A method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry

Abstract

This invention discloses a method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry (GC-MS / MS), comprising: taking 20–200 μL of breast milk or dairy products, adding an isotope mixed standard solution, mixing well, then adding lysis buffer, vortexing thoroughly, centrifuging, taking the supernatant, centrifuging to concentrate and drying, resuspending the dried sample in a mixture of ethyl acetate and trifluoroacetic anhydride, then derivatizing in a water bath at 40–80 °C for 0.5–3 hours, continuing centrifugation to concentrate and dry after derivatization, redissolving in dichloromethane, performing solid-phase extraction purification, blowing the purified solution to near dryness with nitrogen, adding 20–200 μL of a mixture of isopropanol and ethyl acetate to redissolve, and performing GC-MS / MS detection; qualitatively identifying the polyamines based on the qualitative ion pairs and retention times; substituting the ratio of the peak area of ​​the quantitative ion of the polyamine to the peak area of ​​the quantitative ion of the deuterated isotope internal standard into the matrix-matched internal standard curve of the polyamine to obtain the concentration of the polyamine.

Description

Technical Field

[0001] This invention relates to a method for detecting polyamines, specifically a method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry. Background Technology

[0002] Polyamines (typical examples include putrescine, spermidine, and spermine) are a class of compounds containing two or more amino groups. Besides endogenous synthesis in organisms, food is an important source of polyamines. Dietary polyamines are crucial for human health, especially in the early stages of life. Newborns and infants experience rapid cell growth, increasing their polyamine requirements. Breast milk is the first important exogenous source of polyamines for newborns. The polyamine content in breast milk is closely related to the health of newborns and infants.

[0003] Despite the potential health benefits of polyamines in breast milk, reports on their content in breast milk and dairy products are very rare, largely due to the lack of accurate and sensitive analytical methods. Commonly used methods for polyamine detection include high-performance liquid chromatography (HPLC) and gas chromatography (GC), but due to limitations in qualitative capability and sensitivity, these methods are currently mostly coupled with mass spectrometry (MS). Furthermore, due to the polycationic nature of polyamines, sample processing methods often require the use of acidic reagents and chemical derivatization. The Chinese national standard (GB5009.208-2016) specifies the determination of biogenic amines in food using dansyl chloride derivatization-HPLC-UV method, but dairy products are excluded from the applicable sample range. Literature reporting on breast milk polyamine content often uses HPLC-online derivatization-UV / fluorescence methods, but online derivatization methods are easily limited by chromatographic conditions (including derivatization reagents, reaction time, and reaction conditions), and the qualitative and sensitivity of UV / fluorescence methods are insufficient. Recently, Zhanru Yu (Yu Z, Huang H, Zhang H, Kessler BM. Improved profiling of polyamines using two-dimensional gas chromatography-mass spectrometry. Talanta. 2019, 199: 184-188.) et al. used trifluoroacetic acid derivatization and two-dimensional gas chromatography-single quadrupole mass spectrometry to detect polyamines in urine, brain tissue and cells. However, the sample pretreatment used a single-step liquid-liquid extraction, which is not suitable for breast milk and dairy products with complex matrices, especially affecting the determination of putrescine. In addition, the DB-5 capillary separation column used is prone to peak tailing in practical applications, and two-dimensional gas chromatography is not yet widely used in laboratories.

[0004] Based on the characteristic that trifluoroacetic acid reacts with polyamines to form stable and volatile derivatized products, this invention optimizes the best applicable conditions for the detection of polyamines in breast milk and dairy products. In addition, considering the matrix characteristics of breast milk and dairy products, suitable solid-phase extraction materials are selected for necessary purification treatment. At the same time, by optimizing the parameters of gas chromatography capillary column and tandem quadrupole mass spectrometry, an accurate and sensitive quantitative method for polyamines in breast milk and dairy products is established.

[0005] Compared to current online derivatization detection methods for polyamines in breast milk and dairy products, the derivatization reaction time and conditions of this invention are not limited by chromatographic conditions, and the derivatized sample can be further purified and concentrated. At the same time, combined with tandem mass spectrometry, the sensitivity and specificity of detection are greatly improved. Compared with liquid chromatography-mass spectrometry, the instrument and equipment of this invention are cheaper, which significantly reduces the cost of sample detection. Summary of the Invention

[0006] This invention establishes an accurate and sensitive method for the quantitative detection of polyamines in breast milk and dairy products based on gas chromatography-tandem mass spectrometry. The method is very suitable for the study of polyamine content in breast milk and dairy products.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry includes: taking 20–200 μL of breast milk or dairy products, adding 5–50 μL of a mixed isotope standard solution, followed by adding 100–1000 μL of lysis buffer, and vortexing thoroughly.

[0009] Centrifuge, collect the supernatant, centrifuge to concentrate and dry. The dried sample is resuspended in a mixture of 100–500 μL ethyl acetate and trifluoroacetic anhydride, and derivatized in a water bath at 40–80 °C for 0.5–3 hours. After derivatization, centrifuge to concentrate and dry again, redissolve in dichloromethane, and then perform solid-phase extraction for purification. The purified solution is purged with nitrogen until nearly dry, and redissolved in a mixture of 20–200 μL isopropanol and ethyl acetate. Gas chromatography-tandem mass spectrometry is then performed for detection. The polyamine is qualitatively identified based on the qualitative ion pairs and retention times. The ratio of the peak area of ​​the polyamine quantitative ion to the peak area of ​​the isotopic internal standard quantitative ion is substituted into the polyamine matrix to match the internal standard curve to obtain the concentration of the polyamine.

[0010] The gas chromatography detection conditions are as follows: the chromatographic column is a DB-35 capillary column with the following specifications: column length 15–60 m, inner diameter 0.18–0.32 mm, and film thickness 0.14–0.32 μm; the injection volume is 1–3 μL, preferably 1–2 μL; the injection method is splitless injection; the injection port temperature is 200–400℃, preferably 200–300℃; the initial column temperature is 60–150℃, held for 1 min, increased to 180℃ at 6℃ / min, increased to 260℃ at 15℃ / min, and then increased to 325℃ at 30℃ / min and held for 5 min.

[0011] Tandem mass spectrometry detection conditions: electron impact ionization source, 70 eV; temperature of both ion source and transmission line, 300℃; detection mode, MS / MS detection mode.

[0012] Preferably, the polyamine is selected from one or more of putrescine, spermidine, and spermine.

[0013] Preferably, the isotopes are deuterated putrescine (putrescine-d4) and deuterated spermine (spermine-d8).

[0014] Preferably, the isotope mixed standard solution is a mixed solution with a concentration of 6500 nmol / dL for both putrescine-d4 and spermine-d8.

[0015] Preferably, 150 μL of breast milk or dairy product is added to 20 μL of isotope mixed standard solution, followed by 150 μL of lysis buffer, wherein the lysis buffer is a mixed solution composed of 5% trifluoroacetic acid, 15% deionized water and 80% methanol.

[0016] Preferably, the isotopes are deuterated putrescine (putrescine-d4) and deuterated spermine (spermine-d8); the isotope mixed standard solution is a mixed solution with a concentration of 6500 nmol / dL for both putrescine-d4 and spermine-d8.

[0017] Preferably, the centrifugation conditions are: centrifugation at a temperature of 4–40°C and a rotation speed of 5000–15000 rpm / min for 5–30 min.

[0018] More preferably, the centrifugation conditions are: centrifugation at 15000 rpm / min for 20 min at 4°C.

[0019] Preferably, the supernatant is concentrated and dried at a temperature of 25°C.

[0020] More preferably, the centrifugal concentration and drying conditions are: centrifugation at 25°C and 5000-15000 rpm / min until nearly dry.

[0021] Preferably, the ethyl acetate and trifluoroacetic anhydride mixture is a solution with a volume ratio of 1:2 for ethyl acetate and trifluoroacetic anhydride, and the amount added is 300 μL.

[0022] Preferably, the water bath derivatization temperature is 60°C and the derivatization time is 3 hours.

[0023] Preferably, the solid-phase extraction purification conditions are as follows: a dried tube after centrifugation and concentration with 2 mL of dichloromethane; the solid-phase extraction column packing material is Florisil with a specification of 6 mL / 2 g; the purification process involves activating the column with 10 mL of dichloromethane, then adding the reconstituted solution, followed by rinsing the column with 4 mL of dichloromethane and 4 mL of a mixed solution of dichloromethane and ethyl acetate (dichloromethane: ethyl acetate = 8:2), and finally adding 10 mL of ethyl acetate for elution, and collecting all the eluent.

[0024] Preferably, the isopropanol and ethyl acetate mixture is a solution with a volume ratio of isopropanol to ethyl acetate of 1:9, and the amount added is 50-100 μL, preferably 60 μL.

[0025] Preferably, the gas chromatography detection conditions are as follows: the chromatographic column is a DB-35 capillary column with a length of 30m, an inner diameter of 0.25mm, and a film thickness of 0.25μm.

[0026] The gas chromatography detection conditions are as follows: injection volume 1 μL, splitless injection, injection port temperature 300℃; initial column temperature 80℃, held for 1 min, increased to 180℃ at 6℃ / min, increased to 260℃ at 15℃ / min, and then increased to 325℃ at 30℃ / min and held for 5 min; mass spectrometry detection conditions: electron impact ionization source, 70 eV; temperature of both ion source and transfer line 300℃.

[0027] More preferably, the mass spectrometer is a tandem quadrupole mass spectrometer.

[0028] Preferably, the qualitative ion pairs of putrescine are 167-70 m / z and 167-139 m / z; the qualitative ion pairs of spermidine are 364-251 m / z and 364-154 m / z; the qualitative ion pairs of spermine are 517-126 m / z and 517-154 m / z; the qualitative ion pairs of putrescine-d4 are 171-72 m / z and 171-141 m / z; and the qualitative ion pairs of spermine-d8 are 525-126 m / z and 525-154 m / z.

[0029] The quantitative ion pairs of putrescine, spermidine, spermine, putrescine-d4, and spermine-d8 are 167-139 m / z, 364-251 m / z, 517-154 m / z, 171-141 m / z, and 525-154 m / z, respectively. That is, the quantitative daughter ions of putrescine, spermidine, spermine, putrescine-d4, and spermine-d8 are 139 m / z, 251 m / z, 154 m / z, 141 m / z, and 154 m / z, respectively.

[0030] The higher the response, the higher the sensitivity, and the higher the detection rate of the target substance in the sample.

[0031] Preferably, the collision energies of ion pairs at 167-139 m / z, 364-251 m / z, and 171-141 m / z are 5 eV; the collision energy of ion pairs at 517-154 m / z is 10 eV; the collision energy of ion pairs at 167-70 m / z, 171-72 m / z, and 525-154 m / z is 15 eV; the collision energy of ion pairs at 364-154 m / z is 20 eV; the collision energy of ion pairs at 517-126 m / z is 30 eV; and the collision energy of ion pairs at 525-126 m / z is 35 eV.

[0032] Preferably, the retention time of putrescine is 14.00-14.40 min, the retention time of spermidine is 21.80-22.20 min, the retention time of spermine is 24.80-25.10 min; the retention time of putrescine-d4 is 13.90-14.30 min, and the retention time of spermine-d8 is 24.80-25.10 min.

[0033] More preferably, the retention time of putrescine is 14.10-14.30 min, the retention time of spermidine is 21.95-22.10 min, the retention time of spermine is 24.90-25.00 min, the retention time of putrescine-d4 is 14.05-14.25 min, and the retention time of spermine-d8 is 24.85-24.95 min.

[0034] Preferably, the matrix-matched internal standard curve of the breast milk or dairy product is plotted by the following method: 150 μL of mixed breast milk or dairy product, 20 μL of a 6500 nmol / dL isotope mixed standard solution, and polyamine mixed standard solutions of different concentrations are mixed, and then 150 μL of lysis buffer is added to obtain the standard curve solution. The mixture is stirred (preferably vortexed), centrifuged, and the supernatant is dried and resuspended in a mixture of ethyl acetate and trifluoroacetic anhydride. After derivatization, it is centrifuged again, concentrated, and dried. It is then reconstituted in 2 mL of dichloromethane, followed by solid-phase extraction purification. The purified solution is purified by nitrogen blowing until nearly dry, and finally reconstituted in a mixture of isopropanol and ethyl acetate for gas chromatography-tandem mass spectrometry detection. The standard curve is prepared using putrescine, ... With the concentration of spermidine or spermine as the x-axis and the ratio of the corrected peak area of ​​the quantitative ion of putrescine, spermidine, or spermine to the peak area of ​​the quantitative ion of the isotopic internal standard as the y-axis, a matrix-matched internal standard curve for putrescine, spermidine, or spermine is established. The isotopic internal standard corresponding to putrescine is putrescine-d4, and the isotopic internal standards corresponding to spermidine and spermine are spermine-d8. The corrected peak area of ​​the quantitative ion of putrescine, spermidine, or spermine is the peak area of ​​the quantitative ion of putrescine, spermidine, or spermine minus the peak area of ​​the quantitative ion of putrescine, spermidine, or spermine in the matrix background. The matrix-matched internal standard curve range for the three polyamines is 0–5000 nmol / dL.

[0035] The breast milk or dairy product matrix is ​​prepared by randomly taking 100 μL from each of 50 portions of breast milk or dairy product, mixing them thoroughly, and then preparing a mixed breast milk or dairy product matrix. If the dairy product is solid (such as formula milk powder), it should be prepared into a liquid according to the mixing requirements before sampling.

[0036] The matrix background is prepared by mixing 150 μL of mixed breast milk or dairy products with 20 μL of a 6500 nmol / dL isotope mixed standard solution.

[0037] The beneficial effects of this invention are as follows:

[0038] This invention achieves accurate and sensitive determination of polyamine content in breast milk and dairy products by systematically optimizing pretreatment conditions and selecting suitable capillary separation columns and tandem quadrupole monitoring parameters. The method requires small sample sizes, enables batch processing of samples, and has low instrument detection costs.

[0039] The method of this invention is applicable to the study of polyamine content in breast milk and dairy products. Attached Figure Description

[0040] Figure 1 The graphs show the response of putrescine on capillary columns DB-5 and DB-35, respectively.

[0041] Figure 2 These are the primary and secondary mass spectra of putrescine.

[0042] Figure 3 These are the primary and secondary mass spectra of spermidine.

[0043] Figure 4 These are the primary and secondary mass spectra of spermine.

[0044] Figure 5 These are the primary and secondary mass spectra of putrescine-d4.

[0045] Figure 6 These are the primary and secondary mass spectra of spermine-d8.

[0046] Figure 7 Chromatograms of standards for putrescine, putrescine-d4, spermidine, spermine, and spermine-d8.

[0047] Figure 8 Figure showing the optimization effect of derivatization time for putrescine, spermidine, and spermine.

[0048] Figure 9 Chromatograms of putrescine, putrescine-d4, spermidine, spermine, and spermine-d8 in breast milk before and after solid-phase extraction purification.

[0049] Figure 10 The levels of putrescine, spermidine, and spermine in breast milk and formula milk powder at different time periods. Detailed Implementation

[0050] The instruments and reagents used in the examples are as follows:

[0051] Instruments and reagents: Trace1300 gas chromatograph-TSQ8000 tandem quadrupole mass spectrometer (Thermo Fisher Scientific, USA); methanol, ethyl acetate and isopropanol (chromatographic grade, Merck & Co., Germany); trifluoroacetic acid (analytical grade, Aladdin Reagent Co., Ltd., Shanghai), trifluoroacetic anhydride (analytical grade, Maclean's Reagent Co., Ltd., Shanghai); Florisil column (Shanghai Anpu Experimental Technology Co., Ltd., 6 mL / 2 g).

[0052] Standard substances: putrescine, spermidine, and spermine were purchased from Shanghai Maclean Reagent Co., Ltd., as solid powders; deuterated putrescine (putrescine-d4) was purchased from Beijing Manhag Biotechnology Co., Ltd., as solid powders; deuterated spermine (spermine-d8) was purchased from Beijing Bailingwei Technology Co., Ltd., as solid powders.

[0053] Preparation of standard solutions: Accurately weigh putrescine, spermidine, spermine, putrescine-d4, and spermine-d8 solid powders, dissolve them in 0.5M hydrochloric acid, and prepare stock solutions of 100 μmol / dL respectively. Before use, dilute the putrescine, spermidine, and spermine stock solutions with 0.5M hydrochloric acid to prepare application solutions or 10 μmol / dL polyamine mixed standard solutions (polyamine mixed standard 1); dilute the putrescine-d4 and spermine-d8 stock solutions with 0.5M hydrochloric acid to prepare application solutions or 10 μmol / dL isotope mixed standard solutions (isotope mixed standard 1). If necessary, dilute polyamine standard 1 with 0.5M hydrochloric acid to prepare a polyamine mixed standard solution (polyamine standard 2) with a concentration of 1000 nmol / dL; prepare isotope standard 1 with 0.5M hydrochloric acid to prepare an isotope mixed standard solution (isotope standard 2) with a concentration of 6500 nmol / dL.

[0054] The lysis buffer is prepared as a mixed solution consisting of 5% trifluoroacetic acid, 15% deionized water, and 80% methanol.

[0055] Mixing breast milk matrix: Randomly extract 100 μL from each of 50 breast milk samples and mix thoroughly.

[0056] Example 1: Optimization of Instrument Conditions

[0057] 1.1 Selection of Capillary Separation Column

[0058] Taking putrescine as an example, the separation and response effects of DB-5 capillary column (specifications: length 30m, inner diameter 0.25mm, film thickness 0.25μm) and DB-35 capillary column (specifications: length 30m, inner diameter 0.25mm, film thickness 0.25μm) on putrescine were investigated.

[0059] The test was conducted using a putrescine solution with a concentration of 10 μmol / dL, under the following conditions:

[0060] Pretreatment of the application solution: Accurately pipette 150 μL of putrescine application solution, add 150 μL of lysis buffer, mix thoroughly, centrifuge at 15000 rpm / min for 20 min at 4℃, take the supernatant and centrifuge and concentrate at 25℃ to dry, resuspend the dried sample in 300 μL of a 1:2 mixture of ethyl acetate and trifluoroacetic anhydride, then derivatize in a 60℃ water bath for 3 hours, concentrate and dry again by vacuum centrifugation, and reconstitute with 60 μL of a 1:9 mixture of isopropanol and ethyl acetate;

[0061] The gas chromatography conditions used for the capillary column were as follows: injection volume of 1 μL, splitless injection, injection port temperature of 300℃, carrier gas of helium, flow rate of 1.0 mL / min, and temperature program: initial temperature of 80℃, hold for 1 min, increase to 180℃ at 6℃ / min, increase to 260℃ at 15℃ / min, and then increase to 325℃ at 30℃ / min and hold for 5 min;

[0062] The mass spectrometry conditions for both capillary columns were as follows: electron impact ionization source, 70 eV; temperature of ion source and transfer line, 300 °C; first-order full scan mode, scan range of 50-550 m / z, solvent delay of 3 min.

[0063] The results are as follows Figure 1 The results show that, compared with the DB-5 capillary column, putrescine exhibits a significantly improved peak shape on the DB-35 capillary column. Therefore, the DB-35 capillary column was selected.

[0064] Both spermidine and spermine yielded similar results, so the DB-35 capillary column was chosen.

[0065] 1.2 Optimization of mass spectrometry conditions

[0066] MS / MS is characterized by high sensitivity and strong qualitative ability. The experiment optimized the detection parameters of MS / MS mode, including the selection of qualitative and quantitative ion pairs of putrescine, spermidine, spermine and isotopic internal standards (putrescine-d4 and spermine-d8), and the selection of collision energy.

[0067] DB-35 capillary columns were used, and the chromatographic conditions were those described in section 1.1, "Selection of Capillary Separation Columns," for gas chromatography. Mass spectrometry used an electron impact ionization source at 70 eV; the temperature of both the ion source and the transfer line was 300 °C.

[0068] For ion pair selection, polyamine standard 1 and isotope standard 1 were used for testing. The pretreatment of each test solution followed the application solution pretreatment described in section "1.1 Selection of Capillary Separation Column". First, a primary mass spectrometry full scan mode was used for detection, with a primary scan range of 50–550 m / z. The molecular ion with the highest response in the obtained primary mass spectrum, or the base peak ion formed by the loss of a certain group from the molecular ion, was selected as the precursor ion. Secondary daughter ion scanning was then performed based on the selected precursor ion. The primary and secondary mass spectra of putrescine are shown below. Figure 2 The primary and secondary mass spectra of spermidine are shown in [reference needed]. Figure 3 The primary and secondary mass spectra of spermine are shown in [reference needed]. Figure 4 The primary and secondary mass spectra of putrescine-d4 are shown in [reference needed]. Figure 5 The primary and secondary mass spectra of spermine-d8 are shown in [reference needed]. Figure 6Two ions with relatively high response intensities were selected as daughter ions. Based on the results, the ion pairs for putrescine were determined to be: 167-70 m / z and 167-139 m / z; for spermidine, 364-251 m / z and 364-154 m / z; for spermine, 517-126 m / z and 517-154 m / z; for putrescine-d4, 171-72 m / z and 171-141 m / z; and for spermine-d8, 525-126 m / z and 525-154 m / z.

[0069] The selected daughter ions were then optimized to the best response using different collision energies. The collision energies for ion pairs at 167-139 m / z, 364-251 m / z, and 171-141 m / z were 5 eV; for ion pairs at 517-154 m / z, the collision energy was 10 eV; for ion pairs at 167-70 m / z, 171-72 m / z, and 525-154 m / z, the collision energy was 15 eV; for ion pairs at 364-154 m / z, the collision energy was 20 eV; for ion pairs at 517-126 m / z, the collision energy was 30 eV; and for ion pairs at 525-126 m / z, the collision energy was 35 eV. Further, by comparing the response of two pairs of ion pairs for each standard substance and the deuterated isotope internal standard, the pair of ion pairs with higher response was selected as the quantitative ion pairs. The quantitative ion pairs for putrescine, spermine, spermine, putrescine-d4 and spermine-d8 were 167-139 m / z, 364-251 m / z, 517-154 m / z, 171-141 m / z and 525-154 m / z, respectively.

[0070] Take 150 μL of polyamine mixed standard 2 and 20 μL of isotope mixed standard 2, then add 150 μL of lysis buffer and mix thoroughly. Subsequent pretreatment steps are the same as the application solution pretreatment under "1.1 Selection of Capillary Separation Column". Use the gas chromatography conditions of the DB-35 capillary column under "1.1 Selection of Capillary Separation Column" and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions". Results are as follows: Figure 7 The results show that, using optimized gas chromatography and mass spectrometry conditions, putrescine (retention time 14.17 min), spermidine (retention time 22.03 min), and spermine (retention time 24.93 min) can all effectively elute with high sensitivity. The peak elution times of the isotope internal standards (putrescine-d4 and spermine-d8) are 14.12 min and 24.91 min, respectively. All substances elute within 25 min.

[0071] Example 2: Derivatization Time Optimization

[0072] The test was conducted using polyamine standard 1, and the test conditions were as follows:

[0073] Pretreatment of the sample: Take 150 μL of polyamine standard 1, add 150 μL of lysis buffer, mix thoroughly, centrifuge at 15000 rpm / min for 20 min at 4℃, collect the supernatant, centrifuge and concentrate at 25℃, and dry. Resuspend the dried sample in 300 μL of a 1:2 mixture of ethyl acetate and trifluoroacetic anhydride, then derivatize in a 60℃ water bath for 1, 2, or 3 hours (three parallel samples at each time point), and concentrate and dry again by vacuum centrifugation. Reconstitute with 60 μL of a 1:9 mixture of isopropanol and ethyl acetate. Use the gas phase conditions of the DB-35 capillary column under "1.1 Selection of Capillary Separation Column" and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions". Results are as follows: Figure 8 The results show that putrescine, spermidine, and spermine all exhibit the best response and precision after 3 hours of derivatization. Therefore, a derivatization time of 3 hours was chosen.

[0074] Example 3 Sample Purification

[0075] The following tests were conducted using a mixture of breast milk matrix, polyamine standard 2, and isotope standard 2, under the following conditions:

[0076] Sample non-solid phase extraction treatment (before purification): Accurately pipette 150 μL of mixed breast milk matrix, 20 μL of isotope mixed standard 2 and 50 μL of polyamine mixed standard 2, then add 150 μL of lysis buffer, mix thoroughly, centrifuge at 15000 rpm / min for 20 min at 4℃, take the supernatant and centrifuge and concentrate and dry at 25℃, the dried sample is resuspended in 300 μL of ethyl acetate and trifluoroacetic anhydride mixture with a volume ratio of 1:2, then derivatized in a 60℃ water bath for 3 hours, vacuum centrifuge and concentrate and dry again, and reconstituted in 60 μL of isopropanol and ethyl acetate mixture with a volume ratio of 1:9;

[0077] Solid-phase extraction (after purification) of the sample: ① Accurately pipette 150 μL of mixed breast milk matrix, 20 μL of isotope mixed standard 2, and 50 μL of polyamine mixed standard 2, then add 150 μL of lysis buffer and mix thoroughly. ② Centrifuge at 15000 rpm / min for 20 min at 4℃, collect the supernatant and centrifuge at 25℃ to concentrate and dry it. The dried sample is resuspended in 300 μL of a 1:2 mixture of ethyl acetate and trifluoroacetic anhydride, derivatized in a 60℃ water bath for 3 hours, concentrated and dried again by vacuum centrifugation, and reconstituted with 2 mL of dichloromethane. ③ Activate the Florisil column with 8 mL of dichloromethane, add 2 mL of dichloromethane reconstituted solution, then rinse the column with 4 mL of dichloromethane and 4 mL of a mixed solution of dichloromethane and ethyl acetate (dichloromethane: ethyl acetate = 8:2), and finally elute with 10 mL of ethyl acetate. Collect all the eluent, blow the eluent with nitrogen to near dryness, and reconstitute with 60 μL of a mixture of isopropanol and ethyl acetate in a volume ratio of 1:9.

[0078] Both of the above pretreatment methods adopted the gas phase conditions of the DB-35 capillary column under "1.1 Selection of capillary separation column" and the optimized mass spectrometry conditions under "1.2 Optimization of mass spectrometry conditions".

[0079] The results are as follows Figure 9 The results indicate that before purification, due to the influence of the matrix, putrescine and putrescine-d4 did not have clean and obvious chromatographic peaks, while spermidine, spermine, and spermine-d8 had wider peaks and delayed retention times. After purification, putrescine showed obvious chromatographic peaks, especially putrescine-d4, whose chromatographic peak was clean and free of interference. The peaks of spermidine, spermine, and spermine-d8 narrowed, with symmetrical and sharp peak shapes, and their retention times returned to the same level as the standards, indicating that the purification effect was ideal.

[0080] Example 4: Performance Evaluation of the Method

[0081] 4.1 Instrument detection limit assessment: The instrument detection limits of putrescine, spermidine, and spermine were assessed at a signal-to-noise ratio of 3. Putrescine was approximately 5 nmol / dL, spermidine was approximately 1 nmol / dL, and spermine was approximately 0.5 nmol / dL.

[0082] 4.2 Instrument Quantitation Limits: The instrument quantitation limits of putrescine, spermidine, and spermine were determined using a signal-to-noise ratio of 10. Putrescine was approximately 20 nmol / dL, spermidine was approximately 5 nmol / dL, and spermine was approximately 2 nmol / dL.

[0083] 4.3 Internal Standard Curve: The standard curve range was determined based on the content of the three polyamines in the small sample test. Eight matrix-matched standard curve samples were prepared according to Table 1, consisting of mixed breast milk or dairy product matrix, isotope mixed standard 2, polyamine mixed standard solution, and lysis buffer. The final spiking concentrations of the polyamines in samples 2-7 were 50 nmol / dL, 100 nmol / dL, 200 nmol / dL, 500 nmol / dL, 1000 nmol / dL, 2000 nmol / dL, and 5000 nmol / dL, respectively. Steps ② and ③ of "Sample Solid Phase Extraction Treatment (After Purification)" in "Example 3", the gas phase conditions of the DB-35 capillary column under "1.1 Selection of Capillary Separation Column", and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions" were used.

[0084] Table 1. Polyamine matrix matching standard curves

[0085]

[0086] Note: The polyamine mixed standard solutions measured before mixing are as follows: 2, 3, 4, 5, and 6 are polyamine mixed standard 2; 7 and 8 are polyamine mixed standard 1.

[0087] Using the concentrations of putrescine, spermidine, and spermine as the x-axis and the ratio of the corrected peak area of ​​the polyamine quantitative ions to the peak area of ​​the corresponding isotopic internal standard quantitative ions (the peak area of ​​the polyamine quantitative ions at each concentration point is subtracted from the peak area of ​​the polyamine quantitative ions in the matrix background, and then compared with the peak area of ​​the corresponding isotopic internal standard quantitative ions, i.e., subtracting the peak area of ​​concentration point 1; the isotopic internal standard corresponding to putrescine is putrescine-d4, and the isotopic internal standards corresponding to spermidine and spermine are spermine-d8) as the y-axis, polyamine matrix-matched internal standard curves were established. The correlation coefficients were all greater than 0.99, indicating good linearity, and these curves can be used for the accurate quantification of putrescine, spermidine, and spermine in samples.

[0088] 4.4 Method Recovery and Precision:

[0089] A method recovery test was conducted by adding a polyamine mixed standard solution to the mixed breast milk matrix. Polyamine mixed standard 1 was diluted with 0.5M hydrochloric acid to prepare polyamine standard solutions with concentrations of 2000 nmol / dL, 500 nmol / dL, and 100 nmol / dL.

[0090] The spiking test was conducted as follows: For three concentrations of spiked recovery tests, four 150 μL aliquots of mixed breast milk matrix were taken, and 20 μL of isotope mixed standard 2 was added to each. Then, 60 μL of a polyamine mixed standard solution (2000 nmol / dL, 500 nmol / dL, or 100 nmol / dL) was added to the first three aliquots, respectively. The last aliquot was not added to the polyamine mixed standard solution (matrix background). Each of the above solutions was then added to 150 μL of lysis buffer. After thorough vortexing, the samples were analyzed using steps ② to ③ of "Sample Solid-Phase Extraction Treatment (After Purification)" in "Example 3," the gas phase conditions of the DB-35 capillary column under "1.1 Selection of Capillary Separation Column," and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions." Quantification was performed based on the matrix standard curve. The above experiment was repeated three times, i.e., three parallel experiments for each spiked concentration. The recovery rate was calculated using the formula: "Recovery % = (Quantitative Concentration of Spiked Matrix - Quantitative Concentration of Matrix Background) / Spiked Concentration × 100".

[0091] Table 2. Spiked recoveries and relative standard deviations of polyamines (n=3)

[0092]

[0093]

[0094] The test results are shown in Table 2, indicating that the recoveries of putrescine at different concentrations ranged from 98.2% to 108.4%; the recoveries of spermidine at different concentrations ranged from 92.6% to 100.0%; the recoveries of spermine at different concentrations ranged from 82.7% to 91.4%; and the relative standard deviations of the three polyamines were all less than 11%.

[0095] Example 5

[0096] Breast milk samples from different stages (1 month, 4 months, 6 months, and 12 months) and infant formula from different stages (stage 1, stage 2, stage 3, and stage 4) were selected for testing.

[0097] Pretreatment of breast milk or dairy products (sample taken after reconstitution of infant formula according to the mixing ratio): Accurately pipette 150 μL of breast milk or dairy products, 20 μL of isotope mixed standard 2, and 150 μL of lysis buffer, mix thoroughly, and perform detection using steps ② to ③ of "Sample Solid Phase Extraction Treatment (after purification)" in "Example 3", the gas phase conditions of the DB-35 capillary column under "1.1 Selection of Capillary Separation Column", and the optimized mass spectrometry conditions under "1.2 Optimization of Mass Spectrometry Conditions". The acquired data were processed using Thermo Xcalibur software. The raw data were qualitatively analyzed based on the retention time of the compounds and qualitative ion pairs; quantification was accurately performed based on quantitative ion pairs using a matrix-matched internal standard curve.

[0098] The test results show that the method of this invention can sensitively and accurately detect putrescine, spermidine, and spermine in breast milk or dairy products. The test results also show that the polyamine content varies in breast milk at different stages of development, and the polyamine content also differs in formula milk powder at different stages of development. Figure 10 ).

[0099] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry, characterized in that: include: Take 20–200 μL of breast milk and dairy products, add 5–50 μL of isotope mixed standard solution, mix well, then add 100–1000 μL of lysis buffer, mix thoroughly, centrifuge, collect the supernatant, centrifuge to concentrate and dry, resuspend the dried sample, and then derivatize in a water bath at 40–80 °C for 3 hours. After derivatization, continue centrifugation, concentration and drying, redissolve in dichloromethane, and then perform solid-phase extraction purification. Blow the purified solution to near dryness with nitrogen, add 20–200 μL of a mixture of isopropanol and ethyl acetate to redissolve, and perform gas chromatography-tandem mass spectrometry detection; qualitatively identify the polyamine based on the qualitative ion pairs and retention times; substitute the ratio of the peak area of ​​the quantitative ion of the polyamine to the peak area of ​​the quantitative ion of the deuterated isotope internal standard into the matrix matching internal standard curve of the polyamine to obtain the concentration of the polyamine; The pyrolysis solution is a mixed solution consisting of 5% trifluoroacetic acid, 15% deionized water and 80% methanol. The dried sample was resuspended in a mixture of 100-500 μL of ethyl acetate and trifluoroacetic anhydride, wherein the volume ratio of ethyl acetate to trifluoroacetic anhydride was 1:

2. The solid-phase extraction column packing material is Florisil, with a specification of 6mL / 2g. The purification process is as follows: the column is activated with 3-10mL of dichloromethane, then the sample solution is added, followed by rinsing the column with 4-10mL of dichloromethane and 2-8mL of a mixed solution of dichloromethane and ethyl acetate, and finally elution with 6-12mL of ethyl acetate. All eluent is collected. Gas chromatography detection conditions: The chromatographic column is a DB-35 capillary column with the following specifications: column length 15–60 m, inner diameter 0.18–0.32 mm, and film thickness 0.14–0.32 μm; the injection volume is 1–3 μL, the injection method is splitless injection, and the injection port temperature is 200–400 °C; the initial column temperature is 60–150 °C, held for 1 min, increased to 180 °C at 6 °C / min, increased to 260 °C at 15 °C / min, and then increased to 325 °C at 30 °C / min and held for 5 min. Tandem mass spectrometry detection conditions: electron impact ionization source, 70 eV; temperature of both ion source and transfer line, 300℃; detection mode: MS / MS detection mode. The polyamine is selected from one or more of putrescine, spermidine, and spermine.

2. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The deuterated isotopes mentioned are deuterated putrescine and deuterated spermine.

3. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The isotope mixed standard solution is a mixed solution of deuterated putrescine and deuterated spermine, each with a concentration of 6500 nmol / dL.

4. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: Take 150 μL of breast milk or dairy products, add 20 μL of isotope mixed solution, then add 150 μL of lysis buffer, mix thoroughly, centrifuge, and take the supernatant for centrifugation, concentration and drying.

5. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The centrifugation conditions are as follows: at a temperature of 4–40°C, centrifuge at a speed of 5000–15000 rpm / min for 5–30 min.

6. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 5, characterized in that: The centrifugal concentration and drying conditions are as follows: centrifugation at 25°C and 5000-15000 rpm / min until nearly dry.

7. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The derivatization conditions were: derivatization in a 60°C water bath for 3 hours.

8. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The solid-phase extraction purification conditions are as follows: a dried tube after centrifugation and concentration with 2 mL of dichloromethane; the solid-phase extraction column packing material is Florisil with a specification of 6 mL / 2 g; the purification process is as follows: activate the column with 3-10 mL of dichloromethane, then add the sample solution, then rinse the column with 4-10 mL of dichloromethane and 2-8 mL of a mixed solution of dichloromethane and ethyl acetate, and finally add 6-12 mL of ethyl acetate for elution, and collect all the eluent.

9. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The volume ratio of dichloromethane to ethyl acetate in the mixed solution of dichloromethane and ethyl acetate is 8:

2.

10. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The isopropanol and ethyl acetate mixture is a solution with a volume ratio of isopropanol to ethyl acetate of 1:9, and the amount added is 50~100μL.

11. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The gas chromatography detection conditions are as follows: the chromatographic column is a DB-35 capillary column with the following specifications: column length 30m, inner diameter 0.25mm, film thickness 0.25μm; the injection volume is 1μL, the injection method is splitless injection, and the injection port temperature is 300℃; the initial column temperature is 80℃, held for 1min, increased to 180℃ at 6℃ / min, increased to 260℃ at 15℃ / min, and then increased to 325℃ at 30℃ / min and held for 5min.

12. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The qualitative ion pairs of putrescine are 167-70 m / z and 167-139 m / z; the qualitative ion pairs of spermidine are 364-251 m / z and 364-154 m / z; the qualitative ion pairs of spermine are 517-126 m / z and 517-154 m / z; the qualitative ion pairs of deuterated putrescine are 171-72 m / z and 171-141 m / z; and the qualitative ion pairs of deuterated spermine are 525-126 m / z and 525-154 m / z.

13. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 12, characterized in that: The collision energies of ion pairs at 167-139 m / z, 364-251 m / z, and 171-141 m / z are 5 eV; the collision energy of ion pairs at 517-154 m / z is 10 eV; the collision energy of ion pairs at 167-70 m / z, 171-72 m / z, and 525-154 m / z is 15 eV; the collision energy of ion pairs at 364-154 m / z is 20 eV; the collision energy of ion pairs at 517-126 m / z is 30 eV; and the collision energy of ion pairs at 525-126 m / z is 35 eV.

14. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 12, characterized in that: The quantitative ion pairs of putrescine, spermine, spermine, deuterated putrescine, and deuterated spermine are 167-139 m / z, 364-251 m / z, 517-154 m / z, 171-141 m / z, and 525-154 m / z, respectively.

15. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The retention times of putrescine are 14.00-14.40 min, spermidine is 21.80-22.20 min, and spermine is 24.80-25.10 min; the retention times of putrescine-d4 are 13.90-14.30 min, and the retention times of spermine-d8 are 24.80-25.10 min.

16. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 15, characterized in that: The retention times of putrescine are 14.10–14.30 min, spermidine is 21.95–22.10 min, spermine is 24.90–25.00 min, deuterated putrescine is 14.05–14.25 min, and deuterated spermine is 24.85–24.95 min.

17. The method for determining polyamines in breast milk and dairy products by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The matrix-matched internal standard curve of the polyamine was constructed using the following method: 150 μL of mixed breast milk or dairy products, 20 μL of a 6500 nmol / dL isotope mixed standard solution, and mixed standard solutions of different polyamine concentrations were mixed, and then 150 μL of lysis buffer was added to obtain the standard curve solution. The mixture was stirred, centrifuged, and the supernatant was dried and resuspended in a mixture of ethyl acetate and trifluoroacetic anhydride. After derivatization, the solution was concentrated and dried again by vacuum centrifugation, and then reconstituted in 2 mL of dichloromethane. Solid-phase extraction was then performed for purification, and the purified solution was purged to near dryness with nitrogen. Finally, it was reconstituted in a mixture of isopropanol and ethyl acetate for detection by gas chromatography-tandem mass spectrometry. The standard curve was prepared using putrescine, spermidine, or spermine. With the concentration of the polyamines as the x-axis and the ratio of the corrected peak area of ​​the quantitative ion of putrescine, spermidine, or spermine to the peak area of ​​the quantitative ion of the deuterated isotope internal standard as the y-axis, a matrix-matched internal standard curve for putrescine, spermidine, or spermine is established. Specifically, the isotope internal standard corresponding to putrescine is deuterated putrescine, and the isotope internal standards corresponding to spermidine and spermine are deuterated spermine. The corrected peak area of ​​the quantitative ion of putrescine, spermidine, or spermine is the peak area of ​​the quantitative ion of putrescine, spermidine, or spermine minus the peak area of ​​the quantitative ion of putrescine, spermidine, or spermine in the matrix background. The matrix-matched internal standard curve range for the three polyamines is 0–5000 nmol / dL.

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