A high performance liquid chromatography method for efficiently detecting β-alanine in D-calcium pantothenate and its application

Abstract

The present invention relates to a high-performance liquid chromatography method for efficiently separating and detecting beta-alanine in D-calcium pantothenate, wherein the chromatographic column in the method is selected from a zwitterionic column, the mobile phase is composed of a phosphate solution and acetonitrile, the detection wavelength is 200-300nm, the injection volume is 1-100μl, the column temperature is 10-45°C, and the volume ratio of the phosphate solution to acetonitrile in the mobile phase is 1:1-5. The present invention has the advantages of simple operation, high separation, good specificity, high sensitivity, low detection cost, etc., and is conducive to better controlling product quality and achieving quality control.

Description

Technical Field

[0001] The invention relates to the technical field of chromatography analysis, and in particular to a high performance liquid chromatography method for efficiently separating and detecting beta-alanine in D-calcium pantothenate and an application thereof. Background Art

[0002] D-calcium pantothenate is mainly used in medicine, food and feed additives. It is one of the components of coenzyme A in humans and animals and is involved in the metabolism of proteins, fats and sugars in the body. It is clinically used to treat vitamin B deficiency, peripheral neuritis, postoperative colic and other diseases (such as preventing streptomycin and kanamycin side effects, etc.). In the actual production of D-calcium pantothenate, impurities need to be controlled, and the most important impurity is β-alanine. The "National Food Safety Standard" requires that the content of impurity β-alanine in food-grade D-calcium pantothenate should not exceed 0.5%, while the content of impurity β-alanine in pharmaceutical-grade D-calcium pantothenate is more stringent. Therefore, efficient and accurate detection methods are needed to control product quality.

[0003] CN 110082448A discloses a method for detecting impurities in D-calcium pantothenate, wherein the impurities include β-alanine, D-pantolactone and sodium pantoate; the detection method is a high performance liquid chromatography method, and the mobile phase is a mixed solution of phosphate buffer and acetonitrile; the volume ratio of the phosphate buffer to the acetonitrile is 8.5-9.5:1; and the pH of the phosphate buffer is 2.0-4.0. The main defects are: first, D-calcium pantothenate is not easily soluble in the mobile phase; second, D-calcium pantothenate in the literature contains more impurities. When the purity of D-calcium pantothenate is high, the separation degree of β-alanine in the detection result is not high, and the accuracy is not high. Therefore, it is necessary to study a detection method with better detection and separation effect for β-alanine. Summary of the invention

[0004] The object of the present invention is to provide a high performance liquid chromatography method for efficiently separating and detecting beta-alanine in D-calcium pantothenate. The chromatographic column in the method is selected from a zwitterion column, the mobile phase consists of a phosphate solution and acetonitrile, the detection wavelength is 200-300 nm, the injection volume is 1-100 μl, the column temperature is 10-45° C., and the volume ratio of the phosphate solution to acetonitrile in the mobile phase is 1:1-3.

[0005] In the preferred technical solution of the present invention, the purity of the D-calcium pantothenate is not less than 95.0%, preferably not less than 99%.

[0006] In the preferred technical solution of the present invention, the content of β-alanine in D-calcium pantothenate is not higher than 0.5%.

[0007] According to a preferred technical solution of the present invention, the chromatographic column for high performance liquid chromatography is a HILIC chromatographic column.

[0008] According to a preferred technical solution of the present invention, the number of theoretical plates of the chromatographic column is ≥5000, preferably ≥10000.

[0009] In a preferred technical solution of the present invention, the diameter of the chromatographic column is 3-5 mm, the length of the chromatographic column is 100-300 mm, and the particle size of the filler in the chromatographic column is 3-5 μm.

[0010] In the preferred technical solution of the present invention, the phosphate solution is selected from any one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, phosphoric acid, and ammonium dihydrogen phosphate, or a combination thereof.

[0011] In a preferred technical solution of the present invention, the pH of the phosphate solution is 1.0-pH6.0, preferably pH2.0-pH5.0.

[0012] In a preferred technical solution of the present invention, the pH of the phosphate solution is adjusted with an acid, and the acid is phosphoric acid.

[0013] In the preferred technical solution of the present invention, the phosphate solution is potassium dihydrogen phosphate with a concentration of 0.05M.

[0014] In the preferred technical solution of the present invention, the ratio of phosphate solution to acetonitrile in the mobile phase is 25:75.

[0015] In a preferred technical solution of the present invention, the mobile phase elution is selected from any one of isocratic elution and gradient elution.

[0016] In the preferred technical solution of the present invention, the flow rate of the mobile phase is 0.6-1.5 ml / min, preferably 0.8-1.2 ml / min.

[0017] In the preferred technical solution of the present invention, the detection wavelength is 203nm-210nm, preferably 205-208nm.

[0018] In the preferred technical scheme of the present invention, the dilution solvent is used to configure, dissolve, and dilute any one of the test solution, the control solution, and the reference substance solution. The preferred dilution solvent is selected from any one of ultrapure water, methanol, acetonitrile, and mobile phase.

[0019] In the preferred technical solution of the present invention, the column temperature is 20-40°C, preferably any one of 20°C, 25°C, 30°C, and 35°C.

[0020] In a preferred technical solution of the present invention, the injection volume is 5-50 μl, preferably any one of 10 μl, 15 μl, 20 μl, 30 μl, and 40 μl.

[0021] Another object of the present invention is to provide an application of the detection method of the present invention in detecting high-purity D-pantothenate calcium, wherein the purity of the D-pantothenate calcium is not less than 99.0%, and the content of β-alanine in the D-pantothenate calcium is not higher than 0.5%.

[0022] In the preferred technical solution of the present invention, the purity of the D-calcium pantothenate is not less than 99.5%, preferably not less than 99.9%.

[0023] In the preferred technical solution of the present invention, the content of β-alanine is not higher than 0.3%, preferably not higher than 0.1%, and further preferably not higher than 0.05%.

[0024] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects:

[0026] 1. The present invention provides a high performance liquid phase zwitterion chromatography column method, which simultaneously screens the mobile phase and significantly improves the separation degree of high purity D-pantothenate calcium and impurity β-alanine.

[0027] 2. The detection method of the present invention has the advantages of simple operation, high separation, good specificity, high accuracy, and low detection cost, which is conducive to better control of product quality and achieve quality control.

[0028] 3. The present invention provides a high-purity D-calcium pantothenate, strictly controls the limit of impurity β-alanine in D-calcium pantothenate, promotes the quality improvement of D-calcium pantothenate products, and can be used for the detection of pharmaceutical-grade products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Liquid chromatography detection diagram of β-alanine standard solution in Example 1

[0030] Figure 2 Liquid chromatography detection diagram of D-calcium pantothenate solution in Example 1

[0031] Figure 3 Liquid chromatography detection diagram of D-calcium pantothenate solution in Comparative Example 1 DETAILED DESCRIPTION

[0032] The present invention is further described in detail by the following examples. These examples and experimental examples are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0033] Example 1

[0034] 1.1 Instruments and reagents

[0035] High performance liquid chromatograph: Shimadzu LC-16;

[0036] Potassium dihydrogen phosphate buffer: 0.05 M potassium dihydrogen phosphate aqueous solution adjusted to pH 3.0 with phosphoric acid;

[0037] D-Calcium pantothenate: commercially available, purity 99.6%.

[0038] 1.2 Liquid phase conditions

[0039] Chromatographic column: Yuexu HILIC Amphion II (5μm, 4.6X150mm), theoretical plate number ≥10000;

[0040] Column temperature: 30°C;

[0041] Flow rate: 0.8 mL / min;

[0042] Mobile phase: potassium dihydrogen phosphate buffer: acetonitrile = 25:75 (v / v);

[0043] Liquid volume: 10μL.

[0044] 1.3 Linear relationship of the method

[0045] Dissolve the β-alanine standard in ultrapure water to obtain a series of β-alanine standard solutions with concentrations of 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, and 0.05 g / L. Filter the series of standard solutions through a 0.2 μm water membrane, and inject the filtrate according to the instruments and conditions described in 1.1 and 1.2. The peak area-concentration (see Table 1, Figure 1 ) Draw the β-alanine standard curve, and its regression equation is y=500564.8496x-4.0393, R 2 =1.0000, where y is the β-alanine peak area and x is the concentration of the standard solution.

[0046] Table 1 β-alanine standard solution concentration and peak area

[0047] β-Alanine standard concentration / g / L Peak area 0.0101 5021 0.0201 10043 0.0302 15169 0.0402 20152 0.0503 25126

[0048] 1.4 Test methods

[0049] Accurately weigh 0.5g D-calcium pantothenate (purity 99.3%), dissolve in ultrapure water, dilute to 100mL, filter with 0.2μm water membrane, and collect the filtrate as the test solution. The test solution is injected according to the instruments and conditions described in 1.1 and 1.2, and the peak area is recorded and substituted into the regression equation described in 1.3 to obtain the β-alanine content. Take 6 groups of samples in parallel, marked as samples 1-6, calculate the content, average content and relative standard deviation (RSD), and the results are shown in Table 2.

[0050] Table 2. β-Alanine content, average content and RSD in D-calcium pantothenate samples

[0051]

[0052] 1.5 Method recovery

[0053] Blank spike recovery rate: Dissolve 3 different masses of β-alanine standard with ultrapure water, 3 portions each, recorded as groups 1 to 9. Filter each group of solutions through a 0.2 μm water system membrane, inject the filtrate according to the instruments and conditions described in 1.1 and 1.2, record the peak area and substitute it into the regression equation described in 1.3 to obtain the detection concentration of β-alanine standard, calculate the recovery rate, average recovery rate and relative standard deviation (RSD), the results are shown in Table 3.

[0054] The blank spike recovery rate is calculated as follows:

[0055] Blank spike recovery rate = blank spike value / spike amount x 100%

[0056] Table 3

[0057]

[0058] Sample spike recovery rate: Dissolve 3 groups of D-pantothenate calcium samples in ultrapure water, add 50%, 100%, and 150% β-alanine standards of the residual β-alanine in D-pantothenate calcium to each group, 3 portions per group, recorded as groups 1-9, filter each group of solutions through a 0.2 μm water system membrane, inject the filtrate according to the instruments and conditions described in 1.1 and 1.2, record the peak area and substitute it into the regression equation described in 1.3, obtain the detection concentration of β-alanine standards, calculate the recovery rate, average recovery rate and relative standard deviation (RSD), and the results are shown in Table 4. The test shows that the recovery rate meets the requirements, proving that this method has good recovery rate and repeatability.

[0059] The sample spike recovery rate was calculated as follows:

[0060] Spiked recovery rate = (spiked sample measurement value - sample measurement value) / spiked amount x 100

[0061] Table 4

[0062]

[0063] 1.6 Limit of quantitation

[0064] The quantification limit of β-alanine was 1 μg / mL using the above chromatographic conditions, with an RSD of 1.28%, a resolution greater than 11, and an RSD of <10, which met the methodological requirements.

[0065] Comparative Example 1

[0066] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0067] Mobile phase: Buffer (0.02 mol / L potassium dihydrogen phosphate buffer, pH adjusted to 3.0 with phosphoric acid): acetonitrile = 9:1;

[0068] Detection wavelength: 210nm;

[0069] Column temperature: 30°C;

[0070] Mobile phase flow rate: 1.0 mL / min

[0071] Liquid volume: 20μL.

[0072] Accurately weigh 0.5g D-calcium pantothenate (purity 99.3%), dissolve in ultrapure water, dilute to 100mL, filter with 0.2μm water membrane, collect the filtrate as the test solution. The test solution is injected according to the instruments and conditions described in 1.1 and 1.2. The test results are detailed in Figure 3 , Table 5. From Figure 3 It can be seen that the detection conditions cannot effectively separate β-alanine and D-calcium pantothenate.

[0073] Table 5

[0074]

[0075] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A high performance liquid chromatography method for efficiently separating and detecting β-alanine in D-calcium pantothenate, characterized in that: The chromatographic column in the method is selected from a zwitterion HILIC chromatographic column, the mobile phase consists of a phosphate solution and acetonitrile, the detection wavelength is 200-300nm, the injection volume is 1-100μl, the column temperature is 10-45℃, the volume ratio of the phosphate solution to acetonitrile in the mobile phase is 25:75, and the pH of the phosphate solution is 2.0-5.

0.

2. The method according to claim 1, characterized in that The purity of the D-calcium pantothenate is not less than 95%.

3. The method according to any one of claims 1 to 2, characterized in that The theoretical plate number of the chromatographic column is ≥5000.

4. The method according to claim 1, characterized in that The phosphate is selected from any one of potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate, or a combination thereof.

5. The method according to claim 1, characterized in that The concentration of the phosphate solution is not less than 0.1M.

6. The method according to claim 1, characterized in that The diluent solvent is used to prepare any one of the test solution and the reference solution, and is selected from any one of ultrapure water, methanol, acetonitrile, and mobile phase.

7. Use of the method according to any one of claims 1 to 6 for detecting high-purity D-calcium pantothenate, characterized in that: The purity of the D-pantothenate calcium in the D-pantothenate calcium is not less than 99.0%, and the content of β-alanine in the D-pantothenate calcium is not higher than 0.5%.

8. The use according to claim 7, characterized in that The purity of the D-calcium pantothenate is not less than 99.5%.

9. The use according to claim 8, characterized in that The content of β-alanine is not higher than 0.3%.

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