Analysis method of carbohydrates in sialic acid lactose and application of analysis method

Abstract

The invention relates to the technical field of food science and analytical chemistry, in particular to a method for analyzing carbohydrates in sialic acid lactose and application of the method. The analysis method disclosed by the invention comprises the following steps: separating carbohydrates through ultra-high performance liquid chromatography, wherein in the ultra-high performance liquid chromatography, a mobile phase A comprises acetonitrile; the mobile phase B comprises 10-30 mmol / L of an ammonium formate aqueous solution; the elution procedure is a specific gradient elution procedure. According to the analysis method, lactose, N-acetylneuraminic acid, 3 '-sialic acid lactose, 6'-sialic acid lactose, 3 '-sialic acid lactulose, 6'-sialic acid lactulose and other carbohydrates in sialic acid lactose can be completely separated, and the problem that the substances cannot be effectively separated in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of food science and analytical chemistry, and in particular to a method for analyzing sugar substances in sialyllactose and an application thereof. Background Art

[0002] In the production process of 3'-sialyllactose (3'-SL), 3'-sialyllactulose and 6'-sialyllactose (6'-SL) are its common isomers. In the production process of 6'-SL, 6'-sialyllactulose and 3'-SL are its common isomers. For production process samples, it is often necessary to identify and analyze all the miscellaneous sugars and accompanying products.

[0003] In the prior art, the analysis of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) typically involves using a hydrophilic interaction chromatography column to separate the individual sugars in the 3'-SL and 6'-SL systems. High-performance liquid chromatography-electrospray ionization detection (HPLC-CAD) is then used for analysis, with external standard quantification. The HPLC conditions are acetonitrile and 25 mmol / L ammonium formate gradient elution. The column is 150 mm × 4.6 mm, 5 μm; the flow rate is 1 mL / min; the column temperature is 60°C; and the sample analysis time is 50 minutes. However, this analytical method cannot separate 3'-SL from 6'-sialyllactulose.

[0004] The "Public Comments on Six New Food Additives, Including Peroxidase," issued by the National Center for Food Safety Risk Assessment on October 24, 2024, only mentions a detection method for 3'-SL. This method utilizes high-performance liquid chromatography with ultraviolet detection (HPLC-UV) and external standard quantification. The liquid chromatography conditions include isocratic elution with acetonitrile and potassium dihydrogen phosphate buffer, an amide-bonded column (250 mm × 4.6 mm, 3.5 μm), a flow rate of 1 mL / min, a column temperature of 40°C, and a sample analysis time of 30 minutes. However, this method only separates 3'-SL and its lactulose isomer (3'-sialyllactulose), but not 6'-SL and 6'-sialyllactulose. This method also fails to meet the analytical requirements for production process samples.

[0005] Other methods used in other applications to measure 3'-SL and 6'-SL content include HPLC-FLD, HPAEC-PAD, and HPLC-MS. However, none of these methods can separate and analyze the corresponding lactulose isomers. This poses challenges to the raw material production and purification processes of 3'-SL and 6'-SL. Therefore, a new analytical method is needed to fully separate the four isomers and provide guidance for process optimization, component purification, and identification. Summary of the Invention

[0006] The present invention provides a method for analyzing sugar substances in sialyllactose and its application, which are used to solve the problem that existing detection methods cannot separate and analyze corresponding lactulose isomers.

[0007] According to a first aspect of the present invention, the present invention provides a method for analyzing carbohydrate substances in sialyllactose, comprising the following steps: The separation of sugars is carried out by ultra-high performance liquid chromatography, wherein: Mobile phase A includes acetonitrile; mobile phase B includes 10-30 mmol / L ammonium formate aqueous solution; The gradient elution program includes: 0-(9.5-10.5) min, the proportion of mobile phase A gradually decreased from 85-95% to 70-75%, and the proportion of mobile phase B gradually increased from 5-15% to 25-30%; (9.5-10.5)-(11.5-12.5) min, the proportion of mobile phase A gradually decreased from 70-75% to 60-65%, and the proportion of mobile phase B gradually increased from 25-30% to 35-40%; (11.5-12.5)-(13.5-14.5) min, mobile phase A maintained at 60-65%, mobile phase B maintained at 35-40%; (13.5-14.5)-(14.6-15.5) min, the proportion of mobile phase A gradually increased from 60-65% to 85-95%, and the proportion of mobile phase B gradually decreased from 35-40% to 5-15%; (14.6-15.5)-(24.5-25.5) min, mobile phase A is maintained at 85-95%, and mobile phase B is maintained at 5-15%.

[0008] The present invention provides a novel method for analyzing sugar substances in sialyllactose. By using specific ultra-high performance liquid chromatography conditions, utilizing a specific mobile phase composed of ammonium formate and acetonitrile at specific concentrations, and performing elution according to a specific gradient, the method can effectively separate various sugar substances in sialyllactose, thereby solving the problem in the prior art of being unable to completely separate 3'-SL, 6'-SL and their lactulose isomers, and providing a more accurate and reliable analytical method for the production, purification and quality control of sialyllactose.

[0009] Furthermore, the mobile phase A is acetonitrile, and the mobile phase B is a 15-25 mmol / L aqueous ammonium formate solution. This optimization improves the separation effect of the mobile phase, while maintaining separation efficiency and improving analytical stability and repeatability, facilitating more accurate quantitative analysis of sugars in sialyllactose.

[0010] Furthermore, the gradient elution procedure includes: From 0 to 10 min, the proportion of mobile phase A gradually decreased from 90% to 72%, and the proportion of mobile phase B gradually increased from 10% to 28%; From 10 to 12 minutes, the proportion of mobile phase A gradually decreased from 72% to 62%, and the proportion of mobile phase B gradually increased from 28% to 38%; 12-14 min, mobile phase A maintained at 62%, mobile phase B maintained at 38%; At 14-15 min, the proportion of mobile phase A gradually increased from 62% to 90%, and the proportion of mobile phase B gradually decreased from 38% to 10%; 15-25 min, mobile phase A is maintained at 90%, and mobile phase B is maintained at 10%.

[0011] This optimized gradient elution procedure can more effectively separate various sugar substances in sialyllactose, improve resolution, reduce analysis time, and reduce the possibility of baseline drift and peak overlap, further improving the accuracy and reliability of the analytical method.

[0012] Furthermore, the ultra-high performance liquid chromatography method uses a UPLC column, BEH Amide 2.1×100mm 1.7μm. This column features high column efficiency and rapid separation, better meeting the needs of sugar analysis in sialyllactose, improving separation efficiency and analysis speed, and achieving clearer separation results for complex sugar systems.

[0013] Furthermore, the chromatographic column used in the ultra-high performance liquid chromatography method has a flow rate of 0.3-0.5 mL / min and a column temperature of 55-65° C. Setting this parameter range ensures that the chromatographic column maintains good separation performance and stability during operation, while taking into account both analysis speed and separation effect, avoiding poor separation effect due to excessively fast flow rate or high column temperature, or prolonged analysis time due to excessively slow flow rate or low column temperature.

[0014] Furthermore, the chromatographic column used in the ultra-high performance liquid chromatography method has a flow rate of 0.35-0.45 mL / min and a column temperature of 58-62° C. A more precise parameter range helps to further improve separation efficiency and analytical accuracy, reduce chromatographic peak broadening and tailing, and make analytical results more accurate and reliable.

[0015] Furthermore, after separating the sugars using the ultra-high performance liquid chromatography method, analysis was performed using a charged aerosol detector. This detector, with its high sensitivity, wide linear range, and good reproducibility, enables accurate detection and quantitative analysis of the separated sugars, further improving the performance and reliability of the entire analytical method and enabling it to better meet the detection needs of practical applications.

[0016] Furthermore, the sugars in the sialyllactose include 3'-SL, 6'-SL, 3'-sialyllactulose, and 6'-sialyllactulose. This demonstrates the broad applicability of this method, enabling comprehensive analysis of key sugar components in sialyllactose and providing important technical support for studying the structure and function of sialyllactose, as well as for quality control during production.

[0017] Furthermore, the sugar substances in the sialyllactose include LA, SA, 3'-SL, 6'-SL, 3'-sialyllactulose, and 6'-sialyllactulose. This protocol further expands the analysis range by adding lactose (LA) and N-acetylneuraminic acid (SA). This shows that the analysis method of the present invention can not only analyze the main components of sialyllactose, but also detect and analyze other related sugar substances, further enhancing the application value of this method in sialyllactose research and production, enabling it to more comprehensively evaluate the quality and purity of sialyllactose.

[0018] According to a second aspect of the present invention, the present invention also provides the use of the above-mentioned method for analyzing carbohydrate substances in sialyllactose in optimizing the preparation process of sialyllactose, purifying sialyllactose components and improving the accuracy of identification of sialyllactose products.

[0019] The analytical method of the present invention not only has theoretical research value, but also has important practical application significance. It can provide strong technical support for the production, purification and quality control of sialyllactose, help improve the quality and market competitiveness of sialyllactose products, and promote the development of related industries.

[0020] The beneficial effects of the present invention are: The present invention discloses a method for analyzing carbohydrate substances in sialyllactose. By optimizing the mobile phase composition, gradient elution program and chromatographic column parameters of ultra-high performance liquid chromatography, the method can completely separate carbohydrate substances such as lactose (LA), N-acetylneuraminic acid (SA), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3'-sialyllactulose and 6'-sialyllactulose in sialyllactose, thereby solving the problem in the prior art of being unable to effectively separate these substances and providing a more accurate analysis method for structural research and quality control of sialyllactose.

[0021] Compared with existing technologies, the analytical method of the present invention significantly shortens analysis time while maintaining effective separation. For example, compared with ion exchange chromatography (HPAEC-PAD), the analysis time of the analytical method of the present invention is shortened from approximately one hour to less than 25 minutes, significantly improving sample analysis efficiency. This method can better meet the need for timely monitoring of process effects during production, helping to improve production efficiency and the timeliness of product quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a chromatogram obtained by testing a method for analyzing carbohydrate substances in sialyllactose provided in Example 1 of the present invention.

[0024] Figure 2 This is a chromatogram obtained by testing a method for analyzing carbohydrate substances in sialyllactose provided in Comparative Example 1 of the present invention.

[0025] Figure 3 This is a chromatogram obtained by testing a method for analyzing carbohydrate substances in sialyllactose provided in Comparative Example 2 of the present invention.

[0026] Figure 4 This is a chromatogram obtained by testing a method for analyzing carbohydrate substances in sialyllactose provided in Comparative Example 3 of the present invention.

[0027] Figure 5 This is a chromatogram obtained by testing a method for analyzing sugar substances in sialyllactose provided in Comparative Example 4 of the present invention.

[0028] Figure 6 This is a chromatogram obtained by testing a method for analyzing carbohydrate substances in sialyllactose provided in Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0030] The sialyllactose samples involved in the following examples and comparative examples include a mixed standard sample of 0.1 g / L LA, 0.1 g / L SA, 0.1 g / L 3'-SL, 0.1 g / L 6'-SL, 0.1 g / L 3'-sialyllactulose and 0.1 g / L 6'-S sialyllactulose. LA, SA, 3'-SL and 6'-SL are all commercially available samples. 3'-sialyllactulose and 6'-sialyllactulose are samples generated by isomerization of the corresponding sialyllactose under alkaline conditions.

[0031] Example 1 This embodiment provides a method for analyzing carbohydrate substances in sialyllactose, and the analysis method comprises the following steps: Samples were analyzed using an ultra-high performance liquid chromatography (UPLC) instrument equipped with a charged aerosol detector (CAD) detector. A BEH Amide 2.1×100 mm 1.7 μm UPLC column was selected for its excellent resolution, separation efficiency, and short analysis time. The flow rate was 0.4 mL / min and the column temperature was 60°C. Specific elution conditions are shown in Table 1. In Table 1, mobile phase A was acetonitrile, and mobile phase B was 20 mmol / L aqueous ammonium formate. The CAD detector had a data acquisition frequency of 10 Hz and a filter constant of 1.0.

[0032] Table 1 Elution conditions

[0033] Under these experimental conditions, LA and SA were well separated, and 3'-SL, 6'-SL and their corresponding lactulose isomers 3'-sialyllactulose and 6'-sialyllactulose were completely separated. Figure 1 .

[0034] This method was used to repeatedly test 3'-SL and 6'-SL final product samples more than 10 times, and the RSD values ​​were all lower than 1%. Through spiked experiments, it was verified that the recoveries of 3'-SL and 6'-SL were both between 98-101%.

[0035] Comparative Example 1 This comparative example provides a method for analyzing sugar substances in sialyllactose, using ion exchange chromatography (HPAEC-PAD). The specific test conditions are as follows: Column temperature: 30°C, flow rate: 1 mL / min; injection solution concentration: approximately 0.5 g / L. Elution conditions: A: 50 mM NaOH + 25 mM NaOAc, B: 100 mM NaOH + 100 mM NaOAc, gradient elution. Specific elution conditions are shown in Table 2.

[0036] Table 2 Elution conditions

[0037] 6'-SL elutes before 6'-sialyllactulose and 3'-SL, and 6'-sialyllactulose and 3'-SL completely overlap and cannot be separated. Therefore, ion exchange chromatography cannot specifically analyze the lactulose isomers and regioisomers corresponding to SL. For details, see Figure 2 In addition, the ion chromatography method takes a long time to regenerate and balance the ion exchange chromatography column, and each sample analysis time takes about 1 hour. The sample analysis efficiency is low, which is not conducive to timely monitoring of the process treatment effect during the production process.

[0038] Comparative Example 2 This comparative example provides a method for analyzing sugar substances in sialyllactose, using high performance liquid chromatography (HPLC-CAD) in accordance with the method of "Publicly Soliciting Materials Related to 7 New Food Additives Including Hydroxytyrosol" issued by the National Food Safety Risk Assessment Center on March 13, 2024. The specific chromatographic conditions are as follows: Chromatographic column: BEH Amide 4.6×150mm 5μm, column temperature: 60℃, mobile phase A: acetonitrile, mobile phase B: 25mmol / L ammonium formate aqueous solution, elution conditions are shown in Table 3.

[0039] Table 3 Elution conditions

[0040] Under these conditions, 6'-sialyllactulose and 3'-SL cannot be separated, which causes great trouble in the analysis of the 6'-SL purification process. It can be seen that the separation effect of this chromatographic column cannot meet the detection requirements. It takes 50 minutes to analyze one sample, which is very time-consuming. For details, see Figure 3 Considering the separation effect and analysis efficiency, a rapid analysis column with the same filler but smaller particle size and better separation effect was selected.

[0041] Comparative Example 3 This comparative example provides a method for analyzing sugar substances in sialyllactose, using high performance liquid chromatography (HPLC-CAD), which is based on the method of "Publicly Soliciting Materials Related to 7 New Food Additives Including Hydroxytyrosol" issued by the National Food Safety Risk Assessment Center on March 13, 2024, and the specific chromatographic conditions are as follows: A chromatographic column with higher column efficiency was selected. The specific chromatographic conditions were as follows: chromatographic column: BEH Amide 2.1×100mm 1.7μm, column temperature: 60℃, mobile phase A: acetonitrile, mobile phase B: 10 mmol / L / L ammonium formate aqueous solution, and elution conditions were as shown in Table 4.

[0042] Table 4 Elution conditions

[0043] Under these chromatographic conditions, the separation between LA and SA is not high, 3'-SL and 6'-sialyllactulose cannot be separated, and there is a serious baseline drift at 6'-SL, resulting in incomplete peak shape, which seriously affects quantitative analysis. See the specific chromatogram for details. Figure 4 Considering that the insufficient separation may be caused by insufficient buffer salt concentration, the buffer salt concentration needs to be adjusted.

[0044] Comparative Example 4 This comparative example provides a method for analyzing sugar substances in sialyllactose, using high performance liquid chromatography (HPLC-CAD), which is based on the method of "Publicly Soliciting Materials Related to 7 New Food Additives Including Hydroxytyrosol" issued by the National Food Safety Risk Assessment Center on March 13, 2024, and the specific chromatographic conditions are as follows: Chromatographic column: BEH Amide 2.1×100mm 1.7μm, column temperature: 60℃, mobile phase A: acetonitrile, mobile phase B: 50mmoL / L ammonium formate aqueous solution, elution conditions are shown in Table 5.

[0045] Table 5 Elution conditions

[0046] Under this chromatographic condition, LA and SA are still not well separated, and the peak shape is not smooth enough, and the lactulose isomers are still not separated from the system. The overall baseline noise is slightly high, which may be due to the sensitivity of this column to the buffer salt concentration. A high concentration of buffer salt has a negative impact on the column efficiency. See the chromatographic column for details. Figure 5 Therefore, the buffer concentration needs to be lowered.

[0047] Comparative Example 5 This comparative example provides a method for analyzing sugar substances in sialyllactose, using high performance liquid chromatography (HPLC-CAD), which is based on the method of "Publicly Soliciting Materials Related to 7 New Food Additives Including Hydroxytyrosol" issued by the National Food Safety Risk Assessment Center on March 13, 2024, and the specific chromatographic conditions are as follows: Chromatographic column: BEH Amide 2.1×100mm 1.7μm, column temperature: 60℃, mobile phase A: acetonitrile, mobile phase B: 25mmoL / L ammonium formate aqueous solution, elution conditions are shown in Table 6.

[0048] Table 6 Elution conditions

[0049] From the analysis results, it can be seen that under this elution condition, the SA chromatographic peak has a splitting phenomenon, 3'-sialyllactulose can be completely separated from 3'-SL, but the response of 6'-sialyllactulose is extremely low, the baseline at the peak drifts, and the separation degree of 6'-SL and the subsequent solvent peak is not high, which affects the accuracy of quantitative analysis. For details, see the chromatographic Figure 6 The gradient elution conditions and column equilibration time need to be further optimized.

[0050] In summary, the ultra-high performance liquid chromatography method of the present invention, utilizing a specific chromatographic column, mobile phase, and gradient elution conditions, successfully achieved the complete separation of lactose (LA), N-acetylneuraminic acid (SA), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3'-sialyllactulose, and 6'-sialyllactulose in sialyllactose, with excellent separation results and a short analysis time. Comparison shows that the analytical method of the present invention is superior to the other methods described in Comparative Examples 1-5 in terms of separation effect, analytical efficiency, and quantitative accuracy, effectively resolving the problems existing in the prior art and providing a more accurate and reliable analytical method for the production, purification, and quality control of sialyllactose.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for analyzing carbohydrates in sialyllactose, characterized in that: The steps include: The separation of sugars is carried out by ultra-high performance liquid chromatography, wherein: Mobile phase A includes acetonitrile; mobile phase B includes 10-30 mmol / L ammonium formate aqueous solution; The gradient elution program includes: 0-(9.5-10.5) min, the proportion of mobile phase A gradually decreased from 85-95% to 70-75%, and the proportion of mobile phase B gradually increased from 5-15% to 25-30%; (9.5-10.5)-(11.5-12.5) min, the proportion of mobile phase A gradually decreased from 70-75% to 60-65%, and the proportion of mobile phase B gradually increased from 25-30% to 35-40%; (11.5-12.5)-(13.5-14.5) min, mobile phase A maintained at 60-65%, mobile phase B maintained at 35-40%; (13.5-14.5)-(14.6-15.5) min, the proportion of mobile phase A gradually increased from 60-65% to 85-95%, and the proportion of mobile phase B gradually decreased from 35-40% to 5-15%; (14.6-15.5)-(24.5-25.5) min, mobile phase A is maintained at 85-95%, and mobile phase B is maintained at 5-15%.

2. The method for analyzing carbohydrates in sialyllactose according to claim 1, wherein The mobile phase A is acetonitrile; the mobile phase B is a 15-25 mmol / L ammonium formate aqueous solution.

3. The method for analyzing carbohydrates in sialyllactose according to claim 1 or 2, wherein: The gradient elution procedure includes: From 0 to 10 min, the proportion of mobile phase A gradually decreased from 90% to 72%, and the proportion of mobile phase B gradually increased from 10% to 28%; From 10 to 12 minutes, the proportion of mobile phase A gradually decreased from 72% to 62%, and the proportion of mobile phase B gradually increased from 28% to 38%; 12-14 min, mobile phase A maintained at 62%, mobile phase B maintained at 38%; At 14-15 min, the proportion of mobile phase A gradually increased from 62% to 90%, and the proportion of mobile phase B gradually decreased from 38% to 10%; 15-25 min, mobile phase A is maintained at 90%, and mobile phase B is maintained at 10%.

4. The method for analyzing carbohydrates in sialyllactose according to any one of claims 1 to 3, wherein: The chromatographic column used in the ultra-high performance liquid chromatography method is a UPLC chromatographic column, BEH Amide 2.1×100mm 1.7μm.

5. The method for analyzing carbohydrates in sialyllactose according to claim 1 or 4, wherein: The flow rate of the chromatographic column used in the ultra-high performance liquid chromatography method is 0.3-0.5 mL / min, and the column temperature is 55-65°C.

6. The method for analyzing carbohydrates in sialyllactose according to claim 5, wherein: The flow rate of the chromatographic column used in the ultra-high performance liquid chromatography method is 0.35-0.45 mL / min, and the column temperature is 58-62°C.

7. The method for analyzing carbohydrates in sialyllactose according to claim 1, wherein: After the sugar substances were separated by ultra-high performance liquid chromatography, they were analyzed using a charged aerosol detector.

8. The method for analyzing carbohydrates in sialyllactose according to claim 1, wherein: The sugar substances in the sialyllactose include 3'-SL, 6'-SL, 3'-sialyllactulose and 6'-sialyllactulose.

9. The method for analyzing carbohydrates in sialyllactose according to claim 8, wherein: The sugar substances in the sialyllactose include LA, SA, 3'-SL, 6'-SL, 3'-sialyllactulose and 6'-sialyllactulose.

10. Use of the method for analyzing carbohydrates in sialyllactose according to any one of claims 1 to 9 in optimizing the preparation process of sialyllactose, purifying sialyllactose components, and improving the accuracy of identification of sialyllactose products.

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