GC-MS / MS technology-based method for detecting short-chain free fatty acids in infant formula powder

Abstract

The invention belongs to the technical field of fatty acid detection, and particularly relates to a GC-MS / MS (gas chromatography-mass spectrometry / mass spectrometry) technology-based method for detecting short-chain free fatty acid in baby compounding powder, which comprises the following steps: extracting short-chain free fatty acid in a baby compounding powder dissolved sample by using acetonitrile, and removing precipitated protein through centrifugation; adding anhydrous magnesium sulfate into the centrifuged sample, removing water, standing, adding acetyl chloride / butanol into the supernate for esterification reaction, adding an anhydrous sodium carbonate solution for neutralization, cooling, adding normal hexane for layering, and taking an upper organic phase as a to-be-detected sample; and detecting the sample to be detected and the series deuterated substance mixed standard solution by adopting a GC-MS / MS (Gas Chromatography-Mass Spectrometry / Mass Spectrometry) method, and analyzing the detected data to obtain the content of the short-chain free fatty acid in the baby compounding powder. According to the method, interference ion signals generated by a large number of other coexisting substances in a sample matrix can be effectively eliminated, a target compound can be accurately detected in a complex sample matrix, and finally reliable qualitative and quantitative analysis on the short-chain free fatty acid is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of fatty acid detection, and specifically relates to a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology. Background Art

[0002] Lipids, as the second macronutrient in breast milk, provide a lot of energy for infants and young children. Free fatty acids are an important component of lipids. Their main production pathway is fat hydrolysis, such as the hydrolysis of triglycerides under the action of lipase; the other part is synthesized by mammary cells through a series of complex biochemical reactions. Short-chain free fatty acids generally refer to organic fatty acids with less than 6 carbon atoms in the carbon chain, which play an important role in the human body. Studies have shown that short-chain free fatty acids are an important source of energy for the body. For example, butyric acid provides 60%-70% of the energy for intestinal flora; promotes body metabolism, such as regulating gastrointestinal pH and affecting the absorption of electrolyte nutrients; and also has anti-inflammatory, anti-tumor, immune regulation and nervous system protection.

[0003] Infant formula (IF) is a nutritional supplement specially designed for infants, which aims to simulate the nutritional components of breast milk to meet the growth and development needs of infants. At present, the research on lipids in IF mainly focuses on the simulation of lipid structure, the addition of polyunsaturated fatty acids, the adjustment of fatty acid ratios, etc., and there is little research on short-chain free fatty acids. The lack of short-chain free fatty acids will affect the intestinal, immune, metabolic and other body functions of infants. Therefore, a detection method for short-chain free fatty acids in IF is needed to ensure that the ingredients in IF meet the growth and development needs of infants.

[0004] At present, the methods for detecting short-chain free fatty acids mostly use gas chromatography. Most studies extract short-chain free fatty acids through pretreatment and then directly perform GC analysis. The disadvantage of this detection method is that it ignores factors such as the volatility of short-chain fatty acids and the large difference in polarity, which ultimately leads to poor recovery, poor chromatographic separation and low detection sensitivity. In order to improve the problems encountered in these tests, derivatization treatment began to be introduced in the pretreatment. For example, Siciliano et al. used diazomethane and boron trifluoride solution to methylate short-chain free fatty acids. This method has good detection results, but the reagents used are flammable and explosive and dangerous to operate; Jialin Du et al. used trimethylsilane to derive short-chain free fatty acids. The derivatization reagents of this method are sensitive to moisture requirements. Because short-chain fatty acids have a small molecular weight, derivatization with alcohols with larger molecular weight can better separate and quantify them more accurately in chromatography, so we use butanol esterification in pretreatment.

[0005] The short-chain free fatty acid detection method based on GC-MS / MS technology has a good detection effect in infant formula detection. Most studies use the selected ion monitoring mode (SIM) on GC-MS. Our method uses the multiple reaction monitoring mode (MRM), which reduces background interference and noise, and has higher selectivity, sensitivity and quantitative accuracy. When using the short-chain free fatty acid detection method of GC-MS / MS technology for analysis, it is usually necessary to pre-treat the sample, optimize the amount of extraction reagent, esterification reagent selection, esterification temperature and time, etc.; optimize the split ratio, program temperature, electron bombardment energy, collision energy, etc. in the GC-MS / MS part. However, the problems with the existing determination methods in extraction are excessive reagent dosage, dangerous esterification reagents, and long esterification time; the analysis time on the instrument is too long, and the peak shape overlaps and tails. The present invention optimizes in detail the main parameters in the detection method of short-chain free fatty acids in infant formula using GC-MS / MS technology. Compared with the existing method, the improved method has the advantages of small reagent dosage, high safety, short esterification time, short chromatographic analysis time, good chromatographic peak shape, and more accurate quantification, which provides a favorable reference for the quantitative analysis of infant formula.

[0006] Based on this technical background, the present invention studies a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology. Summary of the invention

[0015] In view of the deficiencies in the prior art, the present invention provides a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology. The method uses derivatization treatment as a pretreatment, which can effectively reduce the influence of the small molecular weight, volatility and instability of short-chain free fatty acids. The instrument part can quickly and efficiently analyze and detect short-chain free fatty acids.

[0016] In order to achieve the above object, the present invention provides a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology, the method comprising the following steps:

[0017] (1) Take a sample of infant formula and dissolve it in water;

[0018] (2) extracting short-chain free fatty acids in the dissolved infant formula sample using an extractant, acetonitrile, and removing precipitated protein by centrifugation;

[0019] (3) adding anhydrous magnesium sulfate as a dehydrating agent to the sample after centrifugation to remove water and allowing the sample to stand to promote further transfer of short-chain free fatty acids to the acetonitrile phase;

[0020] (4) taking the supernatant after dehydration and standing to a stoppered tube;

[0021] (5) adding acetyl chloride / butanol as an esterification reagent to the supernatant in the stoppered tube, and placing the tube in a constant temperature water bath for esterification reaction;

[0022] (6) adding anhydrous sodium carbonate solution to the solution after the esterification reaction for neutralization;

[0023] (7) After the neutralized solution is cooled to room temperature, n-hexane is added, and after separation, the upper organic phase is taken as the sample to be tested;

[0024] (8) using gas chromatography-tandem triple quadrupole mass spectrometry to detect the sample to be tested and the series of deuterated isotope mixed standard solutions to obtain detection data;

[0025] (9) performing qualitative analysis and / or quantitative analysis on the test data to obtain the content of short-chain free fatty acids in the infant formula.

[0026] The beneficial effects of the present invention include:

[0027] (1) The present invention proposes a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology. Acetonitrile and anhydrous magnesium sulfate are used for pretreatment. The introduction of the extraction reagent acetonitrile has three advantages: first, protein is precipitated; second, the extraction reagent extracts short-chain free fatty acids; third, acetonitrile is a green, environmentally friendly and less harmful esterification solvent system, which enhances the esterification effect. In addition, the introduction of anhydrous magnesium sulfate has the following benefits for the extraction effect of fatty acids: adding anhydrous magnesium sulfate to the acetonitrile-water-fatty acid mixed system, anhydrous magnesium sulfate quickly absorbs water, promotes the acetonitrile phase and water phase separation, better enriches short-chain free fatty acids in the acetonitrile phase, and improves the extraction recovery rate; removing water from the system is conducive to the esterification reaction in the acetonitrile phase, reducing the influence of water on the esterification reaction, and having a better esterification effect.

[0028] (2) The present invention is based on the GC-MS / MS technology to detect short-chain free fatty acids in infant formula. By using butyl esterification derivatization means, the short-chain free fatty acids are derivatized to change the properties of short-chain free fatty acids due to strong volatility and high polarity, thereby improving the phenomenon of short retention time and poor peak shape in the chromatographic column. Converting short-chain free fatty acids into fatty acid butyl esters increases their molecular weight, reduces their polarity and volatility, and better separates and improves the detection effect.

[0029] (3) The method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology of the present invention can effectively eliminate the interfering ion signals generated by a large number of other substances coexisting in the sample matrix, and can accurately detect the target compounds in a complex sample matrix, ultimately achieving reliable qualitative and quantitative analysis of short-chain free fatty acids.

[0030] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0032] Figure 1 The present invention provides a flow chart of the processing and analysis of infant formula samples according to the method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology.

[0033] Figure 2 This is a chromatogram of the separation of short-chain free fatty acids in the method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology proposed by the present invention.

[0034] Figure 3 This is a graph showing the influence of the amount of extractant on the signal response value of short-chain free fatty acids in the short-chain free fatty acid detection method in infant formula based on GC-MS / MS technology proposed in the present invention.

[0035] Figure 4 This is a graph showing the influence of the amount of anhydrous magnesium sulfate, a dehydrating reagent, on the signal response value of short-chain free fatty acids in the GC-MS / MS technology-based short-chain free fatty acid detection method in the present invention.

[0036] Figure 5 This is a diagram showing the influence of different esterification reagents on the signal response value of short-chain free fatty acids in the detection method of short-chain free fatty acids in infant formula based on GC-MS / MS technology proposed in the present invention.

[0037] Figure 6 This is a graph showing the influence of the amount of esterification reagent on the signal response value of short-chain free fatty acids in the short-chain free fatty acid detection method in infant formula based on GC-MS / MS technology proposed in the present invention.

[0038] Figure 7 This is a diagram showing the influence of esterification temperature on the signal response value of short-chain free fatty acids in the short-chain free fatty acid detection method in infant formula based on GC-MS / MS technology proposed in the present invention.

[0039] Figure 8 This is a diagram showing the influence of esterification time on the signal response value of short-chain free fatty acids in the short-chain free fatty acid detection method in infant formula based on GC-MS / MS technology proposed in the present invention. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0041] Terminology

[0042] The abbreviations of the terms used in the present invention have the following meanings:

[0043] Propionic acid-propionic acid, Isobutyric acid-isobutyric acid, Butyric acid-butyric acid, Isovaleric acid-isovaleric acid, Valeric acid-valeric acid, 2-Methylpentanoic acid-2-methylpentanoic acid, Hexanoic acid-hexanoic acid, SIM-selective ion monitoring mode, MRM-multiple reaction monitoring mode, EI-electron impact ionization source, anhydrous sodium carbonate-Na2CO3, CE-collision energy, ME-matrix effect, GC-MS / MS-gas chromatography-tandem triple quadrupole mass spectrometry.

[0044] The present invention provides a method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology. Figure 1 As shown, the following steps are included:

[0045] (1) Take a sample of infant formula and dissolve it in water;

[0046] (2) extracting short-chain free fatty acids from the infant formula solution using acetonitrile as an extractant, and removing precipitated proteins by centrifugation;

[0047] (3) adding anhydrous magnesium sulfate as a dehydrating agent to the sample after centrifugation to remove water and allowing the sample to stand to promote further transfer of short-chain free fatty acids to the acetonitrile phase;

[0048] (4) taking the supernatant after dehydration and standing to a stoppered tube;

[0049] (5) adding acetyl chloride / butanol as an esterification reagent to the supernatant in the stoppered tube, and placing the tube in a constant temperature water bath for esterification reaction;

[0050] (6) adding anhydrous sodium carbonate solution to the solution after the esterification reaction for neutralization;

[0051] (7) After the neutralized solution is cooled to room temperature, n-hexane is added, and after separation, the upper organic phase is taken as the sample to be tested;

[0052] (8) using gas chromatography-tandem triple quadrupole mass spectrometry to detect the sample to be tested and a series of deuterated isotope mixed standard solutions to obtain detection data;

[0053] (9) Performing qualitative and / or quantitative analysis on the test data to obtain the content of short-chain free fatty acids in the infant formula.

[0054] In the present invention, acetonitrile and anhydrous magnesium sulfate are used in the pretreatment, and the introduction of the extraction reagent acetonitrile has three advantages: first, protein is precipitated; second, the extraction reagent extracts short-chain free fatty acids; third, acetonitrile is a green, environmentally friendly and less harmful esterification solvent system, which enhances the esterification effect. In addition, the introduction of anhydrous magnesium sulfate has the following benefits for the extraction effect of fatty acids: adding anhydrous magnesium sulfate to the acetonitrile-water-fatty acid mixed system, the anhydrous magnesium sulfate quickly absorbs water, promotes the acetonitrile phase and the water phase to better enrich the short-chain free fatty acids in the acetonitrile phase, and improves the extraction recovery rate; removing water in the system is conducive to the esterification reaction in the acetonitrile phase, reducing the influence of water on the esterification reaction, and having a better esterification effect.

[0055] The present invention investigates the use of acetonitrile for protein precipitation and target extraction in pretreatment, adds anhydrous magnesium sulfate to remove water and enrich short-chain free fatty acids in the acetonitrile phase, compares the esterification effects of different esterification reagents in the derivatization stage, and finally selects the acetyl chloride / butanol esterification method to esterify the short-chain free fatty acids.

[0056] According to the present invention, the parameters of the method include the amount of acetonitrile used as the extractant, the amount of anhydrous magnesium sulfate used as the dehydrating agent, the type of esterification agent selected, the amount of esterification agent used, the esterification temperature and the esterification time;

[0057] The amount of acetonitrile used as the extractant is 1-3 mL, and the volume ratio of acetonitrile to the infant formula dissolved sample is 1-3:1;

[0058] The dosage of dehydrating agent anhydrous magnesium sulfate is 700-900 mg;

[0059] The mass concentration of the esterification reagent acetyl chloride / butanol is 5-20%, wherein acetyl chloride is the solute and butanol is the solvent;

[0060] The volume ratio of the esterification reagent to the supernatant is 1-3:1;

[0061] The temperature of the esterification reaction is 50-70°C and the time is 5-15 minutes.

[0062] According to the present invention, the optimal value of the parameter is obtained by a signal response method, which includes the following steps:

[0063] (1) Change the value of any one parameter while keeping the other parameters unchanged;

[0064] (2) using gas chromatography-tandem triple quadrupole mass spectrometry to detect the values ​​of different parameters of the test sample;

[0065] (3) Calculate the signal response peak area based on the chromatogram of the sample to be tested. The maximum value of the peak area corresponds to the optimal value of the parameter.

[0066] In the present invention, the chromatogram of short-chain free fatty acid separation is as follows: Figure 2 shown.

[0067] The present invention investigates the effect of the amount of extractant on the signal response value. Insufficient amount of extractant will make acetonitrile unable to fully extract the target compound, affecting the extraction efficiency; excessive amount of extractant will dilute the target compound, affecting the detection result; The present invention investigates the effect of the amount of extractant on the signal response value of short-chain free fatty acids when the amount of extractant is 2:1, 3:1, 4:1, 6:1, and 8:1. When the amount of extractant is 2:1, the signal value response of the target analyte is the best. The effect of the amount of extractant on the signal response value of short-chain free fatty acids is as follows: Figure 3 shown.

[0068] The amount of anhydrous magnesium sulfate used as a dehydrating agent will affect the detection response of short-chain free fatty acids to a certain extent. Insufficient anhydrous magnesium sulfate used as a dehydrating agent will cause water to affect the subsequent esterification efficiency, and excessive anhydrous magnesium sulfate used as a dehydrating agent may result in adsorption of the target analyte. Therefore, the present invention sets different amounts of anhydrous magnesium sulfate to 600mg, 700mg, 800mg, and 1000mg to evaluate the impact on the signal response value of the target analyte. The response is optimal at 800mg. The impact of the amount of anhydrous magnesium sulfate used as a dehydrating agent on the signal response value of short-chain free fatty acids is shown in the figure. Figure 4 shown.

[0069] The choice of esterification reagent has a certain influence on the response of short-chain free fatty acids. Therefore, the present invention evaluates different esterification reagents: 5% H2SO4 / butanol, 10% HCl / butanol, 10% acetyl chloride / butanol, and 15% boron trifluoride-ether / butanol. By comparing the response values, it is found that the esterification effect of short-chain free fatty acids is best under 10% acetyl chloride / butanol. The influence of different esterification reagents on the signal response value of short-chain free fatty acids is shown in the figure. Figure 5 As shown in Figure 2, the effect of the amount of esterification reagent on the signal response value of short-chain free fatty acids is as follows: Figure 6 shown.

[0070] The esterification time affects the esterification efficiency and pretreatment time, and is crucial to judging the short-chain free fatty acid pretreatment method. The present invention sets different esterification times of 5 min, 10 min, 15 min, 30 min, 60 min, and 90 min to find the optimal esterification time. By comparing the signal response values, it is found that the esterification response intensity is the highest at 10 min. If the esterification time is continuously increased, the response intensity decreases or remains unchanged. The effect of the esterification time on the short-chain free fatty acid signal response value is shown in FIG. Figure 8 shown.

[0071] The esterification temperature affects the progress of the esterification reaction. The present invention sets different esterification temperatures of 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C. The esterification signal response shows that the esterification effect is best at 60°C. The effect of the esterification temperature on the short-chain free fatty acid signal response value is shown in FIG. Figure 7 shown.

[0072] In the present invention, the short-chain free fatty acids are subjected to derivatization treatment by using butyl esterification, thereby changing the properties of short-chain free fatty acids due to strong volatility and high polarity, thereby improving the phenomenon of short retention time and poor peak shape in the chromatographic column. Short-chain free fatty acids are converted into fatty acid butyl esters to increase their molecular weight, reduce their polarity and volatility, and better separate and improve the detection effect.

[0073] According to the present invention, a series of deuterated isotope mixed standard solutions are obtained by mixing the isotopes of short-chain free fatty acids and water;

[0074] The concentrations of the series of deuterated isotope mixed standard solutions are 1, 2, 5, 10, 50, 100, 200, 500, 1000 and 2000 times the limit of quantification (LOQ) of different short-chain free fatty acids.

[0075] According to the present invention, the electron bombardment source parameters in the triple quadrupole mass spectrometry of gas chromatography-tandem triple quadrupole mass spectrometry include:

[0076] The acquisition mode was MRM mode;

[0077] The ion source temperature is 220-240°C;

[0078] MS1 quadrupole temperature is 140-160°C;

[0079] MS2 quadrupole temperature is 140-160°C;

[0080] MSD temperature is 240-260°C;

[0081] The collision gas was nitrogen.

[0082] According to the present invention, the data of retention time, ionization energy, parent ion, quantitative daughter ion, collision energy 1, qualitative daughter ion, collision energy 2, internal standard parent ion, internal standard qualitative daughter ion, and internal standard quantitative daughter ion of short-chain free fatty acids in triple quadrupole mass spectrometry of gas chromatography-tandem triple quadrupole mass spectrometry are as follows:

[0083]

[0084] In the present invention, the internal standard parent ion, the internal standard qualitative daughter ion, and the internal standard quantitative daughter ion are respectively the deuterated isotope parent ion, the deuterated isotope qualitative daughter ion, and the deuterated isotope quantitative daughter ion.

[0085] According to the present invention, the gas chromatography conditions of gas chromatography-tandem triple quadrupole mass spectrometry include:

[0086] The injection port temperature is 270-290°C;

[0087] The carrier gas is helium;

[0088] The flow rate of the carrier gas is 1.4-1.6 mL / min, the injection method adopts the split mode, the split ratio of the split mode is 8-12:1, and the injection volume is 0.8-1.2 μL.

[0089] According to the present invention, the temperature program data of the gas chromatography of the gas chromatography-tandem triple quadrupole mass spectrometry is as follows:

[0090]

[0091] According to the present invention, the detection data are chromatograms and mass spectra of short-chain free fatty acids;

[0092] Qualitative analysis is to identify short-chain free fatty acids by comparing retention times in chromatograms and comparing parent ion and daughter ion fragments in mass spectra;

[0093] The quantitative analysis is to integrate the chromatogram of the series of deuterated isotope mixed standard solutions to obtain the linear equation Y=aX+b for each short-chain free fatty acid, where Y represents the response peak area of ​​the short-chain free fatty acid, and X represents the concentration of the short-chain free fatty acid. The sample to be tested is detected by gas chromatography-tandem triple quadrupole mass spectrometry to obtain the response peak area of ​​the sample to be tested, and the concentration of the short-chain free fatty acid in the sample to be tested is calculated according to the linear equation.

[0094] The present invention can effectively eliminate interfering ion signals generated by a large number of other substances coexisting in the sample matrix, can accurately detect target compounds in complex sample matrices, and ultimately achieve reliable qualitative and quantitative analysis of short-chain free fatty acids.

[0095] The present invention is described in more detail below through embodiments and test examples.

[0096] In the examples, the instruments and reagents used are as follows:

[0097] 7890A-7000 gas chromatograph / triple quadrupole mass spectrometer (Agilent, USA); balance ME614S (Sartorius, Germany); balance XPE56Q (Mettler-Toledo, Switzerland); Vortex-Genie 2 vortex mixer (Scientific Industries, USA); GR21GⅢ centrifuge (Hitachi, Japan); refrigerator (Haier, China).

[0098] Short-chain free fatty acid butyl ester standards and short-chain free fatty acid isotope internal standards: butyl propionate, butyl isobutyrate, butyl butyrate, butyl isovalerate, butyl valerate, butyl 2-methylvalerate, and butyl hexanoate standard solutions were purchased from McLean; propionic acid-d3 and hexanoic acid-d3 were purchased from (MCE, USA); isobutyric acid-d6, butyric acid-d7, isovalerate-d9, and valeric acid-d9 were purchased from (McLean, Shanghai).

[0099] Acetonitrile (chromatographic grade) was purchased from Merck; acetyl chloride, anhydrous magnesium sulfate, and sodium carbonate were purchased from China MacLean Chemical Co., Ltd.; butanol (chromatographic grade) was purchased from Italy Sigma; and purified water was purchased from China Wahaha Company.

[0100] Example

[0101] like Figure 1 As shown, this embodiment proposes a method for determining short-chain free fatty acids in infant formula based on gas chromatography-tandem triple quadrupole mass spectrometry, and the specific steps include:

[0102] (1) Preparation of isotope standards:

[0103] The isotopes of short-chain free fatty acids are mixed with water to obtain isotope mixed standards of the corresponding short-chain free fatty acids. The concentrations of the series of mixed standard solutions are 1, 2, 5, 10, 50, 100, 200, 500, 1000, and 2000 times the quantification limit (LOQ) of different short-chain free fatty acids.

[0104] (2) Pretreatment of infant formula samples:

[0105] Take 0.15g of infant formula sample in a 15mL centrifuge tube, add 1mL of water to the centrifuge tube and vortex for 1min to fully dissolve the infant formula; after it is fully dissolved, add 2mL of acetonitrile to each centrifuge tube, place it in a multi-tube vortex mixer and vortex for 1min, and then centrifuge it in a centrifuge at 5000rpm for 10min; take the supernatant, add 800mg of dehydration reagent anhydrous magnesium sulfate to the supernatant, vortex for 2min, and centrifuge it in a centrifuge at 5000rpm for 3min; take 0.5mL of supernatant to a glass stopper tube, add 1mL of freshly prepared 10% acetyl chloride / butanol to the glass stopper tube, and place the glass stopper tube in a constant temperature water bath at 70℃ for 10min; after cooling, add 2.5mL of 6% anhydrous sodium carbonate solution to the stopper tube for neutralization; after neutralization, add 0.5mL of n-hexane to shake and mix; take 1mL of supernatant to a sampling vial for testing;

[0106] (3) GC-MS / MS detection:

[0107] In this embodiment, the gas chromatograph used is Agilent's 7890A, the chromatographic column is Agilent DB-FastFAME (30m×0.25mm×0.25μm), the injection port temperature is 280°C, the carrier gas is helium, the carrier gas flow rate is 1.5mL / min, the injection mode adopts the split mode, the split ratio is 10:1, the injection volume is 1μL, and the gas chromatograph temperature program is as shown in Table 1:

[0108] Table 1 Gas chromatograph temperature program data

[0109]

[0110] The mass spectrometry detection conditions were as follows: the triple quadrupole mass spectrometer was Agilent 7000, and the electron impact source (EI) parameters were as follows: acquisition mode MRM mode, ion source temperature 230°C, MS1 quadrupole temperature 150°C, MS2 quadrupole temperature 150°C, MSD temperature 250°C, collision gas nitrogen; the data acquisition and processing software was MassHunter;

[0111] This embodiment optimizes the data of retention time, ionization energy, parent ion, quantitative daughter ion, collision energy 1, qualitative daughter ion, collision energy 2, internal standard parent ion, internal standard qualitative daughter ion, and internal standard quantitative daughter ion in the mass spectrometry detection of short-chain free fatty acids by gas chromatography-tandem triple quadrupole mass spectrometry, as shown in Table 2;

[0112] Table 2 Mass spectrometry conditions data

[0113]

[0114]

[0115] Test Example 1

[0116] This test example tests the linear range, detection limit and quantification limit of the method for determining short-chain free fatty acids in infant formula by gas chromatography-tandem triple quadrupole mass spectrometry. The test method is as follows: the sample is prepared by adding low-concentration short-chain free fatty acid isotope mixed standard to infant formula, pre-treated and tested on the machine, the detection limit is determined by 3 times the signal-to-noise ratio, and the quantification limit is determined by 10 times the signal-to-noise ratio; a certain volume of isotope standard stock solution is used to prepare a series of mixed standard working solutions with the same matrix as the infant formula sample to be tested, so that the concentration is divided into The concentrations of the quantification ions of the target analytes were 1, 2, 5, 10, 50, 100, 200, 500, 1000 and 2000 times of the LOQ, respectively. The standard curve was made based on the quantitative ion peak area and concentration of the selected target analytes. The results showed that the linear range of the matrix matching curves of propionic acid, isobutyric acid and butyric acid was 1-2000 times LOQ, and the linear range of the matrix matching curves of valeric acid, isovaleric acid, 2-methylvaleric acid and hexanoic acid was 1-1000 times LOQ, and the linear relationships were all greater than 0.999. The test results are shown in Table 3.

[0117] Test Example 2

[0118] In this test example, the accuracy and precision of the method for determining short-chain free fatty acids in infant formula by gas chromatography-tandem triple quadrupole mass spectrometry were tested. The test method was as follows: a short-chain free fatty acid isotope mixed standard solution was added to the infant formula sample to prepare three samples at different concentration levels (16 times LOQ, 160 times LOQ, and 320 times LOQ), and three parallel tests were performed for each batch at each concentration, and three batches were performed respectively; the analysis and determination were performed according to the sample pretreatment method and chromatographic conditions of the present invention, and the matrix-matched mixed standard working solution was used for external standard quantification. The calculated recovery rate was 78-99%, the intra-day relative standard deviation was less than 3%, and the accuracy and precision met the detection requirements. The test results are shown in Table 3.

[0119] Test Example 3

[0120] This test example tests the matrix effect of the method for determining short-chain free fatty acids in infant formula by gas chromatography-tandem triple quadrupole mass spectrometry. The test method is as follows: adopting the extraction and addition method, taking an infant formula sample, treating the sample according to the sample pretreatment method of the present invention, and nitrogen blowing after obtaining 0.5 mL of supernatant, selecting three samples with different concentration levels (16 times LOQ, 160 times LOQ, 320 times LOQ) for isotope mixed standard solutions for redissolution, and then performing subsequent esterification operations, and finally operating on the machine; after the on-machine test, the peak area ratio of the infant formula re-solution and the mixed standard working solution of the same concentration is calculated to evaluate the influence of the matrix effect on the result; according to the matrix effect calculation formula, the evaluation is performed, and the short-chain free fatty acid matrix effect range is calculated to be -7.7% to 21.9%, indicating that the matrix effect is relatively weak and can be ignored in the test of actual samples. The test results are shown in Table 3;

[0121] In this embodiment, the matrix effect calculation formula is:

[0122] ME (%) = [1-(sample pretreatment nitrogen blowing spike re-dissolution concentration / acetonitrile standard re-dissolution concentration)] × 100%;

[0123] Table 3 Test results of test case 1-3

[0124]

[0125]

[0126] In this embodiment, acetonitrile-water-anhydrous magnesium sulfate extraction and butanol esterification pretreatment technology combined with GC-MS / MS method are used to analyze and detect 7 short-chain free fatty acids in infant formula; the method is fast, simple, highly sensitive, and the matrix effect is negligible; the 7 short-chain free fatty acids have good linearity in the range of 1-2000 times / 1000 times of LOQ, and the linear relationship is greater than 0.999; the accuracy and precision of the method meet the detection requirements of the analysis; it provides an efficient and rapid analytical means for the detection research of short-chain free fatty acids in infant formula.

[0127] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for detecting short-chain free fatty acids in infant formula based on GC-MS / MS technology, characterized in that: The method comprises the following steps: (1) Take a sample of infant formula and dissolve it in water; (2) extracting short-chain free fatty acids in the dissolved infant formula sample using an extractant, acetonitrile, and removing precipitated protein by centrifugation; (3) adding anhydrous magnesium sulfate as a dehydrating agent to the sample after centrifugation to remove water and allowing the sample to stand to promote further transfer of short-chain free fatty acids to the acetonitrile phase; (4) taking the supernatant after dehydration and standing to a stoppered tube; (5) adding acetyl chloride / butanol as an esterification reagent to the supernatant in the stoppered tube, and placing the tube in a constant temperature water bath for esterification reaction; (6) adding anhydrous sodium carbonate solution to the solution after the esterification reaction for neutralization; (7) After the neutralized solution is cooled to room temperature, n-hexane is added, and after separation, the upper organic phase is taken as the sample to be tested; (8) using gas chromatography-tandem triple quadrupole mass spectrometry to detect the sample to be tested and a series of deuterated isotope mixed standard solutions to obtain detection data; (9) performing qualitative analysis and / or quantitative analysis on the test data to obtain the content of short-chain free fatty acids in the infant formula.

2. The method according to claim 1, characterized in that: The parameters of the method include the amount of acetonitrile used as the extractant, the amount of anhydrous magnesium sulfate used as the dehydrating agent, the type of esterification agent selected, the amount of esterification agent used, the esterification temperature and the esterification time; The amount of the extractant acetonitrile used is 1-3 mL, and the volume ratio of the extractant acetonitrile to the infant formula dissolved sample is 1-3:1; The dosage of the dehydrating agent anhydrous magnesium sulfate is 700-900 mg; The mass concentration of the esterification reagent acetyl chloride / butanol is 5-20%, wherein acetyl chloride is the solute and butanol is the solvent; The volume ratio of the esterification reagent to the supernatant is 1-3:1; The temperature of the esterification reaction is 50-70° C. and the time is 5-15 min.

3. The method according to claim 2, characterized in that The optimal value of the parameter is obtained by a signal response method, which includes the following steps: (1) Change the value of any one parameter while keeping the other parameters unchanged; (2) using the gas chromatography-tandem triple quadrupole mass spectrometry to detect the values ​​of different parameters of the sample to be tested; (3) Calculating the signal response peak area according to the chromatogram of the sample to be tested, wherein the maximum value of the peak area corresponds to the optimal value of the parameter.

4. The method according to claim 1, characterized in that: The series of deuterated isotope mixed standard solutions are obtained by mixing the isotopes of the short-chain free fatty acids and water; The concentrations of the series of deuterated isotope mixed standard solutions are 1 times, 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 1000 times, and 2000 times the quantitative limits of different short-chain free fatty acids.

5. The method according to claim 1, characterized in that The electron bombardment source parameters in the triple quadrupole mass spectrometry of the gas chromatography-tandem triple quadrupole mass spectrometry include: The acquisition mode was MRM mode; The ion source temperature is 220-240°C; MS1 quadrupole temperature is 140-160°C; MS2 quadrupole temperature is 140-160°C; MSD temperature is 240-260°C; The collision gas was nitrogen.

6. The method according to claim 1, characterized in that The data of retention time, ionization energy, parent ion, quantitative daughter ion, collision energy 1, qualitative daughter ion, collision energy 2, internal standard parent ion, internal standard qualitative daughter ion, and internal standard quantitative daughter ion of the short-chain free fatty acid in the triple quadrupole mass spectrometry of the gas chromatography-tandem triple quadrupole mass spectrometry are as follows: 。 7. The method according to claim 1, characterized in that The gas chromatography conditions of the gas chromatography-tandem triple quadrupole mass spectrometry include: The injection port temperature is 270-290°C; The carrier gas is helium; The flow rate of the carrier gas is 1.4-1.6 mL / min, the injection method adopts the split mode, the split ratio of the split mode is 8-12:1, and the injection volume is 0.8-1.2 μL.

8. The method according to claim 1, characterized in that The temperature program data of the gas chromatography of the gas chromatography-tandem triple quadrupole mass spectrometry are as follows:

9. The method according to claim 1, characterized in that: The detection data is a chromatogram and a mass spectrum of the short-chain free fatty acids; The qualitative analysis is to qualitatively identify the short-chain free fatty acids by comparing the retention time in the chromatogram and comparing the parent ion and daughter ion fragments in the mass spectrum; The quantitative analysis is to integrate the chromatograms of the series of deuterated isotope mixed standard solutions to obtain a linear equation Y=aX+b for each short-chain free fatty acid, where Y represents the response peak area of ​​the short-chain free fatty acid and X represents the concentration of the short-chain free fatty acid. The gas chromatography-tandem triple quadrupole mass spectrometry is performed on the sample to be tested to obtain the response peak area of ​​the sample to be tested, and the concentration of the short-chain free fatty acid in the sample to be tested is calculated according to the linear equation.

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