Method for determining content of Vc sodium phosphate

Abstract

The invention discloses a method for determining the content of Vc sodium phosphate, and belongs to the field of analytical chemistry. The determination method comprises the following steps: carrying out HPLC (High Performance Liquid Chromatography) detection by using a sulfonated polystyrene-divinylbenzene hydrogen type ion exchange chromatographic column and taking a 0.01-0.1 mol / L sulfuric acid solution as a mobile phase, and calculating the contents of the Vc sodium phosphate and the intermediate thereof through a peak area. According to the method, the hydrogen ion exchange column is used for detecting the Vc sodium phosphate for the first time, the combination of the chromatographic column and the mobile phase is optimized, high-precision detection with RSD smaller than or equal to 0.5% is achieved, and data reliability is improved. According to the method, through innovative chromatographic condition design, the bottlenecks in the prior art are broken through in detection sensitivity, operation stability and application range, and an efficient and environment-friendly integrated solution is provided for quality control of the whole production process of the Vc sodium phosphate.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and relates to a method for the determination of the content of sodium ascorbyl phosphate, specifically to a high performance liquid chromatography (HPLC) method based on a sulfonated polystyrene-divinylbenzene hydrogen ion exchange column for the determination of the content of vitamin C derivative - sodium ascorbyl phosphate (SAP) and its key intermediates (ascorbic acid-2-phosphate, ascorbic acid-2-phosphate tricyclohexylammonium salt). Background Art

[0002] Vitamin C (Vc), as an important water-soluble antioxidant, is widely used in the fields of medicine, food, cosmetics and animal feed. However, the strong reducibility of Vc makes it extremely easy to be oxidized and inactivated, especially with extremely poor stability under conditions of high temperature, light or the presence of metal ions. To overcome this defect, the development of Vc derivatives has become a research hotspot, among which sodium ascorbyl phosphate (SAP) stands out due to its high stability and bioavailability. SAP slowly releases Vc through enzymatic hydrolysis in vivo, and can maintain the concentration of vitamin C in the blood for a long time, and is widely used in injections, oral preparations and functional foods.

[0003] The synthesis process of sodium ascorbyl phosphate (as shown in Figure 1 ) generally consists of three steps: (1) Phosphorylation reaction: L-ascorbic acid reacts with a phosphorylation reagent (such as POCl3) to generate intermediate I (ascorbic acid-2-phosphate, in free acid form); (2) Amination and neutralization: Intermediate I reacts with tricyclohexylamine to generate intermediate II (ascorbic acid-2-phosphate tricyclohexylammonium salt) to improve its water solubility; (3) Ion replacement: Intermediate II is converted into the final product SAP (in sodium salt form) through a sodium-type cation exchange resin.

[0004] Quality control challenges in the production process: (1) The yield of the phosphorylation reaction is significantly affected by temperature, reagent ratio and stirring efficiency, and the concentration of intermediate I may fluctuate violently from 0.1 mg / mL to 5.0 mg / mL; (2) Unreacted phosphorylation reagents, by-products (such as pyrophosphates) and organic amine residues need to be accurately monitored; (3) The sodium salt form of SAP is prone to moisture absorption and may degrade during storage, and rapid and accurate content detection means are required.

[0005] At present, the detection of sodium ascorbyl phosphate mainly relies on the HPLC method based on a C18 reversed-phase chromatographic column. Traditional C18 reversed-phase chromatographic columns (such as Agilent ZORBAX Eclipse XDB-C18) require the use of organic solvents (such as methanol) and buffer salts (such as tetrabutylammonium hydrogen sulfate), resulting in complex mobile phase preparation, high cost, and poor environmental friendliness. Moreover, the existing method has a narrow linear range (0.025–1.2 mg / mL), and there is room for optimization in terms of precision (RSD≤0.68%) and recovery rate (95.5–102.5%).

[0006] Although sulfonated polystyrene-divinylbenzene hydrogen ion exchange columns have been widely used in the detection of organic acids, sugars, and alcohols (such as lactic acid, glucose, etc.), their application in the analysis of sodium ascorbyl phosphate and its derivatives has not been reported. The traditional concept holds that ion exchange columns have a weak retention ability for polar substances and require a mobile phase with high ionic strength, which may have a negative impact on detection sensitivity. However, through systematic experiments, the present inventors have discovered the unique advantages of hydrogen ion exchange columns: in a 0.05 N sulfuric acid mobile phase, the sulfonic acid groups have a strong ion exchange interaction with the phosphate ions of sodium ascorbyl phosphate, significantly enhancing the retention ability; the single aqueous mobile phase system avoids the use of organic solvents, reduces baseline noise (drift < 0.005 AU / 30 min), and simultaneously eliminates the risk of buffer salt precipitation.

[0007] Based on the above background, the present invention aims to provide a method for determining the content of sodium ascorbyl phosphate that is simple to operate, environmentally friendly, has a wide linear range, and high precision, and is suitable for quality control in industrial production. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for determining the content of sodium ascorbyl phosphate, which is particularly suitable for quality control of intermediates and finished products in the industrial production process of sodium ascorbyl phosphate. Through innovative chromatographic condition design and a simplified mobile phase system, the technical bottleneck of the traditional C18 reversed-phase chromatography method is broken through. The specific technical solutions include the following core elements: 1. Chromatographic column selection Use a sulfonated polystyrene-divinylbenzene hydrogen ion exchange column for HPLC detection with an aqueous sulfuric acid solution as the mobile phase.

[0009] Preferably, the model of the chromatographic column is CONCISE Corel 87H3 ICE-99-9861 (250 mm×4.6mm, 5 μm).

[0010] 2 Mobile phase preparation The mobile phase is a 0.04–0.06 mol / L sulfuric acid solution.

[0011] Preferably, the sulfuric acid concentration of the mobile phase is 0.05 mol / L.

[0012] Preferably, the mobile phase is prepared by measuring 2.7 mL of concentrated sulfuric acid, slowly adding it to 800 mL of HPLC-grade pure water to make up to 1 L; filtering through a 0.45 μm filter membrane and ultrasonically degassing for 10 min.

[0013] 3. Setting of chromatographic conditions HPLC detection was performed under the following conditions: column temperature 20–40°C; flow rate 0.5–1.5 mL / min; detection wavelength 250 ± 2 nm; injection volume 1–10 μL, and run time 10–20 min.

[0014] Preferably, the sample is injected and detected at a column temperature of 35° C., a flow rate of 0.5 mL / min, a detection wavelength of 250 nm, and a running time of 15 min.

[0015] 4. Standard and Sample Preparation The detection objects include any one of the following: Vc sodium phosphate finished product, intermediate I (ascorbic acid-2-phosphate), intermediate II (ascorbic acid-2-phosphate tricyclohexyl ammonium salt).

[0016] Preferably, the sample preparation method is as follows: Vc sodium phosphate finished product solution: weigh 50.0 mg of pure water and dilute to 50 mL; intermediate I (ascorbic acid-2-phosphate) solution: absorb 0.5 mL of pure water and dilute to 50 mL; intermediate II (ascorbic acid-2-phosphate tricyclohexylammonium salt) solution: weigh 50.0 mg of pure water and dilute to 50 mL.

[0017] Preferably, all solutions are filtered through a 0.45 μm filter membrane, and 1 mL of the initial filtrate is discarded to eliminate particulate interference.

[0018] 5. Quantitative calculation and verification The content was calculated according to the formula based on the peak area, and the linearity, precision and accuracy of the method were verified.

[0019] Preferably, the calculation formula is: Preferably, when detecting Vc sodium phosphate (molecular weight 319.10), the conversion factor is 319.10 / 553.63; when detecting intermediate I (molecular weight 256.10), the conversion factor is 256.10 / 553.63; when detecting intermediate II (molecular weight 553.63), the conversion factor is 1.0.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly: The method for determining the content of sodium ascorbyl phosphate provided by the present invention uses a hydrogen-type ion exchange column for the detection of sodium ascorbyl phosphate for the first time. It adopts a single sulfuric acid aqueous solution mobile phase, eliminating the organic solvent and pH adjustment steps, simplifying the operation process, reducing the consumption of methanol at the same time, and reducing the burden of waste liquid treatment. Moreover, by optimizing the combination of the chromatographic column and the mobile phase, high-precision detection with RSD ≤ 0.5% is achieved, improving the reliability of the data. Through innovative chromatographic condition design, the present invention breaks through the bottlenecks of the existing technology in terms of detection sensitivity, operation stability and application range, providing an efficient and environmentally friendly integrated solution for the quality control of the entire production process of sodium ascorbyl phosphate. Description of the Drawings

[0021] Figure 1 It is the chromatogram of the reference substance (L-ascorbic acid-2-monophosphate tricyclohexylammonium salt) based on the hydrogen-type ion exchange column; Figure 2 It is the chromatogram of the finished product of sodium ascorbyl phosphate based on the hydrogen-type ion exchange column, corresponding to Example 1 of the present invention; Figure 3 It is the chromatogram of Intermediate I (ascorbic acid-2-phosphate) based on the hydrogen-type ion exchange column, corresponding to Example 2 of the present invention; Figure 4 The chromatogram of Intermediate II (ascorbic acid-2-phosphate tricyclohexylammonium salt) based on the hydrogen-type ion exchange column, corresponding to Example 3 of the present invention; Figure 5 It is the chromatogram of the reference substance based on the C18 reversed-phase column; Figure 6 It is the chromatogram of the finished product of sodium ascorbyl phosphate based on the C18 reversed-phase column; Figure 7 It is the chromatogram of Intermediate I based on the C18 reversed-phase column; Figure 8 It is the chromatogram of Intermediate II based on the C18 reversed-phase column. Detailed Embodiments

[0022] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are all conventional products obtained through commercial channels without special instructions.

[0023] Table 1 Instruments and Reagents Example 1 Determination of the Content of the Finished Product of Sodium Ascorbyl Phosphate 1. Preparation of the mobile phase: Use a graduated cylinder to measure 2.7 mL of 98% concentrated sulfuric acid, add approximately 800 mL of HPLC-grade pure water, stir to dissolve, and then make up the volume to 1 L (corresponding to 0.05 mol / L H2SO4). Filter through a 0.45 μm filter membrane and degas by ultrasonic treatment for 10 min (ultrasonic instrument model: Branson3800).

[0024] 2. Pretreatment of the chromatographic column: Take a sulfonated polystyrene-divinylbenzene hydrogen-type ion exchange column. When the new column is used for the first time, flush it forward with pure water (flow rate 0.5 mL / min) for 15 min and then flush it backward for 15 min; then switch to a 0.05 mol / L sulfuric acid mobile phase and equilibrate at the same flow rate for 30 min (about twice the column volume).

[0025] 3. Sample preparation Reference solution: Weigh 25.0 mg of L-ascorbic acid-2-monophosphate tricyclohexylammonium salt reference substance, place it in a 25 mL volumetric flask, add HPLC-grade pure water to dissolve and make up the volume to the mark. After shaking well, filter through a 0.45 μm filter membrane to obtain a 1.0 mg / mL reference solution.

[0026] Sample solution: Weigh 50.0 mg of the finished product of sodium ascorbyl phosphate, place it in a 50 mL volumetric flask, add HPLC-grade pure water to dissolve and make up the volume to the mark. After shaking well, filter and discard the first 1 mL of the filtrate, and collect the subsequent filtrate for testing.

[0027] 4. HPLC detection conditions Chromatographic column: CONCISE Corel 87H3 ICE-99-9861; Mobile phase: 0.05 mol / L sulfuric acid solution; Flow rate: 0.5 mL / min; Column temperature: 35°C; Detection wavelength: 250 nm; Injection volume: 10 μL; Running time: 15 min.

[0028] 5. Detection procedure System equilibration: Start the HPLC system and continuously pump the mobile phase at a flow rate of 0.5 mL / min. Start injecting samples after the baseline drift is less than 0.005 AU / 30 min.

[0029] Injection sequence: Inject 10 μL of the blank solution (pure water) to confirm no interfering peaks; Inject the reference solution continuously 3 times, record the retention time of the main peak (t0 = 3.8 min) and the average peak area (A0 = 15230); Inject the sample solution, and insert the reference solution every 5 samples to monitor the system stability.

[0030] 6. Result calculation According to the formula: Where: A0 is the peak area of the reference solution; A is the peak area of the sample solution; C0 is the concentration of the reference solution, mg / ml; C is the prepared concentration of the sample solution, mg / ml; 319.10 is the molecular weight of Vc sodium phosphate; 553.63 is the molecular weight of the reference (L-ascorbic acid-2-monophosphate tricyclohexylammonium salt).

[0031] The HPLC results of the reference and the finished product of Vc sodium phosphate are as Figure 1 、 2 shown. Substitute the data (A0 = 15230, A = 15085, C0 = 1.0 mg / mL, C = 1.0 mg / mL), and the calculated content of the finished product of Vc sodium phosphate is 98.7%.

[0032] 7. Method validation Linear range: 0.025–5.0 mg / mL, linear equation Y = 30245X + 12.3, R² = 0.9998; Precision: RSD = 0.45% for 6 repeated injections; Recovery rate: The recovery rate of the spiked samples is 97.5%–101.2%.

[0033] Example 2 Determination of the content of Intermediate I (ascorbic acid-2-phosphate) 1. Preparation of the mobile phase: Measure 2.7 mL of 98% concentrated sulfuric acid with a measuring cylinder, add about 800 mL of HPLC-grade pure water, stir to dissolve and make up the volume to 1 L (corresponding to 0.05 mol / L H2SO4), filter with a 0.45 μm filter membrane, and degas by ultrasound for 10 min (ultrasonic instrument model: Branson3800).

[0034] 2. Pretreatment of the chromatographic column: Take a sulfonated polystyrene-divinylbenzene hydrogen-type ion exchange column. When the new column is used for the first time, flush it forward with pure water (flow rate 0.5 mL / min) for 15 min and backward for 15 min; then switch to the 0.05 mol / L sulfuric acid mobile phase and equilibrate at the same flow rate for 30 min (about twice the column volume).

[0035] 3. Sample preparation Reference solution: Weigh 25.0 mg of L-ascorbic acid-2-monophosphate tricyclohexylammonium salt reference substance, place it in a 25 mL volumetric flask, and dilute it to the mark with HPLC-grade pure water to obtain a 1.0 mg / mL solution (corresponding concentration C0 = 1.0 mg / mL).

[0036] Sample solution: Pipette 0.5 mL of the intermediate of ascorbic acid-2-phosphate reaction solution, place it in a 50 mL volumetric flask, dissolve it with pure water and dilute it to the mark, shake well and filter (0.45 μm) to obtain a solution of about 10.0 mg / mL (C = about 10.0 mg / mL).

[0037] 4. HPLC detection conditions Chromatographic column: CONCISE Corel 87H3 ICE-99-9861; Mobile phase: 0.05 mol / L sulfuric acid solution; Flow rate: 0.5 mL / min; Column temperature: 35 °C; Detection wavelength: 250 nm; Injection volume: 1 μL; Running time: 15 min.

[0038] 5. Result calculation According to the formula: Where: A0 is the peak area of the reference solution; A is the peak area of the sample solution; C0 is the concentration of the reference solution, mg / ml; C is the concentration of the sample solution prepared, mg / ml; 256.10 is the molecular weight of ascorbic acid-2-phosphate; 553.63 is the molecular weight of the reference substance (L-ascorbic acid-2-monophosphate tricyclohexylammonium salt).

[0039] The HPLC results of Intermediate I are as Figure 3 shown. Substitute the data (A0 = 15230, A = 14850, C0 = 1.0 mg / mL, C = 10.0 mg / mL) and calculate that the content of Intermediate I is 45.10%.

[0040] 7. Method validation Linear range: 0.025–5.0 mg / mL (R² = 0.9997); RSD = 0.47% (n = 6); Recovery rate: 96.5%–101.8%.

[0041] Example 3 Determination of the content of Intermediate II (ascorbic acid-2-phosphate tricyclohexylammonium salt) 1. Preparation of mobile phase: Measure 2.7 mL of 98% concentrated sulfuric acid with a graduated cylinder, add approximately 800 mL of HPLC-grade pure water, stir to dissolve, and make up the volume to 1 L (corresponding to 0.05 mol / L H2SO4). Filter through a 0.45 μm filter membrane and perform ultrasonic degassing for 10 min (ultrasonic instrument model: Branson3800).

[0042] 2. Chromatographic column pretreatment: Take a sulfonated polystyrene-divinylbenzene hydrogen-type ion exchange column. When the new column is used for the first time, rinse it forward with pure water (flow rate 0.5 mL / min) for 15 min and then rinse it backward for 15 min. Subsequently, switch to a mobile phase of 0.05 mol / L sulfuric acid and equilibrate at the same flow rate for 30 min (about twice the column volume).

[0043] 3. Sample preparation Reference solution: Weigh 25.0 mg of the reference substance of L-ascorbic acid-2-monophosphate tricyclohexylammonium salt, place it in a 25 mL volumetric flask, add HPLC-grade pure water to make up the volume to the mark, and obtain a 1.0 mg / mL solution (corresponding concentration C0 = 1.0 mg / mL).

[0044] Sample solution: Weigh 50.0 mg of Intermediate II, place it in a 50 mL volumetric flask, dissolve it with pure water and make up the volume to the mark, shake well and filter to obtain a 1.0 mg / mL solution (C = 1.0 mg / mL).

[0045] 4. HPLC detection conditions Chromatographic column: CONCISE Corel 87H3 ICE-99-9861; Mobile phase: 0.05 mol / L sulfuric acid solution; Flow rate: 0.5 mL / min; Column temperature: 35 °C; Detection wavelength: 250 nm; Injection volume: 10 μL; Running time: 15 min.

[0046] 5. Result calculation Since the molecular weights of Intermediate II and the reference substance are the same (553.63), the formula is simplified to: Where: A0 is the peak area of the reference solution; A is the peak area of the sample solution; C0 is the concentration of the reference solution, mg / ml; C is the concentration of the sample solution prepared, mg / ml.

[0047] The HPLC results of Intermediate II are as shown in Figure 4As shown, substituting the data (A0 = 15230, A = 15380, C0 = 1.0 mg / mL, C = 1.0 mg / mL), the content of Intermediate II was calculated to be 101.0% (which may be related to the low purity of the reference substance and the actual value of C0 being slightly less than 1.0 mg / mL).

[0048] 7. Method Validation Linear range: 0.025–5.0 mg / mL (R² = 0.9999); RSD = 0.42% (n = 6); Recovery rate: 98.2%–101.5%.

[0049] Comparative Example Traditional C18 Column Method (Prior Art) 1. Preparation of mobile phase: Measure 150 mL of methanol and 20 mL of tetrabutylammonium hydrogen sulfate solution (5% w / v), add pure water to make up to 1000 mL, and mix evenly; weigh 16.83 g of potassium hydroxide and make up to 100 mL (corresponding to 3 mol / L KOH solution) to adjust the pH to 6.0 ± 0.1; filter through a 0.45 μm filter membrane and degas by ultrasound for 15 min.

[0050] 2. Pretreatment of chromatographic column: Take an Agilent ZORBAX Eclipse XDB-C18 chromatographic column. When the new column is used for the first time, rinse it with methanol (flow rate 1.0 mL / min) for 30 min, and then equilibrate it with the mobile phase until the baseline is stable (about 60 min).

[0051] 3. Sample preparation Reference substance solution: Weigh 25.0 mg of L-ascorbic acid-2-monophosphate tricyclohexylammonium salt reference substance, place it in a 25 mL volumetric flask, add HPLC-grade pure water to make up to the mark, and obtain a 1.0 mg / mL solution (corresponding concentration C0 = 1.0 mg / mL).

[0052] Sodium ascorbyl phosphate finished product solution: Weigh 50.0 mg of sodium ascorbyl phosphate finished product, place it in a 50 mL volumetric flask, dissolve it with pure water and make up to the mark, shake well and filter to obtain a 1.0 mg / mL solution (C = 1.0 mg / mL).

[0053] Intermediate I solution: Pipette 0.4 mL of the intermediate of ascorbic acid-2-phosphate reaction solution, place it in a 50 mL volumetric flask, dissolve it with pure water and make up to the mark, shake well and filter (0.45 μm) to obtain a solution of about 10.0 mg / mL (C = about 10.0 mg / mL).

[0054] Intermediate II solution: Weigh 50.0 mg of Intermediate II, place it in a 50 mL volumetric flask, dissolve it with pure water and make up to the mark, shake well and filter to obtain a 1.0 mg / mL solution (C = 1.0 mg / mL).

[0055] 4. HPLC detection conditions Chromatographic column: Agilent ZORBAX Eclipse XDB-C18; Mobile phase: Methanol - Ammonium hydrogen sulfate - water (15:2:83, v / v / v, pH 6.0); Flow rate: 1.0 mL / min; Column temperature: 35 °C; Detection wavelength: 250 nm; Injection volume: 10 μL for reference substance, finished product of Vc phosphate sodium, and Intermediate II; 1 μL for Intermediate I; Run time: 15 min.

[0056] 5. Detection steps System equilibration: Pump in the mobile phase at a flow rate of 1.0 mL / min, and start injection after the baseline drift is less than 0.01 AU / 30 min.

[0057] Injection sequence: Inject 10 μL of blank solution (pure water) to confirm no interfering peaks; Inject the reference substance solution continuously 3 times, record the retention time of the main peak (t0 = 6.2 min) and the average peak area (A0 = 14210); Inject the sample solution, and insert the reference substance solution every 3 samples to monitor the system stability.

[0058] The HPLC results are as Figures 5 - 8 shown.

[0059] 6. Result calculation According to the formula: Substitute the data (A0 = 15230, A = 13980, C0 = 0.5 mg / mL, C = 0.5 mg / mL), and calculate that the content of the finished product of Vc phosphate sodium is 97.2%.

[0060] 7. Method validation Linear range: 0.025–1.2 mg / mL, linear equation Y = 27890X + 25.6, R² = 0.9995; Precision: RSD = 0.68% for 6 repeated injections; Recovery rate: Recovery rate of spiked samples is 95.5%–102.5%; Table 2 Measurement Results Data comparison in Table 2 shows that the method provided by the present invention for the determination of the content of sodium ascorbyl phosphate (Examples 1 - 3) has significant advantages in detection performance compared with the traditional C18 column method (Comparative Example 1): the linear ranges of Examples 1 - 3 are all extended to 0.025–5.0 mg / mL, far better than 0.025–1.2 mg / mL of the comparative example, indicating that the method of the present invention has stronger adaptability to samples with high and low concentrations and does not require additional dilution or concentration. The RSDs of the examples are all lower than 0.5%, indicating that the present invention significantly improves the detection repeatability by simplifying the mobile phase system (single sulfuric acid solution) and optimizing the chromatographic column (hydrogen-type ion exchange column). The recovery rate ranges of the examples are narrower and closer to the theoretical values (96.5%–101.8%), further verifying that the method of the present invention can still maintain high accuracy in complex matrices (such as salt-containing intermediates) and avoid detection deviations caused by the volatilization of organic solvents or the precipitation of buffer salts in the traditional method.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described with specific implementation schemes above. On the basis of the present invention, some modifications or replacements can be made, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection required by the present invention.

Claims

1. A method for determining the content of sodium ascorbyl phosphate and its intermediate, characterized in that, Use a sulfonated polystyrene-divinylbenzene hydrogen ion exchange chromatographic column, and use a 0.01–0.1 mol / L sulfuric acid solution as the mobile phase; perform HPLC detection under the following chromatographic conditions: column temperature: 20–40 °C; flow rate: 0.5–1.5 mL / min; detection wavelength: 250 ± 2 nm; injection volume: 1–10 μL; running time: 10–20 min; calculate the contents of sodium ascorbyl phosphate and its intermediates by peak area.

2. The method according to claim 1, wherein The detection object includes any one of the following: sodium ascorbyl phosphate finished product, intermediate ascorbic acid-2-phosphate, and intermediate ascorbic acid-2-phosphate tricyclohexylammonium salt.

3. The method according to claim 1, characterized in that, The calculation formula for calculating the content of the target substance by peak area is: Wherein, A is the peak area of the sample, A0 is the peak area of the reference substance, C is the concentration of the sample solution, C0 is the concentration of the reference substance solution, M 目标物 is the molecular weight of the analyte, M 对照品 is the molecular weight of the reference substance.

4. The method according to claim 3, characterized in that, The reference substance is L-ascorbic acid-2-monophosphate tricyclohexylammonium salt, with a purity of ≥99.8% and a molecular weight of 553.

63.

5. The method according to claim 1, wherein The concentration of the sulfuric acid solution in the mobile phase is 0.05 mol / L.

6. The method according to claim 1, wherein The chromatographic conditions are: column temperature: 35 °C; flow rate: 0.5 mL / min; detection wavelength: 250 nm.

7. The method according to claim 1, wherein The running time of the chromatographic conditions is 15 min.

8. The method according to any one of claims 1-7, characterized in that, The linear detection range of the method is 0.025–5.0 mg / mL, and the correlation coefficient R²≥0.9997.

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