Quality control method for synthesizing 3 '-sialic acid lactose through enzymatic process

Abstract

The invention provides a quality control method for synthesizing 3 '-sialic acid lactose by an enzymatic process, which comprises the following steps: A) dissolving and extracting a test sample by using a solvent to obtain a to-be-detected solution; the test sample is a reaction solution or a finished product of 3 '-sialic acid lactose synthesized by enzyme catalysis; b) determining the liquid to be detected by adopting a high performance liquid chromatography to obtain an HPLC (High Performance Liquid Chromatography) spectrum of the 3 '-sialic acid lactose; according to the method, an HPLC-UV-RI combined detection method is adopted, the specificity is high, the sensitivity is high, the linear correlation is good, and the pretreatment process of the sample is simple and rapid; according to the method, the content of 3 '-lactulose sialylate, 6'-lactose sialylate sodium salt, D-lactose, CMP, CMP-SA and SA in the 3 '-SL process development process can be accurately detected, control over the conversion rate and impurities in the process is facilitated, effective detection of a product 3'-SL related substance-isomer can be achieved, and control over the product quality is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a quality control method for synthesizing 3'-sialyllactose by an enzymatic process. Background Art

[0002] HMOs are considered to be part of the innate immune shift and have received increasing attention during breastfeeding due to their unique role in the healthy gut microbiota of infants, in addition, they may promote infant brain development and prevent pathogen adhesion.

[0003] There are various types of HMOs, mainly including 3-fucosyllactose, 2'-fucosyllactose, 6'-sialyllactose, 3'-sialyllactose, lactose-N-neotetraose, lactose-N-tetraose, etc. Among them, 3'-SL (3'-sialyllactose) is an abundant sialylated HMO in breast milk. The specific functional benefits of 3'-SL include reducing the risk of adhesion of harmful bacteria and their proteins. 3'-SL specifically supports the development of the infant brain by providing sialic acid, plays a special role in the growth and development of infants, and has an important impact on the growth, development and health of newborns. 3'-SL is added as a food additive to foods for infants and young children, including non-exempt full-term infant formula milk powder, as well as specific traditional food and beverage products for the general population. It has become an important trend in the high-end formula milk powder industry and the food industry.

[0004] The quality of 3'-SL products is related to the quality of infant formula milk powder, certain traditional food and beverage products, so 3'SL needs to be strictly controlled. Therefore, it is necessary to provide a method for quality control of the 3'SL production process and product testing, which can solve the problem of separation and detection of products, starting materials and intermediate impurities and isomers. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a quality control method for synthesizing 3'-sialyllactose by enzymatic process, and the method of the present invention is accurate and reliable in detection.

[0006] The method provided by the present invention can be used for quality control of the 3'SL production process and product detection, solve the problem of separation and detection of products, starting materials and intermediate impurities, and isomers, and ensure the conversion rate and yield of each step of the production process and the quality of the product.

[0007] The present invention provides a quality control method for synthesizing 3'-sialyllactose by an enzymatic process, comprising:

[0008] A) dissolving a test sample with a solvent, extracting, and obtaining a test solution; the test sample is a reaction solution of enzyme-catalyzed synthesis of 3'-sialyllactose or a finished product of enzyme-catalyzed synthesis of 3'-sialyllactose;

[0009] B) measuring the test solution by high performance liquid chromatography to obtain an HPLC spectrum of 3'-sialyllactose;

[0010] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, and the mobile phase B is a dipotassium hydrogen phosphate trihydrate aqueous solution; and the elution ratio is: the mobile phase A: mobile phase B is 74:26.

[0011] The test sample of the present invention is the reaction solution of enzyme-catalyzed synthesis of 3'-sialyllactose, that is, the substances in each link of enzyme-catalyzed synthesis of 3'-sialyllactose, which exist in liquid form.

[0012] In a preferred embodiment of the present invention, the method for preparing 3'-sialyllactose by enzyme catalysis comprises:

[0013] In the first step of the reaction, sialic acid (SA) and cytidine monophosphate (CMP) are used as substrates, and intermediate 1 (CMP-SA) is generated through the catalysis of CMP-sialic acid synthetase. In the second step of the reaction, intermediate 1 and D-lactose are used as substrates, and the product 3'-SL is generated through the catalysis of 3'-sialyltransferase. In the second step of the reaction, 3'-SL isomers 3'-sialyllactulose and 6'-sialyllactose sodium salt are produced. The content of the two isomers is an important quality control parameter.

[0014] The present invention firstly dissolves the reaction solution of catalytic synthesis of 3'-sialyllactose or the finished product of enzyme-catalyzed synthesis of 3'-sialyllactose with a solvent, extracts, and obtains a test solution.

[0015] The pretreatment method of the present invention is very simple, and can be dissolved by using a solvent. The solvent of the present invention is 75% acetonitrile; after dissolving, dilution is performed and then filtered by using a 22 μm organic phase filter membrane, and the filtrate is taken for detection.

[0016] According to the present invention, the mass volume ratio of the test sample and the solvent is: 1 ml of the enzyme linkage reaction solution is measured into a 10 ml volumetric bottle, and diluted to the scale with 75% acetonitrile;

[0017] The mass volume ratio of the test sample and the solvent for product isomer detection is as follows: weigh 200 mg of the test sample into a 100 mL volumetric flask and dilute to the mark with 75% acetonitrile.

[0018] Since there are a large number of enzyme proteins in the reaction liquid of the bioenzymatic method, which have biological activity, inactivation and protein removal treatment are required after sampling, otherwise the judgment of the detection result will be affected and the liquid chromatography system will fail. The present invention uses 75% acetonitrile aqueous solution to carry out reaction liquid inactivation treatment, which can effectively inactivate the enzyme protein and precipitate the protein, and uses 0.22μm organic filter membrane to filter and remove the protein, thereby protecting the residual protein from the influence of the chromatography system.

[0019] The test solution is measured by high performance liquid chromatography to obtain the HPLC spectrum of 3'-sialyllactose.

[0020] The chromatographic conditions of the high performance liquid chromatography method of the present invention are: the chromatographic column is a C18 column; the chromatographic column is HILICpak VG-50 4E, with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0021] The temperature of the chromatographic column of the present invention is 40°C.

[0022] The inventor creatively discovered that if the AQ-C18 column with a conventional hydrophobic separation mechanism is used for separation and elution, there are problems such as weak compound retention and poor peak separation; using a silica-based amino chromatographic column, in the separation of 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL, the 3'-SL baseline is improved, interfering with the quantification of 6'-sialyllactose sodium salt. The present invention uses a polymer-based HILIC column (HILICpak VG-50 4E), adopts a hydrophilic interaction mechanism, enhances the retention of the target, and effectively separates the isomers 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL. Compared with a silica-based amino column, the chromatographic column has good chemical stability, a long service life, and a small change in separation effect over time.

[0023] According to the present invention, mobile phase A is an acetonitrile solution, and mobile phase B is a dipotassium hydrogen phosphate trihydrate aqueous solution;

[0024] In a specific embodiment, the dipotassium hydrogen phosphate trihydrate aqueous solution is a 10 mmol / L dipotassium hydrogen phosphate trihydrate aqueous solution with a pH value of 8.0.

[0025] At the same time, the inventors found that a boat-shaped chromatographic peak appeared under an acidic mobile phase. The present invention adopts a mobile phase system of pH 8.0, which can effectively avoid the boat-shaped peak and accurately quantify CMP-SA.

[0026] In a specific embodiment, the elution ratio: mobile phase A: mobile phase B is 74:26.

[0027] The present invention finds that the pH value of the mobile phase also affects the separation and peak shape of the peaks. Through attempts at different pH values, it is found that a pH value of 8.0 for a dipotassium hydrogen phosphate trihydrate aqueous solution is more conducive to the separation of the peaks and obtaining a good peak shape.

[0028] The target substances for quality control of the present invention include CMP-SA, CMP, SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL. The above-mentioned substances, including reaction raw materials, reaction intermediates and reaction by-product isomers, pose a great challenge to the separation of the above-mentioned substances. The present invention finally achieves effective separation of the above-mentioned substances through the selection of chromatographic columns and the adjustment of other parameters, and can perform quality control well.

[0029] In the specific implementation of the present invention, the flow rate of the mobile phase is 0.8 mL / min

[0030] In a specific implementation of the present invention, the wavelength of the ultraviolet detector is 210 nm; the temperature of the differential refractometer detector is 40°C ± 1°C.

[0031] There is no chromophore in the lactose structure and no absorption under the ultraviolet detector. The present invention innovatively adopts HPLC-UV-RI for analysis and detection, which can simultaneously and accurately quantify the contents of SA, CMP, CMP-SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL in the reaction process and in the product.

[0032] The detector of the present invention is a UV-RI combined detector, which enhances the applicability of quantitative substance detection, so that one detection method can quantify seven substances.

[0033] The detection method of the invention can simultaneously detect two isomers of 3'-sialyllactulose and 6'-sialyllactose sodium salt.

[0034] In a specific implementation of the present invention, the injection volume is 25 μL;

[0035] In a specific implementation of the present invention, the sample tray temperature of the sample to be tested is 15° C.; the running time is 35 minutes.

[0036] The present invention also includes preparing a reference solution: taking CMP-SA, CMP, SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL reference substance respectively, dissolving them in 75% acetonitrile to obtain a reference solution;

[0037] The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of 3'-sialyllactose are qualitatively and quantitatively determined according to the chromatogram of the reference.

[0038] According to the present invention, the injection concentration, standard curve and quantitative limit of the reference solution are specifically:

[0039] CMP-SA is 0.0015mg / mL~2.0mg / ml, the standard curve is y=56.731x-0.6091, and the correlation coefficient is R 2 =0.9998; the limit of quantification concentration is 0.0015 mg / mL;

[0040] CMP is 0.001mg / mL~2.0mg / ml, standard curve is y=340.9942x+0.1722, correlation coefficient R 2 =0.9999; the limit of quantification concentration is 0.001 mg / mL;

[0041] SA is 0.00125mg / mL~2.0mg / ml, standard curve is y=153.5071x-0.1863; correlation coefficient R 2 =1.0000; the quantitative limit concentration is 0.0012 mg / mL;

[0042] D-lactose is 0.05mg / mL~2.0mg / ml, the standard curve is y=9.0672x+0.1255, and the correlation coefficient is R 2 =0.9997; the limit of quantification concentration is 0.009 mg / mL;

[0043] 3'-sialyllactulose is 0.002mg / mL~2.0mg / ml, the standard curve is y=42.211x-0.3845, and the correlation coefficient is R 2 =0.9999; the limit of quantification concentration is 0.002 mg / mL;

[0044] 6'-sialyllactose sodium salt is 0.002mg / mL~2.0mg / ml, the standard curve is y=50.63x+0.0938, the correlation coefficient is R 2 =0.9998; the limit of quantification concentration is 0.002 mg / mL;

[0045] 3'-SL is 0.002mg / mL~2.0mg / ml, the standard curve is y=49.7438x+0.1081, and the correlation coefficient is R 2 =0.9997; the limit of quantification concentration is 0.002 mg / mL;

[0046] It can be seen that the correlation coefficient of each standard curve is not less than 0.999, and the standard curves have a good linear relationship.

[0047] The invention provides a quality control method for synthesizing 3'-sialyllactose by an enzymatic process, comprising: A) dissolving a test sample by a solvent, extracting, and obtaining a test solution; the test sample is a reaction solution of synthesizing 3'-sialyllactose by enzyme catalysis or a finished product of synthesizing 3'-sialyllactose by enzyme catalysis; B) determining the test solution by high performance liquid chromatography to obtain an HPLC spectrum of 3'-sialyllactose; the invention adopts an HPLC-UV-RI combined detection method, which has strong specificity, high sensitivity, good linear correlation, and a simple and fast sample pretreatment process, and can accurately detect the contents of 3'-sialyllactulose, 6'-sialyllactose sodium salt, D-lactose, CMP, CMP-SA, and SA in the process of 3'-SL process development, which is helpful for controlling the conversion rate and impurities in the process, and can realize effective detection of product 3'-SL related substances-isomers, which is helpful for controlling product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Different pH investigation-pH4.5-CMP-SA;

[0049] Figure 2 Different pH investigation-pH6.0-CMP-SA;

[0050] Figure 3 Different pH investigation-pH8.0-CMP-SA;

[0051] Figure 4 Initial reaction solution-UV spectrum;

[0052] Figure 5 End of combined reaction - UV spectrum;

[0053] Figure 6 Initial reaction solution - RI spectrum;

[0054] Figure 7 Combined reaction ends - RI spectrum;

[0055] Figure 8 Specificity investigation-UV spectrum;

[0056] Fig. 9 Specificity investigation-RI spectrum;

[0057] Fig.10 SA / 3'-sialyllactulose / 3'-SL / 6'-sialyllactose sodium salt / CMP-SA / CMP limit of quantitation spectrum;

[0058] Fig.11 Lactose quantification limit spectrum (RI spectrum);

[0059] Fig.12 SA linear graph;

[0060] Fig.13 3′-sialyllactulose linear profile;

[0061] Fig.14 3'-SL linear map;

[0062] Fig.15 6'-SL linear map;

[0063] Fig.16 For: CMP-SA linear graph;

[0064] Fig.17 D-lactose linear graph;

[0065] Fig.18 CMP linear graph;

[0066] Fig.19 AQ-C18 column separated SA, CMP, CMP-SA, 6'-SL, 3'-SL, and 3'-sialyllactulose spectra, where 1 was SA, 2 was 6'-SL+CMP-SA, and 3 was 3'-SL+3'-sialyllactulose+CMP;

[0067] Fig. 20 AQ-C18 column initial reaction liquid spectrum (RI+UV);

[0068] Fig.21 AQ-C18 column combined reaction end spectrum (RI+UV);

[0069] Fig. 22 Amino column reaction liquid spectrum (RI+UV). DETAILED DESCRIPTION

[0070] The present invention provides a quality control method for synthesizing 3'-sialyllactose by an enzymatic process. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0071] In order to further illustrate the present invention, the construction method of HPLC content determination of Fructus Carthami provided by the present invention is described in detail below in conjunction with the examples.

[0072] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0073] The English abbreviations and Chinese names involved in the present invention are:

[0074] 3'-SL: 3'-sialyllactose; 6'-SL: 6'-sialyllactose; HMOs: human milk oligosaccharides; SA: N-acetyl-D-neuraminic acid; CMP: cytidine-1-phosphate; CMP-2Na: cytidine-1-phosphate disodium salt; CMP-SA: 5'-monophosphate adenosine-sialic acid disodium salt; HPLC: high performance liquid chromatography;

[0075] HPLC-UV: high performance liquid chromatography-ultraviolet spectrophotometry; RI: differential refractive index detector; nm: nanometer; mL / min: milliliter / minute; μg / mL: microgram / milliliter; μL: microliter; ℃: degree Celsius; h: hour; μm: micrometer.

[0076] Example 1

[0077] This embodiment provides a method for monitoring the process of enzyme-catalyzed synthesis of 3'-SL linkage reaction. The substances to be monitored are SA, CMP-SA, CMP, D-lactose, 3'-sialyllactulose, 3'-SL, and 6'-sialyllactose sodium salt. The content of each substance and the conversion rate of the reaction are calculated.

[0078] The method comprises the following steps:

[0079] ① Chromatographic conditions: mobile phase A is acetonitrile, mobile phase B is 10 mmol / L potassium dihydrogen phosphate trihydrate aqueous solution (pH 8.0); the elution ratio of mobile phase A: mobile phase B is 74:26; the flow rate is 0.8 ml / min; the chromatographic column is HILICpak VG-50 4E (length 250 mm, inner diameter 4.6 mm, particle size 5 μm); the wavelength of the ultraviolet detector is 210 nm; the temperature of the differential detector is 40 ° C; the injection volume is 25 μ L; the column temperature is 40 ° C; the sample plate temperature is controlled at 15 ° C; the running time is 35 min.

[0080] ②Solvent: 75% acetonitrile aqueous solution, filtered with a 22μm organic phase filter membrane, and the filtrate was taken for detection.

[0081] ③Sample solution preparation:

[0082] Accurately measure 1 ml of the reaction solution after 0 h of the enzyme-catalyzed synergy reaction to synthesize 3'-sialyllactose by CMP-sialic acid synthetase using sialic acid (SA), cytidine monophosphate (CMP) and D-lactose as substrates, and the reaction solution after 6 h of the enzyme-catalyzed synergy reaction, respectively, and place them in 10 ml volumetric flasks, dilute to the scale with 75% acetonitrile aqueous solution, shake well, filter with a 22 μm organic phase filter membrane, and take the filtrate for detection.

[0083] ④ Preparation of reference solution: Accurately weigh 20 mg of CMP-SA, CMP, SA, lactose, 3'-sialyllactulose, and 6'-sialyllactose sodium salt reference substances, and 200 mg of 3'-SL in a 100 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to the scale, shake well, filter with a 0.22 μm organic phase filter membrane, and take the filtrate for detection.

[0084] ⑤ Determination: Use the chromatographic conditions in ① to detect the solution to be tested in ②, ③, and ④ to obtain a chromatogram. Figure 4-Figure 5 ), the contents of CMP-SA, CMP, SA, 3'-sialyllactulose, 6'-sialyllactose sodium salt, and 3'-SL were calculated using the external standard method. Figure 6-Figure 7 ) The D-lactose content was calculated using the external standard method. The test results are shown in Table 1.

[0085] Table 1

[0086] Quantitative substances Initial reaction solution mmol Combined reaction ends mmol S 150mmol 0.63mmol CMP 2mmol 0.22mmol CMP-SA NA 0.08mmol lactose 200mmol 10.12mmol 3'-sialyllactulose NA 0.15mmol 6'-Sialyl lactose sodium salt NA 0.33mmol 3'-SL NA 110.8mmol Reaction conversion rate (%) NA 90.6

[0087] Example 1 shows that the method of the present invention can effectively and accurately monitor the content and conversion rate of each substance in the enzyme catalytic process linkage reaction.

[0088] Example 2

[0089] This embodiment provides a liquid phase detection method for isomeric impurities in a 3'-SL product synthesized by an enzyme catalysis method, and the method comprises the following steps:

[0090] ① Chromatographic conditions: mobile phase A was acetonitrile, mobile phase B was 10 mmol / L potassium dihydrogen phosphate aqueous solution (pH 8.0); the elution ratio of mobile phase A: mobile phase B was 74:26; the flow rate was 0.8 ml / min; the chromatographic column was HILICpak VG-50 4E (length 250 mm, inner diameter 4.6 mm, particle size 5 μm); the wavelength of the UV detector was 210 nm; the temperature of the differential detector was 40 °C; the injection volume was 25 μL; the column temperature was 40 °C; the sample plate temperature was controlled at 15 °C; and the running time was 35 min.

[0091] ②Solvent: 75% acetonitrile aqueous solution, filtered with a 22μm organic phase filter membrane, and the filtrate was taken for detection.

[0092] ③Sample solution preparation:

[0093] The 3'-SL product produced by our company was used as the sample to be tested.

[0094] Accurately weigh 200 mg of the sample and place it in a 50 mL volume, add 75% acetonitrile aqueous solution to dissolve and dilute to the scale, shake well, filter with a 0.22 μm organic phase filter membrane, and take the filtrate for detection.

[0095] ④ Preparation of reference solution: weigh 20 mg of 3'-sialyllactose sodium salt reference substance and place it in a 100 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to the mark, shake well and filter with a 0.22 μm organic phase filter membrane, and take the filtrate for detection.

[0096] ⑤ Sensitivity solution: Accurately pipette 1 ml of the reference solution into a 50 ml volumetric flask, add 75% acetonitrile water

[0097] Dilute to scale, shake well and filter with a 0.22 μm organic phase filter membrane, and take the filtrate for detection.

[0098] ⑥ Separation solution: Weigh 20 mg of 3'-sialyllactulose, 20 mg of 6'-sialyllactose sodium salt reference substance, and 200 mg of 3'-sialyllactose sodium salt, accurately weigh, place in a 100 mL volumetric flask, add 25 ml of water to dissolve, add acetonitrile to dilute to the scale, shake well, and obtain.

[0099] ⑦ Determination: Use the chromatographic conditions in ① to detect the test solutions in ②, ③, ④, ⑤, and ⑥, use 3'-sialyllactose sodium salt as the reference substance, and use the external standard method to calculate the contents of 3'-sialyllactulose and 6'-sialyllactose sodium salt.

[0100] The following experiments were conducted to investigate the methodology of the 3'-SL control and product detection methods provided by the present invention, and the results are as follows:

[0101] ① System applicability inspection

[0102] Accurately weigh 20 mg of SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt, CMP-SA, CMP reference substance, and 200 mg of 3'-SL in a 100 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to the scale, shake well and filter with a 0.22 μm organic phase filter membrane, inject continuously 6 times using the detection method provided in Example 2, calculate the peak area RSD value, which should be ≤2.0%. Specific results are shown in Table 2

[0103] Table 2

[0104]

[0105]

[0106] It can be seen that the method of the present invention has good system adaptability, and the RSD of the peak area of ​​each substance after repeated injection is less than 2.0%, which meets the quantitative accuracy requirement.

[0107] ②Specificity inspection

[0108] A mixed solution of SA, CMP-SA, CMP, D-lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt, and 3'-SL and a diluent were prepared and analyzed using the detection method provided in Example 2. The results showed that the diluent had no interference in the peak positions of 3'-SL and other substances, and the separation of the seven compounds was greater than 1.5, meeting the baseline separation requirements, indicating that the detection method of the present invention has strong specificity. The peak elution time and separation degree of each peak are shown in Table 3 (see the spectrum Figure 8 , Fig. 9 )

[0109] Table 3

[0110]

[0111] ③Sensitivity investigation

[0112] By using the stepwise dilution method and the detection method provided in Example 1, different concentrations of SA, CMP-2Na, CMP-SA, D-lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt, and 3'-SL solutions were analyzed. When the signal-to-noise ratio was not less than 10, the minimum quantitative detection concentrations of SA, CMP-SA, CMP, D-lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt, and 3'-SL were measured. The specific results are shown in Table 4 (see the graph Fig.10 , Fig.11 ), the results show that the detection method of the present invention has high sensitivity.

[0113] Table 4

[0114] Substance name Limit of Quantitation Signal-to-Noise Ratio S 0.0012mg / mL 31.1 3'-sialyllactulose 0.002mg / mL 24.1 3'-SL 0.002mg / mL 19.7 D-Lactose 0.009mg / mL 22.2 6'-Sialyl lactose sodium salt 0.002mg / mL 28.1 CMP-SA 0.0015mg / mL 24.5 CMP 0.001mg / mL 28.7

[0115] ④Linear investigation

[0116] SA linear solution: Accurately weigh 20 mg of SA reference substance, place in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with SA concentration in the range of 0.00125 mg / mL to 2.0 mg / ml.

[0117] 6'-Sialyl lactulose linear solution: accurately weigh 20 mg of 6'-Sialyl lactulose, place in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with a 6'-Sialyl lactulose concentration in the range of 0.002 mg / mL to 2.0 mg / ml.

[0118] 3'-SL linear solution: accurately weigh 20 mg of 3'-sialyllactose sodium salt reference substance, place it in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with a 3'-SL concentration in the range of 0.002 mg / mL to 2.0 mg / ml.

[0119] D-lactose linear solution: Accurately weigh 20 mg of lactose reference substance, place in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with a D-lactose concentration in the range of 0.05 mg / mL to 2.0 mg / ml.

[0120] 6'-Sialyl lactose sodium salt linear solution: accurately weigh 20 mg of 6'-Sialyl lactose sodium salt reference substance, place it in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with a concentration of 6'-Sialyl lactose sodium salt in the range of 0.002 mg / mL to 2.0 mg / ml.

[0121] CMP-SA linear solution: accurately weigh 20 mg of CMP-SA reference substance, place it in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with a CMP-SA concentration in the range of 0.0015 mg / mL to 2.0 mg / ml.

[0122] CMP linear solution: accurately weigh 20 mg of CMP-2Na reference substance, place it in a 10 mL volumetric flask, add 75% acetonitrile aqueous solution to dissolve and dilute to volume, shake well. Dilute step by step to make a linear solution with CMP concentration in the range of 0.001 mg / mL to 2.0 mg / ml.

[0123] The analysis was performed using the detection method provided in Example 1, and a standard curve was drawn with concentration as the abscissa and peak area as the ordinate (see Figure 12-Figure 18 ), the standard curve of SA is y=153.5071x-0.1863, and the correlation coefficient is R 2 =1.0000; the standard curve of 3'-sialyllactulose is y=42.211x-0.3845, and the correlation coefficient is R 2 =0.9999; the standard curve of 3'-SL is y=49.7438x+0.1081, and the correlation coefficient R 2 =0.9997; the standard curve of D-lactose is y=9.0672x+0.1255, and the correlation coefficient R 2 =0.9997; the standard curve of 6'-sialyllactose sodium salt is y=50.63x+0.0938, and the correlation coefficient R 2=0.9998; the standard curve of CMP-SA is y=56.731x-0.6091, and the correlation coefficient R 2 =0.9998; the standard curve of CMP is y=340.9942x+0.1722, and the correlation coefficient R 2 =0.9999; thus, the correlation coefficients of the standard curves are not less than 0.999, and the standard curves all have good linear relationships.

[0124] ⑤Precision inspection

[0125] The same batch of 3'-SL obtained by enzyme catalysis synthesis method was taken as a sample, and 6 test solutions were prepared. Each sample was injected with 1 injection using the detection method provided in Example 2. The results showed that the RSD of 3'-sialyllactulose and 6'-sialyllactose sodium salt in the six samples were ** and **, respectively, indicating that the isomer impurity detection method of this product has high precision. The specific results are shown in Table 5.

[0126] Table 5

[0127]

[0128] Comparative Example 1:

[0129] The difference between the detection method in Comparative Example 1 and Example 1 is that an AQ-C18 column (length 250 mm, inner diameter 4.6 mm, particle size 5 μm) is used, the mobile phase is 10 mmol potassium dihydrogen phosphate, the elution ratio is 100%, and the other chromatographic parameters are consistent with the example. The enzyme reaction solution is detected using this chromatographic condition, and the HPLC chromatogram is recorded as follows: Figure 19-21 As shown, the chromatographic conditions have the following problems:

[0130] 1. The baselines of the two substrates SA and CMP were not effectively separated, and the chromatographic peaks of lactose, SA and CMP overlapped on the differential chromatographic peaks, making it impossible to accurately quantify the starting material;

[0131] 2. The chromatographic peak of the intermediate product CMP-SA is between the substrates SA and CMP, and the chromatographic peaks of the intermediate product CMP-SA and the isomer 6'-SL completely overlap, making it impossible to confirm whether the reaction process is proceeding normally and whether there are isomers;

[0132] 3. The chromatographic peaks of substrate CMP, product 3'-SL and isomer 3'-sialyllactulose almost completely overlap, and the product and starting material cannot be quantified, and it is impossible to distinguish whether isomer 3'-sialyllactulose exists during the reaction.

[0133] In summary, Comparative Example 1 shows that the chromatographic conditions cannot accurately calculate the content and conversion rate of each substance in the enzyme-catalyzed process linkage reaction.

[0134] Comparative Example 2:

[0135] The difference between the detection method in Example 2 and Example 1 and Example 2 is that a silica-based amino chromatographic column (length 250 mm, inner diameter 4.6 mm, particle size 5 μm) is used, and the mobile phase, elution ratio and other chromatographic parameters are consistent with those in the examples. The enzyme catalytic reaction solution is detected using this chromatographic condition, and the HPLC chromatogram is recorded as follows: Fig. 22 As shown, the chromatographic conditions have the following problems:

[0136] 1. The starting material SA and the impurities in the reaction solution failed to achieve baseline separation, and the concentration of the substrate SA could not be accurately quantified;

[0137] 2. The chromatographic peaks of the intermediate product CMP-SA and the isomer 6'-SL overlap, and it is impossible to determine whether CMP-SA or the isomer 6'-SL is detected in the reaction solution;

[0138] 3. The isomer 6'-SL and the product 3'-SL failed to achieve baseline separation, and the isomer 6'-SL content in the finished product could not be accurately quantified;

[0139] In summary, Comparative Example 2 shows that the chromatographic conditions cannot accurately calculate the content of each substance in the enzyme-catalyzed process linkage reaction, and cannot accurately detect the content of the isomer 6'-SL in the finished product.

[0140] Comparative Example 3:

[0141] The difference between the detection method in Comparative Example 3 and Example 1 is that, under the conditions that the chromatographic column, mobile phase, elution ratio and other chromatographic parameters are consistent with those in the example, the mobile phase pH is changed to 4.5 and pH to 6.0, and the intermediate product is detected using the chromatographic conditions of mobile phase pH 4.5 and pH 6.0, and the chromatogram is recorded. The chromatographic conditions have the following problems:

[0142] 1. When the pH of the mobile phase of the intermediate product CMP-SA is 4.5, the acetamide or carboxyl group in the compound undergoes a reversible nucleophilic addition reaction with water in the acidic mobile phase, resulting in a boat-shaped chromatographic peak that is low at the beginning and high at the end (such as Figure 1 As shown), the intermediate product CMP-SA cannot be accurately quantified;

[0143] 2. When the mobile phase pH is increased to 6.0, the possible nucleophilic addition reaction is inhibited, and the intermediate product CMP-SA forms a boat-shaped chromatographic peak with a high front and a low back (such as Figure 2 As shown), the intermediate product CMP-SA cannot be accurately quantified;

[0144] 3. When the mobile phase pH is raised to 8.0 again, the reversible nucleophilic addition reaction is inhibited under high pH conditions, and the chromatographic peak obtained by the intermediate product CMP-SA overlaps with the chromatographic peak in front of the boat-shaped peak at pH 4.5 and pH 6.0 (e.g. Figure 3 As shown in the figure, the peak shape can be effectively improved and the intermediate product CMA-SA can be accurately quantified.

[0145] In summary, Comparative Example 3 shows that the chromatographic conditions of mobile phase pH 4.5 and pH 6.0 cannot accurately calculate the content of each substance in the enzyme-catalyzed process linkage reaction, and cannot accurately monitor the content and conversion rate of each substance in the enzyme-catalyzed process linkage reaction.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quality control method for synthesizing 3'-sialyllactose by an enzymatic process, comprising: A) Dissolve the test sample in a solvent, extract, and obtain a test solution; The test sample is the reaction solution or finished product of enzyme-catalyzed synthesis of 3'-sialyllactose; B) measuring the test solution by high performance liquid chromatography to obtain an HPLC spectrum of 3'-sialyllactose; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is an acetonitrile solution, and the mobile phase B is a dipotassium hydrogen phosphate trihydrate aqueous solution; and the elution ratio is: the mobile phase A: mobile phase B is 74:

26.

2. The method according to claim 1, characterized in that The dipotassium hydrogen phosphate trihydrate aqueous solution is a 10 mmol / L dipotassium hydrogen phosphate trihydrate aqueous solution with a pH value of 8.

0.

3. The method according to claim 1, characterized in that: The target substances for quality control include CMP-SA, CMP, SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL.

4. The method according to claim 3, characterized in that The method also includes preparing a reference solution: taking CMP-SA, CMP, SA, lactose, 3'-sialyllactulose, 6'-sialyllactose sodium salt and 3'-SL reference substances respectively, dissolving them in 75% acetonitrile to obtain a reference solution; The reference solution is measured by high performance liquid chromatography to obtain a chromatogram of the reference; and the components of 3'-sialyllactose are qualitatively and quantitatively determined according to the chromatogram of the reference.

5. The method according to claim 4, characterized in that The injection concentrations of the reference solution are as follows: CMP-SA is 0.0015 mg / mL to 2.0 mg / ml, CMP is 0.001 mg / mL to 2.0 mg / ml, SA is 0.00125 mg / mL to 2.0 mg / ml, D-lactose is 0.05 mg / mL to 2.0 mg / ml, 3'-sialyllactulose is 0.002 mg / mL to 2.0 mg / ml, 6'-sialyllactose sodium salt is 0.002 mg / mL to 2.0 mg / ml, and 3'-SL is 0.002 mg / mL to 2.0 mg / ml.

6. The method according to claim 1, characterized in that The chromatographic column is HILICpak VG-50 4E, with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

7. The method according to claim 1, characterized in that The wavelength of the ultraviolet detector is 210 nm; the temperature of the differential refractive index detector is 40°C.

8. The method according to claim 1, characterized in that The injection volume was 25 μL; the chromatographic column temperature was 40° C.; and the mobile phase flow rate was 0.8 mL / min.

9. The method according to claim 1, characterized in that: The sample tray temperature of the sample to be tested is 15°C; the running time is 35 minutes.

10. The method according to claim 1, characterized in that The solvent in step A) is 75% acetonitrile; after dissolution, the solvent is filtered using a 22 μm organic phase filter membrane.

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