Method for detecting content of ascorbic acid in rosa roxburghii tratt fruit product

Abstract

The invention relates to the technical field of roxburgh rose component detection, in particular to a method for detecting the content of ascorbic acid in a roxburgh rose product. The invention provides a detection method which is good in separation effect of L (+)-ascorbic acid and D (-)-ascorbic acid, convenient to operate and accurate and reliable in determination result. A C18 column is used as a chromatographic column, and mobile phase gradient elution is adopted; a mobile phase A is methanol; a mobile phase B: 10% methanol water; and a mobile phase C: 7.0 g of potassium dihydrogen phosphate and 1.0 g of cetyltrimethylammonium bromide are dissolved in 900 ml of water, 20 ml of methanol is added, the mixture is uniformly mixed, the pH is adjusted to 2.6 with phosphoric acid, the volume is fixed to 1 L, the mixture passes through a 0.45 [mu] m filter membrane, and ultrasonic degassing is performed. The method is based on a high performance liquid chromatography method and a first method for determining ascorbic acid in food in national food safety standard GB 5009.86-2016, the defects in the prior art are optimized and improved through a large number of experiments, the detection effect of ascorbic acid in the roxburgh rose juice is improved, and therefore quality control over effective components of the roxburgh rose juice is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of detection of roxburghii components, in particular to a method for detecting ascorbic acid content in roxburghii products. Background Art

[0002] Rosa roxburghii Tratt. Ros. Monogr. belongs to the genus Rosa of the Rosaceae family. It is a perennial deciduous shrub. The nutritional value of Rosa roxburghii is high. It mainly contains ascorbic acid, total acid, polysaccharides, flavonoids, superoxide dismutase (SOD), rososterol (β-sitosterol) and triterpenoid compounds. Ascorbic acid, also known as vitamin C, is a substance necessary for the human body to maintain normal growth and development. L(+)-ascorbic acid is left-handed right-handed ascorbic acid, which has reducing properties, high human absorption rate, and biological activity to the human body. D(-)-ascorbic acid has basically no biological activity to the human body.

[0003] In the process of testing L(+)-ascorbic acid and D(-)-ascorbic acid in sea buckthorn products, there are problems such as difficulty in separating L(+)-ascorbic acid and D(-)-ascorbic acid, poor results, and retention time drift.

[0004] Therefore, it is currently urgent to find a detection method with good separation effect of L(+)-ascorbic acid and D(-)-ascorbic acid and accurate and reliable measurement results. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for detecting the content of ascorbic acid in a roxburghii product, which is as follows:

[0006] The invention discloses a method for detecting the content of ascorbic acid in a roxburghii product, which adopts a high performance liquid chromatography method for determination, and the specific parameters are as follows: a C18 column is used as a chromatographic column, mobile phase A is methanol, mobile phase B is 10% methanol water, and mobile phase C is 7.0g of potassium dihydrogen phosphate and 1.0g of hexadecyltrimethylammonium bromide, which are dissolved in 900ml of water, and 20ml of methanol is added, mixed, the pH value is adjusted to 2.6 with phosphoric acid, and the volume is fixed to 1L, filtered through a 0.45-mu m filter membrane, and ultrasonically degassed.

[0007] Furthermore, the C18 column is a column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm, or a chromatographic column with equivalent performance, such as the Yuexu Technology XB-C18 column, with a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the Agilent 5HC-C18 (2) column, with a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0008] Furthermore, the specific parameters of the high performance liquid chromatography method are as follows: injection volume: 10 μL; column temperature: 25° C.; flow rate: 0.8 ml / min; detection wavelength: 245 nm.

[0009] Furthermore, the high performance liquid chromatography method adopts gradient elution of mobile phase.

[0010] The mobile phase gradient elution, the specific elution procedure is as follows:

[0011] time Mobile phase A Mobile phase B Mobile phase C 0-9 minutes 0 0 100 9-11 minutes 80 20 0 11-15 minutes 80 20 0 15.1-16 minutes 0 0 100 16-22 minutes 0 0 100

[0012] Furthermore, the method simultaneously separates L(+)-ascorbic acid and D(-)-ascorbic acid and detects them simultaneously.

[0013] Furthermore, the method specifically involves drawing standard curves of L(+)-ascorbic acid and D(-)-ascorbic acid respectively, and after measuring the sample to be tested, obtaining the concentration of the sample to be tested according to the standard curves.

[0014] Furthermore, the drawing of the standard curves of L(+)-ascorbic acid and D(-)-ascorbic acid is specifically carried out by measuring the mixed standard series of ascorbic acid working solutions, with the mass concentration of the L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions as the abscissa and the peak height or peak area of ​​the L(+)-ascorbic acid and D(-)-ascorbic acid as the ordinate, drawing the standard curve, and calculating the regression equation.

[0015] Further, the preparation method of the test solution is as follows: weigh 0.1g to 2g of the sample, so that the sample contains 0.03mg to 6mg of L(+)-ascorbic acid, operate in a dark environment, transfer the sample to a 100mL volumetric flask with a 2% metaphosphoric acid solution, adjust the volume, shake well, perform ultrasonic extraction for 5min, control the water temperature at 5 to 15°C, filter through a 0.45μm aqueous phase filter membrane, and filter the filtrate for testing. The sample solution is measured, and the concentration (μg / mL) of L(+)-ascorbic acid or [D(-)-ascorbic acid] in the test solution is obtained according to the standard curve, and the chromatogram of the test sample is recorded.

[0016] Further, the preparation method of the ascorbic acid mixed standard series working solution is as follows: weigh 10 mg of L(+)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution; weigh 10 mg of D(-)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution, ultrasonicate for 2 minutes, take out and place for 10 minutes, shake and respectively absorb 0 mL, 0.05 mL, 0.50 mL, 1.0 mL, 2.5 mL, 5.0 mL of L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, and dilute to 100 mL with 20 g / L metaphosphoric acid solution. The concentrations of L(+)-ascorbic acid and D(-)-ascorbic acid in the standard series working solution are 0 μg / mL, 0.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL, respectively.

[0017] Furthermore, the preparation method of the ascorbic acid mixed standard series working solution is as follows: weigh 10 mg of L(+)-ascorbic acid standard, and dilute to 10 mL with 2% metaphosphoric acid solution; weigh 10 mg of D(-)-ascorbic acid standard, and dilute to 10 mL with 2% metaphosphoric acid solution, ultrasonicate for 2 minutes, take out and place for 10 minutes, shake well, respectively absorb L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, dilute to 25 μg / mL standard working solution, and the injection volumes are 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, and 30 μL, respectively.

[0018] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0019] (1) The present invention proposes a detection method with good separation effect of L(+)-ascorbic acid and D(-)-ascorbic acid, and the determination is easy to operate, and the determination result is accurate and reliable. The present invention is based on the first method of determination of ascorbic acid in food of GB 5009.86-2016 National Food Safety Standard, high performance liquid chromatography, and through a large number of experiments, optimizes and improves the deficiencies of the prior art, improves the detection effect of ascorbic acid in roxburghii juice, thereby achieving quality control of roxburghii juice.

[0020] (2) The method of the present invention adopts a gradient elution procedure, which improves the separation degree of L(+)-ascorbic acid and D(-)-ascorbic acid. At the same time, the system suitability test indicators such as the symmetry factor, plate number, and selectivity of each component peak meet the requirements, the retention time repeatability is good, and the standard curve linearity is good. The separation effect of L(+)-ascorbic acid and D(-)-ascorbic acid is also good in the test sample. The peak area of ​​the test sample is within the range of the standard curve, and the relative deviation meets the requirements.

[0021] (3) The durability, precision, stability, repeatability and recovery rate of the determination method obtained in the present invention are good, the separation effect of L(+)-ascorbic acid and D(-)-ascorbic acid is good, and the ascorbic acid content in the test sample has a good linear relationship in the range of 0 to 150 μg / mL.

[0022] (4) The method of the present invention adopts a relatively simple instrument, and the sample processing is easy to operate, which improves efficiency, and has high detection sensitivity and good stability. The measurement results are accurate and reliable, and the decomposition of ascorbic acid at high temperature is avoided. It is of great significance to the quality control and evaluation of ascorbic acid in roxburghii juice and roxburghii products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the chromatogram of standard solution.

[0024] Figure 2 It is the standard curve chromatogram information.

[0025] Figure 3 It is the chromatogram of standard solution.

[0026] Figure 4 It is the standard curve chromatogram information.

[0027] Figure 5 The chromatograms of the standard solutions are displayed in an overlapping manner.

[0028] Figure 6 It is an overlay and enlarged view of the chromatogram of the standard solution.

[0029] Figure 7 is the regression equation and correlation coefficient of the L(+)-ascorbic acid standard curve.

[0030] Figure 8 is the regression equation and correlation coefficient of the D(-)-ascorbic acid standard curve.

[0031] Fig. 9 It is the chromatogram of the test product of Example 1.

[0032] Fig.10 These are chromatograms of standard solutions with different injection volumes.

[0033] Fig.11 It is the standard curve chromatogram information.

[0034] Fig.12 The chromatograms of the standard solutions are displayed as an overlay.

[0035] Fig.13 is the regression equation and correlation coefficient of the D(-)-ascorbic acid standard curve.

[0036] Fig.14 is the regression equation and correlation coefficient of the D(-)-ascorbic acid standard curve.

[0037] Fig.15 The chromatograms of six parallel injections are overlapped. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0039] 1. Instruments and Materials

[0040] 1.1 Instrument

[0041] Instruments: Agilent 1260 InfinityⅡ liquid chromatograph, UV detector, autosampler; chromatographic column: Agilent 5HC-C18(2) column, column length 25 cm, column inner diameter 4.6 mm, particle size 5 μm; filter membrane: 0.45 μm aqueous membrane; Ph meter FE28; HH-4 digital display constant temperature water bath [Changzhou Putian Instrument Manufacturing Co., Ltd. (Jintan Jingbo Experimental Instrument Factory)], ME204TE / 02 electronic balance [Mettler-Toledo International Trade (Shanghai) Co., Ltd.]; KQ-500E CNC ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0042] 1.2 Drug testing

[0043] L(+)-ascorbic acid reference substance (batch number: 100425-202105, content: 100%) was purchased from China Food and Drug Inspection Institute; D(-)-ascorbic acid reference substance (batch number: C1447587, content: 99.5%) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; metaphosphoric acid (batch number: 20230715, purchased from Tianjin Komio Chemical Reagent Co., Ltd.), hexadecyltrimethylammonium bromide (batch number: RH42658, purchased from Shanghai Yien Chemical Technology Co., Ltd.), potassium dihydrogen phosphate (batch number: 20240103, purchased from Sinopharm Chemical Reagent Co., Ltd.), methanol (batch number: F24070201, purchased from Thermo Fisher Scientific (China) Co., Ltd.); water was homemade purified water.

[0044] 1.3 Materials

[0045] The samples were provided by Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd. and Sinopharm Great Health Industry Development Co., Ltd. The sources of the samples are shown in Table 1.

[0046] Table 1 Sample source information

[0047]

[0048] Comparative Example 1

[0049] The first method of GB 5009.86 is to use high performance liquid chromatography (equal concentration elution with mobile phase)

[0050] 1. Instrument conditions are as follows:

[0051] a) Chromatographic column: C18 column, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, or chromatographic column with equivalent performance.

[0052] b) Mobile phase: A: 6.8g potassium dihydrogen phosphate and 0.91g hexadecyltrimethylammonium bromide, dissolved in water and fixed to 1L (adjust pH to 2.5-2.8 with phosphoric acid); B: 100% methanol. Mix in the ratio of A:B=98:2, filter through a 0.45μm filter membrane, and degas by ultrasonic.

[0053] c) Detector: diode array detector or ultraviolet detector.

[0054] d) Flow rate: 0.7 mL / min;

[0055] e) Column temperature: 25℃;

[0056] f) Detection wavelength: 245nm;

[0057] g) Injection volume: 20 μL.

[0058] 2. Preparation of standard solution

[0059] L(+)-ascorbic acid standard stock solution (1.000 mg / mL): Accurately weigh 0.01 g (accurate to 0.01 mg) of L(+)-ascorbic acid standard and dilute to 10 mL with 20 g / L metaphosphoric acid solution. The stock solution can be stored at 2°C to 8°C in the dark for one week.

[0060] D(-)-ascorbic acid standard stock solution (1.000 mg / mL): Accurately weigh 0.01 g (accurate to 0.01 mg) of D(-)-ascorbic acid standard and dilute to 10 mL with 20 g / L metaphosphoric acid solution. The stock solution can be stored at 2°C to 8°C in the dark for one week.

[0061] Ascorbic acid mixed standard series working solution: Take 0mL, 0.05mL, 0.50mL, 1.0mL, 2.5mL, 5.0mL of L(+)-ascorbic acid and D(-)-ascorbic acid standard stock solutions respectively, and dilute to 100mL with 20g / L metaphosphoric acid solution. The concentrations of L(+)-ascorbic acid and D(-)-ascorbic acid in the standard series working solutions are 0μg / mL, 0.5μg / mL, 5.0μg / mL, 10.0μg / mL, 25.0μg / mL, 50.0μg / mL respectively. Prepare immediately before use.

[0062] Measure the mixed standard series of ascorbic acid working solutions respectively, with the mass concentration (μg / mL) of the L(+)-ascorbic acid [or D(-)-ascorbic acid] standard solution as the abscissa and the peak height or peak area of ​​the L(+)-ascorbic acid [or D(-)-ascorbic acid] as the ordinate, draw a standard curve or calculate the regression equation. Record the peak area, as shown in Figure (1).

[0063] Depend on Figure 2 It can be obtained that: as the concentration of the reference substance increases, the corresponding peaks of L(+)-ascorbic acid and D(-)-ascorbic acid in the reference substance are more difficult to separate. When the concentration of the reference substance is 50.0 μg / mL (the actual injection volume is 10 μl), the separation degree becomes 1.38, which is less than the standard 1.5. The system suitability test does not meet the requirements.

[0064] Example 1

[0065] 1. The preparation method of standard solution is as follows: weigh 10 mg of L(+)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution; weigh 10 mg of D(-)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution, ultrasonicate for 2 minutes, take out and place for 10 minutes, shake well, respectively absorb 0 mL, 0.05 mL, 0.50 mL, 1.0 mL, 2.5 mL, 5.0 mL of L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, and dilute to 100 mL with 20 g / L metaphosphoric acid solution. The concentrations of L(+)-ascorbic acid and D(-)-ascorbic acid in the standard series working solution are 0 μg / mL, 0.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, respectively. Prepare immediately before use.

[0066] 2. Chromatographic parameters: Instrument: Agilent 1260 InfinityⅡ liquid chromatograph, UV detector, autosampler; Chromatographic column: Yuexu Technology XB-C18 column, column length 25cm, column inner diameter 4.6mm, particle size 5μm; Injection volume: 10μL Column temperature: 25℃, flow rate: 0.8ml / min; Detection wavelength: 245nm;

[0067] Mobile phase A: methanol; Mobile phase B: 10% methanol water; Mobile phase C: 7.0g potassium dihydrogen phosphate and 1.0g hexadecyltrimethylammonium bromide, dissolved in 900ml water, add 20ml methanol, mix well, adjust pH to 2.6 with phosphoric acid, and make up to 1L, filter through 0.45μm filter membrane, and degas by ultrasonication; Mobile phase gradient elution:

[0068] time Mobile phase A Mobile phase B Mobile phase C 0-9 minutes 0 0 100 9-11 minutes 80 20 0 11-15 minutes 80 20 0 15.1-16 minutes 0 0 100 16-22 minutes 0 0 100

[0069] 3. Measure the mixed standard series of ascorbic acid working solutions, using the mass concentration of L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions as the horizontal coordinate and the peak height or peak area of ​​L(+)-ascorbic acid and D(-)-ascorbic acid as the vertical coordinate. Draw the standard curve and calculate the regression equation. Take the test solution with an injection volume of 10 μL and record the peak area, as shown in Figure (3).

[0070] 4. Preparation of test solution: Weigh 0.1g~2g of sample, so that the sample contains 0.03mg~6mg of L(+)-ascorbic acid. Operate in a dark place, transfer the sample to a 50mL volumetric flask with 2% metaphosphoric acid solution, make up to volume, shake well, perform ultrasonic extraction for 5 minutes, control the water temperature at 5~15℃, filter through a 0.45μm aqueous filter membrane, and the filtrate is ready for testing. Measure the sample solution and obtain the concentration (μg / mL) of L(+)-ascorbic acid or [D(-)-ascorbic acid] in the test solution based on the standard curve. Record the chromatogram of the test sample at Fig. 9 .

[0071] According to the standard curve and the peak area of ​​the test sample, the concentration (μg / mL) of L(+)-ascorbic acid or [D(-)-ascorbic acid] in the test solution was obtained, and the content of L(+)-ascorbic acid or [D(-)-ascorbic acid] in each test sample was calculated. The results are shown in Tables 2 and 3 below:

[0072] Table 2

[0073]

[0074]

[0075] Table 3

[0076]

[0077]

[0078] Summary: Compared with Comparative Example 1, Example 1 adopts a gradient elution procedure to improve the separation degree of L(+)-ascorbic acid and D(-)-ascorbic acid. At the same time, the system suitability test indicators such as the symmetry factor, plate number, and selectivity of each component peak meet the requirements, the retention time repeatability is good, and the standard curve has good linearity. The separation effect of L(+)-ascorbic acid and D(-)-ascorbic acid is also good in the test sample, the test sample peak area is within the standard curve range, and the relative deviation meets the requirements.

[0079] Example 2

[0080] 1. The preparation method of standard solution is as follows: weigh 10 mg of L(+)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution; weigh 10 mg of D(-)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution, ultrasonicate for 2 minutes, take out and let stand for 10 minutes, shake well, respectively absorb L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, dilute to 25 μg / mL standard working solution, and the injection volumes are 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, and 30 μL respectively.

[0081] 2. Chromatographic parameters: Instrument: Agilent 1260 InfinityⅡ liquid chromatograph, UV detector, autosampler; Chromatographic column: Agilent 5HC-C18(2) column, column length 25 cm, column inner diameter 4.6 mm, particle size 5 μm; Column temperature: 25°C, flow rate: 0.8 ml / min; Detection wavelength: 245 nm; Mobile phase A: methanol; Mobile phase B: 10% methanol water; Mobile phase C: 7.0 g potassium dihydrogen phosphate and 1.0 g hexadecyltrimethylammonium bromide, dissolved in 900 ml water, added with 20 ml methanol, mixed, adjusted to pH 2.6 with phosphoric acid, and fixed to 1 L, filtered through a 0.45 μm filter membrane, and degassed by ultrasonication;

[0082] Mobile phase gradient elution:

[0083] time Mobile phase A Mobile phase B Mobile phase C 0-9 minutes 0 0 100 9-11 minutes 80 20 0 11-15 minutes 80 20 0 15.1-16 minutes 0 0 100 16-22 minutes 0 0 100

[0084] 3. Measure the mixed standard solution of ascorbic acid according to the above injection volume and record the chromatograms under different injection volumes, such as Fig.10 shown.

[0085] With the mass concentration of L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions as the abscissa and the peak area of ​​L(+)-ascorbic acid and D(-)-ascorbic acid as the ordinate, a standard curve was drawn and the regression equation was calculated, such as Fig.13 , Fig.14 .

[0086] 4. Preparation of test solution: Weigh 0.1g~2g of sample, so that the sample contains 0.03mg~6mg of L(+)-ascorbic acid, operate in dark, transfer the sample to a 100mL volumetric flask with 2% metaphosphoric acid solution, make up to volume, shake well, ultrasonically extract for 5min, control the water temperature at 5~15℃, filter through 0.45μm aqueous phase membrane, and filtrate for testing. Determine the sample solution, obtain the concentration (μg / mL) of L(+)-ascorbic acid or [D(-)-ascorbic acid] in the test solution according to the standard curve, and record the chromatogram of the test sample.

[0087] 4.1 Durability

[0088] In the laboratory environment that meets the experimental conditions, different analysts were replaced, different instruments and chromatographic columns were used, and the organic sea buckthorn juice (50 ml / bag) batch number 240506 sample was analyzed. The calculated result was 1213.27 mg / 100 g, which was compared with the previous experimental result of 1214.34 mg / 100 g, with a deviation of 0.1%. This method has good durability.

[0089] 4.2 Precision experiment

[0090] Take the 25 μg / mL standard working solution prepared in item "1" of this embodiment above, inject 6 parallel samples, inject each sample continuously once, and analyze the sample according to the parameter conditions in item "2" of this embodiment, and record the peak areas of L(+)-ascorbic acid and D(-)-ascorbic acid. The results are shown in Table 4 and Fig.15 As shown. This shows that the instrument has a high precision and is suitable for detection.

[0091] Table 4

[0092]

[0093] 4.3 Stability

[0094] Fresh roxburgh fruit 240904, fresh roxburgh fruit 240905, roxburgh lutein natural vitamin C tablets 240801, yellow roxburgh ginger juice drink 240301, conscience fruit organic roxburgh juice 240501, and roxburgh freeze-dried fruit crisps 240301 were used as test samples and processed according to the "2.3.4" method. Each sample was placed in 12 injection vials numbered 1-1, 1-2, 2-1, 2-2, 3-1, 3-2, 4-1, 4-2, 5-1, 5-2, 6-1, and 6-2 in sequence as the test sample solution. The samples were injected and analyzed at 0h, 2h, 4h, 6h, 8h, and 24h according to the parameter conditions in "2.3.2". The specific results are shown in Table 5 below.

[0095] Table 5

[0096]

[0097] The peak area of ​​L(+)-ascorbic acid was recorded, and the peak area RSD% obtained in the six time periods were: L(+)-ascorbic acid 1.72%, D(-)-ascorbic acid 1.49%, indicating that the sample had good stability after 24 hours.

[0098] Summary: The results show that the relative retention time of the chromatographic peaks of L(+)-ascorbic acid and D(-)-ascorbic acid has good repeatability, the accuracy meets the requirements, the instrument precision is good, the method has good durability, the separation of the chromatographic peaks of L(+)-ascorbic acid and D(-)-ascorbic acid meets the requirements, and the test solution is stable within 24 hours. Example 2 overcomes the difficulties of low separation of the chromatographic peaks of L(+)-ascorbic acid and D(-)-ascorbic acid, many reference sample dilution steps, and poor retention time repeatability. By changing the gradient elution conditions, optimizing the reference sample and sample processing methods, simplifying the operation, and improving the efficiency, the detection sensitivity is high, the stability is good, and the measurement results are accurate and reliable.

[0099] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for detecting ascorbic acid content in a roxburghii product, characterized in that: The determination was carried out by high performance liquid chromatography, and the specific parameters were as follows: C18 column was used as the chromatographic column, mobile phase A: methanol; mobile phase B: 10% methanol water; mobile phase C: 7.0g potassium dihydrogen phosphate and 1.0g hexadecyltrimethylammonium bromide, dissolved in 900ml water, added with 20ml methanol, mixed, adjusted to pH 2.6 with phosphoric acid, and fixed to 1L, filtered through a 0.45μm filter membrane, and degassed by ultrasound.

2. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: The C18 column is a chromatographic column with a length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 μm, or a chromatographic column with equivalent performance.

3. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that, The specific parameters of the high performance liquid chromatography method are as follows: injection volume: 10 μL; column temperature: 25° C.; flow rate: 0.8 ml / min; detection wavelength: 245 nm.

4. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: The high performance liquid chromatography method adopts mobile phase gradient elution.

5. The method for detecting ascorbic acid content in the roxburghii product according to claim 4, characterized in that, The mobile phase gradient elution, the specific elution procedure is as follows:

6. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: L(+)-ascorbic acid and D(-)-ascorbic acid were separated and detected simultaneously.

7. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: The standard curves of L(+)-ascorbic acid and D(-)-ascorbic acid were drawn respectively, and after the sample to be tested was measured, the concentration of the sample to be tested was obtained according to the standard curves.

8. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: The preparation method of the test solution is as follows: weigh 0.1g to 2g of the sample, so that the sample contains 0.03mg to 6mg of L(+)-ascorbic acid, operate in a dark environment, transfer the sample to a 100mL volumetric flask with a 2% metaphosphoric acid solution, adjust the volume, shake well, perform ultrasonic extraction for 5min, control the water temperature at 5 to 15°C, filter through a 0.45μm aqueous phase filter membrane, and filter the filtrate for testing. The sample solution is measured, and the concentration (μg / mL) of L(+)-ascorbic acid or [D(-)-ascorbic acid] in the test solution is obtained according to the standard curve, and the chromatogram of the test sample is recorded.

9. The method for detecting ascorbic acid content in the roxburghii product according to claim 1, characterized in that: The preparation method of the ascorbic acid mixed standard series working solution is as follows: weigh 10 mg of L(+)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution; weigh 10 mg of D(-)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution, put in ultrasonic for 2 minutes, take out and place for 10 minutes, shake and respectively absorb 0 mL, 0.05 mL, 0.50 mL, 1.0 mL, 2.5 mL, 5.0 mL of L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, and dilute to 100 mL with 20 g / L metaphosphoric acid solution. The concentrations of L(+)-ascorbic acid and D(-)-ascorbic acid in the standard series working solution are 0 μg / mL, 0.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 25.0 μg / mL, and 50.0 μg / mL, respectively.

10. The method for detecting ascorbic acid content in roxburghii products according to claim 1, characterized in that: The preparation method of the ascorbic acid mixed standard series working solution is as follows: Weigh 10 mg of L(+)-ascorbic acid standard and dilute to 10 mL with 2% metaphosphoric acid solution; Weigh 10 mg of D(-)-ascorbic acid standard, dilute to 10 mL with 2% metaphosphoric acid solution, place under ultrasound for 2 minutes, take out and let stand for 10 minutes, shake well, respectively absorb L(+)-ascorbic acid and D(-)-ascorbic acid standard solutions, dilute to 25 μg / mL standard working solution, the injection volumes are 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 30 μL respectively.

Images