 1, 2-oleic acid-3-palmitic acid (rac-OOP), has a physiological function, so that fat digestion and absorption are facilitated.

Therefore, the research on triglyceride isomers in the mother milk fat is beneficial to further research on the relationship between the triglyceride structure and the function. The triglyceride with the highest content in Chinese breast milk is oleic acid-palmitic acid-linoleic acid (OPL), and OPO. OPL is the ABC type triglyceride with the most abundant content in breast milk and contains three positional isomers of rac-OPL, rac-PLO and rac-POL. Lauric acid is a medium-chain fatty acid contained in breast milk at the highest content, and the composition of positional isomers of triglycerides constituting lauric acid is also important. Lauric dioleate (LaOO), a triglyceride containing lauric acid that is most abundant in breast milk. LaOO contains two positional isomers of rac-OLaO and rac-OOLa.

At present, in the breast milk, the chromatographic detection methods of other position isomers of triglyceride are not reported, except that the position isomers of the dipalmitoyl palmitate (OOP) and the dipalmitoyl-glyceryl oleate (PPO) can be detected by a chromatographic technique. 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 simultaneous detection method for multiple positional isomers breaks through the bottleneck of the detection of the components of the breast milk triglyceride, and the operation is simple, thereby being beneficial to the component monitoring in the production process.

Disclosure of Invention

The purpose of the invention is as follows: provides a method for simultaneously detecting the position isomers of the triglyceride PPO (rac-POP/rac-PPO), OPO (rac-OOP/rac-OPO), LaOO (rac-LaOO/rac-OLaO) and OPL (rac-OPL/rac-PLO/rac-POL) in breast milk by using single-column two-dimensional liquid chromatography-APCI/MS. The method is simple to operate, and can realize simultaneous detection of the 9 triglyceride position isomers in breast milk and infant formula milk powder.

The technical problem is as follows: the technical problems to be solved by the invention are as follows: 1) because the breast milk contains dozens of triglyceride molecules; 2) the phenomenon that target triglyceride is easily overlapped with other triglyceride in the process of detecting position isomers by silver ion chromatography is eliminated by the two-dimensional chromatography; 3) chromatographic peaks are extracted from APCI/MS, so that the overlapping phenomenon of rac-OPO and rac-LaOO in the chromatographic peaks is solved, and the interference of other triglyceride molecules is avoided; 4) nine triglycerides including rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO and rac-OPL/rac-PLO/rac-POL are detected simultaneously in the silver ion chromatogram, so that the detection steps are simplified, the detection time is shortened, and convenience is provided for the detection of components in breast milk.

The technical scheme is as follows: the invention relates to a method for simultaneously detecting nine positional isomers in breast milk. By this method, interference of triglycerides other than nine target triglycerides (rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO, and rac-OPL/rac-PLO/rac-POL) can be excluded. And the overlapping problem of rac-OPO and rac-LaOO peaks can be solved. And simultaneously monitoring the content of positional isomers of nine triglycerides including rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO and rac-OPL/rac-PLO/rac-POL. The requirements of simple operation and easy quantification are met. The technical scheme adopted by the invention is as follows.

1. Accurately obtaining molecular components of OPO, PPO, LaOO and OPL triglyceride: 1) fat extraction: and extracting the total lipid of the sample by a Folch method, drying by nitrogen, then fixing the volume by n-hexane, and passing through a membrane for later use. 2) And (3) reverse-phase chromatographic separation of triglyceride: HPLC C18 column, separating triglyceride in sample according to different ECN (carbon atom equivalent), separating triglyceride in sample primarily. The liquid phase gradient elution conditions are shown in table 1:

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

3) 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 molecules (OPO, PPO, LaOO, OPL) is determined. The mass spectrometry conditions were as follows: ESI ion source, fragment: 70V, scanning range: 350-1200. 4) Targeted collection of target triglyceride molecules. Fractions from the C18 reverse phase chromatography were collected using an autosampler at a rate of 2 min/tube, tubes with corresponding retention times for OPO, PPO, LaOO and OPL triglyceride molecules were collected and dried with nitrogen for further use.

2, building a method for simultaneously detecting nine positional isomers of rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO and rac-OPL/rac-PLO/rac-POL. 1) Preparing a standard solution: dissolving nine standard products of 25mg rac-OPO, rac-OOP, rac-OLaO, rac-LaOO, rac-POP, rac-PPO, rac-OPL, rac-PLO and rac-POL by using n-hexane into a 25mL volumetric flask to prepare a standard stock solution of 1 mg/mL; 2) optimizing the liquid phase condition of the silver ion chromatography. The silver ion 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/L. 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. The flow rate and the mobile phase gradient are respectively optimized by adopting a dichloromethane system and an acetone system, and the injection volume is 10 mu l. The final silver ion chromatogram has the following liquid phase gradient elution conditions: 0-20 min 70% A +30% B, 20-21min 70% A +30% -57% A +43% B, 21-45min 57% A +43% B, wherein A is n-hexane, B is n-hexane: isopropanol (99:2, v/v), and the flow rate is 0.5 ml/min; 3) and (5) detecting silver ion chromatography HPLC-APCI/MS. The 1 mg/L mixed standard was again tested using anion chromatography in conjunction 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. 1) The invention can simultaneously and effectively detect nine position isomers of rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO and rac-OPL/rac-PLO/rac-POL. Facilitates simultaneous monitoring of nine triglyceride positional isomers. And by utilizing a two-dimensional liquid chromatography technology, target triglyceride components can be effectively and quickly obtained, the interference of other triglyceride molecules is eliminated, and the detection of nine triglyceride position isomers in breast milk and other complex samples is realized. 2) The invention can simultaneously separate nine position isomers of rac-OPO/rac-OOP, rac-PPO/rac-POP, rac-LaOO/rac-OLaO and rac-OPL/rac-PLO/rac-POL (figure 1), and utilizes the extraction chromatographic function in APCI/MS to distinguish the overlapped rac-OPO and rac-LaOO (figure 1 and figure 2).

Description of the drawings in which: FIG. 1 is a total ion flow graph of a mixed standard under silver ion chromatography HPLC APCI/MS under elution conditions of the present invention. FIG. 2 is a chromatogram of the extracted rac-OPO and rac-OOP of mixed standards on silver ion chromatography HPLC APCI/MS under elution conditions of the present invention. FIG. 3 is a chromatogram of the extracted rac-LaOO and rac-OLaO of mixed standards on silver ion chromatography HPLC APCI/MS under elution conditions of the present invention. FIG. 4 is a total ion flow graph of a sample of breast milk in an example of silver ion chromatography HPLC APCI/MS under elution conditions of the present invention. FIG. 5 is a chromatogram of extracted rac-PPO and rac-POP of a sample of breast milk from an example under silver ion chromatography HPLC APCI/MS under elution conditions according to the invention. FIG. 6 is a chromatogram of the extracted rac-OPO and rac-OOP of a sample of breast milk in an example under silver ion chromatography, HPLC, APCI/MS, under elution conditions of the present invention. FIG. 7 is a chromatogram of the extracted rac-LaOO and rac-OLaO of the breast milk samples of the examples under silver ion chromatography HPLC APCI/MS under elution conditions of the present invention. FIG. 8 is a chromatogram of the extracted rac-PLO, rac-POL and rac-OPL of a sample of breast milk from an example under silver ion chromatography HPLCAPCI/MS under elution conditions of the invention.

The specific implementation mode is as follows: 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.