Breast milk phospholipid detection method

Abstract

The invention belongs to the technical field of breast milk phospholipid detection, and provides a breast milk phospholipid detection method. The breast milk phospholipid detection method is developed on the basis of the UPLC-QQQ-MS / MS technology, the method combines the high separation capacity of the ultra-high performance liquid chromatography and the high sensitivity and the high selectivity of the triple quadrupole mass spectrometry, and the detection efficiency and the quantitative accuracy of phospholipid molecules can be effectively improved. The method can be used for determining six phospholipid subclasses including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and sphingomyelin and 151 phospholipid molecules. According to the present invention, the chromatographic peak column shape and the separation degree of each phospholipid molecule are good, the analysis time is short, the absolute quantification is performed through the internal and external calibration method, all phospholipid subclasses show the good linear relationship, the R2 is in the range of 0.9908-0.9997, the specificity of the method is good, the recovery rate and the precision meet the requirements of the precise instrument, and the important technical support is provided for the detection of the phospholipid in the breast milk.

Description

Technical Field

[0001] This invention belongs to the field of breast milk phospholipid detection technology, specifically, it relates to a method for detecting breast milk phospholipids. Background Technology

[0002] Phospholipids are the most abundant polar lipids in breast milk, accounting for 0.2%–2.0% of total lipids. Of these, 60%–65% are distributed on the milk fat globule membrane, while the remaining 35%–40% are present in the aqueous phase of the emulsion. Based on the main chain structure and the organic groups attached to the phosphate groups, phospholipids can be classified into six types: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and sphingomyelin (SM). Breast milk phospholipids not only play a crucial role in maintaining the stability of milk fat globule structure but are also essential for infant growth and development, particularly in cognitive, immune, and lipid absorption functions.

[0003] Among methods for phospholipid detection, spectroscopic methods have limitations. For example, spectrophotometry can only determine phosphorus content; ultraviolet detectors are difficult to distinguish specific subclasses of phospholipids; electro-fogging detectors and evaporative light scattering detectors cannot identify phospholipid molecule types; and Fourier transform infrared spectroscopy has limited separation capabilities for complex samples. 31P NMR and LC-MS are the most advanced methods for phospholipid analysis. Although NMR is powerful, its high sample purity requirement (>95%) makes it unsuitable for routine sample analysis. In mass spectrometry, shotgun mass spectrometry, while flexible, lacks a separation step and struggles with complex samples; high-resolution mass spectrometry (such as TOF-MS and QTOF-MS) provides accurate mass information, but is susceptible to background noise and matrix effects in complex matrices, leading to inaccurate quantitative results. Furthermore, its poor instrument stability affects the repeatability and accuracy of quantitative analysis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting phospholipids in breast milk.

[0005] To achieve the objective of this invention, this invention provides a method for detecting phospholipids in breast milk, which uses UPLC-QQQ-MS / MS technology to detect phospholipids in breast milk and performs absolute quantification of phospholipids in breast milk through internal and external calibration methods. In this invention, the phospholipids include six subclasses: PC (phosphatidylcholine), PE (phosphatidylethanolamine), PS (phosphatidylserine), PI (phosphatidylinositol), PG (phosphatidylglycerol), and SM (sphingomyelin). Includes the following steps: (1) Preparation of a series of mixed solutions of internal and external phospholipid standards: The preparation method for the PC internal and external standard series mixed solutions is as follows: Dissolve the PC external standard stock solution in the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of PC internal standard stock solution; and containing 20 µL of... The preparation method of the PE internal and external standard series mixed solution is as follows: Dilute the PE external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of PE internal standard stock solution; The preparation method of the SM internal and external standard series mixed solution is as follows: Dilute the SM external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of SM internal standard stock solution; The preparation method for the PI internal and external standard series mixed solutions is as follows: Dilute the PI external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg / mL. Each concentration gradient has a volume of 200 µL and contains 8 µL of PI internal standard stock solution. The preparation method for the PS internal and external standard series mixed solutions is as follows: Dilute the PS external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 8 µL of PS internal standard stock solution; The preparation method of the PG internal and external standard series mixed solution is as follows: Dilute the PG external standard stock solution with the reconstitution solution to concentrations of 0.001, 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg / mL, with each concentration gradient having a volume of 200 µL and containing 4 µL of PG internal standard stock solution; The external standard stock solutions are as follows: 40 mg / L PC (18:0 / 18:2), 40 mg / L PE (18:0 / 18:2), 40 mg / L SM (d18:1 / 24:0), 10 mg / L PS (18:0 / 18:2), 10 mg / L PI (16:0 / 18:1) and 10 mg / L PG (16:0 / 18:1). Among the external standard stock solutions, PC, PE, SM, PS and PI are prepared with reconstitution solutions, and PG is obtained directly from the procurement. The internal standard stock solutions are as follows: 500 mg / L SM (d18:1 / 17:0), 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0), and 9.81 mg / L PG (14:1 / 17:0). Of these internal standard stock solutions, SM is prepared using a reconstituted solution, while PC, PE, PS, PI, and PG are directly purchased. The internal standard working solutions are as follows: 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 10 mg / L SM (d18:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) and 9.81 mg / L PG (14:1 / 17:0). Among them, the internal standard stock solution of PC is the same as the internal standard working solution of PC, the internal standard stock solution of PE is the same as the internal standard working solution of PE, the internal standard stock solution of PS is the same as the internal standard working solution of PS, the internal standard stock solution of PI is the same as the internal standard working solution of PI, and the internal standard stock solution of PG is the same as the internal standard working solution of PG. The complex solution is a mixture of dichloromethane and methanol in a 1:1 volume ratio and contains 5 mM ammonium acetate; (2) The series of mixed solutions of internal and external phospholipid standards were analyzed by UPLC-QQQ-MS / MS: The Ultimate 3000 chromatograph was coupled with a TSQ Quantiva mass spectrometer equipped with an ESI ion source for phospholipid detection; PC, PE, PS, PI, and PG were analyzed in negative ion mode, and SM was analyzed in positive ion mode. 1) Chromatographic conditions: Chromatographic columns: PC, PE, PS, PI and PG were analyzed using an ACQUITY UPLC BEH Amide Column with dimensions of 2.1 mm × 150 mm and 1.7 µm; SM was analyzed using a CORTECS HILIC Column with dimensions of 2.1 mm × 150 mm and 1.6 µm. Mobile phase A: An aqueous solution containing 0.1% formic acid and 10 mM ammonium formate; Mobile phase B: An acetonitrile-water solution containing 0.1% formic acid and 10 mM ammonium formate; wherein the volume ratio of acetonitrile to water in the acetonitrile-water solution is 95:5; Flow rate 0.4 mL / min, injection volume 2 μL, column temperature 25℃; Gradient elution was used, and the total SM analysis time was 15 min, with the following specific conditions: Table 1 UPLC elution procedure for SM

[0006] The total analysis time for PC, PE, PS, PI, and PG is 17 minutes, with the following specific conditions: Table 2 UPLC elution procedures for PC, PE, PS, PI, and PG

[0007] 2) Mass spectrometry conditions: Selective reaction monitoring was used for data acquisition in both positive and negative ion modes, under the following specific conditions: Table 3 Conditional parameters for QQQ-MS / MS

[0008] (3) Using the peak area of ​​the external standard / peak area of ​​the internal standard as the ordinate and the concentration of the external standard as the abscissa, standard curves for the six phospholipid subclasses are plotted respectively. (4) Establishment of a local phospholipid database: Referring to the characteristic phospholipids disclosed in CN116106231A and related literature, a local phospholipid database of high-content phospholipids in breast milk was established. The specific phospholipid molecular types are as follows: (5) Detection of phospholipids in breast milk samples: After pretreatment, breast milk samples from different lactation stages were tested according to step (2). The peak area was substituted into the standard curve of step (3) to obtain the phospholipid content in the breast milk sample to be tested.

[0009] Furthermore, the preprocessing method is as follows: ① Extraction of phospholipids (PLs) from breast milk: Take 200 µL of breast milk and add the following internal standard working solutions: 10.38 mg / L PC (14:1 / 17:0) 20 µL, 9.85 mg / L PE (14:1 / 17:0) 20 µL, 10 mg / L SM (d18:1 / 17:0) 20 µL, 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) 8 µL and 9.81 mg / L PG (14:1 / 17:0) 4 µL; add 200 µL of ultrapure water, 900 µL of dichloromethane and 2 mL of methanol, vortex and sonicate for 5 min; then add another 200 µL of ultrapure water and 900 µL of dichloromethane, and incubate at 6000 °C. Centrifuge at 15 min at 1000 rpm to separate the organic and aqueous phases. Mix the separated organic phase with 1 mL of ultrapure water, 2.2 mL of methanol and 600 µL of dichloromethane, and centrifuge at 3000 × g for 10 min to separate the organic phase, which is designated as organic phase I. Add 1.8 mL of dichloromethane to the aqueous phase, centrifuge at 6000 rpm for 15 min to separate the organic phase, which is designated as organic phase II. Combine organic phases I and II, dry them under nitrogen, and dissolve them in 1 mL of a reconstituted solution to obtain a reconstituted solution containing breast milk lipids. ② Phospholipid separation using solid-phase extraction: First, activate the normal-phase silica gel adsorbent solid-phase extraction column (ProElut Silica gel bonded cartridges, 1 g / 6 ml, purchased from Dikma Technologies) with n-hexane, and discard the eluent; then, add the reconstituted solution containing breast milk lipids to the solid-phase extraction column and let it stand for 5 minutes; elute the nonpolar lipids with 3 mL of diethyl ether-n-hexane mixture I (8:2, v / v) and 3 mL of diethyl ether-n-hexane mixture II, and discard the eluent; then, elute the phospholipids in the solid-phase extraction column in two steps: first, add 4 mL of methanol and collect eluent I, then add 2 mL of methanol and 2 mL of chloroform-methanol-water mixture and collect eluent II; combine eluents I and II, dry them under nitrogen, reconstitute them with 200 µL of reconstituted solution, and then perform phospholipid detection.

[0010] In the diethyl ether-n-hexane mixture I, the volume ratio of diethyl ether to n-hexane is 8:2.

[0011] The volume ratio of diethyl ether to n-hexane in the diethyl ether-n-hexane mixture II is 1:1.

[0012] The volume ratio of chloroform, methanol, and water in the chloroform-methanol-water mixture is 3:5:2.

[0013] Furthermore, the equations and correlation coefficients corresponding to the standard curves of SM, PC, PE, PS, PI, and PG are as follows: SM: Y = -0.1295 + 0.8697X, R 2 = 0.9964; PC: Y = -0.0693 + 1.7142X, R 2 = 0.9908; PE: Y = 0.0908 + 1.2248X, R 2 = 0.9997; PS: Y = -0.6798 + 6.3168X, R 2 = 0.9937; PI: Y = -0.0507 + 3.1100X, R 2 = 0.9953; PG: Y = -1.3174 + 28.9250X, R 2 = 0.9969.

[0014] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention develops a method for detecting phospholipids in breast milk based on UPLC-QQQ-MS / MS technology. This method combines the high separation capability of ultra-high performance liquid chromatography (UPLC) with the high sensitivity and selectivity of triple quadrupole mass spectrometry (TQMS), effectively improving the detection efficiency and quantitative accuracy of phospholipid molecules. This method can determine 151 phospholipid molecules from six phospholipid subclasses: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and sphingomyelin (SM). The chromatographic peaks of each phospholipid molecule show good column shape and resolution, and the analysis time is short. Absolute quantification is achieved through internal and external calibration methods. All phospholipid subclasses show good linearity, with R0... 2 The values ​​were all between 0.9908 and 0.9997, and the method showed good specificity, with recovery and precision meeting the requirements of precision instruments. This invention provides important technical support for the detection of phospholipids in breast milk. Attached Figure Description

[0015] Figure 1 The images show phospholipid spectra obtained under different chromatographic columns and ion modes in a preferred embodiment of the present invention. a) Phospholipid spectra obtained using a HILIC column and negative ion mode mass spectrometry; b) Phospholipid spectra obtained using a HILIC column and positive ion mode mass spectrometry.

[0016] Figure 2The images show phospholipid spectra under different chromatographic columns and ion modes in a preferred embodiment of the present invention. c) Phospholipid spectra obtained using an Amide column and negative ion mode mass spectrometry; d) Phospholipid spectra obtained using an Amide column and positive ion mode mass spectrometry.

[0017] Figure 3 The effect of column temperature on the total ion current response intensity is shown in a preferred embodiment of the present invention. a) Positive ion mode, b) Negative ion mode. This indicates a significant difference between groups ( P <0.05, P <0.01, P <0.001 and P <0.0001).

[0018] Figure 4 The total ion current response in positive and negative ion modes is shown in the preferred embodiments of the present invention when the injection volume is 2 µL, 5 µL, and 10 µL. a) Positive ion mode, b) Negative ion mode. This indicates a significant difference between groups ( P <0.05, P <0.01, P <0.001 and P <0.0001).

[0019] Figure 5 The mass-to-charge ratio and retention time of breast milk phospholipids in a preferred embodiment of the present invention are as follows: a) Mass-to-charge ratio and retention time of SM under positive ion mass spectrometry detection; b) Mass-to-charge ratio and retention time of PC, PE, PS, PI, and PG under negative ion mass spectrometry detection.

[0020] Figure 6 These are phospholipid spectra of a blank sample and a mixed breast milk sample in a preferred embodiment of the present invention. a) Mass spectrum of the blank sample in positive ion mode, b) Mass spectrum of the mixed breast milk sample in positive ion mode.

[0021] Figure 7 These are phospholipid spectra of a blank sample and a mixed breast milk sample in a preferred embodiment of the present invention. c) Mass spectrum of the blank sample in negative ion mode; d) Mass spectrum of the mixed breast milk sample in negative ion mode.

[0022] Figure 8 This is a preferred embodiment of the phospholipid content of breast milk at different lactation stages. Different superscript letters indicate significant differences between groups. P <0.05, unless otherwise specified, indicates no significant difference between groups. Colostrum milk (CM) (days 1-5 postpartum), transitional milk (TM) (days 6-15 postpartum), and mature milk (MM) (day 15 postpartum). Phospholipids (PLs). Detailed Implementation

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0024] Example 1: Method for Detecting Phospholipids in Breast Milk This embodiment provides an efficient and accurate detection method for 151 phospholipid molecules from six phospholipid subclasses in breast milk: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and sphingomyelin (SM).

[0025] (a) Methods for pre-processing breast milk Phospholipid extraction from breast milk: 200 µL of breast milk sample was mixed with the following internal standard working solutions: 20 µL PC (14:1 / 17:0), 20 µL PE (14:1 / 17:0), 20 µL SM (d18:1 / 17:0), 8 µL PS (14:1 / 17:0), 8 µL LPI (14:1 / 17:0), and 4 µL PG (14:1 / 17:0), all at a concentration of 10 mg / L. 200 µL of ultrapure water, 900 µL of dichloromethane, and 2 mL of methanol were added to the sample, followed by vortexing and sonication for 5 min. Then, another 200 µL of ultrapure water and 900 µL of dichloromethane were added, and the mixture was centrifuged at 6000 r / min for 15 min to separate the organic and aqueous phases. The organic phase was mixed with 1 mL of ultrapure water, 2.2 mL of methanol, and 600 µL of dichloromethane. The mixture was centrifuged at 3000 × g for 10 min to separate the organic phase, which was designated as organic phase I. 1.8 mL of dichloromethane was added to the aqueous phase, and the mixture was centrifuged at 6000 rpm for 15 min to separate the organic phase, which was designated as organic phase II. Organic phases I and II were combined and dried under nitrogen. The mixture was dissolved in 1 mL of a reconstitution solution, which consisted of a 1:1 volume ratio of dichloromethane and methanol and contained 5 mM ammonium acetate.

[0026] PLs were separated using solid-phase extraction (SPE). First, a normal-phase silica gel adsorbent SPE column (ProElut Silica gel bonded cartridges, 1 g / 6 ml, Dikma Technologies) was activated with 3 mL of n-hexane, and the eluent was discarded. Then, a reconstituted solution containing breast milk lipids was added to the SPE column and allowed to stand for 5 minutes. Nonpolar lipids were eluted with 3 mL of diethyl ether:n-hexane (8:2, v / v) and 3 mL of diethyl ether:n-hexane (1:1, v / v), respectively, and the eluent was discarded. Next, PLs were eluted from the SPE column in two steps: first, 4 mL of methanol was added, and the eluent was collected (referred to as eluent I); then, 2 mL of methanol and 2 mL of chloroform:methanol:water (3:5:2, v / v / v) were added, and the eluent was collected (referred to as eluent II). Eluents I and II were combined, dried under nitrogen, reconstituted with 200 µL of the reconstituted solution, and then analyzed for phospholipids.

[0027] (II) Phospholipid Detection Methods The Ultimate 3000 chromatograph was coupled with a TSQ Quantiva mass spectrometer equipped with an ESI ion source for qualitative and quantitative analysis of phospholipids in the samples. PC, PE, PS, PI, and PG were analyzed in negative ion mode, while SM was analyzed in positive ion mode.

[0028] (1) Chromatographic conditions: Chromatographic columns: ACQUITY UPLC BEH Amide Column (1.7 µm 2.1 × 150 mm, purchased from Waters) was used to analyze PC, PE, PS, PI and PG; CORTECS HILIC Column (1.6 µm, 2.1 × 150 mm, purchased from Waters) was used to analyze SM. Mobile phase A: water containing 0.1% formic acid and 10 mM ammonium formate; Mobile phase B: acetonitrile / water (v:v, 95:5), containing 0.1% formic acid and 10 mM ammonium formate; The flow rate was 0.4 mL / min, the injection volume was 2 μL, and the column temperature was 25℃.

[0029] Gradient elution was used, and the total SM analysis time was 15 min, as detailed in Table 1.

[0030] Table 1 UPLC elution procedure for SM

[0031] The total analysis time for PC, PE, PS, PI, and PG was 17 minutes, as detailed in Table 2.

[0032] Table 2 UPLC elution procedures for PC, PE, PS, PI, and PG

[0033] (2) Mass spectrometry conditions: Selective reaction monitoring was used to collect data in both positive and negative ion modes. Specific conditions and parameters are shown in Table 3.

[0034] Table 3 Conditional parameters for QQQ-MS / MS

[0035] Example 2: Development of a method for detecting phospholipids in breast milk 1. Selection of mass spectrometry analysis parameters A mixed solution containing PC, PE, PS, PI, PG, and internal and external SM standards was directly injected into the mass spectrometer for analysis, initiating the SMR optimization process. During this process, the instrument automatically adjusted the lens radio frequency voltage (RF) and collision energy (CE) parameters to achieve maximum ion transport efficiency. The optimized mass spectrometry parameters for each phospholipid are shown in Table 4. In negative ion mode, the adduct ions of PE, PS, PI, and PG are "-H". + The adduct ion of PC is +CH3COO. - In positive ion mode, the adduct ion of SM is +H+. + ".

[0036] Table 4. Parent and daughter ions and corresponding mass spectrometry parameters of each PL standard.

[0037] 2. Selection of chromatographic conditions 2.1 Selection of Chromatographic Column Based on the mass spectrometry optimization results, SM was analyzed in positive ion mode, while PC, PE, PS, PI, and PG were analyzed in negative ion mode.

[0038] Figure 1 and Figure 2 This paper presents the mass spectrometry results of different phospholipid external standard mixtures on HILIC and Amide columns in both positive and negative ion modes. In the negative ion mode of the HILIC column, PC (18:0 / 18:2) and PI (16:0 / 18:1) showed good peak shapes, but PE (18:0 / 18:2) and PG (16:0 / 18:1) both showed double peaks, and PS (18:0 / 18:2) had a long tailing peak. In contrast, in the negative ion mode of the Amide column, the peak shapes of PC, PE, PS, PI, and PG were complete, with only PC (18:0 / 18:2) showing a tailing peak. This indicates that the Amide column is more suitable for separating PC, PE, PS, PI, and PG; therefore, the negative ion mode of the Amide column should be used for the detection of PC, PE, PS, PI, and PG. In the positive ion mode of the HILIC column, the peak shape of SM (d18:1 / 24:0) was symmetrical and tall, while the peak shape in the positive ion mode of the Amide column showed multiple shoulders and a wider peak width. Therefore, the positive ion mode of the HILIC column was selected for the detection of the SM subclass.

[0039] 2.2 Selection of Column Temperature The experiment selected 20, 25, 30, 35, 40, and 45°C to test the mixture of internal and external standards of phospholipids, and repeated the test 5 times at each temperature.

[0040] Figure 3The effect of column temperature on the total ion current response intensity in positive and negative ion modes is shown. With increasing column temperature, the total ion current response intensity initially increases, then decreases, and then increases again, but overall shows a decreasing trend. This indicates that at lower temperatures, although chromatographic resolution is better, excessively high column pressure may limit ion transport efficiency, leading to a decrease in response intensity, since column temperature is inversely proportional to column pressure. As temperature increases and column pressure decreases, ion transport efficiency improves, and response intensity increases. However, excessively high column temperatures may lead to reduced separation selectivity, decreasing the target signal detected by mass spectrometry and affecting qualitative and quantitative results. As shown in the figure, the total ion current response intensity reaches its maximum in both positive and negative ion modes at a column temperature of 25℃, and the total ion current response intensity is significantly higher than at other column temperatures. Therefore, 25℃ was selected as the column temperature for this study.

[0041] 2.3 Selection of Injection Volume In this experiment, injection volumes of 2 µL, 5 µL, and 10 µL were used to detect the internal and external standard mixture of phospholipids. Each injection volume was repeated five times. The results are as follows: Figure 4 As shown, when detecting SM in positive ion mode, the response intensity increases with increasing injection volume; however, when detecting PC, PE, PS, PI, and PG in negative ion mode, the response intensity decreases with increasing injection volume, reaching its maximum at an injection volume of 2 µL. Furthermore, at an injection volume of 2 µL, the average response intensity of SM in positive ion mode is 124000±16175, significantly higher than the average response intensity of PC, PE, PS, PI, and PG in negative ion mode (65350±3944), thus meeting the requirements for SM detection. Based on these results and the standardized injection volume for the experimental method, this experiment selected 2 µL as the injection volume for both positive and negative ion modes. In addition, a smaller injection volume may not only improve detection sensitivity and peak clarity but also avoid peak diffusion, reduce separation efficiency, and minimize contamination and damage to the mass spectrometer.

[0042] 3. Mass spectrometry analysis parameter settings Since a complete set of standards for high-content phospholipids in breast milk is unavailable, it is impossible to accurately optimize the mass spectrometry parameters for each type of phospholipid using these standards to obtain the most precise parameter settings. Therefore, this study integrates the internal and external standard mass spectrometry parameters of various phospholipids to set the mass spectrometry parameters for phospholipid detection. Based on the information provided in Table 4, mass spectrometry parameters were set for high-content phospholipids in breast milk, including the corresponding precursor and daughter ion pairs, RF, and CE, thereby improving the selectivity and sensitivity of detection.

[0043] Nine breast milk samples from different lactation stages were pooled, pre-processed, and then analyzed five times. The peak times of different phospholipids in breast milk were as follows: Figure 5As shown, the peak time of SM is between 9 and 13.5 min, that of PC is between 3 and 4.5 min, that of PE is between 3 and 5 min, that of PS is between 11 and 12.5 min, that of PI is between 11.5 and 13 min, and that of PG is between 3 and 4.5 min.

[0044] Setting the mass spectrometry detection window based on the elution times of different phospholipid subclasses ensures that mass spectrometry analysis is performed when PL molecules are eluted from the column, thereby optimizing signal intensity and data quality. Detection at the molecular concentration peak not only effectively reduces background noise and improves the signal-to-noise ratio (S / N), resulting in more accurate detection results, but also enhances analytical selectivity, allowing the mass spectrometer to focus on detecting target compounds within a specific time period, reducing potential interference from other compounds. This is particularly important when processing complex samples. Specific mass spectrometry parameters for phospholipid analysis are shown in Tables 5-7.

[0045] Table 5 Mass spectrometry analysis of 151 phospholipid parameters

[0046] Table 6. Mass spectrometry analysis of 151 phospholipid parameters

[0047] Table 7 Mass spectrometry analysis of 151 phospholipid parameters

[0048] 4. Method Validation 4.1 Limit of Quantitation and Linear Range External standard stock solutions: Prepare 40 mg / L PC (18:0 / 18:2), PE (18:0 / 18:2), SM (d18:1 / 24:0), 10 mg / L PS (18:0 / 18:2) and PI (16:0 / 18:1) standard stock solutions. Purchase 10 mg / L PG (16:0 / 18:1) standard stock solution (purchased from Avanti Polar Lipids). Seal and store at -20℃ for later use.

[0049] Internal standard stock solutions: Prepare 500 mg / L SM (d18:1 / 17:0) standard stock solutions, seal and store at -20℃ for later use. The following internal standard stock solutions were directly purchased (from Avanti Polar Lipids): 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) and 9.81 mg / L PG (14:1 / 17:0).

[0050] Internal standard working solutions: Prepare 10 mg / L SM (d18:1 / 17:0), and purchase and obtain standard working solutions of 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) and 9.81 mg / L PG (14:1 / 17:0). Internal standard working solutions should be prepared fresh for use.

[0051] Standard working solutions: External standard stock solutions were diluted with reconstituted solutions to prepare 200 µL standard working solutions for each concentration gradient. The concentrations of the series of standard working solutions were as follows: PC, PE, SM: 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, 25 mg / mL, each containing 20 µL of PC, PE, or SM internal standard working solution. PI, PS: 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 mg / mL, each containing 8 µL of PI or PS internal standard working solution. PG: 0.001, 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg / mL, each containing 4 µL of PG internal standard working solution. Standard working solutions should be prepared fresh before use.

[0052] Standard working solutions of PC, PE, PS, PI, PG, and SM at gradient concentrations were prepared. Based on the optimization results of mass spectrometry parameters and chromatographic conditions in the experiment, the optimal conditions were selected for the determination of standard curves. The ordinate represents the external standard peak area / internal standard peak area, and the abscissa represents the concentration of the external standard. The linear equations and correlation coefficients for the six phospholipid subclasses were obtained. The standard curves, limits of quantitation, and corresponding S / N are shown in Table 8. All phospholipid subclasses showed good linearity, with R... 2All values ​​are between 0.9908 and 0.9997, and all are greater than 0.99, indicating that the established method has good linear correlation.

[0053] Table 8 Standard curves and sensitivity of the detection methods

[0054] 4.2 Specificity Blank (ultrapure water instead of breast milk) and breast milk samples were prepared separately, pretreated, and then analyzed. The experimental results are as follows: Figure 6 and Figure 7 As shown, taking PC (18:0 / 18:2), PE (18:0 / 18:2), SM (d18:1 / 24:0), PS (18:0 / 18:2), PI (16:0 / 18:1) and PG (16:0 / 18:1) as examples, there are no endogenous phospholipids in the blank samples, no "false positive" phospholipid peaks appear, and the total ion current response intensity is low when the blank samples are detected by UPLC-QQQ-MS / MS. Therefore, this method has good specificity.

[0055] 4.3 Recovery and precision of UPLC-QQQ-MS / MS method Deionized water was selected as the blank matrix. Low, medium, and high concentrations of phospholipid standards were added to all samples, with the following specific concentrations: PC, PE, SM: 2, 4, 6 mg / L; PI, PS: 0.8, 0.4, 0.2 mg / L; PG: 0.4, 0.2, 0.1 mg / L. Six parallel samples were analyzed for each concentration. The recovery rate was calculated based on the concentration of the external standard in the blank samples with only internal standard and the blank samples with both internal and external standards. Injections were performed continuously for three days, and the inter-day and intra-day repeatability of this method was calculated.

[0056] The accuracy of the analytical method was assessed by evaluating recovery rates, and the precision was assessed by evaluating intra-day and inter-day repeatability RSD values. These validation methods collectively ensured the reliability and validity of the analytical method. As shown in Table 9, the recovery rates for the PC detection method ranged from 102.56% to 105.07%, for the PE detection method from 92.27% to 103.64%, for the PS detection method from 94.16% to 104.94%, for the PI detection method from 91.59% to 101.31%, for the PG detection method from 91.66% to 98.26%, and for the SM detection method from 81.19% to 96.35%. At high, medium, and low spiking concentrations, the recoveries of the phospholipid subclass detection methods remained consistently within the range of 89% to 106%, meeting the recovery rate requirements for precision instruments. This demonstrates that the detection method can guarantee the accuracy and reliability of the results at different spiking concentrations. The intra-day and inter-day RSDs obtained by the detection method for phospholipid subclasses were both within 10%, meeting the precision requirements of precision instruments. This further demonstrates that the detection method has good repeatability and stability, and can achieve relatively consistent detection results under different times and conditions. These results indicate that the established detection method is suitable for the quantitative analysis of phospholipids and can provide reliable data support for related research.

[0057] Table 9 Recovery and precision of UPLC-QQQ-MS / MS method

[0058] Example 3: Detection of actual breast milk samples UPLC-QQQ-MS / MS was used to analyze breast milk from 56 mothers at different lactation stages. A total of 131 phospholipid molecules were identified, including 41 SM, 21 PC, 27 PE, 14 PS, 17 PI, and 11 PG. Breast milk phospholipids were absolutely quantified using internal and external calibration methods, and the total phospholipid content was 204.133 ± 7.205 mg / L. Figure 8 ).

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting phospholipids in breast milk, characterized in that, UPLC-QQQ-MS / MS technology was used to detect phospholipids in breast milk, and absolute quantification of phospholipids in breast milk was performed by internal and external calibration methods. The phospholipids include six subclasses: PC, PE, PS, PI, PG, and SM. Includes the following steps: (1) Preparation of a series of mixed solutions of internal and external phospholipid standards: The preparation method for the PC internal and external standard series mixed solutions is as follows: dissolve the PC external standard stock solution in the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of PC internal standard stock solution; The preparation method of the PE internal and external standard series mixed solution is as follows: Dilute the PE external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of PE internal standard stock solution; The preparation method of the SM internal and external standard series mixed solution is as follows: Dilute the SM external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 5.0, 10, 15, 20, and 25 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 20 µL of SM internal standard stock solution; The preparation method for the PI internal and external standard series mixed solutions is as follows: Dilute the PI external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg / mL. Each concentration gradient has a volume of 200 µL and contains 8 µL of PI internal standard stock solution. The preparation method for the PS internal and external standard series mixed solutions is as follows: Dilute the PS external standard stock solution with the reconstitution solution to concentrations of 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg / mL, with each concentration gradient having a volume of 200 µL, and containing 8 µL of PS internal standard stock solution; The preparation method of the PG internal and external standard series mixed solution is as follows: Dilute the PG external standard stock solution with the reconstitution solution to concentrations of 0.001, 0.0025, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 mg / mL, with each concentration gradient having a volume of 200 µL and containing 4 µL of PG internal standard stock solution; The external standard stock solutions are as follows: 40 mg / L PC (18:0 / 18:2), 40 mg / L PE (18:0 / 18:2), 40 mg / L SM (d18:1 / 24:0), 10 mg / L PS (18:0 / 18:2), 10 mg / L PI (16:0 / 18:1) and 10 mg / L PG (16:0 / 18:1). Among the external standard stock solutions, PC, PE, SM, PS and PI are prepared with reconstitution solutions, and PG is obtained directly from the procurement. The internal standard stock solutions are as follows: 500 mg / L SM (d18:1 / 17:0), 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0), and 9.81 mg / L PG (14:1 / 17:0). Of these internal standard stock solutions, SM is prepared using a reconstituted solution, while PC, PE, PS, PI, and PG are directly purchased. The internal standard working solutions are as follows: 10.38 mg / L PC (14:1 / 17:0), 9.85 mg / L PE (14:1 / 17:0), 10 mg / L SM (d18:1 / 17:0), 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) and 9.81 mg / L PG (14:1 / 17:0). The complex solution is a mixture of dichloromethane and methanol in a 1:1 volume ratio and contains 5 mM ammonium acetate; (2) The series of mixed solutions of internal and external phospholipid standards were analyzed by UPLC-QQQ-MS / MS: The Ultimate 3000 chromatograph was coupled with a TSQ Quantiva mass spectrometer equipped with an ESI ion source for phospholipid detection; PC, PE, PS, PI, and PG were analyzed in negative ion mode, and SM was analyzed in positive ion mode. 1) Chromatographic conditions: Chromatographic columns: PC, PE, PS, PI and PG were analyzed using an ACQUITY UPLC BEH Amide Column with dimensions of 2.1 mm × 150 mm and 1.7 µm; SM was analyzed using a CORTECS HILIC Column with dimensions of 2.1 mm × 150 mm and 1.6 µm. Mobile phase A: An aqueous solution containing 0.1% formic acid and 10 mM ammonium formate; Mobile phase B: An acetonitrile-water solution containing 0.1% formic acid and 10 mM ammonium formate; wherein the volume ratio of acetonitrile to water in the acetonitrile-water solution is 95:5; Flow rate 0.4 mL / min, injection volume 2 μL, column temperature 25℃; Gradient elution was used, and the total SM analysis time was 15 min, with the following specific conditions: The total analysis time for PC, PE, PS, PI, and PG is 17 minutes, with the following specific conditions: 2) Mass spectrometry conditions: Selective reaction monitoring was used for data acquisition in both positive and negative ion modes, under the following specific conditions: (3) Using the peak area of ​​the external standard / peak area of ​​the internal standard as the ordinate and the concentration of the external standard as the abscissa, standard curves for the six phospholipid subclasses are plotted respectively. (4) Establishment of a local phospholipid database: Referring to the characteristic phospholipids disclosed in CN116106231A and related literature, a local phospholipid database of high-content phospholipids in breast milk was established. The specific phospholipid molecular types are as follows: (5) Detection of phospholipids in breast milk samples: After pretreatment, breast milk samples from different lactation stages were tested according to step (2). The peak area was substituted into the standard curve of step (3) to obtain the phospholipid content in the breast milk sample to be tested.

2. The method according to claim 1, characterized in that, The preprocessing method is as follows: ① Extraction of phospholipids from breast milk: Take 200 µL of breast milk and add the following internal standard working solutions: 10.38 mg / L PC (14:1 / 17:0) 20 µL, 9.85 mg / L PE (14:1 / 17:0) 20 µL, 10 mg / L SM (d18:1 / 17:0) 20 µL, 9.90 mg / L PS (14:1 / 17:0), 10 mg / L PI (14:1 / 17:0) 8 µL and 9.81 mg / L PG (14:1 / 17:0) 4 µL; add 200 µL of ultrapure water, 900 µL of dichloromethane and 2 mL of methanol, vortex and sonicate for 5 min; then add another 200 µL of ultrapure water and 900 µL of dichloromethane, and incubate at 6000 °C. Centrifuge at 15 min at 1000 r / min to separate the organic and aqueous phases; mix the separated organic phase with 1 mL of ultrapure water, 2.2 mL of methanol and 600 µL of dichloromethane, centrifuge at 3000 × g for 10 min to separate the organic phase, and designate it as organic phase I; add 1.8 mL of dichloromethane to the aqueous phase, centrifuge at 6000 r / min for 15 min to separate the organic phase, and designate it as organic phase II; combine organic phases I and II, dry them under nitrogen, and dissolve them in 1 mL of reconstituted solution to obtain a reconstituted solution containing breast milk lipids; ② Phospholipid separation using solid-phase extraction: First, activate the normal-phase silica gel adsorbent solid-phase extraction column (ProElutSilica gel bonded cartridges) with n-hexane, and discard the eluent; then, add the reconstituted solution containing breast milk lipids to the solid-phase extraction column and let it stand for 5 minutes; elute nonpolar lipids with 3 mL of diethyl ether-n-hexane mixture I and 3 mL of diethyl ether-n-hexane mixture II, and discard the eluent; then, elute the phospholipids in the solid-phase extraction column in two steps: first, add 4 mL of methanol and collect eluent I; then add 2 mL of methanol and 2 mL of chloroform-methanol-water mixture and collect eluent II; combine eluents I and II, dry them under nitrogen, reconstitute them with 200 µL of reconstituted solution, and then perform phospholipid detection. In the diethyl ether-n-hexane mixture I, the volume ratio of diethyl ether to n-hexane is 8:2; The volume ratio of diethyl ether to n-hexane in the diethyl ether-n-hexane mixture II is 1:1; The volume ratio of chloroform, methanol, and water in the chloroform-methanol-water mixture is 3:5:

2.

3. The method according to claim 1 or 2, characterized in that, The equations and correlation coefficients corresponding to the standard curves of SM, PC, PE, PS, PI, and PG are as follows: SM:Y = -0.1295+0.8697X,R 2 = 0.9964; PC:Y = -0.0693+1.7142X,R 2 = 0.9908; PE:Y = 0.0908+1.2248X,R 2 = 0.9997; PS:Y = -0.6798+6.3168X,R 2 = 0.9937; PI:Y = -0.0507+3.1100X,R 2 = 0.9953; PG:Y = -1.3174+28.9250X,R 2 = 0.9969。

Images