Method for determining sugar in dairy product by using ultra-high performance liquid chromatography-mass spectrometry

Abstract

The invention relates to the technical field of sugar detection in dairy products, and discloses a method for determining sugar in dairy products by using ultra-high performance liquid chromatography-mass spectrometry, and the method comprises the following steps: establishing a regression equation of various sugar standard substances by using ultra-high performance liquid chromatography-mass spectrometry; removing protein in the dairy product sample by using acetic acid to obtain a dairy product sample without the protein; removing lipids in the dairy product sample without the proteins by using a solid-phase extraction small column to obtain a dairy product sample without the proteins and the lipids; detecting the dairy product sample without proteins and lipids by using ultra-high performance liquid chromatography-mass spectrometry, and carrying out qualitative and quantitative analysis on sugar in the dairy product sample according to the regression equation; wherein in the ultra-high performance liquid chromatography-mass spectrometry, two amino chromatographic columns are connected in series. According to the method provided by the invention, as many as 20 saccharides and sugar alcohols in the dairy product can be accurately determined, and the 20 saccharides and sugar alcohols in the dairy product can be qualitatively and quantitatively determined.

Description

Technical Field

[0001] The present application relates to the technical field of sugar detection in dairy products, and in particular to a method for determining sugar in dairy products using ultra-high performance liquid chromatography-mass spectrometry. Background Art

[0002] Carbohydrates are important functional ingredients in food and are the main source of energy required for all living organisms to maintain life activities. Carbohydrates are polyhydroxy aldehydes, polyhydroxy ketones, and organic compounds that can be hydrolyzed to form polyhydroxy aldehydes or polyhydroxy ketones. They can be divided into monosaccharides, disaccharides, polysaccharides, etc. Among carbohydrates, fructose, glucose, sucrose, maltose, lactose, etc. are often used in various food industries.

[0003] Sugar alcohols are substances that result from the reduction of the aldehyde or ketone groups of sugars. Sugar alcohols are highly stable to acid and heat, are not prone to Maillard reaction, and have low calories, so they will not cause blood sugar to rise, making them the main choice of sweeteners for low-calorie foods.

[0004] At present, sugars and sugar alcohols are widely used in dairy products. For example, sugars and / or sugar alcohol sweeteners are usually added to flavored milk, yogurt, milk drinks, ice cream and other foods. However, excessive intake of sugars or sugar alcohols may cause gastrointestinal intolerance and even diarrhea in some people. Therefore, it is of great significance to detect sugars and sugar alcohols in dairy products.

[0005] In the prior art, the detection methods for sugars and sugar alcohols mainly include high performance liquid chromatography, ion chromatography, high performance liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, gas chromatography, etc. In recent years, there have been reports on the use of high performance liquid chromatography-mass spectrometry to analyze sugars and sugar alcohols in food, but most of the existing methods are aimed at detecting one type of sugar or sugar alcohol, or a few types of sugars and sugar alcohols, which cannot meet the needs of the future sweetener market. Summary of the invention

[0006] To solve the above problems, the present application provides a method for determining sugars in dairy products using ultra-high performance liquid chromatography-mass spectrometry, which can accurately determine up to 20 types of sugars and sugar alcohols in dairy products.

[0007] This application adopts the following technical solutions:

[0008] The present application provides a method for determining sugar in dairy products using ultra-high performance liquid chromatography-mass spectrometry, the method comprising:

[0009] Using ultra-high performance liquid chromatography-mass spectrometry, regression equations for multiple sugar standards were established;

[0010] Using acetic acid to remove proteins from the dairy product sample, thereby obtaining a dairy product sample from which proteins have been removed; using a solid phase extraction column to remove lipids from the dairy product sample from which proteins have been removed, thereby obtaining a dairy product sample from which proteins and lipids have been removed;

[0011] Ultra-high performance liquid chromatography-mass spectrometry was used to detect dairy samples after removing proteins and lipids, and the sugars in the dairy samples were qualitatively and quantitatively analyzed according to the regression equation;

[0012] Among them, two amino chromatographic columns are connected in series in ultra-high performance liquid chromatography-mass spectrometry.

[0013] Optional, multiple sugar standards, including: erythritol, xylose, ribitol, xylitol, rhamnose, fucose, allulose, fructose, glucose, inositol, mannitol, sorbitol, sucrose, maltose, lactose, lactitol, maltitol, maltotriose, isomaltotriose, isomalt.

[0014] Optionally, ultra-high performance liquid chromatography-mass spectrometry is used to establish regression equations for multiple sugar standards, including:

[0015] Each sugar standard was prepared into solutions with different mass concentrations; the solvents in the solutions were acetonitrile and water, and the volume ratio of acetonitrile to water was 1:9;

[0016] Each solution was analyzed by ultra-performance liquid chromatography-mass spectrometry, and the regression equations of various sugar standards were obtained using mass concentration as the independent variable and the peak area of ​​the chromatographic peak as the dependent variable.

[0017] Optionally, acetic acid can be used to remove proteins from dairy samples, including:

[0018] Weigh the dairy product sample into a centrifuge tube, add water and vortex to mix;

[0019] After adding acetic acid and mixing, ultrasonic extraction was performed;

[0020] Transfer to a volumetric flask and dilute to volume with water, shake well, let stand, and then centrifuge at low temperature;

[0021] The supernatant was collected by filtration to obtain a protein-removed dairy product sample.

[0022] Optionally, acetic acid can be used to remove proteins from dairy samples, including:

[0023] If the dairy sample is solid, weigh 2 g of the dairy sample into a 50 mL centrifuge tube, add 40 mL of water, and vortex to mix; add 50-140 μL of acetic acid and mix well, then ultrasonically extract for 15 min; transfer to a volumetric flask and dilute to 50 mL with water, shake well, let stand for 30 min, and centrifuge at 5000 r / min and -10°C for 3 min; filter and collect the supernatant to obtain a deproteinized dairy sample;

[0024] If the dairy sample is liquid or semi-solid, weigh 5 g of the dairy sample into a 50 mL centrifuge tube, add 40 mL of water, and vortex to mix; add 50-140 μL of acetic acid and mix, then ultrasonically extract for 15 min; transfer to a volumetric flask and dilute to 50 mL with water, shake well and let stand for 30 min, then refrigerate centrifuge at 5000 r / min and -10°C for 3 min; filter and collect the supernatant to obtain a protein-removed dairy sample.

[0025] Optionally, lipids can be removed from deproteinized dairy samples using solid phase extraction cartridges, including:

[0026] Methanol and water were used to activate the C18 solid phase extraction column and the NH2 extraction column respectively;

[0027] The first straight-through cleanup of the deproteinized dairy sample was performed using a C18 solid phase extraction cartridge;

[0028] The dairy product samples after the first purification were purified for the second time using NH2 solid phase extraction cartridges;

[0029] The initial part of the filtrate after the second straight-through purification is discarded, and the subsequent filtrate is filtered to obtain a dairy product sample with protein and lipid removed.

[0030] Optionally, the conditions for ultra-high performance liquid chromatography-mass spectrometry are:

[0031] Chromatographic conditions:

[0032] The chromatographic column was an amino column 4.6×150 mm, 5 μm, and two amino columns were connected in series; the injection volume was 5 μL, the flow rate was 1.0 mL / min, and the column temperature was 30°C;

[0033] Mobile phase A was acetonitrile and mobile phase B was water;

[0034] The mobile phase gradient is: 0 min, mobile phase A is 84%, mobile phase B is 16%; 4 min, mobile phase A is 84%, mobile phase B is 16%; 7 min, mobile phase A is 80%, mobile phase B is 20%; 15 min, mobile phase A is 80%, mobile phase B is 20%; 16.5 min, mobile phase A is 82%, mobile phase B is 18%; 19 min, mobile phase A is 79%, mobile phase B is 21%; 20 min, mobile phase A is 65%, mobile phase B is 35%; 25 min, mobile phase A is 65%, mobile phase B is 35%; 25.1 min, mobile phase A is 84%, mobile phase B is 16%; 30 min, mobile phase A is 84%, mobile phase B is 16%.

[0035] Mass spectrometry conditions:

[0036] The ion source was an atmospheric pressure chemical ionization source, the scanning mode was a multiple reaction monitoring negative ion mode, the curtain gas was 30 psi, the nebulizer gas was 45 psi, the collision gas was medium intensity, the heating temperature was 400°C, and the ionization current was 2.00 μA.

[0037] Optionally, the conditions for ultra-high performance liquid chromatography-mass spectrometry are determined by the following method:

[0038] Weigh each sugar standard and prepare a standard solution of each sugar standard respectively, and use the standard solution of each sugar standard to determine the mass spectrometry parameters by mass spectrometry full scan, secondary mass spectrometry scan, collision energy and declustering voltage optimization process;

[0039] A variety of sugar standards were prepared into a mixed standard solution, and the chromatographic parameters were determined using the mixed standard solution and a total ion current optimization process.

[0040] Optionally, weigh each sugar standard and prepare a standard solution of each sugar standard separately, including:

[0041] Weigh each sugar standard and prepare a standard solution with a mass concentration of 50 mg / mL;

[0042] Prepare a variety of sugar standards as mixed standard solutions, including:

[0043] A variety of sugar standards were prepared into a mixed standard solution with a mass concentration of 0.5 mg / mL;

[0044] The solvents in the standard solution and the mixed standard solution are both acetonitrile and water, and the volume ratio of acetonitrile to water is 1:9.

[0045] Optionally, qualitative and quantitative analysis of sugars in dairy samples can be performed based on regression equations, including:

[0046] Determine the type of sugar in the dairy sample according to the retention time of the sample chromatogram obtained by detecting the dairy sample from which protein and lipids are removed by ultra-high performance liquid chromatography-mass spectrometry;

[0047] The chromatographic peak area of ​​each type of sugar was substituted into the regression equation to calculate the content of each type of sugar in the dairy sample.

[0048] The above technical solution adopted in this application can achieve the following beneficial effects:

[0049] This application proposes a method for determining sugars in dairy products using ultra-high performance liquid chromatography-mass spectrometry, which can provide technical support for the detection of up to 20 types of sugars and sugar alcohols in milk and dairy products.

[0050] The present application uses acetic acid to remove proteins from dairy samples, and the removal effect is good. It can avoid the interference of inorganic salts, strong acids, and strong oxidizing substances introduced by heavy metal precipitation methods with the determination or instrument contamination, and it can also avoid the organic phase in the extraction solvent of the organic solvent precipitation method affecting the subsequent lipid removal.

[0051] The present application adopts C18 solid phase extraction cartridge and NH2 solid phase extraction cartridge for two straight-through purifications, and uses water as solvent, which can reduce the loss of sugar and sugar alcohol target analytes during the purification process, effectively improve the recovery rate of target analytes, and improve detection sensitivity.

[0052] In the ultra-high performance liquid chromatography-mass spectrometry of the present application, two amino chromatographic columns (4.6×150 mm, 5 μm) are used in series, which has good specificity, high sensitivity and high accuracy. The detection of 20 target analytes of sugars and sugar alcohols can be completed in 30 minutes, which improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0054] Figure 1 A schematic diagram showing a process of a method for determining sugar in dairy products using ultra-high performance liquid chromatography-mass spectrometry according to one embodiment of the present application;

[0055] Figure 2 The total ion current diagram of various sugars according to one embodiment of the present application is shown. DETAILED DESCRIPTION

[0056] The exemplary embodiments of the present application will be described in more detail below. However, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0057] First, the reagents and consumables involved in the embodiments of the present application are described.

[0058] Reagents and consumables

[0059] Xylose standard, xylitol standard, maltose standard, maltitol standard, lactose standard, lactitol standard, maltotriose standard, isomaltotriose standard, isomaltose standard, rhamnose standard, fucose standard, fructose standard, glucose standard, sucrose standard, allulose standard, erythritol standard, ribitol standard, inositol standard, mannitol standard, sorbitol standard, Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.;

[0060] Acetic acid (analytical grade, 500 mL), China Resources Chemical Materials Technology Co., Ltd.;

[0061] Water (primary), prepared by milliopore pure water instrument;

[0062] Methanol (chromatographic grade), CNW Technology Company, Germany;

[0063] Acetonitrile (chromatographic grade), CNW Technology Company, Germany;

[0064] Straight-through C18 solid phase extraction cartridge (500 mg, 6 mL), CNW Technology, Germany;

[0065] Straight-through NH2 solid phase extraction cartridge (500 mg, 6 mL), CNW Technology, Germany;

[0066] Secondly, the instruments and equipment involved in the embodiments of the present application are described.

[0067] AB4500 ultra-high performance liquid chromatography-mass spectrometry, SCIEX, USA;

[0068] Ultrasonic cleaner, Tianjin Aotesense Instrument Co., Ltd.;

[0069] ME203E electronic analytical balance, METTLER TOLEDO, Switzerland;

[0070] High-speed refrigerated centrifuge, Sichuan Shuke Instrument Co., Ltd.;

[0071] Solid phase extraction device, Shanghai Anpu Experimental Technology Co., Ltd.

[0072] The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry proposed in the present application is described in detail below.

[0073] Figure 1 The schematic diagram of the process of determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to one embodiment of the present application is shown. Figure 1 As shown, the method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry proposed in the present application comprises the following steps:

[0074] Step S100, establishing regression equations for multiple sugar standards using ultra-high performance liquid chromatography-mass spectrometry.

[0075] The present application aims to provide an efficient determination method that can simultaneously perform qualitative and quantitative detection of up to 20 types of sugars and sugar alcohols in dairy products. Dairy products include: various foods made from milk or processed milk products as the main raw materials, with or without other auxiliary materials. For example: dairy products can include pure cow's milk, pure goat's milk, flavored milk, yogurt, milk drinks, ice cream, etc.

[0076] The 20 sugars and sugar alcohols include: erythritol, xylose, ribitol, xylitol, rhamnose, fucose, allulose, fructose, glucose, inositol, mannitol, sorbitol, sucrose, maltose, lactose, lactitol, maltitol, maltotriose, isomaltotriose, and isomalt.

[0077] In order to obtain the regression equations for various sugar standards, the chromatographic conditions and mass spectrometric conditions of ultra-high performance liquid chromatography-mass spectrometry need to be determined first.

[0078] First, determine the mass spectrometry conditions

[0079] (I) Preparation of standard solution

[0080] Weigh appropriate amounts of xylose standard, xylitol standard, maltose standard, maltitol standard, lactose standard, lactitol standard, maltotriose standard, isomaltotriose standard, isomaltulose standard, rhamnose standard, fucose standard, fructose standard, glucose standard, sucrose standard, allulose standard, erythritol standard, ribitol standard, inositol standard, mannitol standard, and sorbitol standard, and prepare them into 50 mg / mL standard solutions.

[0081] The solvents of each standard solution are acetonitrile and water, wherein the volume ratio of acetonitrile to water is 1:9.

[0082] (II) Determination of mass spectrometry conditions

[0083] Each standard solution was subjected to full mass spectrometry scanning by needle pump flow injection continuous sampling to obtain a primary mass spectrum, thereby determining the parent ion of each sugar.

[0084] Then a secondary mass spectrometry scan was performed to obtain the product ions corresponding to each sugar.

[0085] Characteristic ion pairs were found, and the collision energy and declustering voltage were optimized to obtain the mass spectrometry parameters of each sugar.

[0086] Table 1 shows the mass spectrum parameters of each sugar, where * indicates a quantitative ion.

[0087] Table 1:

[0088]

[0089]

[0090]

[0091] Sugar molecules contain only C, H, and O atoms and are of medium polarity. Atmospheric pressure chemical ionization (APCI) is suitable for medium polarity and small molecular compounds. Therefore, the ion source is determined to be an atmospheric pressure chemical ionization source. Since sugars have low thermal stability and are easily decomposed and carbonized at high temperatures, the heating temperature is determined to be 400°C. Only the response value of isomalt in positive mode is better than that in negative mode. The response values ​​of other sugars in negative mode are due to the positive mode, so the scanning mode is determined to be multiple reaction monitoring negative ion mode.

[0092] It was comprehensively determined that the mass spectrometry conditions were: the ion source was an atmospheric pressure chemical ionization source, the scanning mode was a multiple reaction monitoring negative ion mode, the curtain gas was 30 psi, the nebulizer gas was 45 psi, the collision gas was medium intensity, the heating temperature was 400°C, and the ionization current was 2.00 μA.

[0093] Second, determine the chromatographic conditions

[0094] (I) Preparation of mixed standard solution

[0095] Weigh equal amounts of xylose standard, xylitol standard, maltose standard, maltitol standard, lactose standard, lactitol standard, maltotriose standard, isomaltotriose standard, isomaltulose standard, rhamnose standard, fucose standard, fructose standard, glucose standard, sucrose standard, allulose standard, erythritol standard, ribitol standard, inositol standard, mannitol standard, and sorbitol standard to prepare a 0.5 mg / mL mixed standard solution.

[0096] The solvents of the mixed standard solution are acetonitrile and water, wherein the volume ratio of acetonitrile to water is 1:9.

[0097] (II) Determination of chromatographic conditions

[0098] In the prior art, hydrophilic interaction chromatography (HILIC), amino chromatography columns, and sugar analysis columns can be used to determine a small number of sugars or sugar alcohols.

[0099] HILIC (2.1×100mm, 2.6μm), amino chromatography column (2.1×150mm, 1.9μm), sugar analysis column SUGARKS-820 (8×300mm, 6μm), etc., have good separation for a few sugars or sugar alcohols. However, among various sugars, sugars and sugar alcohols are isomers of each other, and the above methods are not good for the separation of multiple sugars including sugars and sugar alcohols at the same time.

[0100] The amino column (4.6×150mm, 5μm) has a better separation effect on isomers, but the resolution is not good.

[0101] Therefore, the present application uses two amino chromatographic columns connected in series, and optimizes the mobile phase ratio and determines the chromatographic conditions based on the total ion current chromatogram.

[0102] The chromatographic conditions were determined to be: an amino column (4.6×150 mm, 5 μm), two amino columns connected in series, an injection volume of 5 μL, a flow rate of 1.0 mL / min, and a column temperature of 30°C; mobile phase A was acetonitrile, and mobile phase B was water; the mobile phase gradient was shown in Table 2.

[0103] Table 2:

[0104] Time (min) Acetonitrile (%) water(%) 0 84 16 4 84 16 7 80 20 15 80 20 16.5 82 18 19 79 21 20 65 35 25 65 35 25.1 84 16 30 84 16

[0105] Figure 2 The total ion flow diagram of various sugars in the method of determining sugars in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to one embodiment of the present application is shown. Figure 2 As shown, 20 kinds of sugars and sugar alcohols are well separated, especially isomers (psicose, fructose, glucose, inositol), (ribitol, xylitol), (rhamnose, fucose), (mannitol, sorbitol), (sucrose, maltose, lactose), (maltitol, lactitol) are well separated. The ultra-high performance liquid chromatography-mass spectrometry used in this application can accurately quantify items with different molecular weights, thereby improving the reliability of the test results.

[0106] Third, determine the regression equation

[0107] (I) Preparation of solutions with different mass concentrations

[0108] Each sugar standard was prepared into solutions with different mass concentrations. The solvents in the solutions were acetonitrile and water, and the volume ratio of acetonitrile to water was 1:9.

[0109] The standard solution of each sugar standard can be diluted step by step to different mass concentrations. For example, for a standard solution of xylose standard with a mass concentration of 50 mg / mL, it can be diluted step by step to 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.13 mg / mL, 1.56 mg / mL, etc. using a solvent with a volume ratio of acetonitrile to water of 1:9.

[0110] 2. Determination of regression equation

[0111] The solutions of different mass concentrations of each sugar standard were sampled and analyzed according to the chromatographic conditions and mass spectrometry conditions, with mass concentration (mg / mL) as the independent variable and the peak area of ​​the chromatographic peak as the dependent variable, and the regression equation, linear correlation coefficient, linear range, detection limit and quantification limit of each sugar standard were obtained. Among them, the detection limit was calculated and determined when the signal-to-noise ratio of the quantitative ion chromatographic peak was 3, and the quantification limit was calculated and determined when the signal-to-noise ratio of the quantitative ion chromatographic peak was 10.

[0112] The regression equations for each sugar are shown in Table 3, where the linear range is in mg / L, the detection limit is in mg / kg, and the quantification limit is in mg / kg.

[0113] Table 3:

[0114]

[0115]

[0116] As can be seen from Table 3, the linear relationships of the 20 sugars and sugar alcohols were good, all reaching above 0.998. The precision of the ultra-high performance liquid chromatography-mass spectrometry was good.

[0117] Step S200, using acetic acid to remove proteins from the dairy sample to obtain a dairy sample from which proteins have been removed; and using a solid phase extraction column to remove lipids from the dairy sample from which proteins have been removed to obtain a dairy sample from which proteins and lipids have been removed.

[0118] This application is to determine sugar in dairy products. Dairy products have complex ingredients, including proteins, fats, phospholipids and other substances. These substances are prone to irreversible damage to the amino chromatographic column during the analysis process, and are also prone to interfere with the ionic strength of the target ions, thereby affecting the measurement results. Therefore, it is necessary to purify the dairy samples before treatment to remove proteins and lipids.

[0119] First, remove the protein

[0120] In the prior art, methods for removing protein from dairy products include heavy metal precipitation, pH adjustment, organic solvent precipitation, etc. For the heavy metal precipitation method, although the precipitation effect is good, when ultra-high performance liquid chromatography-mass spectrometry is used for analysis, the introduced inorganic salts, strong acids, and strong oxidizing substances will interfere with the measurement or contaminate the instrument. For the organic solvent precipitation method, the organic phase in the extraction solvent will affect the subsequent lipid removal, making the pre-treatment operation more cumbersome. Therefore, the present application adopts the pH adjustment method.

[0121] Specifically, the dairy sample is weighed and placed in a centrifuge tube, and water is added and vortexed to mix; acetic acid is added and mixed, and ultrasonic extraction is performed; the sample is transferred to a volumetric flask, the volume is fixed with water, the sample is shaken and allowed to stand, and then frozen and centrifuged; the supernatant is collected by filtration to obtain a dairy sample from which protein has been removed.

[0122] Due to the different states of dairy samples, proteins can be removed by the following methods for dairy samples in different states.

[0123] For dairy samples in a solid state, 2 g of dairy sample can be weighed into a 50 mL centrifuge tube, and about 40 mL of water is added, and vortexed to mix; 50-140 μL of acetic acid is added to mix, and ultrasonic extraction is performed for 15 minutes; the sample is transferred to a 50 mL volumetric flask and made up to 50 mL with water, and the sample is shaken and allowed to stand for 30 minutes; the sample is centrifuged at 5000 r / min and -10°C for 3 minutes, and the supernatant is collected after filtering with filter paper to obtain a dairy sample from which protein has been removed.

[0124] For dairy samples in liquid or semi-solid state, 5 g of dairy sample can be weighed into a 50 mL centrifuge tube, and about 40 mL of water is added, and vortexed to mix; 50-140 μL of acetic acid is added and mixed, and ultrasonic extraction is performed for 15 minutes; the sample is transferred to a 50 mL volumetric flask and made up to 50 mL with water, and the volume is allowed to stand for 30 minutes after shaking; the sample is centrifuged at 5000 r / min and -10°C for 3 minutes, and the supernatant is collected after filtering with filter paper to obtain a dairy sample from which protein has been removed.

[0125] In the protein removal step, after adding about 40 mL of water, 10 mL of n-hexane may be added and vortexed for 1 min, centrifuged at 5000 r / min for 3 min, the n-hexane layer may be discarded, and 50 to 140 μL of acetic acid may be added to the remaining portion.

[0126] The amount of acetic acid should be between 50 and 140 μL. When it is less than 50 μL or greater than 140 μL, the protein removal effect is poor. Since the acidic environment has a certain inhibitory effect on the generation and detection of negative ions, the preferred amount of acetic acid added is 50 μL.

[0127] Second, remove lipids

[0128] In order to reduce the influence of lipid substances on the determination, a solid phase extraction column is used to remove lipids. Table 4 shows the purification recovery rate of each sugar standard by C18 solid phase extraction column (elution purification), C18 solid phase extraction column (straight-through purification) and NH2 solid phase extraction column (straight-through purification). The purification effect of C18 solid phase extraction column (straight-through purification) and NH2 solid phase extraction column (straight-through purification) is better. Therefore, the present application adopts C18 solid phase extraction column straight-through purification to remove oil and weak polar impurities, and then further purifies by NH2 solid phase extraction column straight-through purification to obtain a dairy sample with protein and lipid removed.

[0129] Table 4:

[0130]

[0131] Specifically, methanol and water are used to activate the C18 solid phase extraction column and the NH2 extraction column respectively; the C18 solid phase extraction column is used to perform the first direct purification on the dairy sample after protein removal; the NH2 solid phase extraction column is used to perform the second direct purification on the dairy sample after the first purification; the initial part of the filtrate after the second direct purification is discarded, and the subsequent filtrate is filtered to obtain the dairy sample after protein and lipid removal.

[0132] In actual situations, the C18 solid phase extraction column can be activated with 5 mL of methanol and 5 mL of water in sequence, and the NH2 solid phase extraction column can be activated with 5 mL of methanol and 5 mL of water in sequence.

[0133] The protein-removed dairy sample was purified twice by direct purification through a C18 solid phase extraction column and an NH2 solid phase extraction column, the first 6 mL of filtrate was discarded, and the subsequent 2 mL of filtrate was collected and filtered through a 0.22 μm filter membrane to obtain a protein and lipid-removed dairy sample for determination by ultra-performance liquid chromatography-mass spectrometry.

[0134] To determine the accuracy and precision, a spike recovery experiment was performed to evaluate the results.

[0135] After the dairy product samples were treated to remove protein and lipid using the method proposed in the embodiment of the present application, three different levels of added concentrations were prepared by the addition method, and each added level was measured in parallel 6 times. Among them, only one added concentration of lactose was prepared, and the parallel measurement was 6 times. The measurement results are shown in Table 5 below.

[0136] In Table 5, the unit of spiked amount is mg / kg, the unit of average recovery is %, and the unit of RSD is %.

[0137] Table 5:

[0138]

[0139]

[0140] As can be seen from Table 5, under different spiked conditions, the average recoveries of the 20 sugars and sugar alcohols were between 80% and 120%, and the RSD values ​​of the 20 sugars and sugar alcohols were all less than 10%. Therefore, the above method proposed in this application can be used for the detection and analysis of sugars in dairy samples.

[0141] Step S300, using ultra-high performance liquid chromatography-mass spectrometry to detect the dairy sample from which proteins and lipids have been removed, and performing qualitative and quantitative analysis of sugars in the dairy sample according to a regression equation.

[0142] Specifically, the types of sugars in the dairy sample are determined based on the retention time of the sample chromatogram obtained by ultra-high performance liquid chromatography-mass spectrometry when detecting the dairy sample from which proteins and lipids have been removed; the chromatographic peak area of ​​each type of sugar is substituted into the regression equation to calculate the content of each type of sugar in the dairy sample.

[0143] The dairy sample with protein and lipid removed is injected and analyzed according to the chromatographic conditions and mass spectrometry conditions to obtain the sample chromatogram. The type of sugar in the dairy sample is determined based on the retention time of the sample chromatogram. The chromatographic peak area of ​​each type of sugar is inserted into the corresponding regression equation of each sugar standard to determine the concentration of each type of sugar, thereby determining the content of various sugars in the dairy sample.

[0144] According to the method proposed in this application, the sugar content of a certain yogurt, a certain milk, and a certain milk powder on the market was measured. The measurement results are shown in Table 6 below.

[0145] In Table 6, the unit of each numerical value is g / 100g.

[0146] Table 6:

[0147]

[0148]

[0149] Note: Other sugars and sugar alcohols (erythritol, xylose, ribitol, rhamnose, fucose, allulose, mannitol, lactitol, maltotriose, isomaltotriose, isomaltose) were not detected.

[0150] According to GB28050-2011 "National Food Safety Standard - General Rules for Nutrition Labeling of Prepackaged Foods", the allowable error range of sugar (lactose only) in foods is ≥80% of the labeled value. According to GB10767-2021 "National Food Safety Standard - Infant Formula", for milk-based infant formula (lactose-free), the lactose content of carbohydrates should be ≥50%. This type of milk powder meets the requirements according to the value stated on the product label.

[0151] This application proposes a method for determining sugars in dairy products using ultra-high performance liquid chromatography-mass spectrometry, which can provide technical support for the detection of up to 20 types of sugars and sugar alcohols in milk and dairy products.

[0152] The present application uses acetic acid to remove proteins from dairy samples, and the removal effect is good. It can avoid the interference of inorganic salts, strong acids, and strong oxidizing substances introduced by heavy metal precipitation methods with the determination or instrument contamination, and it can also avoid the organic phase in the extraction solvent of the organic solvent precipitation method affecting the subsequent lipid removal.

[0153] The present application adopts C18 solid phase extraction cartridge and NH2 solid phase extraction cartridge for two straight-through purifications, and uses water as solvent, which can reduce the loss of sugar and sugar alcohol target analytes during the purification process, effectively improve the recovery rate of target analytes, and improve detection sensitivity.

[0154] In the ultra-high performance liquid chromatography-mass spectrometry of the present application, two amino chromatographic columns (4.6×150 mm, 5 μm) are used in series, which has good specificity, high sensitivity and high accuracy. The detection of 20 target analytes of sugars and sugar alcohols can be completed in 30 minutes, which improves the detection efficiency.

[0155] The above is only a specific implementation of the present application. Under the above teachings of the present application, those skilled in the art can make other improvements or modifications based on the above embodiments. Those skilled in the art should understand that the above specific description is only to better explain the purpose of the present application, and the protection scope of the present application shall be based on the protection scope of the claims.

[0156] In addition, those skilled in the art will appreciate that, although some embodiments described herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the claims below, any one of the claimed embodiments may be used in any combination.

Claims

1. A method for determining sugar in dairy products using ultra-high performance liquid chromatography-mass spectrometry, characterized in that: The method comprises: Using ultra-high performance liquid chromatography-mass spectrometry, regression equations for multiple sugar standards were established; Using acetic acid to remove protein from the dairy sample, to obtain the dairy sample from which protein has been removed; using a solid phase extraction column to remove lipids from the dairy sample from which protein has been removed, to obtain the dairy sample from which protein and lipid have been removed; Using the ultra-high performance liquid chromatography-mass spectrometry to detect the dairy product sample from which proteins and lipids have been removed, so as to perform qualitative and quantitative analysis of sugars in the dairy product sample according to a regression equation; Wherein, the ultra-high performance liquid chromatography-mass spectrometry method uses two amino chromatographic columns connected in series.

2. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The various sugar standards include: erythritol, xylose, ribitol, xylitol, rhamnose, fucose, allulose, fructose, glucose, inositol, mannitol, sorbitol, sucrose, maltose, lactose, lactitol, maltitol, maltotriose, isomaltotriose, and isomalt.

3. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The method of establishing a regression equation for a plurality of sugar standards by ultra-high performance liquid chromatography-mass spectrometry comprises: Each sugar standard was prepared into solutions with different mass concentrations; the solvents in the solutions were acetonitrile and water, and the volume ratio of acetonitrile to water was 1:9; The ultra-high performance liquid chromatography-mass spectrometry was used to analyze each solution, and the regression equations of various sugar standards were obtained by taking the mass concentration as the independent variable and the peak area of ​​the chromatographic peak as the dependent variable.

4. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The method of removing protein from a dairy product sample by using acetic acid comprises: Weigh the dairy product sample and place it in a centrifuge tube, add water and vortex mix; After adding acetic acid and mixing, ultrasonic extraction was performed; Transfer to a volumetric flask and dilute to volume with water, shake well, let stand, and then centrifuge at low temperature; The supernatant was collected by filtration to obtain the protein-removed dairy product sample.

5. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 4, characterized in that: The method of removing protein from a dairy product sample by using acetic acid comprises: If the dairy sample is solid, weigh 2 g of the dairy sample into a 50 mL centrifuge tube, add 40 mL of water, and vortex to mix; add 50-140 μL of acetic acid and mix, then ultrasonically extract for 15 min; transfer to a volumetric flask and dilute to 50 mL with water, shake well, let stand for 30 min, and refrigerate centrifuge at 5000 r / min and -10°C for 3 min; filter and collect the supernatant to obtain the dairy sample from which protein has been removed; If the dairy sample is liquid or semi-solid, weigh 5 g of the dairy sample into a 50 mL centrifuge tube, add 40 mL of water, and vortex to mix; add 50-140 μL of acetic acid and mix, then ultrasonically extract for 15 min; transfer to a volumetric flask and dilute to 50 mL with water, shake well and let stand for 30 min, and refrigerate centrifuge at 5000 r / min and -10°C for 3 min; filter and collect the supernatant to obtain the dairy sample from which protein has been removed.

6. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The method of removing lipids from the protein-removed dairy sample by using a solid phase extraction column comprises: Methanol and water were used to activate the C18 solid phase extraction column and the NH2 extraction column respectively; A C18 solid phase extraction cartridge is used to perform a first straight-through purification on the dairy product sample from which protein has been removed; Using an NH2 solid phase extraction column to perform a second straight-through purification on the dairy product sample after the first purification; The initial part of the filtrate after the second straight-through purification is discarded, and the subsequent filtrate is filtered to obtain the dairy product sample with protein and lipid removed.

7. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The conditions of the ultra-high performance liquid chromatography-mass spectrometry are: Chromatographic conditions: The chromatographic column was an amino column 4.6×150 mm, 5 μm, and two amino columns were connected in series; the injection volume was 5 μL, the flow rate was 1.0 mL / min, and the column temperature was 30°C; Mobile phase A was acetonitrile and mobile phase B was water; The mobile phase gradient was as follows: 0 min, mobile phase A was 84%, mobile phase B was 16%; 4 min, mobile phase A was 84%, mobile phase B was 16%; 7 min, mobile phase A was 80%, mobile phase B was 20%; 15 min, mobile phase A was 80%, mobile phase B was 20%; 16.5 min, mobile phase A: 82%, mobile phase B: 18%; 19 min, mobile phase A is 79%, mobile phase B is 21%; 20 min, mobile phase A is 65%, mobile phase B is 35%; 25 min, mobile phase A is 65%, mobile phase B is 35%; 25.1min, mobile phase A was 84% ​​and mobile phase B was 16%; 30min, mobile phase A was 84% ​​and mobile phase B was 16%. Mass spectrometry conditions: The ion source was an atmospheric pressure chemical ionization source, the scanning mode was a multiple reaction monitoring negative ion mode, the curtain gas was 30 psi, the nebulizer gas was 45 psi, the collision gas was medium intensity, the heating temperature was 400°C, and the ionization current was 2.00 μA.

8. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 7, characterized in that: The conditions of the ultra-high performance liquid chromatography-mass spectrometry were determined by the following method: Weigh each sugar standard and prepare a standard solution of each sugar standard respectively, and use the standard solution of each sugar standard to determine the mass spectrometry parameters by mass spectrometry full scan, secondary mass spectrometry scan, collision energy and declustering voltage optimization process; A variety of sugar standards were prepared into a mixed standard solution, and the chromatographic parameters were determined using the mixed standard solution and a total ion current optimization process.

9. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 8, characterized in that: The step of weighing each sugar standard and preparing a standard solution of each sugar standard comprises: Weigh each sugar standard and prepare a standard solution with a mass concentration of 50 mg / mL; The method comprises preparing a plurality of sugar standards into a mixed standard solution, comprising: A variety of sugar standards were prepared into a mixed standard solution with a mass concentration of 0.5 mg / mL; The solvents in the standard solution and the mixed standard solution are both acetonitrile and water, and the volume ratio of acetonitrile to water is 1:

9.

10. The method for determining sugar in dairy products by ultra-high performance liquid chromatography-mass spectrometry according to claim 1, characterized in that: The qualitative and quantitative analysis of sugars in the dairy product sample according to the regression equation comprises: Determining the type of sugar in the dairy product sample according to the retention time of the sample chromatogram obtained by detecting the dairy product sample after removing protein and lipids using the ultra-high performance liquid chromatography-mass spectrometry; The chromatographic peak areas of various types of sugars were substituted into the regression equation to calculate the contents of various sugars in the dairy product samples.

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