Non-derivatization HPLC (High Performance Liquid Chromatography) method for simultaneously measuring beta-aminobutyric acid and crotonic acid

Abstract

The invention discloses a non-derivatization HPLC (High Performance Liquid Chromatography) method for simultaneously determining beta-aminobutyric acid and crotonic acid. According to the method provided by the invention, determination is carried out through reversed-phase liquid chromatography, a high-purity spherical porous silica gel 5C18-PAQ chromatographic column suitable for separating hydrophilic compounds is selected as a chromatographic column, a 20 mmol / L dipotassium phosphate aqueous solution is adopted, the pH value is adjusted to 7.0 through phosphoric acid to serve as a single mobile phase, the detection time is 6 min, the detection wavelength is 200-210 nm, the column temperature is 30-40 DEG C, and the flow velocity is 0.5-0.6 mL / min. The detection technology provided by the invention is simple, convenient and efficient, does not need complex derivatization steps, and can realize rapid elution, separation and quantification of beta-aminobutyric acid and crotonic acid. The method is suitable for rapid detection and analysis of beta-aminobutyric acid synthesized by biological catalysis crotonic acid, and a convenient solution is provided for research and application in related fields.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to an HPLC method for simultaneously determining beta-aminobutyric acid and crotonic acid without derivatization. Background Art

[0002] β-aminobutyric acid (BABA), also known as 3-aminobutyric acid, has a chemical formula of C4H9NO2. It consists of two functional groups, amino and carboxyl, and four carbon atoms. It is a simple non-protein β-amino acid. β-aminobutyric acid is a starter that plays a vital role in plants' resistance to adverse environments such as drought and disease. In addition, β-aminobutyric acid can be used as a precursor of the pharmaceutical intermediate β-aminobutanol, which can be used to synthesize the anti-tumor drug 4-methylcyclophosphamide and penicillin antibiotics. In addition, it is a key intermediate (a chiral six-membered ring) for the drug dolutegravir used to treat HIV. Therefore, β-aminobutyric acid has excellent production and application value.

[0003] At present, the main production method of β-aminobutyric acid still relies on traditional chemical synthesis, especially the synthesis of β-aminobutyric acid from (r)-3-amino-3-methylpropionic acid tert-butyl ester or methyl butyrate. The chemical synthesis method uses metal or organic catalysts, which does not meet the requirements of safe production and environmental protection, and usually requires the addition of protective agents and contact protective agents during the reaction. The conditions are harsh and cumbersome, and the yield is low.

[0004] As a new technology, biocatalysis has been widely evaluated due to its mild reaction conditions, reduced operating costs and the benefits of generating no hazardous waste. Therefore, the production of β-aminobutyric acid by biocatalysis is very ideal and has become the most commercially viable way to produce β-aminobutyric acid. Among them, the production of high value-added β-aminobutyric acid by biocatalytic technology using cheap and readily available crotonic acid as a substrate has been favored by more and more researchers due to its high yield, high specificity and simple process.

[0005] As for the detection method of biocatalytic synthesis of β-aminobutyric acid from crotonic acid, most of the existing technologies only focus on the detection of a single component. β-aminobutyric acid needs to be derivatized with derivatization reagents such as o-phthalaldehyde (OPA) or 2,4-dinitrofluorobenzene before HPLC detection. The derivatization process is complicated, cumbersome and time-consuming.

[0006] "Mu Yumin, Han Guangxin, Zhang Haoyue, et al. Study on HPLC method for R-3-aminobutyric acid related substances in aspartase system [J]. Coal and Chemical Industry, 2023, 46(5): 122-125." It mentioned that β-aminobutyric acid and crotonic acid can be determined simultaneously, but the chromatogram of crotonic acid was not given. When we used the method described in it for detection, we found that it had the following obvious disadvantages: Because the mobile phase contained 5% acetonitrile, the baseline fluctuated greatly when detecting crotonic acid, the retention time drifted seriously, the peak shape was poor, and the crotonic acid could not be effectively quantified, and the detection time was long.

[0007] Therefore, the prior art has not found an analytical method that can quickly separate and accurately quantify β-aminobutyric acid and crotonic acid at the same time, and it is impossible to calculate the conversion rate and verify the enzyme activity in a timely manner. Therefore, developing a method that can effectively separate and determine β-aminobutyric acid and crotonic acid at the same time will help promote the industrial production process of biocatalytic synthesis of β-aminobutyric acid. Summary of the invention

[0008] In view of this, one of the objects of the present invention is to provide a method for quickly and simply separating β-aminobutyric acid and crotonic acid. According to the properties of β-aminobutyric acid and crotonic acid, a 5C18-PAQ chromatographic column which has good retention for both and is suitable for separating hydrophilic compounds and can use 100% water as the mobile phase is selected. By direct injection and isocratic elution in a single aqueous phase, a HPLC method for simultaneously determining β-aminobutyric acid and crotonic acid without derivatization is finally established.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0010] The present invention provides an HPLC method for simultaneously determining β-aminobutyric acid and crotonic acid without derivatization. In the method, the structural formulas of the β-aminobutyric acid and crotonic acid are:

[0011]

[0012] A non-derivatized HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid, the chromatographic conditions are:

[0013] Chromatographic column: high-purity spherical porous silica gel 5C18-PAQ chromatographic column;

[0014] Chromatographic column temperature: 30-40℃;

[0015] Mobile phase: The mobile phase is 20 mmol of dipotassium hydrogen phosphate aqueous solution, and the pH is adjusted to 7.0 with phosphoric acid;

[0016] Flow rate: 0.5-0.6mL / min;

[0017] Detection wavelength: 200-210nm.

[0018] As a preferred embodiment of the above technical solution of the present invention, the separation column uses COSMOSIL 5C18-PAQ, which is suitable for separating hydrophilic compounds, and 100% water can be used as the mobile phase. The column temperature is 35°C; the flow rate is 0.5 mL / min; and the detection wavelength is 210 nm.

[0019] As a preferred embodiment of the above technical solution of the present invention, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

[0020] As a preferred embodiment of the above technical solution of the present invention, the HPLC method adopts isocratic elution.

[0021] As a preferred embodiment of the above technical solution of the present invention, a 20 mmol / L potassium hydrogen phosphate aqueous solution (the pH value is adjusted to 7.0 with phosphoric acid) is used as a single mobile phase, isocratic elution is performed, and the running time is 6 minutes.

[0022] The above-mentioned HPLC method for simultaneously determining β-aminobutyric acid and crotonic acid without derivatization comprises the following steps:

[0023] S1. Accurately weigh the standard products of β-aminobutyric acid and crotonic acid, dissolve them in ultrapure water and make up to volume to obtain a mixed standard solution of β-aminobutyric acid and crotonic acid;

[0024] S2. Performing high performance liquid chromatography on the mixed standard solution using the chromatographic conditions, and drawing standard curves of β-aminobutyric acid and crotonic acid respectively according to the obtained peak areas, and obtaining concentration-peak area linear regression equations of β-aminobutyric acid and crotonic acid standards;

[0025] S3. Detection of the sample to be tested: Detection of the sample catalytic solution using the chromatographic conditions, substitute the obtained peak area into the concentration-peak area linear regression equation, multiply by the dilution factor, and calculate the content of β-aminobutyric acid and crotonic acid in the sample to be tested.

[0026] As a preferred embodiment of the above technical solution of the present invention, the mixed standard solution of β-aminobutyric acid and crotonic acid in step S1 is at least 5 portions, wherein the concentration range of β-aminobutyric acid is 0.4-2.2 mg / mL, and the concentration range of crotonic acid is 0.004-0.02 mg / mL.

[0027] As a preferred embodiment of the above technical scheme of the present invention, the sample to be tested in step S3 is a catalytic liquid for whole-cell biocatalysis of crotonic acid to synthesize β-aminobutyric acid (for example, the fermentation liquid of bacteria expressing aspartase in a fermentation medium containing crotonic acid, from which the bacteria can be removed before detection), and is diluted with ultrapure water before detection.

[0028] As a preferred embodiment of the above technical solution of the present invention, in step S3, the sample to be tested needs to be diluted to a concentration range of 0.4-2.2 mg / mL of β-aminobutyric acid and a concentration range of 0.004-0.02 mg / mL of crotonic acid.

[0029] Preferably, in steps S2 and S3, the injection volume of the standard solution and the sample to be tested is 10 μL.

[0030] The significant advantages of the present invention are:

[0031] (1) The present invention provides a novel method for simultaneously determining β-aminobutyric acid and crotonic acid. The method can simultaneously separate and determine β-aminobutyric acid and crotonic acid and their related substances.

[0032] (2) In the technical solution provided by the present invention, a COSMOSIL 5C18-PAQ chromatographic column is used, which is suitable for separating hydrophilic compounds and can use a 100% aqueous mobile phase. It has good retention for β-aminobutyric acid and crotonic acid, and can achieve effective separation of β-aminobutyric acid and crotonic acid in the sample to be tested, while also achieving effective separation of other related impurities.

[0033] (3) In the technical solution provided by the present invention, the high performance liquid chromatography method using a single aqueous mobile phase for isocratic elution can achieve effective elution, separation and quantification of β-aminobutyric acid and crotonic acid in one go, with a running time of 6 minutes, so that the chromatographic peaks of the two are completely separated, thereby achieving rapid quantitative analysis of β-aminobutyric acid and crotonic acid.

[0034] (3) The detection technology provided by the present invention has simple pre-treatment of the sample to be tested, no derivatization treatment is required, and the sample can be directly injected for detection. It is simple to operate, has a high recovery rate, low detection cost, high detection efficiency, and has high-throughput sample detection capabilities, and can dynamically monitor the conversion rate of whole-cell biocatalytic crotonic acid to β-aminobutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0036] Figure 1 The chromatogram of the mixed standard solution of β-aminobutyric acid and crotonic acid established by the present invention in Example 1 without derivatization is shown;

[0037] Figure 2 The chromatogram is a chromatogram of a mixed standard solution of β-aminobutyric acid and crotonic acid detected in Example 2 of the present invention;

[0038] Figure 3 The chromatogram is a chromatogram of a mixed standard solution of β-aminobutyric acid and crotonic acid detected in Example 3 of the present invention;

[0039] Figure 4 The chromatogram is a chromatogram of a mixed standard solution of β-aminobutyric acid and crotonic acid detected in Example 4 of the present invention;

[0040] Figure 5 This is a chromatogram of the mixed standard solution of β-aminobutyric acid and crotonic acid detected in Example 5 of the present invention;

[0041] Figure 6 The chromatogram is a chromatogram of a mixed standard solution of β-aminobutyric acid and crotonic acid detected in Example 6 of the present invention;

[0042] Figure 7 The standard curve for the non-derivatized determination of β-aminobutyric acid established by the present invention in Example 6;

[0043] Figure 8 The standard curve for the non-derivatized determination of crotonic acid established by the present invention in Example 6;

[0044] Fig. 9 The chromatogram of the whole-cell biocatalytic solution sample without derivatization established by the present invention in Example 6;

[0045] Fig.10 This is a graph showing the precision investigation results of the non-derivatized determination of β-aminobutyric acid and crotonic acid established by the present invention in Example 6;

[0046] Fig.11 This is a graph showing the results of the non-derivatized stability determination of β-aminobutyric acid and crotonic acid established by the present invention in Example 6. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] Example 1

[0049] 1.1 Instruments and reagents

[0050] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0051] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), dipotassium hydrogen phosphate (analytical grade), phosphoric acid (analytical grade), ultrapure water.

[0052] 1.2 Chromatographic conditions

[0053] Chromatographic column: COSMOSIL 5C18-PAQ, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm;

[0054] Chromatographic column temperature: 35°C;

[0055] Mobile phase: The mobile phase was 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), isocratic elution, running time 6 min.

[0056] Flow rate: 0.5 mL / min;

[0057] Injection volume: 10 μL;

[0058] Detection wavelength: 210nm.

[0059] 1.3 Operation steps

[0060] Accurately weigh 13.72 mg of β-aminobutyric acid standard and 0.06 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute to volume with ultrapure water, and shake well to obtain a mixed standard solution with a β-aminobutyric acid concentration of 1.3720 mg / mL and a crotonic acid concentration of 0.006 mg / mL.

[0061] Take the mixed standard solution, filter it into a sample injection bottle through a 0.22μm water filter membrane, perform HPLC analysis according to the chromatographic conditions described in Section 1.2, and record the chromatogram.

[0062] See attached for the results Figure 1 In the figure, β-aminobutyric acid is at 3.687min and crotonic acid is at 4.802min. It can be seen that under this condition, the baseline is stable, the peaks of β-aminobutyric acid and crotonic acid are sharp, and the separation effect is good.

[0063] Example 2

[0064] The main difference from Example 1 is that the mobile phase is a mixed solution of 20 mmol / L dipotassium hydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid): acetonitrile = 95:5 (volume ratio).

[0065] 2.1 Instruments and reagents

[0066] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0067] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), dipotassium hydrogen phosphate (analytical grade), phosphoric acid (analytical grade), acetonitrile (chromatographic grade), ultrapure water.

[0068] 2.2 Chromatographic conditions

[0069] Chromatographic column: COSMOSIL 5C18-PAQ, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm;

[0070] Chromatographic column temperature: 35°C;

[0071] Mobile phase: The mobile phase is a mixed solution of 20 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 7.0 with phosphoric acid): acetonitrile = 95:5 (volume ratio), running time 6 min.

[0072] Flow rate: 0.5 mL / min;

[0073] Injection volume: 10 μL;

[0074] Detection wavelength: 210nm.

[0075] 2.3 Operation steps

[0076] Accurately weigh 13.72 mg of β-aminobutyric acid standard and 0.06 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute to volume with ultrapure water, and shake well to obtain a mixed standard solution with a β-aminobutyric acid concentration of 1.3720 mg / mL and a crotonic acid concentration of 0.006 mg / mL.

[0077] Take the mixed standard solution, filter it into a sample injection bottle using a 0.22 μm water filter membrane, perform HPLC analysis according to the chromatographic conditions described in Section 2.2, and record the chromatogram.

[0078] See attached for the results Figure 2 The results showed that when a small amount of acetonitrile was added to the mobile phase, the baseline fluctuated greatly and the separation effect of β-aminobutyric acid and crotonic acid became worse.

[0079] Example 3

[0080] The main difference from Example 1 is that the mobile phase is a mixed solution of water:acetonitrile=95:5 (volume ratio).

[0081] 3.1 Instruments and reagents

[0082] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0083] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), acetonitrile (chromatographic grade), ultrapure water.

[0084] 3.2 Chromatographic conditions

[0085] Chromatographic column: COSMOSIL 5C18-PAQ, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm;

[0086] Chromatographic column temperature: 35°C;

[0087] Mobile phase: The mobile phase was a mixed solution of water:acetonitrile = 95:5 (volume ratio), and the running time was 6 min.

[0088] Flow rate: 0.5 mL / min;

[0089] Injection volume: 10 μL;

[0090] Detection wavelength: 210nm.

[0091] 3.3 Operation steps

[0092] Accurately weigh 13.72 mg of β-aminobutyric acid standard and 0.06 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute to volume with ultrapure water, and shake well to obtain a mixed standard solution with a β-aminobutyric acid concentration of 1.3720 mg / mL and a crotonic acid concentration of 0.006 mg / mL.

[0093] Take the mixed standard solution, filter it into a sample injection bottle through a 0.22μm water filter membrane, perform HPLC analysis according to the chromatographic conditions described in Section 3.2, and record the chromatogram.

[0094] See attached for the results Figure 3 ,The results showed that when the buffered salt solution in the mobile phase was replaced with an aqueous solution, the retention time of crotonic acid could not be reproduced, the retention time was delayed, and eventually it overlapped with β-aminobutyric acid and could not be separated.

[0095] Example 4

[0096] The main difference from Example 1 is that the chromatographic column is ZORBAX Eclipse XDB-C18, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

[0097] 4.1 Instruments and reagents

[0098] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0099] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), dipotassium hydrogen phosphate (analytical grade), phosphoric acid (analytical grade), ultrapure water.

[0100] 4.2 Chromatographic conditions

[0101] Chromatographic column: ZORBAX Eclipse XDB-C18, 150 mm in length, 4.6 mm in inner diameter, and 5 μm in particle size;

[0102] Chromatographic column temperature: 35°C;

[0103] Mobile phase: The mobile phase was 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), isocratic elution, running time 10 min.

[0104] Flow rate: 0.5 mL / min;

[0105] Injection volume: 10 μL;

[0106] Detection wavelength: 210nm.

[0107] 4.3 Operation steps

[0108] Accurately weigh 13.72 mg of β-aminobutyric acid standard and 0.06 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute to volume with ultrapure water, and shake well to obtain a mixed standard solution with a β-aminobutyric acid concentration of 1.3720 mg / mL and a crotonic acid concentration of 0.006 mg / mL.

[0109] Take the mixed standard solution, filter it into a sample injection bottle using a 0.22μm water filter membrane, perform HPLC analysis according to the chromatographic conditions described in Section 4.2, and record the chromatogram.

[0110] See attached for the results Figure 4 In the figure, β-aminobutyric acid is at 2.173min and crotonic acid is at 2.392min. The results show that the chromatographic column has poor retention for β-aminobutyric acid and crotonic acid, early peak elution, large baseline fluctuations, and poor separation effect of the two.

[0111] Example 5

[0112] The main difference from Example 1 is that the chromatographic column is Luna@5μm NH2 The length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

[0113] 5.1 Instruments and reagents

[0114] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0115] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), dipotassium hydrogen phosphate (analytical grade), phosphoric acid (analytical grade), ultrapure water.

[0116] 5.2 Chromatographic conditions

[0117] Chromatographic column: Luna@5μm NH2 The length of the chromatographic column is 250 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm;

[0118] Chromatographic column temperature: 35°C;

[0119] Mobile phase: The mobile phase was 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), isocratic elution, running time 10 min.

[0120] Flow rate: 1 mL / min;

[0121] Injection volume: 10 μL;

[0122] Detection wavelength: 210nm.

[0123] 5.3 Operation steps

[0124] Accurately weigh 13.72 mg of β-aminobutyric acid standard and 0.06 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute to volume with ultrapure water, and shake well to obtain a mixed standard solution with a β-aminobutyric acid concentration of 1.3720 mg / mL and a crotonic acid concentration of 0.006 mg / mL.

[0125] Take the mixed standard solution, filter it into a sample injection bottle through a 0.22μm water filter membrane, perform HPLC analysis according to the chromatographic conditions described in Section 5.2, and record the chromatogram.

[0126] See attached for the results Figure 5 In the figure, the β-aminobutyric acid and crotonic acid peak times completely overlap, and the results show that although Luna@5μm NH2 The column is also suitable for separating hydrophilic compounds and can use 100% water as the mobile phase, but it cannot separate β-aminobutyric acid and crotonic acid.

[0127] Example 6

[0128] The catalytic solution of whole-cell biocatalysis of crotonic acid to β-aminobutyric acid was detected by the chromatographic method of Example 1, and a standard curve was drawn to quantitatively analyze crotonic acid and β-aminobutyric acid by the external standard method.

[0129] 6.1 Instruments and reagents

[0130] High performance liquid chromatograph: Agilent 1260 Infinity II high performance liquid chromatograph;

[0131] β-Aminobutyric acid standard (98%), crotonic acid standard (98%), dipotassium hydrogen phosphate (analytical grade), phosphoric acid (analytical grade), ultrapure water.

[0132] 6.2 Chromatographic conditions

[0133] Chromatographic column: COSMOSIL 5C18-PAQ, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm;

[0134] Chromatographic column temperature: 35°C;

[0135] Mobile phase: The mobile phase was 20 mmol / L potassium dihydrogen phosphate aqueous solution (pH adjusted to 7.0 with phosphoric acid), isocratic elution, running time 6 min.

[0136] Flow rate: 0.5 mL / min;

[0137] Injection volume: 10 μL;

[0138] Detection wavelength: 210nm.

[0139] 6.3 Operation steps

[0140] 6.3.1 Drawing a standard curve

[0141] Accurately weigh 22.74 mg of β-aminobutyric acid standard and 0.21 mg of crotonic acid standard into a 10 mL volumetric flask, dissolve and dilute with ultrapure water, shake well, and obtain a mixed standard solution stock with a β-aminobutyric acid concentration of 2.2285 mg / mL and a crotonic acid concentration of 0.0205 mg / mL.

[0142] Take the above mixed standard solution mother liquor and dilute it with ultrapure water with the initial mobile phase according to the following concentrations: β-aminobutyric acid concentrations of 0.4457 mg / ml, 0.8914 mg / ml, 1.3371 mg / ml, 1.7828 mg / ml, 2.2285 mg / ml; crotonic acid concentrations of 0.0041 mg / ml, 0.0082 mg / ml, 0.0123 mg / ml, 0.0164 mg / ml, 2.0205 mg / ml, to obtain gradient standard solutions of 5 concentration points each.

[0143] Take the above-mentioned 5 concentration point gradient standard solutions of β-aminobutyric acid and crotonic acid, respectively, and filter them into injection bottles using 0.22μm water filter membranes, and perform high performance liquid chromatography analysis according to the chromatographic conditions described in Section 6.2.

[0144] The results of the concentration and peak area of ​​the mixed standard solution are shown in Tables 1 and 2. The theoretical plate number of each chromatogram is greater than 5000, and the separation is good. The chromatogram of the mixed standard solution with a β-aminobutyric acid concentration of 0.8914 mg / mL and a crotonic acid concentration of 0.0082 mg / mL is shown in Figure 6 shown.

[0145] Table 1 β-aminobutyric acid standard curve detection results

[0146] Concentration (mg / mL) Peak area (mAU·s) 0.4457 426.573 0.8914 785.511 1.3371 1190.151 1.7828 1561.522 2.2285 1991.676

[0147] Table 2 Crotonic acid standard curve detection results

[0148] Concentration (mg / mL) Peak area (mAU·s) 0.0041 763.425 0.0082 1563.611 0.0123 2315.345 0.0164 3035.827 0.0205 3689.081

[0149] The standard curves of β-aminobutyric acid and crotonic acid standards were drawn according to the concentration of the standards and the corresponding peak areas, with the concentration as the horizontal axis and the peak area as the vertical axis, and the concentration-peak area linear regression equation of β-aminobutyric acid standard was obtained: y=882.6372x+8.9466, R 2 =0.99945; the concentration-peak area linear regression equation of the crotonic acid standard was obtained: y=181045.5578x+32.7964, R 2 =0.99918, Figure 7 is the standard curve of β-aminobutyric acid, Figure 8 This is the standard curve of crotonic acid.

[0150] The results of this example show that, through the determination of β-aminobutyric acid and crotonic acid standard solutions, the method of the present invention has a good linear relationship (R 2 =0.99945), and crotonic acid also has a good linear relationship between 0.04 and 0.02 mg / mL (R 2 =0.99918).

[0151] 6.3.2 Testing of samples to be tested

[0152] The preparation method of the catalytic solution (i.e., fermentation solution) for whole-cell biocatalysis of crotonic acid to synthesize β-aminobutyric acid refers to the method described in Chapter 2 of Wang Yaling's master's thesis: Rational Transformation of Aspartase and Its Efficient Conversion to Synthesize β-Aminobutyric Acid.

[0153] Take 1 ml of the catalytic solution of whole-cell biocatalysis of crotonic acid to β-aminobutyric acid, centrifuge at 3000 rpm for 10 min, take the supernatant, dilute it to 5 ml with ultrapure water, and microfilter it into a sample injection bottle with a 0.22 μm water filter membrane to obtain the sample to be tested.

[0154] Under the same detection conditions as the standard solution, the sample to be tested is tested, and the chromatogram is as follows: Fig. 9 As shown, the peak area of ​​β-aminobutyric acid is 402.254, the peak area is substituted into the above β-aminobutyric acid concentration-peak area linear regression equation, multiplied by the dilution factor 5, and the content of β-aminobutyric acid in the sample to be tested is calculated to be 2.22 mg / mL; the peak area of ​​crotonic acid is 3547.525, the peak area is substituted into the above crotonic acid concentration-peak area linear regression equation, multiplied by the dilution factor 5, and the content of guanidine acetic acid in the sample to be tested is calculated to be 0.097 mg / mL.

[0155] 6.3.3 Precision investigation

[0156] Prepare a mixed standard solution with a concentration of 1.1143 mg / mL of β-aminobutyric acid and 0.006 mg / mL of crotonic acid, and perform HPLC analysis under the above chromatographic conditions. Repeat 6 injections. The results are shown in Fig.10 The obtained β-aminobutyric acid peak areas were 1037.418, 1041.811, 1038.252, 1036.065, 1035.839, and 1034.244, with an average of 1037.272 and an RSD of 0.252%; the obtained crotonic acid peak areas were 1066.309, 1074.990, 1082.894, 1088.036, 1096.692, and 1098.481, with an average of 1084.567 and an RSD of 1.152%, indicating that the HPLC detection method for β-aminobutyric acid and crotonic acid provided in this embodiment has good precision.

[0157] 6.3.4 Stability test

[0158] Prepare a mixed standard solution with a concentration of 1.1143 mg / mL of β-aminobutyric acid and 0.08 mg / mL of crotonic acid. Perform HPLC analysis under the above chromatographic conditions at 0 h, 4 h, 8 h, 12 h, and 24 h after preparation. The results are shown in Fig.11 The peak areas of β-aminobutyric acid were 1113.895, 1112.617, 1112.426, 1114.493, and 1109.986, with an average of 1112.683 and an RSD of 0.156%. The retention times were 3.692min, 3.692min, 3.693min, 3.692min, and 3.692min, with an average of 3.692min and an RSD of 0.007%. The peak areas of crotonic acid were 1418.4 37, 1417.169, 1411.387, 1426.500, 1433.616 with an average value of 1421.422 and an RSD of 0.611%. The retention times were 4.818min, 4.818min, 4.818min, 4.818min, 4.818min, and 4.818min, respectively. The average value was 4.818min and the RSD was 0.008%, indicating that the HPLC detection method for β-aminobutyric acid and crotonic acid provided in this embodiment has good stability.

[0159] 6.3.5 Limit of quantitation

[0160] Accurately weigh 13.37 mg of β-aminobutyric acid standard in a 10 mL volumetric flask, dissolve it in ultrapure water and make up the volume, perform HPLC analysis according to the above chromatographic conditions, and measure the peak height to be 152.007 and the baseline noise to be 0.390; dilute the β-aminobutyric acid standard solution to 400 times, and test it under the same detection conditions as the standard solution, and measure the peak height to be 3.94. Therefore, the quantitative limit of this method is 13.37 / 10 / 400=0.003342 mg / mL. It shows that the HPLC detection method of β-aminobutyric acid provided in this embodiment has a lower detection limit.

[0161] 0.205 mg of the crotonic acid standard was accurately weighed in a 10 mL volumetric flask, dissolved in ultrapure water and fixed to volume, and analyzed by high performance liquid chromatography according to the above chromatographic conditions, and the peak height was 510.736 and the baseline noise was 0.617; the crotonic acid standard solution was diluted to 200 times, and the peak height was measured to be 6.899 under the same detection conditions as the standard solution. Therefore, the quantitative limit of this method is 0.205 / 10 / 200=0.000103 mg / mL. It is shown that the high performance liquid phase detection method of crotonic acid provided in this embodiment has a lower detection limit.

[0162] 6.3.6 Recovery determination

[0163] Take 10ml of the catalytic solution of the whole cell biocatalysis of crotonic acid to synthesize β-aminobutyric acid, centrifuge at 3000rpm for 10min, take the supernatant, dilute to 100ml with ultrapure water, and microfilter into the injection bottle with a 0.22μm water filter membrane to obtain the sample to be tested. Take out 9 portions, each 10mL; weigh 3 portions of 2mg, 4mg, and 8mg of β-aminobutyric acid standard, respectively, add them to the above 9 portions of sample solution, dissolve and mix, microfilter into the injection bottle with a 0.22μm water filter membrane, perform high performance liquid chromatography analysis according to the above chromatographic conditions, detect the content of β-aminobutyric acid standard in each solution, calculate the recovery rate, and the results are shown in Table 3.

[0164] Take 10ml of the catalytic solution of the whole cell biocatalysis of crotonic acid to synthesize β-aminobutyric acid, centrifuge at 3000rpm for 10min, take the supernatant, dilute to 100ml with ultrapure water, and filter into the injection bottle with a 0.22μm water filter membrane to obtain the sample to be tested. Take out 9 portions, each 10mL; weigh 3 portions of 0.02mg, 0.04mg, and 0.08mg of crotonic acid standard, respectively, add them to the above 9 sample solutions, dissolve and mix, filter into the injection bottle with a 0.22μm water filter membrane, perform high performance liquid chromatography analysis according to the above chromatographic conditions, detect the content of crotonic acid standard in each solution, calculate the recovery rate, and the results are shown in Table 4.

[0165] Table 3 β-aminobutyric acid recovery and relative standard deviation

[0166]

[0167] Table 4 Recovery rate and relative standard deviation of crotonic acid

[0168]

[0169] It can be seen from Table 3 / 4 that in 9 recovery tests, the average recovery rate of β-aminobutyric acid was 99.07%, RSD=0.82 (n=9); the average recovery rate of crotonic acid was 99.23%, RSD=2.56 (n=9), and the recovery rates were all greater than 98%, indicating that the HPLC detection method for β-aminobutyric acid and crotonic acid provided in this embodiment has a good recovery rate.

[0170] Data analysis

[0171] (1) Example 1 and Example 2-3 optimize the mobile phase. Example 1 uses 20mmol / L dipotassium hydrogen phosphate solution (adjusting pH to 7.0 with phosphoric acid) as the mobile phase, β-aminobutyric acid and crotonic acid are effectively separated, and the peak shapes of the two are sharp and symmetrical, while Example 2 adds 5% acetonitrile in the mobile phase, resulting in large baseline fluctuations, and the separation effect of β-aminobutyric acid and crotonic acid is poor. Example 3 uses a mixed solution of water: acetonitrile = 95:5 (volume ratio) as the mobile phase, and the retention time of crotonic acid cannot be reproduced, and it cannot be effectively separated from β-aminobutyric acid, which has a greater impact on the accuracy and sensitivity of the detection, and is not conducive to accurate quantification. Therefore, the mobile phase of this method invention is most preferably 20mmol / L dipotassium hydrogen phosphate solution (adjusting pH to 7.0 with phosphoric acid).

[0172] (2) Examples 1 and 4-5 compare the detection results of different chromatographic columns. Example 1 uses COSMOSIL 5C18-PAQ chromatographic column, which is suitable for separating hydrophilic compounds, can use 100% water as the mobile phase, has good retention for β-aminobutyric acid and crotonic acid, β-aminobutyric acid and crotonic acid are effectively separated, the baseline is stable and the chromatographic peak shape is symmetrical; while Example 4 uses Agilent Ecipse XDB-C18 chromatographic column. Since β-aminobutyric acid and crotonic acid have high polarity, ordinary C18 chromatographic column has almost no retention for them and cannot be detected; Example 5 uses amino-bonded silica gel chromatographic column: Luna@5μm NH2 The chromatographic column has good retention for polar compounds, but due to the similar structures of β-aminobutyric acid and crotonic acid, the two peaks elute at the same time and cannot be separated.

[0173] (3) It can be seen from Examples 1 and 6 that the HPLC detection method provided by the present invention can effectively separate β-aminobutyric acid and crotonic acid, and the peaks of the two are symmetrical and sharp, and β-aminobutyric acid and crotonic acid in the whole cell catalytic solution can be simultaneously determined and accurately quantified. In addition, the high-performance liquid phase detection method of β-aminobutyric acid and crotonic acid provided by the present invention has a low detection limit, good precision, stability and recovery rate.

[0174] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization, characterized in that: The chromatographic conditions of the HPLC method are: Chromatographic column: high-purity spherical porous silica gel 5C18-PAQ chromatographic column; Chromatographic column temperature: 30-40℃; Mobile phase: The mobile phase is 20mmol / L potassium dihydrogen phosphate aqueous solution, and the pH is adjusted to 7.0 with phosphoric acid; Flow rate: 0.5-0.6mL / min; Detection wavelength: 200-210nm.

2. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 1, characterized in that: The separation column used was COSMOSIL 5C18-PAQ, the column temperature was 35°C, the flow rate was 0.5 mL / min, and the detection wavelength was 210 nm.

3. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 1, characterized in that: The length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

4. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 1, characterized in that: A 20 mmol / L potassium hydrogen phosphate aqueous solution was used as the single mobile phase for isocratic elution.

5. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 4, characterized in that: The elution run time was 6 min.

6. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: S1. Accurately weigh the standard products of β-aminobutyric acid and crotonic acid, dissolve them in ultrapure water and make up to volume to obtain a mixed standard solution of β-aminobutyric acid and crotonic acid; S2. Performing high performance liquid chromatography on the mixed standard solution using the chromatographic conditions, and drawing standard curves of β-aminobutyric acid and crotonic acid respectively according to the obtained peak areas, and obtaining concentration-peak area linear regression equations of β-aminobutyric acid and crotonic acid standards; S3. Detection of the sample to be tested: Detection of the sample catalytic solution using the chromatographic conditions, substitute the obtained peak area into the concentration-peak area linear regression equation, multiply by the dilution factor, and calculate the content of β-aminobutyric acid and crotonic acid in the sample to be tested.

7. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 6, characterized in that: The mixed standard solution of β-aminobutyric acid and crotonic acid in step S1 is at least 5 portions, wherein the concentration range of β-aminobutyric acid is 0.4-2.2 mg / mL, and the concentration range of crotonic acid is 0.004-0.02 mg / mL.

8. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 6, characterized in that: The sample to be tested in step S3 is a catalytic solution for whole-cell biocatalysis of crotonic acid to synthesize β-aminobutyric acid, which is diluted with ultrapure water before testing.

9. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 6, characterized in that: In step S3, the sample to be tested is diluted to a concentration range of 0.4-2.2 mg / mL of β-aminobutyric acid and a concentration range of 0.004-0.02 mg / mL of crotonic acid.

10. The HPLC method for simultaneous determination of β-aminobutyric acid and crotonic acid without derivatization according to claim 6, characterized in that: In steps S2 and S3, the injection volume of the standard solution and the sample to be tested is 10 μL.

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