A method for detecting carnosine and its application

Abstract

The invention discloses a detection method of carnosine and its application, the method comprising: (1) preparing samples: preparing control samples and test samples; (2) chromatographic conditions: detecting instrument: high performance liquid phase system; chromatographic column: octadecylsilane bonded silica gel column; mobile phase: inorganic phase containing phosphate and sodium heptane sulfonate, adjusting pH value to 2.5-6, organic phase is methanol; (3) determination method: taking control sample solution and test sample solution and filtering them, injecting into a sample injector, and calculating the purity of carnosine in the test sample according to a formula. The detection method of the invention has fast analysis speed, high sensitivity and high separation degree; good linear relationship, high retention time repeatability, and is suitable for accurate quantification of carnosine; adopting isocratic elution, a single pump liquid phase can be detected, convenient to use, strong practicality, and suitable for industrial production; no need for pre-column derivatization, simple operation, and time saving; and is suitable for the production process of enzymatic production of carnosine.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting carnosine and application thereof. Background Art

[0002] L-Carnosine, also known as carnosine, is mainly found in animal skeletal muscle and brain. It is an active dipeptide composed of β-alanine and L-histidine. Its molecular weight is 226.24 and its molecular formula is C9H 14 N4O3. It can maintain the pH balance of the body, remove free radicals to make cells anti-oxidant, and maintain cell life, so it is widely used in food additives, cosmetics, health products and pharmaceuticals.

[0003] At present, the production methods of carnosine mainly include extraction method, chemical synthesis method and enzymatic method. The extraction method mainly uses protease to hydrolyze muscle tissue to extract trace amounts of carnosine contained therein; the chemical synthesis method mainly includes acyl azide method, acyl chloride ammonolysis method, mixed acid anhydride method and trimethylsilyl protected histidine method, etc., and the generated products are complex; the enzymatic method uses adenosine triphosphate ATP as an energy donor and uses enzymes to catalyze β-alanine and L-histidine to synthesize carnosine.

[0004] No matter which of the above methods is used, impurities will be produced more or less in the process of preparing pure products. At present, there are few detection methods for carnosine and related impurities, and there is no unified standard. If the detection method is inappropriate, the related substances cannot be effectively separated, and some risk impurities will be included in the main peak. Therefore, it is of great significance to develop an analytical detection method that can effectively separate carnosine and its related impurities, which can greatly reduce the risk of its use. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting carnosine and its application, which can overcome the defects of the prior art, can quickly, stably and accurately detect the content of carnosine and the content of related impurities, and is particularly suitable for the detection of carnosine produced by enzymatic method.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A detection method for carnosine comprises the following steps:

[0008] (1) Sample preparation:

[0009] Control sample: Weigh the carnosine standard sample as the control sample, place it in a volumetric flask, add water to dissolve it by ultrasonication and dilute it to the scale to obtain the control sample;

[0010] Test sample: Weigh the carnosine crystal sample as the test sample, place it in a volumetric flask, add water to dissolve it by ultrasonic and dilute it to the scale;

[0011] (2) Chromatographic conditions:

[0012] Detection instrument: high performance liquid phase system;

[0013] Chromatographic column: octadecylsilane bonded silica column (C18);

[0014] Mobile phase: Inorganic phase contains phosphate and sodium heptane sulfonate, and the pH value is adjusted to 2.5-6; the organic phase is methanol;

[0015] Detection wavelength: 204nm;

[0016] Detection temperature: 30℃;

[0017] Injection volume: 20 μl;

[0018] Detection flow rate: 1.0ml / min;

[0019] (3) Determination method: Take the control sample solution and the test sample solution, filter them, inject them into the sample injector, and calculate the purity of carnosine in the test sample according to the following formula:

[0020]

[0021] Wherein, c in the formula represents the purity of the test sample, c SD represents the purity of the control sample, A represents the peak area of ​​the test sample, and A SD represents the control peak area, m represents the mass of the test sample, and m SD Represents the quality of the control sample.

[0022] Preferably, in the above technical solution, in step (2), the concentration of inorganic phosphate in the mobile phase is 5-50 mM, the concentration of sodium heptane sulfonate is 1-2 mM, the pH value is adjusted to 2.5-6, the organic phase is methanol, and the volume ratio of the inorganic phase to the organic phase is 95:5-99:1.

[0023] Preferably, in the above technical solution, in step (2), the phosphate concentration of the inorganic phase is 10 mM, the sodium heptane sulfonate concentration is 1 mM, the pH value is adjusted to 3.5-4.5, the organic phase is methanol, and the volume ratio of the inorganic phase to the organic phase is 97:3.

[0024] Preferably, in the above technical solution, in step (2), the mobile phase is prepared by preparing an aqueous solution containing 10 mM KH2PO4 and 1 mM sodium heptane sulfonate, adjusting the pH value to 3.5-4.5, mixing and degassing as the inorganic phase, the organic phase is methanol, and the volume ratio of the inorganic phase to the organic phase is 97:3.

[0025] Preferably, in the above technical solution, during the chromatographic detection in step (2), the detection temperature is 30° C., the injection volume is 20 μl, and the detection flow rate is 1.0 ml / min.

[0026] Preferably, in the above technical solution, the step (2) adopts isocratic elution and uses a single pump liquid phase for detection.

[0027] Application of a method for detecting carnosine.

[0028] Preferably, the above technical solution is applied to the detection of carnosine produced by enzymatic method.

[0029] Preferably, the above technical solution is applied to the detection of β-alanine and / or L-histidine.

[0030] The above technical solution of the present invention has the following beneficial effects:

[0031] (1) This detection method has a fast analysis speed and uses a conventional octadecylsilane bonded silica gel column (C18). The retention time of carnosine is 5-10 minutes.

[0032] (2) This detection method has a better peak shape for carnosine, high sensitivity, and a high degree of separation from other substances, effectively avoiding the influence of other impurities; and has a good linear relationship within a certain range and high retention time repeatability, which is suitable for accurate quantification of carnosine.

[0033] (3) This detection method can adopt isocratic elution and can be detected using a single liquid phase pump. It is easy to use, highly practical, and suitable for industrial production.

[0034] (4) This detection method does not require pre-column derivatization, is simple to operate, and saves time.

[0035] (5) This detection method can also detect substances such as β-alanine and L-histidine, and is particularly suitable for the production process of carnosine by enzymatic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a wavelength scan of carnosine, with a scanning band of 190-370nm.

[0037] Figure 2 This is the HPLC chromatogram of the control sample carnosine of Example 3.

[0038] Figure 3 This is the carnosine external standard method curve.

[0039] Figure 4 This is the HPLC chromatogram of the test sample carnosine in Example 3.

[0040] Figure 5This is the HPLC chromatogram of the enzymatic reaction liquid of Example 4, which contains carnosine and other reaction impurities. DETAILED DESCRIPTION

[0041] The specific embodiments of the present invention are described in detail below to facilitate a further understanding of the present invention. All experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained through commercial channels.

[0042] Example 1 Chromatographic Conditions

[0043] (1) Carnosine wavelength scanning

[0044] Pure carnosine was prepared into a 1 mg / ml solution and the wavelength was scanned using a Shimadzu LC-16 high performance liquid chromatography system in the range of 190-370 nm. Figure 1 The spectrum shows that carnosine has a maximum absorption peak at 204nm, so 204nm was selected as the detection wavelength.

[0045] (2) Column selection

[0046] The experiments were conducted using a reverse C18 column, a sulfonic acid strong cation column, and a forward amino-bonded silica column. The retention time of carnosine was less than 2 minutes when using a forward amino-bonded silica column. When using a sulfonic acid strong cation column, carnosine was not easily separated from L-His and ATP. After using an ion pair reagent, the retention time on the reverse C18 column was more than 5 minutes, with a tailing factor of 0.96, a basically symmetrical peak shape, and a tailing factor of 2.6 that effectively separated it from impurities. Therefore, a reverse C18 column was selected. Figure 2 (Kromasil C18 column data).

[0047] Reverse C18 columns of various brands such as Shimadzu WondaSil C18-WR, Waters C18-ODS and Kromasil C18 were selected. The retention times of the substances were slightly different, but no significant effect on the results was found.

[0048] (3) Column temperature selection

[0049] The detection temperature is good within the conventional detection temperature range of 20-40℃, with better separation from other impurities and better peak shape. The retention time of carnosine shortens with increasing temperature.

[0050] (4) Mobile phase selection

[0051] a. Selection of Inorganic Phase

[0052] Phosphate and acetate were selected to prepare buffer for the experiment. Increasing the salt concentration shortened the retention time and reduced the separation between the main peak and each impurity. The baseline was poor and the peak shape of carnosine was poor when using acetate, while the peak shape was symmetrical and did not tail when using phosphate. The retention time of carnosine was shown in Table 1 when the phosphate ratio was adjusted (the buffer contained 1mM sodium heptane sulfonate). In the range of phosphate concentration of 5-50mM, the separation of carnosine from other impurities was good and the peak shape was better.

[0053] Table 1. Phosphate concentration selection

[0054] Phosphate concentration [mM] 5 10 20 50 80 Carnosine retention time [min] 11.8 8.1 5.9 4.8 3.3 Separation 3.3 2.6 2.2 1.5 0.43 Tailing Factor 0.892 0.959 1.107 1.193 1.308

[0055] The separation of carnosine was good when the phosphate pH value was changed in the pH range of 2.5-6.0. The peak shape was better in the pH range of 3.5-4.5.

[0056] Table 2. pH selection (10 mM phosphate)

[0057] pH 2.5 3.5 4.5 6.0 Carnosine retention time [min] 13.2 10.8 8.1 5.4 Separation 3.0 3.2 2.6 1.8 Tailing Factor 1.566 1.218 0.959 1.401

[0058] b. Selection of organic phase

[0059] Methanol and acetonitrile were used as organic phases for the experiment. Methanol gave a better peak shape, so methanol was selected. Table 3 records the retention time of carnosine when different methanol ratios were adjusted (buffer containing 10 mM phosphate, 1 mM sodium heptane sulfonate). When the methanol ratio was 1-5%, carnosine had better separation from other impurities and better peak shape.

[0060] Table 3. Methanol concentration selection

[0061] Methanol ratio [%] 1 3 5 10 20 Carnosine retention time [min] 10.8 8.1 5.3 3.0 2.7 Separation 2.9 2.6 2.2 1.0 0.38 Tailing Factor 0.899 0.959 1.193 1.318 1.068

[0062] c. Selection of ion-pair reagents

[0063] Sodium heptane sulfonate, sodium octane sulfonate, tetrabutylammonium hydroxide, tetrabutylammonium bromide, etc. were selected as ion pair reagents for detection. Sodium heptane sulfonate and sodium octane sulfonate can prolong the retention time of carnosine. Considering factors such as peak shape and separation degree from impurities, sodium heptane sulfonate was selected as the ion pair reagent. When the concentration of sodium heptane sulfonate was 1-2mM, carnosine had a good separation degree from other impurities and a better peak shape, as shown in Table 4.

[0064] Table 4. Effects of ions on reagents

[0065] Sodium heptane sulfonate concentration [mM] 0 1 2 3 Carnosine retention time [min] 1.9 8.1 10.7 16.0 Separation 0.24 2.6 2.2 3.2 Tailing Factor 1.442 0.959 0.862 Poor peak shape

[0066] In summary, the composition of the mobile phase was determined to be phosphate, sodium heptane sulfonate and methanol, with a phosphate concentration of 5-50 mM, a methanol concentration of 1-5%, and a sodium heptane sulfonate concentration of 1-2 mM. See Table 5.

[0067] Table 5. Mobile phase composition

[0068]

[0069] Example 2 Methodology Establishment

[0070] (1) Exclusivity

[0071] Pure water and carnosine hydrolyzate solution were sampled and the spectrum showed that there was no interference from solvent water and other impurities, and the separation degree met the requirements, with the theoretical plate number being more than 3000.

[0072] (2) Linear range

[0073] Weigh 250.09 mg of carnosine standard sample (control sample) accurately into a 50 ml volumetric flask and adjust the volume to prepare a standard solution. Accurately measure the standard solution to prepare samples of different concentrations for testing. Plot the concentration as the horizontal axis and the corresponding peak area as the vertical axis. The results show that the linear relationship of carnosine concentration is good when it is 1-1000 μg / ml, and the regression equation is: y=61335.48x-25015.10, R 2 The value is 0.99960, and the standard curve is shown in Figure 3 .

[0074] (3) Recovery rate

[0075] Five portions of carnosine samples were accurately weighed, each with 5-10 mg, and the solutions were prepared using 50 ml volumetric flasks. Liquid phase detection was used and the recovery rates were in the range of 98.0-102%, with an average of 100.26%. Details are shown in Table 6.

[0076] Table 6. Carnosine recovery rate

[0077] Sample No. 1 2 3 4 5 Theoretical value [μg / ml] 0.1392 0.1139 0.1158 0.1595 0.1342 Measured value [μg / ml] 0.1411 0.1147 0.1159 0.1602 0.1325 Recovery rate [%] 101.38 100.67 100.12 100.41 98.70

[0078] (4) Sample stability study

[0079] A test solution (control sample) was prepared, and the test was performed every 1 hour, and the peak area was recorded. See Table 7. The test RSD was 0.429%.

[0080] Table 7. Sample stability

[0081]

[0082] (5) Durability

[0083] Three chromatographic columns from three manufacturers (Shimadzu WondaSil C18-WR column, Waters C18-ODS and Kromasil C18 column) were used for detection, and no significant deviations in purity, separation and retention time were found.

[0084] Example 3 Carnosine Sample Detection

[0085] Preparation of test samples: Accurately weigh 5.14 mg of carnosine crystal dry sample of unknown concentration, place it in a 50 ml volumetric flask, add water to ultrasonically dissolve and dilute to the scale.

[0086] Preparation of control sample: Accurately weigh 5.05 mg (purity 99.3%) of known carnosine control sample, place it in a 50 ml volumetric flask, add water to dissolve by ultrasonication and dilute to the scale.

[0087] Prepare the mobile phase: prepare an aqueous solution containing 10 mM KH2PO4 and 1 mM sodium heptane sulfonate, mix and degas as the inorganic phase, and the organic phase is methanol, accounting for 3%.

[0088] The liquid phase detection conditions are as follows:

[0089] Detection instrument: Shimadzu LC-16 high performance liquid chromatography system;

[0090] Column: Kromasil C18;

[0091] Detection wavelength: 204nm;

[0092] Detection temperature: 30℃;

[0093] Injection volume: 20 μl;

[0094] Detection flow rate: 1.0ml / min.

[0095] Take the above test sample and control sample and inject them into the injector, with the injection volume of 20μl each. The test results of the control sample are as follows Figure 2 The retention time is 8.061 minutes, which is the peak of carnosine. The test results of the test samples are as follows: Figure 4 The retention time of carnosine peak is 8.062 minutes, which is basically consistent with the retention time of the test sample.

[0096] The purity of carnosine in the test sample was calculated according to the following formula:

[0097]

[0098] In the formula, c represents the purity of the test sample, c SD represents the purity of the control sample, A represents the peak area of ​​the test sample, and A SD represents the control peak area, m represents the mass of the test sample, and m SD Represents the mass of the control sample. According to the formula, the purity of the test sample is 96.08%.

[0099] Example 4 Detection of carnosine enzymatic reaction solution sample

[0100] Preparation of test samples: diluted carnosine enzymatic reaction solution.

[0101] The control sample and detection conditions are the same as those in Example 3.

[0102] Take the carnosine enzymatic reaction solution to dilute the sample and the control sample and inject them into the injector, with the injection volume of 20μl each. Figure 5 The peak at 8.1 minutes is carnosine, which is basically consistent with the retention time of the control carnosine sample and has good separation from other impurities. The peaks at 1.2, 1.3, 1.9, 2.5 and 5.6 minutes are ATP, ADP, β-alanine, AMP and L-histidine respectively. This shows that carnosine can be effectively separated from other impurities in the reaction solution using this detection method, which can be applied to the reaction process and result detection.

[0103] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for detecting carnosine produced by an enzymatic method, It is characterized in that The method is applied to detecting β-alanine and L-histidine in carnosine, and comprises the following steps: (1) Sample preparation: Control sample: Weigh the carnosine standard sample as the control sample, place it in a volumetric flask, add water to dissolve it by ultrasonication and dilute it to the scale to obtain the control sample; Test sample: Weigh the carnosine crystal sample as the test sample, place it in a volumetric flask, add water to dissolve it by ultrasonic and dilute it to the scale; (2) Chromatographic conditions: Detection instrument: high performance liquid phase system; Chromatographic column: octadecylsilane bonded silica column C18; Mobile phase: The inorganic phase contains phosphate and sodium heptane sulfonate, and the pH value is adjusted to 2.5-6; the organic phase is methanol; the inorganic phase phosphate concentration in the mobile phase is 5-50 mM, the sodium heptane sulfonate concentration is 1-2 mM, the pH value is 2.5-6, the organic phase is methanol, and the volume ratio of the inorganic phase to the organic phase is 95:5-99:1; the phosphate is KH 2 PO 4 ; Detection wavelength: 204 nm; During the chromatographic detection in step (2), the detection temperature is 30°C; the injection volume is 20 μl; the detection flow rate is 1.0 ml / min; and isocratic elution is used; (3) Determination method: Take the control sample solution and the test sample solution, filter them, inject them into the injector, and calculate the purity of carnosine in the test sample according to the following formula: Wherein, c in the formula represents the purity of the test sample, c SD represents the purity of the control sample, A represents the peak area of ​​the test sample, and A SD represents the control peak area, m represents the mass of the test sample, and m SD Represents the quality of the control sample.

2. A method for detecting carnosine according to claim 1, It is characterized in that In the step (2), the phosphate concentration in the inorganic phase is 10 mM, the sodium heptane sulfonate concentration is 1 mM, and the pH value is 2.5-6; the organic phase is methanol, and the volume ratio of the inorganic phase to the organic phase is 97:

3.

3. A method for detecting carnosine produced by an enzymatic method according to claim 1, It is characterized in that In step (2), the mobile phase is prepared by preparing a mobile phase containing 10 mM KH 2 PO 4 and 1 mM sodium heptane sulfonate aqueous solution, adjusted to pH 3.5-4.5, mixed and degassed as the inorganic phase, the organic phase was methanol, and the volume ratio of the inorganic phase to the organic phase was 97:

3.

4. The detection method of carnosine produced by an enzymatic method according to claim 1, It is characterized in that The step (2) uses a single pump liquid phase for detection.

5. Application of the detection method of carnosine according to claim 1-4 in industrial production.

6. The use according to claim 5, It is characterized in that Applied to the detection of carnosine produced by industrial enzymatic method.

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