Method for identifying beta-alanine

Abstract

The invention discloses a beta-alanine identification method, which comprises: S1, sample solution preparation: adding water to a sample, and carrying out ultrasonic treatment to completely dissolve the sample so as to obtain a sample solution; s2, preparing a standard solution: adding water into a beta-alanine standard substance, and performing ultrasonic treatment to completely dissolve the beta-alanine standard substance to obtain the standard solution; s3, HPLC (High Performance Liquid Chromatography) determination: taking the standard solution as a reference substance, and identifying the sample solution by adopting an HPLC method; wherein a chromatographic column is Ultimate OAA, a mobile phase liquid A and a mobile phase liquid B are used for elution, and the mobile phase liquid A is 10mM monopotassium phosphate; the mobile phase liquid B is methanol. The specific mobile phase combination and the specific chromatographic column are adopted, beta-alanine can be rapidly identified, the method has the advantages of being easy and convenient to operate, good in specificity, high in accuracy, low in detection cost and the like, product quality can be better controlled, and quality controllability is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical detection, and more particularly, relates to a method for identifying β-alanine. Background Art

[0002] β-alanine, also known as 3-aminopropionic acid, with the molecular formula C3H7NO2, is a kind of non-natural amino acid. β-alanine is a precursor for the synthesis of vitamin B5 and an important component of coenzyme A. According to research, β-alanine can increase the concentration of carnosine in human muscles, and carnosine can scavenge reactive oxygen species and act as an effective buffer to prevent the production of fatigue toxins in working muscles. Therefore, β-alanine has attracted much attention in the fields of food health care, biomedicine, and feed, and the market demand for β-alanine has also increased significantly.

[0003] In addition, as the only β-type amino acid existing in nature, β-alanine is an important precursor or intermediate for the synthesis of hydroxypropionic acid, poly-3-hydroxypropionate, pantothenic acid, pamidronate sodium, balsalazide, etc., and is widely used in the fields of medicine, food, chemical engineering, and environment. The purity or content of β-alanine directly affects its efficacy or physiological effects.

[0004] However, due to the limitations of the production process, there will be more or less some impurities in the product of β-alanine production. In the existing literature, the content of β-alanine in the β-alanine conversion solution is determined by liquid phase detection using a C18 column after pre-column derivation. This method requires pre-treatment of the sample, the steps are relatively complex, and the effect is not very satisfactory. Therefore, there is a need for a simple and accurate analytical detection method to meet the identification of β-alanine. Summary of the Invention

[0005] In order to identify β-alanine faster and more accurately, the present invention provides a method for identifying β-alanine, comprising the following steps:

[0006] S1. Preparation of sample solution: Add water to the sample and treat it with ultrasonic waves to completely dissolve it to obtain a sample solution;

[0007] S2. Preparation of standard solution: Add water to the β-alanine standard product and treat it with ultrasonic waves to completely dissolve it to obtain a standard solution;

[0008] S3. HPLC determination: Using the above standard solution as a reference substance, identify the sample solution by HPLC method; wherein the chromatographic column is Ultimate OAA, and it is eluted with mobile phase A liquid and mobile phase B liquid. The mobile phase A liquid is 10 mM potassium dihydrogen phosphate; the mobile phase B liquid is methanol.

[0009] In one or more embodiments, the chromatographic conditions for the HPLC determination are:

[0010] Flow rate: 1.0 mL / min

[0011] Column temperature: 15 °C

[0012] Wavelength: 210 nm

[0013] Sample injection volume: 5 μL

[0014] Running time: 18 min.

[0015] In one or more embodiments, the pH of the potassium dihydrogen phosphate in mobile phase A is adjusted to 2.5 with phosphoric acid.

[0016] In one or more embodiments, the volume ratio of mobile phase A to mobile phase B is 98:2.

[0017] In one or more embodiments, in the sample solution, the concentration of the sample is 2 - 2.2 mg / mL; in the standard solution, the concentration of the β-alanine standard is 2 - 2.2 mg / mL.

[0018] In one or more embodiments, the injection sequence for HPLC determination is: blank aqueous solution, standard solution, sample solution.

[0019] In one or more embodiments, during HPLC determination, the retention time of β-alanine is approximately 3.77 min.

[0020] The present invention adopts a specific mobile phase combination and a specific chromatographic column, and can quickly identify β-alanine. The detection method of the present invention has the advantages of simple operation, good specificity, high precision, low detection cost, etc., which is conducive to better controlling the product quality and realizing quality control. Description of the Drawings

[0021] Figure 1 It is the liquid chromatography detection chart of the β-alanine standard solution in Example 1.

[0022] Figure 2 It is the liquid chromatography detection chart of the sample solution in Example 1.

[0023] Figure 3 It is the liquid chromatography detection chart of the β-alanine standard solution in Comparative Example 1.

[0024] Figure 4 It is the liquid chromatography detection chart of the sample solution in Comparative Example 1.

[0025] Figure 5 It is the liquid chromatography detection chart of the β-alanine standard solution in Comparative Example 2.

[0026] Figure 6It is the liquid chromatography detection chart of the sample solution in Comparative Example 2. Detailed implementation mode

[0027] In order to identify β-alanine faster and more accurately, the present invention provides a method for identifying β-alanine, which includes the following steps:

[0028] S1. Preparation of sample solution: Add water to the sample and treat it with ultrasonic waves to completely dissolve it to obtain a sample solution;

[0029] S2. Preparation of standard solution: Add water to the β-alanine standard product and treat it with ultrasonic waves to completely dissolve it to obtain a standard solution;

[0030] S3. HPLC determination: Using the above standard solution as a reference substance, identify the sample solution by HPLC method; among them, the chromatographic column is Ultimate OAA, and it is eluted with mobile phase A liquid and mobile phase B liquid. The mobile phase A liquid is 10 mM potassium dihydrogen phosphate; the mobile phase B liquid is methanol.

[0031] The Ultimate OAA (abbreviation: Ultimate OAA) special chromatographic column adopted by the present invention is a reversed-phase chromatographic column based on silica gel, which has the characteristics of good chromatographic performance, high resolution, and uniform chromatographic peaks.

[0032] In one or more embodiments, the chromatographic conditions for the HPLC determination are as follows:

[0033] Flow rate: 1.0 mL / min

[0034] Column temperature: 15 °C

[0035] Wavelength: 210 nm

[0036] Injection volume: 5 μL

[0037] Running time: 18 min.

[0038] In one or more embodiments, the pH of the potassium dihydrogen phosphate in the mobile phase A liquid is adjusted to 2.5 with phosphoric acid.

[0039] In one or more embodiments, the volume ratio of the mobile phase A and the mobile phase B is 98:2.

[0040] In one or more embodiments, in the sample solution, the concentration of the sample is 2 - 2.2 mg / mL; in the standard solution, the concentration of the β-alanine standard product is 2 - 2.2 mg / mL.

[0041] In one or more embodiments, the injection sequence for the HPLC determination is: blank aqueous solution, standard solution, sample solution.

[0042] In one or more embodiments, during HPLC determination, the retention time of β-alanine is about 3.77 min.

[0043] In one or more embodiments, the specific analysis steps are as follows:

[0044] (1) Preparation of standard solution

[0045] Weigh 0.10 g to 0.11 g (accurate to 0.0001 g) of Beta-alanine standard into a 50 mL volumetric flask, add an appropriate amount of ultrapure water, sonicate in an ultrasonic cleaner for 5 to 10 min until completely dissolved, take it out and let it cool to room temperature, make up the volume and shake well, filter with a 0.22 μm water-based syringe filter, and set aside.

[0046] (2) Preparation of sample solution

[0047] Weigh 0.10 g to 0.11 g (accurate to 0.0001 g) of the sample into a 50 mL volumetric flask, add an appropriate amount of ultrapure water, sonicate in an ultrasonic cleaner for 5 to 10 min until completely dissolved, take it out and let it cool to room temperature, make up the volume and shake well, filter with a 0.22 μm water-based syringe filter, and set aside.

[0048] (3) Injection procedure

[0049] Put the prepared sample into the sample tray, edit the sequence and save it.

[0050] Set the chromatographic parameters. After the liquid phase baseline is stable, inject the sequence once for the blank solution, once for the standard solution, and once for the sample solution, and record the chromatogram for comparison.

[0051] (4) Results

[0052] Using the method of the present invention, the retention time of the sample in the high performance liquid chromatogram is consistent with the standard retention time.

[0053] Retention time: Beta-alanine is about 3.77 min.

[0054] The present invention uses a specific mobile phase combination and a specific chromatographic column, which can quickly identify β-alanine. The detection method of the present invention has the advantages of simple operation, good specificity, high precision, low detection cost, etc., which is conducive to better controlling the product quality and realizing quality control.

[0055] Embodiment

[0056] The following further describes the present invention in detail with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0057] In the examples, the addition amounts, contents, and concentrations of various substances are involved. Unless otherwise specified, the percentage content refers to the mass percentage content.

[0058] Example 1

[0059] 1. Instruments and Reagents

[0060] High-performance liquid chromatograph: Agilent high-performance liquid chromatograph 1260 (or of the same type);

[0061] Electronic balance: accurate to 0.0001 g;

[0062] Methanol (HPLC), phosphoric acid (AR), potassium dihydrogen phosphate (AR or GR), ultrapure water;

[0063] 10 mM potassium dihydrogen phosphate (adjust the pH to 2.5 ± 0.03 with phosphoric acid): Weigh 1.36 g ± 0.01 g (accurate to 0.0001 g) of potassium dihydrogen phosphate into a 1000 mL reagent bottle, add an appropriate amount of ultrapure water to dissolve and make up the volume to 1000 mL, shake well, adjust the pH to 2.50 ± 0.03 with phosphoric acid, and filter three times with a 0.45 μm inorganic microporous filter membrane;

[0064] Sample: Huarui Biotechnology (Chuzhou) Co., Ltd., β-alanine is the concentrated distillate (the concentration in the cooled water vapor during sample concentration generally does not exceed 0.00558 g / L)

[0065] 2. Liquid Phase Conditions

[0066] Chromatographic column: Ultimate OAA, 4.6 × 300 mm, 5 μm;

[0067] Mobile phase: 10 mM potassium dihydrogen phosphate: methanol = 98:2

[0068] Flow rate: 1.0 mL / min

[0069] Column temperature: 15 °C

[0070] Wavelength: 210 nm

[0071] Injection volume: 5 μL

[0072] Running time: 18 min

[0073] Diluent: ultrapure water

[0074] Blank solution: ultrapure water.

[0075] 3. Analysis Steps:

[0076] (1) Preparation of Standard Solution

[0077] Weigh 0.10 g to 0.11 g (accurate to 0.0001 g) of Beta-alanine standard into a 50 mL volumetric flask, add an appropriate amount of ultrapure water, and sonicate in an ultrasonic cleaner for 5 to 10 minutes until completely dissolved. Take it out and let it cool to room temperature, make up the volume and shake well, then filter with a 0.22 μm hydrophilic syringe filter for standby.

[0078] (2) Preparation of sample solution

[0079] Weigh 0.10 g to 0.11 g (accurate to 0.0001 g) of the sample into a 50 mL volumetric flask, add an appropriate amount of ultrapure water, and sonicate in an ultrasonic cleaner for 5 to 10 minutes until completely dissolved. Take it out and let it cool to room temperature, make up the volume and shake well, then filter with a 0.22 μm hydrophilic syringe filter for standby.

[0080] (3) Injection procedure

[0081] Put the prepared sample into the sample tray, edit the sequence and save it.

[0082] Set the chromatographic parameters. After the liquid phase baseline is stable, inject the sequence once for the blank solution, once for the standard solution, and once for the sample solution, and record the chromatogram for comparison.

[0083] 4. Experimental results

[0084] Figure 1 and Figure 2 are respectively the liquid chromatography detection diagrams of the Beta-alanine standard solution and the sample solution.

[0085] It can be seen from the figure that the retention time of the sample in the high performance liquid chromatogram is basically the same as that of the standard. Retention time: Beta-alanine is about 3.77 min.

[0086] Therefore, even in the case of extremely low Beta-alanine content, the method of the present invention can still identify Beta-alanine.

[0087] Comparative example 1:

[0088] Compared with Example 1, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, and the others are the same as in Example 1.

[0089] Experimental results:

[0090] Figure 3 and Figure 4 are respectively the liquid chromatography detection diagrams of the Beta-alanine standard solution and the sample solution.

[0091] As can be seen from the figure, although the retention time of the sample in the high-performance liquid chromatography (HPLC) chromatogram is basically the same as the standard retention time, after replacing the chromatographic column Ultimate OAA with an octadecylsilyl-bonded silica gel chromatographic column, its peak emergence time is nearly three times delayed compared with Comparative Example 1, the detection efficiency is greatly reduced, wasting time and reagents. In addition, its peak shape changes from a sharp and symmetric peak shape to an asymmetric and abnormally trailing peak, which is unacceptable for detection in this field. Therefore, the selection of the chromatographic column in the present invention is crucial, and replacing it with other chromatographic columns cannot be used for the identification of β-alanine.

[0092] Comparative Example 2:

[0093] Compared with Example 1, the volume ratio of 10 mM potassium dihydrogen phosphate to methanol in the mobile phase is 95:5, and the others are the same as in Example 1.

[0094] Experimental results:

[0095] Figure 5 and Figure 6 are respectively the HPLC detection chromatogram of the β-alanine standard solution and the HPLC detection chromatogram of the sample solution.

[0096] As can be seen from the figure, Figure 5 and Figure 6 The peak emergence times are 31.168 min and 31.739 min respectively, indicating that the retention time of the sample in the HPLC chromatogram is basically the same as the standard retention time. However, compared with Example 1, after adjusting the ratio of 10 mM potassium dihydrogen phosphate to methanol to 98:2, the peak emergence time of β-alanine is about 3.77 min, and the detection efficiency is about eight times faster than that of this example in terms of detection time. The detection time is shortened by a multiple, and the detection efficiency is accelerated. Using the same method, it takes about 31.1 min to detect one sample with a volume ratio of 95:5 of 10 mM potassium dihydrogen phosphate to methanol. If the volume ratio of 98:2 of 10 mM potassium dihydrogen phosphate to methanol is used, 8 samples can be detected, and the cost of the mobile phase will also be reduced, and the instrument loss will also be smaller. Therefore, the ratio of 10 mM potassium dihydrogen phosphate to methanol in the mobile phase in the present invention is crucial.

[0097] Based on the above examples and comparative examples, the detection method described in the present invention can quickly identify β-alanine. The detection method of the present invention has the advantages of simple operation, good specificity, high precision, low detection cost, etc., which is conducive to better controlling the product quality and achieving quality control.

[0098] It is apparent to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Accordingly, all changes that fall within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claim concerned.

[0099] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for identifying β-alanine, characterized in that, It includes the following steps: S1. Sample solution preparation: Add water to the sample and treat it with ultrasonic waves to completely dissolve it, obtaining a sample solution; S2. Standard solution preparation: Add water to the β-alanine standard and treat it with ultrasonic waves to completely dissolve it, obtaining a standard solution; S3. HPLC determination: Using the above standard solution as a reference substance, identify the sample solution by HPLC method; the chromatographic column is Ultimate OAA, and it is eluted with mobile phase A liquid and mobile phase B liquid. The mobile phase A liquid is 10 mM potassium dihydrogen phosphate; the mobile phase B liquid is methanol.

2. The identification method of β-alanine according to claim 1, wherein The chromatographic conditions for the HPLC determination are as follows: Flow rate: 1.0 mL / min Column temperature: 15 °C Wavelength: 210 nm Injection volume: 5 μL Running time: 18 min.

3. The identification method of β-alanine according to claim 1, wherein The pH of the potassium dihydrogen phosphate in the mobile phase A liquid is adjusted to 2.5 with phosphoric acid.

4. The method for identifying β-alanine according to claim 1, characterized in that, The volume ratio of the mobile phase A and the mobile phase B is 98:

2.

5. The method for identifying β-alanine according to claim 1, wherein, In the sample solution, the concentration of the sample is 2 - 2.2 mg / mL; in the standard solution, the concentration of the β-alanine standard is 2 - 2.2 mg / mL.

6. The identification method of β-alanine according to claim 1, wherein, The injection sequence for the HPLC determination is: blank aqueous solution, standard solution, sample solution.

7. The identification method of β-alanine according to claim 1, characterized in that, During HPLC determination, the retention time of β-alanine is approximately 3.77 min.

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