High performance liquid chromatography method for simultaneously determining organic acid and vitamin C in kiwi fruit

Abstract

The invention belongs to the field of fruit quality analysis, and particularly relates to a high performance liquid chromatography method for simultaneously determining organic acid and vitamin C in kiwi fruit, which comprises the following steps: grinding kiwi fruit into powder in liquid nitrogen, extracting with metaphosphoric acid solution, centrifuging, and collecting supernate to obtain test solution; a high performance liquid chromatograph is utilized, and under the chromatographic conditions that a C18 column is selected as a chromatographic column, the column temperature is 25-35 DEG C, the sample injection volume is 10 microliters, a 0.01 mol / L monopotassium phosphate buffer solution is used as a mobile phase for isocratic elution, and the organic acid and the vitamin C in a test solution are detected under the conditions that the flow speed is 0.5-0.8 mL / min and the wavelengths are 210 nm and 245 nm. The detection method provided by the invention can realize qualitative and quantitative analysis of flavor and nutritional ingredients of kiwi fruits, and has good specificity, accuracy, durability and stability and high detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of fruit quality analysis, and particularly relates to a high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit. Background Art

[0002] Kiwifruit contains rich organic acids and vitamin C (VC). According to relevant reports, the vitamin C content in kiwifruit is 5 - 10 times that of lemon, 20 - 80 times that of apple, and 10 - 20 times that of pineapple, making it a veritable "king of vitamin C". During the growth and development of kiwifruit, organic acids will accumulate. The main types of organic acids are citric acid, quinic acid, malic acid, etc., and there are also small amounts of oxalic acid, lactic acid, tartaric acid, etc.

[0003] At present, there are few methods for simultaneously determining organic acids and vitamin C in kiwifruit by liquid chromatograph. Existing technologies use liquid chromatography to determine organic acids and VC, but there are problems such as large fluctuations in accuracy, low sensitivity, poor resolution, and easy salting - out phenomenon when mixing the salt phase (phase A) and organic phase (phase B) as the mobile phase, which is likely to damage the chromatographic column and liquid chromatograph during long - term use. Liquid chromatography - mass spectrometry (LC - MS) technology has also been used in the research of organic acids and VC, but its high cost and complex operation limit its wide application. Therefore, it is extremely important to establish a method for determining organic acids and VC in kiwifruit with simple operation, good resolution, and high accuracy. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit. The detection method provided by the present invention can achieve qualitative and quantitative analysis of the flavor and nutritional components of kiwifruit, and has good specificity, durability, and stability, with high detection sensitivity.

[0005] To achieve the above object, the specific technical solution of the present invention is as follows: The present invention provides a high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit, comprising the following steps: Mix kiwifruit with metaphosphoric acid solution, centrifuge and collect the supernatant to obtain the test solution; Using a high performance liquid chromatograph, under the chromatographic conditions: selecting a C18 column as the chromatographic column, column temperature of 25°C - 35°C, injection volume of 10 μL, isocratic elution with 0.01 mol / L potassium dihydrogen phosphate buffer as the mobile phase, and flow rate of 0.5 mL / min - 0.8 mL / min, detect the organic acids and vitamin C in the test solution at wavelengths of 210 nm and 245 nm.

[0006] Further, the organic acid is any one or more of oxalic acid, tartaric acid, quinic acid, malic acid, lactic acid, citric acid, and succinic acid.

[0007] Further, the pH value of the potassium dihydrogen phosphate buffer solution is 2.0 - 3.0.

[0008] Further, the concentration of the metaphosphoric acid solution is 18 g / L - 22 g / L.

[0009] Further, the mixing ratio of the kiwifruit to the metaphosphoric acid solution is 1 g : 10 mL - 20 mL.

[0010] Further, the packing material of the C18 column is octadecylsilyl-bonded silica gel, and its specifications are: 5 μm, 4.6 mm × 250 mm.

[0011] Further, the C18 column is selected from: Ultimate® LP-C18, SupersilAQ C18, or Shim-packGIST C18-AQ.

[0012] Further, the retention time of oxalic acid is 5.15 min - 5.25 min; the retention time of tartaric acid is 5.93 min - 6.03 min; the retention time of quinic acid is 6.20 min - 6.30 min; the retention time of malic acid is 7.90 min - 8.00 min; the retention time of lactic acid is 9.80 min - 9.90 min; the retention time of citric acid is 16.00 min - 17.00 min; the retention time of succinic acid is 19.05 min - 19.15 min.

[0013] Further, the retention time of vitamin C is 9.20 min - 9.30 min.

[0014] Further, the temperature of the centrifugation is 3°C - 5°C, the rotation speed of the centrifugation is 10000 r / min - 12000 r / min, and the time of the centrifugation is 10 min - 15 min.

[0015] Further, the kiwifruit is kiwifruit ground into powder in liquid nitrogen.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit: First, grind kiwifruit into powder in liquid nitrogen, extract it with metaphosphoric acid solution, centrifuge, and collect the supernatant to obtain a test solution; then use a high performance liquid chromatograph under the chromatographic conditions: select a C18 column as the chromatographic column, the column temperature is 25°C to 35°C, the injection volume is 10 μL, isocratic elution is carried out with 0.01 mol / L potassium dihydrogen phosphate buffer solution as the mobile phase, and the flow rate is 0.5 mL / min to 0.8 mL / min. Under these conditions, detect the organic acids and vitamin C in the test solution at wavelengths of 210 nm and 245 nm. (1) The prior art usually processes kiwifruit samples by ultrasonic extraction + centrifugation, but vitamin C is easily oxidized and lost during long-term ultrasonic treatment. Based on the easily degradable characteristics of vitamin C in kiwifruit, the present invention proposes a pretreatment method of "low-temperature grinding + rapid solvent extraction", combined with metaphosphoric acid solution as a stabilizer, which can dissolve the target components efficiently, ensure the extraction purity and efficiency to the greatest extent, and other components of the test solution do not interfere with the detection of organic acids and vitamin C; (2) For the detection of organic acids and vitamin C, a relatively complex mobile phase system is usually used, such as gradient elution or a combination of multiple buffer solutions. The present invention uses potassium dihydrogen phosphate buffer solution as the mobile phase for isocratic elution to avoid the problem of salting out easily generated by mixed mobile phases; (3) The present invention realizes the simultaneous determination of 7 organic acids and vitamin C in kiwifruit for the first time, and the total analysis time < 20 min, while the traditional method requires two separate detections. (4) When the high performance liquid chromatography method provided by the present invention is used for the simultaneous determination of 7 organic acids and vitamin C in kiwifruit, the resolution (R≥1.5), linear range (r>0.999), and precision (RSD≤1.5%) of each component from adjacent interfering peaks can be achieved for the qualitative and quantitative analysis of the flavor and nutritional components of kiwifruit, and the specificity, accuracy, durability, and stability are good, and the detection sensitivity is high. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is the chromatogram at a wavelength of 210 nm when the mobile phase is 0.1 v / v% phosphoric acid solution.

[0019] Figure 2 It is the chromatogram at a wavelength of 210 nm when the mobile phase is a mixed solution of 0.1 v / v% phosphoric acid solution - methanol at a ratio of 95:5.

[0020] Figure 3 Chromatograms of 7 organic acid standard solutions when the extractant is a mixed solution of 0.1 v / v% formic acid - 10 v / v% methanol.

[0021] Figure 4 Chromatograms of 7 organic acids and VC in the mixed standard solution. Figure 4 Figure A shows the chromatograms of each component at a wavelength of 210 nm. Figure 4 Figure B shows the chromatogram of VC at a wavelength of 245 nm.

[0022] Figure 5 Chromatograms of 7 organic acids and VC in the spiked test sample solution. Figure 5 Figure A shows the chromatograms of each component at a wavelength of 210 nm. Figure 5 Figure B shows the chromatogram of VC at a wavelength of 245 nm. Detailed implementation manners

[0023] The following is a detailed description of the specific implementation manners of the present invention. However, it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0024] Kiwi fruits are rich in organic acids and vitamin C. However, there are currently few methods for simultaneously determining organic acids and vitamin C in kiwi fruits by liquid chromatograph, and there are problems such as large fluctuations in accuracy, low sensitivity, poor resolution, and easy salting-out phenomenon when mixing the aqueous phase (phase A) and the organic phase (phase B) as the mobile phase, which is likely to damage the chromatographic column and liquid phase instrument after long-term use. Liquid chromatography - mass spectrometry (LC - MS) technology has also been used in the research of organic acids and VC, but its high cost and complex operation limit its wide application. Therefore, it is very important to establish a method for determining organic acids and VC in kiwi fruits with simple operation, good resolution, and high accuracy.

[0025] The present invention provides a high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit, comprising the following steps: grinding kiwifruit into powder in liquid nitrogen, extracting with metaphosphoric acid solution, centrifuging, collecting the supernatant to obtain a test solution; using a high performance liquid chromatograph under the chromatographic conditions: selecting a C18 column as the chromatographic column, column temperature of 25°C to 35°C, injection volume of 10 μL, isocratic elution with 0.01 mol / L potassium dihydrogen phosphate buffer as the mobile phase, and flow rate of 0.5 mL / min to 0.8 mL / min, detecting the organic acids and vitamin C in the test solution at wavelengths of 210 nm and 245 nm.

[0026] Example 1: A high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit 1. Preparation of solutions Standard curve solution: Weigh appropriate amounts of oxalic acid, quinic acid, tartaric acid, malic acid, lactic acid, citric acid, succinic acid, and VC, dissolve and dilute with an extraction agent to prepare a standard curve solution in the range of 5 μg / mL to 2 mg / mL. Prepare freshly before use and store in the dark.

[0027] Test solution: Weigh 0.5 g of an appropriate amount of ground kiwifruit sample into a 2 mL centrifuge tube, add 5 mL of 20 g / L metaphosphoric acid solution, vortex for 3 min, centrifuge at 12000 r / min at 4°C for 10 min, let stand for 30 min, then aspirate the supernatant, recentrifuge the supernatant under the same conditions for 10 min, and take the supernatant for storage in the dark at 4°C for standby.

[0028] Spiked test solution: Weigh 20 mg of oxalic acid, 40 mg of quinic acid, 200 mg of tartaric acid, 200 mg of malic acid, 200 mg of lactic acid, 400 mg of citric acid, 400 mg of succinic acid, and 60 mg of VC, place them in a 50 mL volumetric flask, dissolve and dilute to the scale with an extraction agent, shake well, and use as a stock solution. Accurately measure 100 μL of the stock solution and 900 μL of the test solution, shake well, and store in the dark.

[0029] 2. Chromatographic conditions 2.1 Selection of detection wavelength Prepare standard solutions of oxalic acid, quinic acid, tartaric acid, malic acid, lactic acid, citric acid, succinic acid, and VC with appropriate concentrations, and perform a full wavelength scan on the solutions using an ultraviolet-visible spectrophotometer.

[0030] The results showed that oxalic acid, quinic acid, tartaric acid, malic acid, lactic acid, citric acid, and succinic acid all showed end absorption. Considering that the detection wavelength of end absorption in liquid chromatography is easily interfered with, affecting the detection accuracy, the present invention selected 210 nm as the detection wavelength for organic acids. Under this condition, there was no interference from unknown peaks in the determination of target components; VC had a maximum absorption peak at 245 nm. To ensure the detection accuracy and convenience and improve the detection efficiency, this study used dual wavelengths (210 nm and 245 nm) to simultaneously determine organic acids and VC in kiwifruit.

[0031] 2.2, Selection of mobile phase Based on 0.1 v / v% phosphoric acid solution and 0.01 moL / L potassium dihydrogen phosphate solution, the present invention investigated the separation of organic acids and VC under different mobile phase conditions, and the results are shown in Table 1.

[0032] Table 1 Resolution of each component under different mobile phases (at 210 nm wavelength) Note: N / A in the table indicates that there is no resolution between the relevant component and the previous component.

[0033] Figure 1 is the chromatogram at 210 nm wavelength when the mobile phase is 0.1 v / v% phosphoric acid solution. Figure 2 is the chromatogram at 210 nm wavelength when the mobile phase is a mixed solution of 0.1 v / v% phosphoric acid solution - methanol at 95:5. From Table 1, Figure 1 and Figure 2 it can be seen that when the mobile phase is 0.1 v / v% phosphoric acid solution, the resolution between the tartaric acid peak and the quinic acid peak is 1.10 (<1.5), and the separation effect is not ideal; when the mobile phase is a mixed solution of 0.1 v / v% phosphoric acid solution - methanol at 95:5, the tartaric acid peak and the quinic acid peak completely overlap, and effective separation cannot be achieved; when the mobile phase is 0.01 moL / L potassium dihydrogen phosphate solution (pH 2.5), the resolution between each target component is >1.5, and the separation situation is good. Therefore, the present invention selected 0.01 moL / L potassium dihydrogen phosphate solution (pH 2.5) as the elution mobile phase.

[0034] 2.3, Selection of chromatographic column Since the Agilent ZORBAX Eclipse XDB-C18 (4.6 mm * 250 mm, 5 μm) chromatographic column has poor tolerance under 100 v / v% aqueous phase conditions, and the mobile phase of this study is a pure aqueous phase, a chromatographic column of the same specification with stronger tolerance, Ultimate® LP-C18, was replaced for subsequent experiments.

[0035] 2.4, Selection of column temperature Organic acids and VC are separated in the chromatographic column. Therefore, the column temperature of the chromatographic column is one of the factors affecting the separation effect. When the column temperature increases, the viscosity of the mobile phase decreases, and the interaction between the mobile phase carrying organic acids and VC and the chromatographic column packing is stronger, resulting in a faster separation speed. However, under high column temperature conditions, the mobile phase carrying organic acids and VC will volatilize, affecting the content determination.

[0036] According to the instruction manual of the Ultimate® LP-C18 chromatographic column, the maximum tolerable temperature of this chromatographic column is 75 °C. In the conventional detection methods of organic acids and VC, the column temperature is generally in the range of 20 °C to 40 °C. The detection condition with a column temperature of 30 °C is selected in the present invention.

[0037] 2.5. Flow rate condition The flow rate of the mobile phase is another factor affecting the peak emergence time and separation effect. Usually, when the flow rate increases, the peak emergence time of each substance advances, the separation rate increases, but the resolution decreases.

[0038] Table 2 Resolution of each component at different flow rates (wavelength: 210 nm) Note: N / A in the table indicates that there is no resolution between the relevant component and the previous component.

[0039] From the results in Table 2, it can be seen that under different flow rate conditions, the resolution between each component is greater than 1.5. However, at a flow rate of 0.6 mL / min, the resolution between quinic acid and tartaric acid is significantly improved compared to the flow rates of 0.7 mL / min and 0.8 mL / min. Although the resolution between each component is better at a flow rate of 0.5 mL / min, the peak emergence time is later. Considering comprehensively to improve the separation efficiency, the optimal flow rate is 0.6 mL / min.

[0040] 2.6. Selection of extractant VC is sensitive to changes in pH value. Therefore, 0.1 v / v% phosphoric acid solution, a mixed solution of 0.1 v / v% formic acid - 10 v / v% methanol, and 20 g / L metaphosphoric acid solution are used as extractants to investigate the stability of organic acids and VC under different extractants.

[0041] Table 3 Changes in the peak areas of each component in different extractant environments (%) Note: NND in the table indicates that there is no change in the peak area of the relevant component at 0 h.

[0042] From Figure 3As can be seen from Table 3, when a mixed solution of 0.1 v / v% formic acid - 10 v / v% methanol is used as the extractant, the resolution between the formic acid peak and the quinic acid peak is 1.04 (<1.5), so it will interfere with the detection of quinic acid; when the extractant is 0.1 v / v% phosphoric acid solution or a mixed solution of 0.1 v / v% formic acid - 10 v / v% methanol, the peak area change rate of VC in 4 hours is greater than 30%, and the stability is poor; while in 20 g / L metaphosphoric acid solution, the peak area change rate of VC in 12 hours is less than 5%, the peak area change rate in 24 hours is less than 10%, and the peak area change rate of 7 organic acids in 24 hours is less than 5%, indicating that the 7 organic acids and VC have good stability in this solution. Therefore, 20 g / L metaphosphoric acid solution is selected as the extractant.

[0043] 3. Results Verification 3.1 Specificity and System Precision Tests 10 μL of the positioning solutions of 7 organic acids and VC, the mixed standard solution, and the spiked test solution were respectively injected under the chromatographic conditions. Figure 4 is the chromatogram of 7 organic acids and VC in the mixed standard solution. Figure 5 is the chromatogram of 7 organic acids and VC in the spiked test solution. As can be seen from Table 4, Figure 4 , Figure 5 it can be known that the method provided by the present invention has good specificity: the resolution between substances is greater than 1.5, the peak purity is higher than 9000, and there is no interference from unknown components in the spiked test solution to the detection of target substances.

[0044] The retention time of oxalic acid is 5.15 min - 5.25 min, the retention time of tartaric acid is 5.93 min - 6.03 min, the retention time of quinic acid is 6.20 min - 6.30 min, the retention time of malic acid is 7.90 min - 8.00 min, the retention time of lactic acid is 9.80 min - 9.90 min, the retention time of citric acid is 16.00 min - 17.00 min, the retention time of succinic acid is 19.05 min - 19.15 min, and the retention time of vitamin C is 9.20 min - 9.30 min.

[0045] Table 4 Results of Specificity Tests Note: N / A in the table indicates that there is no resolution data for the relevant components.

[0046] The standard mixed solution was continuously injected 6 times to verify the system precision. As can be seen from Table 5, the method provided by the present invention has good system precision: the RSDs of the retention times and peak areas of 8 target components are all less than 2.0%.

[0047] Table 5 Results of System Precision Tests 3.2, Sensitivity and Linearity Experiments Inject mixed standard solutions with different concentrations. Plot a standard curve with the concentration including the quantitative limit concentration as the abscissa and the peak area as the ordinate. As can be seen from Table 6, the 8 target substances have good linear relationships within the standard curve range, and the correlation coefficients (r) are all greater than 0.999.

[0048] Table 6 Linear Test Results Determine the quantitative limit and detection limit of each substance according to the signal-to-noise ratio (S / N) results. The results show (Table 7) that this method has high sensitivity: the quantitative limit of oxalic acid is 8.2 ng, and the detection limit is 4.1 ng; the quantitative limit of tartaric acid is 79.4 ng, and the detection limit is 39.7 ng; the quantitative limit of quinic acid is 235.3 ng, and the detection limit is 78.4 ng; the quantitative limit of malic acid is 122.2 ng, and the detection limit is 81.5 ng; the quantitative limit of VC is 1.1 ng, and the detection limit is 0.40 ng; the quantitative limit of lactic acid is 395.4 ng, and the detection limit is 118.6 ng; the quantitative limit of citric acid is 242.8 ng, and the detection limit is 80.9 ng; the quantitative limit of succinic acid is 831.2 ng, and the detection limit is 249.4 ng.

[0049] Table 7 Quantitative Limit and Detection Limit Test Results 3.3, Solution Stability Experiment Take the spiked test solution and inject it for analysis at 0 h, 2 h, 4 h, 8 h, and 12 h at room temperature respectively. Calculate the peak area change rate. As can be seen from Table 8, the spiked test solution has good stability at room temperature for 12 hours: the peak area change rates of the 8 substances are all less than 5%.

[0050] Table 8 Peak Area Change of Spiked Test Solution Note: ND in the table indicates that there is no peak area change for the relevant component at 0 h.

[0051] 3.4, Accuracy Experiment Use the test solution as the background, add an appropriate concentration of mixed standard solution, and conduct accuracy test determination. The test results are shown in Table 9. From the test results, it can be seen that this method has good accuracy: the spiked recovery rates of the 8 substances are all in the range of 93.5% - 109.6%.

[0052] Table 9 Accuracy Test Determination Results Note: In the table, ND indicates that the background value concentration of the relevant component is lower than the detection limit, so no specific value is recorded.

[0053] 3.5 Detection of Organic Acids and VC in Kiwifruit of Different Varieties Take the mature fruits of 4 kiwifruit varieties, namely 'Taishan No. 1', 'Jintao', 'Longcheng No. 2', and 'Cuixiang', and analyze and evaluate their flavor nutrients by this method.

[0054] Table 10 Contents of Organic Acids and VC in Kiwifruit of Different Varieties Note: In the table, ND indicates that the corresponding organic acid component is not detected in the corresponding kiwifruit variety.

[0055] The results are shown in Table 10. Tartaric acid, lactic acid, and succinic acid are not detected in the 4 varieties of kiwifruit, and quinic acid and citric acid are the main organic acid components. The VC content is relatively high in 'Jintao' and 'Cuixiang', and the contents of 7 organic acids and VC are relatively low in 'Longcheng No. 2'. Based on the comprehensive experimental data, the contents of organic acids and VC in 'Jintao' kiwifruit are relatively high among the 4 varieties.

[0056] It should be noted that when the present invention involves a numerical range, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. Since the steps and methods adopted are the same as those in the embodiments, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit, characterized in that, It includes the following steps: Mix kiwifruit with metaphosphoric acid solution, centrifuge and collect the supernatant to obtain the test solution; Using a high-performance liquid chromatograph, under the chromatographic conditions: select a C18 column as the chromatographic column, the column temperature is 25°C - 35°C, the injection volume is 10 μL, isocratic elution is carried out with 0.01 mol / L potassium dihydrogen phosphate buffer as the mobile phase, and the flow rate is 0.5 mL / min - 0.8 mL / min. Detect organic acids and vitamin C in the test solution at wavelengths of 210 nm and 245 nm.

2. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, wherein The organic acid is any one or several of oxalic acid, tartaric acid, quinic acid, malic acid, lactic acid, citric acid and succinic acid.

3. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, characterized in that, The pH value of the potassium dihydrogen phosphate buffer is 2.0 - 3.

0.

4. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, characterized in that, The concentration of the metaphosphoric acid solution is 18 g / L - 22 g / L.

5. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, characterized in that, The mixing ratio of the kiwifruit to the metaphosphoric acid solution is 1 g:10 mL - 20 mL.

6. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, wherein The packing material of the C18 column is octadecylsilyl-bonded silica gel, and its specifications are: 5 μm, 4.6 mm × 250 mm.

7. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 6, characterized in that, The C18 column is selected from: Ultimate® LP-C18, SupersilAQ C18 or Shim-pack GIST C18-AQ.

8. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, characterized in that, The temperature of the centrifugation is 3°C - 5°C, the rotation speed of the centrifugation is 10000 r / min - 12000 r / min, and the time of the centrifugation is 10 min - 15 min.

9. The high performance liquid chromatography method for simultaneously determining organic acids and vitamin C in kiwifruit according to claim 1, characterized in that, The kiwifruit is kiwifruit ground into powder in liquid nitrogen.

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