A method for detecting a b-complex vitamin

Abstract

The application discloses a detection method of B vitamins, wherein the detection method comprises the following steps: preparation of a control sample solution: B vitamin control samples are respectively dissolved to obtain the control sample solution; preparation of a sample solution: a sample solution containing B vitamins is diluted to obtain the sample solution; determination: high performance liquid chromatography is used to determine the content of B vitamins in the sample solution containing B vitamins. The method has good repeatability, high accuracy, high stability, good separation effect and high separation degree for detecting VB1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, nicotinic acid and pterin, and can be simultaneously used for common detection of multiple B vitamins in a hyaluronic acid complex solution, thereby providing a good means for research and development and quality evaluation of the hyaluronic acid complex solution.

Description

Technical Field

[0001] This application relates to the field of vitamin detection, and more particularly to a method for detecting B vitamins. Background Technology

[0002] B vitamins are a class of water-soluble small molecule compounds, and common examples include vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid.

[0003] In the field of medical aesthetics, sodium hyaluronate is a good biocompatible polymer material and is very popular among medical aesthetic researchers. With people's continuous pursuit of beauty, B vitamins, as water-soluble vitamins that are essential for maintaining normal human functions and metabolic activities, are often used in combination with sodium hyaluronate in the field of medical aesthetics. For example, B vitamins can be added to a compound solution containing sodium hyaluronate.

[0004] However, current methods cannot simultaneously determine multiple B vitamins. Although existing technologies have reported or established standards for quality control of B vitamins, these methods can only determine a limited number of vitamins, or require multiple different methods for each vitamin to be tested separately. This makes it difficult to comprehensively reflect the quality of B vitamins in sodium hyaluronate composite solutions and thus hinders the simultaneous determination of multiple vitamins. Therefore, providing a method for determining vitamin B using high-performance liquid chromatography (HPLC) is particularly important. Summary of the Invention

[0005] To address the problems of existing technologies, such as the inability to simultaneously detect multiple B vitamins, complex pretreatment, insensitivity of mixed sample detection, and high consumption of detection reagents, this application provides a method for detecting B vitamins in sodium hyaluronate composite solution. The method of this application can completely separate 10 B vitamins and has specificity, repeatability, and accuracy, with a low detection limit.

[0006] The specific technical solution of this application is as follows:

[0007] 1. A method for detecting B vitamins, wherein the detection method includes the following steps:

[0008] Preparation of reference solutions: Dissolve the B vitamin reference standards separately to obtain reference solutions;

[0009] Preparation of the test solution: The test solution is obtained by diluting the sample solution containing B vitamins;

[0010] Determination: The content of B vitamins in samples containing B vitamins was determined by high performance liquid chromatography;

[0011] The high-performance liquid chromatography method employs gradient elution, which includes at least two sequential gradients, specifically a first gradient elution and a second gradient elution; the composition of the mobile phase in the gradient elution is as follows:

[0012] The volume ratio of mobile phase A to mobile phase B for the first gradient elution is (92-98):(8-2);

[0013] The volume ratio of mobile phase A to mobile phase B for the second gradient elution is (88-82):(12-18).

[0014] 2. According to the method described in item 1, wherein the elution times in the two gradients are respectively:

[0015] The first elution time is 6.8–8 minutes;

[0016] The second-gradient elution time is more than 16 minutes.

[0017] 3. The method according to item 1 or 2, wherein the chromatographic column of the high performance liquid chromatography is a cyano-bonded octadecylsilane-bonded silica column.

[0018] 4. The method according to any one of items 1-3, wherein the mobile phase A is an ion-pairing reagent solution with a concentration of 0.03-0.10 mol / L, and the pH is adjusted to 3-4 with phosphoric acid; the mobile phase B is acetonitrile.

[0019] 5. The method according to item 4, wherein the ion-pairing reagent is selected from tetrabutylammonium hydroxide, tetrabutylammonium bromide, dodecyltrimethylammonium chloride, sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, and sodium decanesulfonate.

[0020] 6. The method according to any one of items 1-5, wherein the flow rate of the mobile phase in the high performance liquid chromatography is 0.6 to 1.0 mL / min.

[0021] 7. The method according to any one of items 1 to 6, wherein the column temperature of the chromatographic column of the high performance liquid chromatogram is 30 to 40°C.

[0022] 8. The method according to any one of items 1 to 7, wherein the detection wavelength of the high performance liquid chromatography is 205 to 215 nm.

[0023] 9. The method according to any one of items 1 to 8, wherein the B vitamins include one or more of vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid.

[0024] 10. The method according to item 9, wherein the separation degree among vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin and pteroic acid is greater than 1.5.

[0025] The effects of the invention

[0026] (1) The detection method of this application can simultaneously detect multiple B vitamins, is simple to operate, and has high accuracy of the test results;

[0027] (2) The detection method of this application not only has high precision, but also good repeatability;

[0028] (3) The method of this application has broad application prospects and can provide reliable experimental basis for the development and quality detection of sodium hyaluronate composite solution;

[0029] (4) The method of this application does not require special treatment of the sample. One injection can simultaneously perform qualitative and quantitative analysis of the compounds in the sample. It is simple to operate and has wide applicability.

[0030] (5) The mobile phase of the high performance liquid chromatography used in this application further employs gradient elution, which can effectively separate VB1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, nicotinic acid and pteroic acid completely, with a resolution of more than 1.5 for each, and the separation effect is good. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the spectrum of the blank solvent.

[0032] Figure 2 This is a schematic diagram of the spectrum of VB1 reference standard.

[0033] Figure 3 This is a schematic diagram of the spectrum of riboflavin reference standard.

[0034] Figure 4 This is a schematic diagram of the spectrum of nicotinamide reference standard.

[0035] Figure 5 This is a schematic diagram of the spectrum of pantothenic acid reference standard.

[0036] Figure 6 This is a schematic diagram of the spectrum of VB6 reference standard.

[0037] Figure 7 This is a schematic diagram of the spectrum of biotin reference standard.

[0038] Figure 8 This is a schematic diagram of the spectrum of folic acid reference standard.

[0039] Figure 9 This is a schematic diagram of the spectrum of VB12 reference standard.

[0040] Figure 10 This is a schematic diagram of the chromatogram of nicotinic acid reference standard.

[0041] Figure 11 This is a schematic diagram of the spectral composition of pteroic acid reference standard.

[0042] Figure 12 This is a schematic diagram of the spectrum of the test solution. Detailed Implementation

[0043] The present application will now be described in detail with reference to specific embodiments. It should be understood that the present application can be implemented in various forms and should not be limited to the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art. It should be noted that the terms "comprising" or "including" as used throughout the specification and claims are open-ended terms and should be interpreted as "comprising but not limited to". The following descriptions in the specification are preferred embodiments for carrying out the present application; however, these descriptions are for the purpose of illustrating the general principles of the present application and are not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the appended claims.

[0044] Since sodium hyaluronate complex solution is a complex solution containing multiple B vitamins, conventional methods for detecting B vitamins are difficult to simultaneously detect the content of B vitamins in sodium hyaluronate complex solution. Therefore, this application provides a method for detecting B vitamins, which can be used to detect B vitamins in samples containing B vitamins, such as sodium hyaluronate complex solution. The method includes:

[0045] Preparation of reference solutions: Dissolve the B vitamin reference standards separately to obtain reference solutions;

[0046] Preparation of the test solution: The test solution is obtained by diluting the sample solution containing B vitamins;

[0047] Determination: The content of B vitamins in the sample solution containing B vitamins was determined by high performance liquid chromatography.

[0048] In this application, there are no restrictions on the preparation of the reference solution, which can be prepared according to conventional methods in the art. For example, each B vitamin can be dissolved in water to obtain the reference solution.

[0049] In this application, there are no restrictions on the preparation of the test solution. It can be prepared in accordance with conventional methods in the art. For example, a sample solution containing B vitamins can be diluted with water to obtain the test solution. Preferably, the filtrate obtained after dilution with water is used as the test solution.

[0050] In this application, the high-performance liquid chromatography (HPLC) method is developed based on gas chromatography and classical chromatography. There is no fundamental difference between modern HPLC and classical HPLC. The only difference is that modern HPLC has higher efficiency and achieves automated operation. In classical HPLC, the mobile phase is transported at atmospheric pressure, the stationary phase column has low efficiency, and the analysis cycle is long. Modern HPLC, however, adopts the theory of gas chromatography, and the mobile phase is transported under high pressure (the maximum transport pressure can reach 4.9 × 10⁻⁶). 7 High-performance liquid chromatography (HPLC) columns are packed with small-particle-size packing material using a special method, resulting in column efficiency significantly higher than classical liquid chromatography (tens of thousands or hundreds of thousands of plates per meter). Furthermore, a highly sensitive detector is connected downstream of the column for continuous monitoring of the eluent. Therefore, HPLC is characterized by its fast analysis speed, high separation efficiency, and automation. It is thus referred to as high-pressure, high-speed, high-efficiency, or modern liquid chromatography.

[0051] The high performance liquid chromatography method employs gradient elution, also known as gradient washing or programmed elution. Gradient elution involves continuously changing the concentration ratio of the mobile phase to a certain extent within the same analysis cycle.

[0052] In this application, the gradient elution includes at least two gradients performed sequentially, specifically a first gradient elution and a second gradient elution; the composition of the mobile phase in the gradient elution is as follows:

[0053] The volume ratio of mobile phase A to mobile phase B for the first gradient elution is (92-98):(8-2);

[0054] The volume ratio of mobile phase A to mobile phase B for the second gradient elution is (88-82):(12-18).

[0055] For example, the volume ratio of mobile phase A to mobile phase B for the first gradient elution is 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, etc.; the volume ratio of mobile phase A to mobile phase B for the second gradient elution is 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, etc.

[0056] In some embodiments, the elution times in the two gradients are respectively:

[0057] The first elution time is 6.8–8 minutes;

[0058] The second-gradient elution time is more than 16 minutes.

[0059] For example, the elution time of the first gradient is 6.8 min, 7.0 min, 7.2 min, 7.4 min, 7.6 min, 7.8 min, and 8.0 min; the elution time of the second gradient is 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 35 min, 40 min, 50 min, and 60 min, etc., preferably 16 to 30 min.

[0060] In some embodiments, the gradient elution process is as follows: during the first gradient elution, the mobile phase consists of mobile phase A and mobile phase B; subsequently, the volume fraction of mobile phase A decreases and the volume fraction of mobile phase B increases; during the second gradient elution, the volume fractions of mobile phase A and mobile phase B remain unchanged.

[0061] After the two gradient elutions described above are completed, other elution processes may be included, such as processes for eluting impurities from the chromatographic column and / or equilibrating the column. For example, a third gradient elution may also be included, in which the volume ratio of mobile phase A to mobile phase B is (92-98):(8-2), and the elution time is 0.1 min or more, which can be 0.1 min, 0.5 min, 1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, etc., preferably 5-30 min.

[0062] In some embodiments, the gradient elution process is as follows: during the first gradient elution, the mobile phase consists of mobile phase A and mobile phase B; subsequently, the volume fraction of mobile phase A decreases and the volume fraction of mobile phase B increases; during the second gradient elution, the volume fractions of mobile phase A and mobile phase B remain unchanged; subsequently, the volume fraction of mobile phase A increases and the volume fraction of mobile phase B decreases; during the third gradient elution, the volume fractions of mobile phase A and mobile phase B remain unchanged.

[0063] In some preferred embodiments, the gradient elution process is as follows:

[0064] From 0 min to (6.8–8) min, the volume fraction of mobile phase A was 92–98%, and the volume fraction of mobile phase B was 8–2%.

[0065] Subsequently, the volume fraction of mobile phase A decreased from 92-98% to 88-82%, while the volume fraction of mobile phase B increased from 8-2% to 12-18%.

[0066] From (6.9–8.1) min to (22.9–38.1) min, the volume fraction of mobile phase A was 88–82%, and the volume fraction of mobile phase B was 12–18%.

[0067] Subsequently, the volume fraction of mobile phase A increased from 88-82% to 92-98%, while the volume fraction of mobile phase B decreased from 12-18% to 8-2%.

[0068] From (23 to 38.2) min to (28 to 68.2) min, the volume fraction of mobile phase A was 92% to 98%, and the volume fraction of mobile phase B was 8% to 2%.

[0069] In some preferred embodiments, the gradient elution process is as follows:

[0070] From 0 to 8 minutes, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%.

[0071] Between 8 and 8.1 minutes, the volume fraction of mobile phase A decreased from 95% to 85%, while the volume fraction of mobile phase B increased from 5% to 15%.

[0072] From 8.1 to 26 minutes, the volume fraction of mobile phase A was 85%, and the volume fraction of mobile phase B was 15%.

[0073] From 26 to 26.1 min, the volume fraction of mobile phase A increased from 85% to 95%, while the volume fraction of mobile phase B decreased from 15% to 5%.

[0074] From 26.1 to 40 minutes, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%.

[0075] The gradient elution process described in this application enables the various B vitamins in the sodium hyaluronate composite solution to achieve good separation according to their respective suitable capacity factors k, thereby improving the accuracy of detection.

[0076] The high-performance liquid chromatography column is a cyano-bonded octadecylsilane-bonded silica column, including but not limited to ZORBX SB-CN columns and ZORBAX Eclipse XDB-CN columns. For example, it can be an Agilent ZORBX SB-CN (4.6x250mm, 5μm) column or an Agilent ZORBAX Eclipse XDB-CN (4.6x250mm, 5μm) column.

[0077] The mobile phase A is an ion-pairing reagent solution with a concentration of 0.03–0.10 mol / L, and the pH is adjusted to 3–4 with phosphoric acid; the mobile phase B is acetonitrile.

[0078] The ion-pairing reagent is selected from tetrabutylammonium hydroxide, tetrabutylammonium bromide, dodecyltrimethylammonium chloride, sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, and sodium decanesulfonate, with sodium octanesulfonate being preferred.

[0079] In some embodiments, mobile phase A is a sodium octane sulfonate solution, and mobile phase B is acetonitrile; preferably, the concentration of the sodium octane sulfonate solution is 0.03–0.10 mol / L, and the pH is adjusted to 3–4 with phosphoric acid; more preferably, it is a sodium octane sulfonate solution of 0.03–0.07 mol / L, with the pH adjusted to 3–4 with phosphoric acid; even more preferably, it is a sodium octane sulfonate solution of 0.05 mol / L, with the pH adjusted to 3–4 with phosphoric acid. For example, mobile phase A can be a sodium octane sulfonate solution of 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or 0.10 mol / L, with the pH adjusted to 3–4 with phosphoric acid.

[0080] In some embodiments, the flow rate of the mobile phase in the high-performance liquid chromatography is 0.6 to 1.0 mL / min, for example, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, etc.

[0081] In some embodiments, the column temperature of the high-performance liquid chromatography column is 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0082] In some embodiments, the injection volume of the chromatographic column of the high-performance liquid chromatograph is 5 to 40 μL, for example, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, etc., preferably 10 to 30 μL.

[0083] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 205-215 nm, for example, 205 nm, 207 nm, 210 nm, 212 nm, 215 nm, etc.

[0084] In some embodiments, the B vitamins include vitamin B1, riboflavin, niacinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid. In some embodiments, the B vitamins are one or more of vitamin B1, riboflavin, niacinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid.

[0085] In some embodiments, the separation degree between vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid is greater than 1.5.

[0086] In some embodiments, the external standard method is used to determine the content of B vitamins. In this application, the external standard method is one of the commonly used methods in instrumental analysis and is a type of comparative method. Compared with the internal standard method, the external standard method does not add the standard substance to the sample being tested; instead, it measures the sample separately under the same chromatographic conditions as the sample being tested, and compares the obtained chromatographic peak area with the chromatographic peak area of ​​the analyte to determine the content of the analyte. The external standard and the analyte are the same substance, but it is required to have a certain purity. During analysis, the concentration of the external standard should be close to the concentration of the analyte to facilitate the accuracy of quantitative analysis.

[0087] In some embodiments, the detection limits for the B vitamins are much lower than the content of B vitamins in the sample solution.

[0088] The method described in this application has high separation efficiency, which can completely separate vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin and pteroic acid. It also has good repeatability, high accuracy, simple operation and no need for sample pretreatment.

[0089] Example

[0090] This application provides a general and / or specific description of the materials and methods used in the experiments. In the following examples, reagents or instruments whose manufacturers are not specified are all commercially available reagents or instruments.

[0091] The effectiveness of each embodiment was compared by measuring the detection limit of the results.

[0092] Example 1

[0093] 1. Preparation of the reference solution:

[0094] Accurately weigh the reference standards for vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid. Dissolve and quantitatively dilute them in 0.05 mol / L sodium octanesulfonate solution (adjusted to pH 3.5 with concentrated phosphoric acid) to prepare solutions containing 1.0 μg vitamin B1, 1.0 μg riboflavin, 3.0 μg nicotinamide, 1.0 μg pantothenic acid, 0.5 μg vitamin B6, 0.5 μg biotin, 0.1 μg folic acid, 0.4 μg vitamin B12, 0.05 μg niacin, and 0.05 μg pteroic acid per mL. Shake well and set aside.

[0095] 2. Test solution:

[0096] Accurately measure 1 mL of sodium hyaluronate composite solution (batch number: 20230113, Bloomage Biotechnology Co., Ltd.), place it in a 10 mL volumetric flask, add 0.05 mol / L sodium octanesulfonate solution (adjust pH to 3.5 with concentrated phosphoric acid) to quantitatively dilute to the mark, and shake well; prepare two parallel portions.

[0097] 3. Blank solution: Use 0.05 mol / L sodium octane sulfonate solution (adjust pH to 3.5 with concentrated phosphoric acid) as the blank solution.

[0098] 4. Chromatographic conditions are as follows:

[0099] Agilent ZORBX SB-CN column, Agilent, 4.6x250mm, 5μm;

[0100] Flow rate: 0.8 mL / min;

[0101] Column temperature 35℃;

[0102] Detection wavelength: 210nm;

[0103] Mobile phase: Mobile phase A (0.05 mol / L sodium octane sulfonate phosphoric acid solution, pH adjusted to 3.5 with concentrated phosphoric acid), mobile phase B (acetonitrile), gradient elution program as follows:

[0104] From 0 to 8 min, the volume fraction of mobile phase A was 95% and that of mobile phase B was 5%; from 8 to 8.1 min, the volume fraction of mobile phase A decreased from 95% to 85%, and that of mobile phase B increased from 5% to 15%; from 8.1 to 26 min, the volume fraction of mobile phase A was 85% and that of mobile phase B was 15%; from 26 to 26.1 min, the volume fraction of mobile phase A increased from 85% to 95%, and that of mobile phase B decreased from 15% to 5%; from 26.1 to 40 min, the volume fraction of mobile phase A was 95% and that of mobile phase B was 5%.

[0105] Injection volume: 20 μL.

[0106] 5. Content determination

[0107] The blank solution, reference solution, and test solution were injected into the sodium hyaluronate composite solution. The contents of vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid in the solution were determined using the external standard method. The spectra of the blank solvent, reference solution, and test solution are shown below. Figures 1-12 As shown.

[0108] The content is determined using the following formula:

[0109] The formulas for calculating the content of vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid in the test sample are as follows:

[0110]

[0111] Note: P i ---The content of each B vitamin in the test sample, μg / mL;

[0112] C i ---Content of each B vitamin in the reference solution, μg / mL;

[0113] A i ---Peak areas of each B vitamin in the test solution;

[0114] A r ---Peak areas of each B vitamin in the reference solution;

[0115] Z---Purity of each B vitamin reference standard. (The purities of vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid are 97.9%, 98.2%, 98.5%, 99.0%, 99.9%, 99.5%, 89.3%, 90.5%, 99.9%, and 99.9%, respectively.)

[0116] According to the test results, Example 1 can completely separate each B vitamin in the sodium hyaluronate composite solution (batch number: 20230113), with a separation degree greater than 1.5. The content results are shown in Table 1 below.

[0117] Table 1

[0118]

[0119]

[0120] Example 2

[0121] The difference between this embodiment and Example 1 is that the concentration of mobile phase A is 0.07 mol / L. The content of B vitamins is shown in Table 2. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0122] Table 2

[0123] VB1 143.56 141.53 9.94 0.18 Riboflavin 122.89 126.37 10.01 0.32 Niacinamide 239.23 234.54 29.48 0.49 pantothenic acid 147.32 148.24 10.16 0.22 VB6 58.45 57.33 4.57 0.31 Biotin 88.38 88.28 4.71 0.04 folic acid 16.44 16.32 0.89 0.03 VB12 77.85 77.22 3.84 0.03 niacin 7.32 7.46 0.28 0.004 Pteric acid 6.47 6.83 0.26 0.006

[0124] Example 3

[0125] The difference between this embodiment and Example 1 is that the concentration of mobile phase A is 0.03 mol / L. The content of B vitamins is shown in Table 3. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0126] Table 3

[0127] VB1 140.32 139.68 9.76 0.21 Riboflavin 128.91 131.46 10.46 0.29 Niacinamide 242.54 241.42 30.11 0.56 pantothenic acid 142.53 149.52 10.04 0.17 VB6 62.67 62.58 4.94 0.37 Biotin 88.33 89.68 4.74 0.06 folic acid 17.57 17.28 0.95 0.03 VB12 81.34 79.64 3.98 0.03 niacin 7.30 6.95 0.27 0.006 Pteric acid 6.84 6.42 0.26 0.004

[0128] Example 4

[0129] The difference between this embodiment and Example 1 is that the concentration of mobile phase A is 0.10 mol / L. The content of B vitamins is shown in Table 4. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0130] Table 4

[0131] VB1 126.64 127.64 9.87 0.20 Riboflavin 122.78 123.67 9.90 0.23 Niacinamide 220.54 218.68 27.33 0.53 pantothenic acid 142.89 146.65 9.95 0.33 VB6 60.77 59.45 4.74 0.34 Biotin 85.33 87.68 4.61 0.06 folic acid 16.54 16.85 0.91 0.09 VB12 76.98 77.66 3.83 0.04 niacin 6.56 6.62 0.25 0.008 Pteric acid 6.44 6.47 0.25 0.008

[0132] Example 5

[0133] The difference between this embodiment and Example 1 is that the mobile phase flow rate was 0.7 mL / min. The content of B vitamins was measured as shown in Table 5. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0134] Table 5

[0135] VB1 139.55 136.39 9.62 0.18 Riboflavin 128.75 127.76 10.30 0.27 Niacinamide 232.57 234.67 29.07 0.49 pantothenic acid 146.72 147.24 10.10 0.22 VB6 63.65 61.67 4.94 0.28 Biotin 88.89 91.03 4.80 0.06 folic acid 18.27 19.87 1.04 0.03 VB12 76.79 79.68 3.87 0.04 niacin 7.85 7.78 0.29 0.002 Pteric acid 6.52 6.54 0.26 0.004

[0136] Example 6

[0137] The difference between this embodiment and Example 1 is that the mobile phase flow rate was 0.9 mL / min. The content of B vitamins was measured as shown in Table 6. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0138] Table 6

[0139]

[0140]

[0141] Example 7

[0142] The difference between this embodiment and Example 1 is that the mobile phase flow rate was 1.0 mL / min. The content of B vitamins was measured as shown in Table 7. The complete separation of each B vitamin was achieved, with a resolution greater than 1.5.

[0143] Table 7

[0144] VB1 123.5 125.63 9.69 0.35 Riboflavin 119.45 116.42 9.47 0.32 Niacinamide 231.11 228.27 28.58 0.54 pantothenic acid 147.42 145.78 10.08 0.28 VB6 51.89 52.11 4.50 0.26 Biotin 67.32 65.98 4.55 0.07 folic acid 15.77 15.89 0.86 0.02 VB12 75.93 77.16 3.79 0.06 niacin 6.65 6.75 0.25 0.007 Pteric acid 5.45 5.53 0.22 0.003

[0145] Example 8

[0146] The difference between this embodiment and Example 1 is that the column temperature was 30℃. The content of B vitamins was determined as shown in Table 8. All B vitamins were completely separated, with a resolution greater than 1.5.

[0147] Table 8

[0148] VB1 136.56 141.78 9.71 0.19 Riboflavin 127.87 126.39 10.21 0.27 Niacinamide 235.91 237.38 29.45 0.58 pantothenic acid 150.22 148.38 10.26 0.17 VB6 61.37 64.82 4.98 0.31 Biotin 91.05 93.37 4.92 0.02 folic acid 17.06 17.94 0.95 0.07 VB12 78.05 79.03 3.89 0.03 niacin 6.73 7.54 0.27 0.006 Pteric acid 6.64 6.71 0.26 0.002

[0149] Example 9

[0150] The difference between this embodiment and Example 1 is that the column temperature was 40℃. The content of B vitamins was determined as shown in Table 9. All B vitamins were completely separated, with a resolution greater than 1.5.

[0151] Table 9

[0152] VB1 138.11 141.6 9.75 0.16 Riboflavin 131.09 130.22 10.49 0.24 Niacinamide 243.81 239.72 30.08 0.61 pantothenic acid 151.24 149.43 10.33 0.16 VB6 63.03 61.92 4.93 0.37 Biotin 93.89 94.39 5.02 0.03 folic acid 16.32 16.78 0.90 0.05 VB12 76.55 79.16 3.85 0.03 niacin 6.79 6.71 0.25 0.007 Pteric acid 6.59 6.62 0.26 0.003

[0153] Example 10

[0154] The difference between this embodiment and Example 1 is that the injection volume is 10 μL. The content of B vitamins is shown in Table 10. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0155] Table 10

[0156] VB1 71.43 75.48 10.24 0.16 Riboflavin 64.95 63.37 10.31 0.24 Niacinamide 121.49 119.42 29.98 0.49 pantothenic acid 73.62 76.35 10.31 0.17 VB6 31.57 30.63 4.91 0.24 Biotin 47.66 46.17 5.00 0.04 folic acid 8.90 9.23 0.99 0.02 VB12 39.67 40.51 3.97 0.01 niacin 4.01 4.23 0.31 0.002 Pteric acid 3.82 3.79 0.30 0.003

[0157] Example 11

[0158] The difference between this embodiment and Example 1 is that the injection volume is 30 μL. The content of B vitamins determined is shown in Table 11. The B vitamins were completely separated, and the resolution was greater than 1.5.

[0159] Table 11

[0160] VB1 215.43 216.58 10.04 0.17 Riboflavin 204.11 203.29 10.91 0.25 Niacinamide 366.27 359.01 30.08 0.44 pantothenic acid 224.29 222.97 10.25 0.19 VB6 95.53 91.17 4.91 0.22 Biotin 142.98 140.27 5.03 0.05 folic acid 29.13 28.35 1.04 0.04 VB12 121.37 121.53 4.01 0.01 niacin 11.42 12.36 0.30 0.004 Pteric acid 10.49 10.51 0.28 0.006

[0161] Example 12

[0162] The difference between this embodiment and Example 1 is that the chromatographic column used was an Agilent ZORBAX Eclipse XDB-CN, 4.6 x 250 mm, 5 μm. The content of B vitamins was determined as shown in Table 12, and each B vitamin was completely separated with a resolution greater than 1.5.

[0163] Table 12

[0164] VB1 123.43 124.71 9.65 0.21 Riboflavin 114.73 115.26 9.94 0.43 Niacinamide 198.69 196.4 29.58 0.79 pantothenic acid 125.69 126.32 9.66 0.29 VB6 54.25 54.67 4.89 0.41 Biotin 82.76 84.6 4.96 0.08 folic acid 17.54 17.11 0.94 0.07 VB12 71.54 72.54 3.87 0.04 niacin 4.33 4.56 0.28 0.005 Pteric acid 5.12 5.23 0.27 0.007

[0165] Comparative Example 1

[0166] The difference between this comparative example and Example 1 is that the elution program of the mobile phase was isocratic elution, and the ratio of mobile phase A to mobile phase B was 85:15. The content of B vitamins was determined as shown in Table 12. Among them, VB1, riboflavin, pantothenic acid, and niacin were not separated, and the resolution of biotin was less than 1.5.

[0167] Table 13

[0168] VB1 0 0 0.00 0 —— Riboflavin 0 0 0.00 0 —— Niacinamide 232.7 229.7 28.77 0.76 2.1 pantothenic acid 142.55 138.28 9.65 0.27 —— VB6 56.47 61.43 4.65 0.38 2.4 Biotin 92.11 95.43 5.00 0.07 1.3 folic acid 16.36 15.99 0.88 0.03 1.6 VB12 76.44 77.95 3.82 0.05 4.7 niacin 0 0 0.00 0 —— Pteric acid 5.61 6.37 0.24 0.006 4.3

[0169] Comparative Example 2

[0170] The difference between this comparative example and Example 1 is that the chromatographic column used in this example is a non-cyano-bonded octadecylsilane-bonded silica column, Agilent TC-C18(2), 4.6 x 250 mm, 5 μm. The contents of B vitamins were determined as shown in Table 14. Among them, VB1, pantothenic acid, VB6, VB12, and niacin were not separated, and the resolution of nicotinamide and biotin was less than 1.5.

[0171] Table 14

[0172] VB1 0.00 0.00 0.00 0 —— Riboflavin 14.66 113.32 9.74 0.49 2.3 Niacinamide 165.35 167.98 29.74 0.57 1.3 pantothenic acid 100.65 98.54 9.85 0.36 —— VB6 0.00 0.00 0.00 0 —— Biotin 82.76 84.6 4.86 0.06 1.4 folic acid 16.46 17.22 0.92 0.11 3.5 VB12 0.00 0.00 0.00 0 —— niacin 0.00 0.00 0.00 0 —— Pteric acid 4.89 5.11 0.27 0.015 5.5

[0173] The experimental parameters for the above embodiments are summarized in Table 16 below.

[0174] Table 16

[0175]

[0176] Linear relationship between peak area and concentration of each B vitamin in Experiment Example 1

[0177] The reference solutions from Example 1 were diluted with 0.05 mol / L sodium octane sulfonate solution to the linear gradient ranges shown in Table 17. The linear equations for each analyte were obtained by plotting the peak area (Y) of the reference standard on the ordinate and the concentration (X) of the reference standard on the abscissa. The results are shown in Table 17. The results show that there is a good linear relationship between the peak area and concentration of VB1, riboflavin, nicotinamide, pantothenic acid, VB6, biotin, folic acid, VB12, nicotinic acid, and pteroic acid, with correlation coefficients r greater than 0.99.

[0178] Table 17

[0179] VB1 Y = 141.62X + 1.5889 0.1～2 0.997 Riboflavin Y = 135.48X - 0.3861 0.1～2 0.998 Niacinamide Y = 86.93X - 9.01 1～5 0.994 pantothenic acid Y = 146.12X + 1.8763 0.5～2 0.995 VB6 Y = 111.14X + 3.5268 0.1～1 0.992 Biotin Y = 145.4X + 0.7976 0.1～1 0.997 folic acid Y = 180.51X + 0.0679 0.01～0.2 0.998 VB12 Y = 200.62X + 1.078 0.1～1 0.994 niacin Y = 264.96X + 0.078 0.01～0.1 0.994 Pteric acid Y = 265.38X - 0.3 0.01～0.1 0.994

[0180] As can be seen from Table 17, using the detection method of this application, the correlation coefficient r of the linear equation for each B vitamin is greater than 0.99, indicating that the linear relationship between the peak area and concentration of each B vitamin is good.

[0181] Test Example 2: Limit of Detection and Limit of Quantification

[0182] The detection limit was set at a signal-to-noise ratio (S / N) of 3, and the quantitation limit was set at a S / N of 10. The experiment was conducted using the method of Example 1, and the results are shown in Table 8.

[0183] Table 18

[0184] VB1 2.73 0.15 9.12 0.46 Riboflavin 3.09 0.24 9.94 0.75 Niacinamide 3.74 0.46 8.92 1.36 pantothenic acid 2.89 0.14 9.76 0.53 VB6 3.02 0.27 10.23 0.83 Biotin 3.21 0.02 9.34 0.06 folic acid 2.92 0.02 10.28 0.06 VB12 3.11 0.01 10.65 0.03 niacin 2.94 0.002 9.55 0.006 Pteric acid 2.97 0.001 10.69 0.004

[0185] As can be seen from Table 18, using the detection method of this application, the detection limit and quantitation limit of B vitamins are much lower than the content of each B vitamin in the sample, indicating that the detection method of this application has high sensitivity.

[0186] Experiment Example 3 Recovery Test

[0187] Following the sample processing method and testing conditions as in Example 1, nine samples from the same batch as in Example 1 were prepared in parallel. Three samples from each batch were taken out, and the reference solution was added at 80%, 100%, and 120% of the corresponding contents of VB1, riboflavin, nicotinamide, pantothenic acid, VB6, biotin, folic acid, VB12, nicotinic acid, and pteroic acid in the sample. The results are shown in Table 19.

[0188] Table 19

[0189]

[0190] As can be seen from Table 19, the spiked recovery rate of vitamin B standards using the detection method of this application is between 91% and 102%, indicating that the detection method of this application has high accuracy.

[0191] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A method for detecting B vitamins, wherein, The detection method includes the following steps: Preparation of reference solutions: Dissolve the B vitamin reference standards separately to obtain reference solutions; Preparation of the test solution: The test solution is obtained by diluting the sample solution containing B vitamins; Determination: The content of B vitamins in samples containing B vitamins was determined by high performance liquid chromatography; The high-performance liquid chromatography method employs gradient elution, which includes at least two sequential gradients, specifically a first gradient elution and a second gradient elution; the composition of the mobile phase in the gradient elution is as follows: The volume ratio of mobile phase A to mobile phase B for the first gradient elution is (92-98):(8-2); The volume ratio of mobile phase A to mobile phase B for the second gradient elution is (88-82):(12-18); The high-performance liquid chromatography column is a cyano-bonded octadecylsilane-bonded silica column; The mobile phase A is an ion-pairing reagent solution with a concentration of 0.03~0.10 mol / L; The flow rate of the mobile phase in the high-performance liquid chromatography is 0.6~1.0 mL / min.

2. The method according to claim 1, wherein, The elution times in the two gradients are as follows: The first elution time is 6.8–8 minutes; The second-gradient elution time is more than 16 minutes.

3. The method according to claim 1 or 2, wherein, The pH of the ion-pairing reagent solution was adjusted to 3-4 with phosphoric acid; mobile phase B was acetonitrile.

4. The method according to claim 3, wherein, The ion-pairing reagents are selected from tetrabutylammonium hydroxide, tetrabutylammonium bromide, dodecyltrimethylammonium chloride, sodium pentanesulfonate, sodium hexanesulfonate, sodium heptanesulfonate, sodium octanesulfonate, and sodium decanesulfonate.

5. The method according to claim 1 or 2, wherein, The column temperature of the high-performance liquid chromatography column is 30~40℃.

6. The method according to claim 1 or 2, wherein, The detection wavelength of the high-performance liquid chromatography is 205~215nm.

7. The method according to claim 1 or 2, wherein, The B vitamins include one or more of the following: vitamin B1, riboflavin, niacinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid.

8. The method according to claim 7, wherein, The separation degree between the vitamin B1, riboflavin, nicotinamide, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, niacin, and pteroic acid is greater than 1.5.

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