Method for identifying different processed products of fresh bamboo juice by detecting mass concentration ratio of sugar component based on HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector) method and application

Abstract

The invention discloses a method for identifying different processed products of fresh bamboo juice by detecting the mass concentration ratio of sugar components based on an HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector) method and application. The method comprises the following steps: taking a processed fresh bamboo juice stock solution, and adding acetonitrile to prepare a fresh bamboo juice test solution; taking reference substances such as fructose, glucose and cane sugar, and adding acetonitrile to prepare a sugar mixed reference substance solution; injecting the fresh bamboo juice test solution into an HPLC-ELSD system, and detecting the content of sugar components; and calculating the mass concentration ratio of fructose to glucose to cane sugar, and identifying the processing technology of the fresh bamboo juice test solution according to the mass concentration ratio range of sugar components in the fresh bamboo juice stock solution obtained by different processing methods. According to the sugar content detection method, the variable spinning isomerism of glucose is eliminated, the analysis time is shortened to 15 minutes, the content difference of solid contents of different processed products can be eliminated by using the mass concentration ratio of sugar components, different processing technologies of the fresh bamboo juice can be quickly and effectively distinguished, and a support is provided for improving the quality standard of the fresh bamboo juice and guaranteeing the curative effect of the fresh bamboo juice.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and in particular relates to a method and application thereof for detecting the mass concentration ratio of sugar components based on an HPLC-ELSD method to identify different processed products of fresh bamboo sap. Background Art

[0002] Fresh bamboo sap refers to the light yellow clear liquid that naturally leaches out from the fresh stems of Phyllostachys glauca MeGlure, Phyllostachys nuda MeGlure and plants of the same genus after heating. It has the effect of clearing away heat and resolving phlegm. Fresh bamboo sap was first recorded as "bamboo juice" in the "Shennong Bencao Jing" of the Han Dynasty, and later called "bamboo sap", "light bamboo sap", "sweet bamboo sap" and "bamboo oil". It is called "fresh bamboo sap" in the 1977 edition of the "Chinese Pharmacopoeia" in modern times. Fresh bamboo sap is cold in nature, sweet and bitter in taste, enters the heart and stomach meridians, has the effects of clearing heat and resolving phlegm, calming the nerves and benefiting the orifices, and can be used to treat symptoms such as lung heat cough and sputum, asthma and chest tightness, tongue stiffness in stroke, phlegm-heat convulsions in children, etc., and is known as the "holy medicine for phlegm". In addition, it is also the main raw material for common Chinese medicine preparations such as fresh bamboo sap, compound fresh bamboo sap liquid, Qutanling oral liquid, and Zhuli Datan pills. Clinical observations have shown that fresh bamboo sap preparations have therapeutic effects on pharyngitis, respiratory tract infections, bronchitis, chronic obstructive pulmonary disease, and lung lesions caused by stroke. They can also improve the prognosis of patients after surgery and craniocerebral trauma.

[0003] There are two main traditional methods for preparing fresh bamboo sap. One is the direct burning method, which involves cutting the bamboo into pieces and placing them on two opposing bricks, roasting the sap with fire, and then collecting it on a plate. The other is the charcoal fire forced method, which involves hanging the bamboo inverted on a plate, cutting the bamboo into pieces five or six inches long, hanging them inverted in a bottle, and using a container to force the charcoal fire around them, so that the oil can flow into the container. However, in order to increase production and reduce costs, the market is currently flooded with fresh bamboo sap that is prepared by boiling water instead of the dry distillation method or direct fire burning method specified in the standard. The inconsistency in the processing technology of fresh bamboo sap has seriously affected the product quality and clinical efficacy of fresh bamboo sap. Therefore, clarifying the material basis and unifying the processing methods will help standardize the fresh bamboo sap market, while effectively controlling its quality and efficacy, and promoting the better inheritance and application of the ancient processing technology.

[0004] Sugars are considered to be the main components of fresh bamboo sap, accounting for 33.1% to 82.7% of the solid content, which is much higher than the content of other compounds. The sugar content in fresh bamboo sap prepared by different processing techniques varies greatly. Studies have shown that the processing process will have a great impact on the sugar content and monosaccharide mass concentration ratio of Chinese medicinal materials. Therefore, the control of the sugar component of fresh bamboo sap is expected to become a quality control indicator reflecting the processing technology of fresh bamboo sap. Research on the quality control method of sugar-solid ratio of fresh bamboo sap and fresh bamboo extract (Chen Chengyu et al., Chinese Pharmaceutical Standards, 2021.22(06):p.503-509) established an analytical method for sugar-solid ratio. The sugar-solid ratio was used to identify fresh bamboo sap prepared by legal process (burning, dry distillation method) and fresh bamboo extract prepared by non-legal process (water extraction method). It was proposed that sugars are potential markers for identifying fresh bamboo sap. The disadvantages of this method are that the sample pretreatment method is cumbersome and has poor specificity, and it cannot effectively distinguish between processed products processed by the firing method and the dry distillation method. Since fresh bamboo sap is both a preparation and a decoction piece, the solid content between different batches of samples varies greatly. If the sugar content is used directly as a quality control indicator, it is necessary to eliminate the difference in solid content caused by the different degree of concentration during the preparation of fresh bamboo sap.

[0005] In addition, the existing HPLC-ELSD method for detecting the sugar components in fresh bamboo sap concentrate takes a long time to analyze, and the mutarotation isomerization of glucose on the chromatographic column interferes with the detection results. It is impossible to efficiently confirm the processing technology of the fresh bamboo sap concentrate, and thus cannot provide strong support for the quality control and efficacy assurance of fresh bamboo sap products on the market.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In order to solve the problem that the existing fresh bamboo sap has different sugar contents due to different concentration levels, which requires additional calculation of the solid content and thus causes operational errors, and at the same time to solve the problems in the prior art of long analysis time when detecting sugar components in fresh bamboo sap stock solution by HPLC-ELSD, and interference of glucose mutarotation isomers on the chromatographic column on the detection results, the present invention provides a method for detecting the mass concentration ratio of sugar components based on the HPLC-ELSD method to identify different processed fresh bamboo sap products.

[0008] The content of free sugar in fresh bamboo sap was detected by HPLC-ELSD, and the proportion of each sugar component was calculated to identify and distinguish the different processing techniques of fresh bamboo sap stock solution. This method can effectively distinguish the fresh bamboo sap stock solutions prepared by three processing techniques: dry distillation, burning and water extraction.

[0009] To achieve the above object, the present invention provides a method for detecting the mass concentration ratio of sugar components based on HPLC-ELSD method to identify different processed products of fresh bamboo sap, comprising the following steps:

[0010] (1) Preparation of fresh bamboo sap test solution: Take the fresh bamboo sap stock solution obtained by the processing method, add acetonitrile as a solvent, and prepare the fresh bamboo sap test solution;

[0011] (2) Preparation of mixed sugar reference solution: Take reference substances fructose, glucose and sucrose, add acetonitrile as solvent, and prepare mixed sugar reference solution;

[0012] (3) HPLC-ELSD detection: The fresh bamboo sap test solution is injected into the HPLC-ELSD system to detect the sugar component content in the fresh bamboo sap test solution, wherein the sugar component is fructose, glucose and sucrose;

[0013] (4) Calculation of the mass concentration ratio of sugar components: Calculate the mass concentration ratio of fructose: glucose: sucrose in the fresh bamboo sap stock solution. According to the mass concentration ratio range of sugar components in the fresh bamboo sap stock solution obtained by different processing methods, identify the processing technology of the fresh bamboo sap test solution, and thus determine the processing method of the fresh bamboo sap processed product.

[0014] In the above step (3), the peak areas of glucose, fructose and sucrose in the fresh bamboo sap sample are obtained by HPLC-ELSD detection. The contents of glucose, fructose and sucrose in the fresh bamboo sap stock solution, the dry distillation method fresh bamboo sap stock solution, the burning method fresh bamboo sap stock solution and the water extraction method fresh bamboo sap stock solution are calculated according to the linear relationship between glucose, fructose and sucrose, thereby calculating the mass concentration ratio of fructose, glucose and sucrose. Among them, the linear relationship of each sugar component is:

[0015] The standard curve of fructose is y=1.4043x+4.0247, R 2 =0.9991, linear range is 0.06520~4.170mg·mL -1 ;

[0016] The standard curve of glucose is y=1.3666x+3.9596, R 2 =0.9994, linear range is 0.06415~4.102mg·mL -1 ;

[0017] The standard curve of sucrose is y=1.4151x+4.0659, R 2 =0.9993, linear range is 0.06275~4.014mg·mL -1 .

[0018] In one or more preferred embodiments, the above-mentioned processing method is any one of dry distillation, firing or water extraction.

[0019] In one or more preferred embodiments, the specific preparation steps of the fresh bamboo sap test solution include: measuring the fresh bamboo sap stock solution into a volumetric flask, adding acetonitrile, passing through a PVDF filter membrane, and taking the filtrate to obtain the fresh bamboo sap test solution; wherein the volume ratio of the fresh bamboo sap stock solution to acetonitrile is (0.5-1.5):1.

[0020] In one or more preferred embodiments, the acetonitrile in the above step (1) is 100% acetonitrile by volume.

[0021] In one or more preferred embodiments, the volume ratio of the fresh bamboo sap stock solution to acetonitrile is 1:1.

[0022] In one or more preferred embodiments, the acetonitrile in the above step (2) has a volume percentage concentration of 50%.

[0023] The purpose of using 50% acetonitrile to dissolve the sample is to remove macromolecular substances in the sample, such as proteins, polysaccharides, etc., to prevent the precipitation of macromolecular substances due to the increase in the proportion of organic phase when the sample is injected into the liquid phase system. After multiple injections for a long time, the precipitate is deposited on the column, which will cause certain damage to the column; at the same time, it can also reduce the solvent effect to a certain extent.

[0024] In one or more preferred embodiments, the chromatographic conditions in the above-mentioned HPLC-ELSD system include:

[0025] Chromatographic column: using triple-bonded amide groups based on ethylene bridge hybrid particles (BEH) as filler; preferably, the chromatographic column is a Waters Amide Column chromatographic column (4.6 mm×250 mm, 3.5 μm);

[0026] Mobile phase: 75% acetonitrile-water;

[0027] Isocratic elution: flow rate 1.0 mL / min;

[0028] Detector: Evaporative Light Scattering Detector (ELSD).

[0029] In one or more preferred embodiments, the column temperature of the chromatographic column is 35° C., and the injection volume is 10 μL.

[0030] In one or more preferred embodiments, the 75% acetonitrile-water contains triethylamine at a volume percentage concentration of 0.1%.

[0031] In one or more preferred embodiments, the evaporation temperature of the evaporative light scattering detector is 50°C.

[0032] In one or more preferred embodiments, the drift tube temperature of the evaporative light scattering detector is 50° C., the carrier gas flow rate is 1.6 L·min-1, and the gain is 1.

[0033] In one or more preferred embodiments, the mass concentration ratio of the sugar components in the fresh bamboo sap solution obtained by the above processing method is as follows:

[0034] When the preparation method is dry distillation, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.0852-0.456) : 1 : (0-0.116);

[0035] When the processing method is the burning method, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.753-1.04) : 1 : (0.297-4.73);

[0036] When the preparation method is water extraction, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.640-0.709):1:(0.133-0.284).

[0037] The present invention also provides an application of the method in identifying different processed products of fresh bamboo sap.

[0038] The above identification method can identify the processing method of the fresh bamboo sap sample to be tested, and can especially identify the three processing techniques of fresh bamboo sap: dry distillation, burning and water extraction.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The present invention measures the ratio of fructose, glucose and sucrose contained in fresh bamboo sap, determines the ratio range of fructose, glucose and sucrose in fresh bamboo sap obtained by dry distillation, burning and water extraction, and then distinguishes the processing methods of fresh bamboo sap according to the ratio range; compared with the prior art, the method has simple operation steps, and the difference in solid content between different processed products can be eliminated by calculating the concentration ratio of fructose, glucose and sucrose, and there is no need to calculate the solid content additionally, thus avoiding the introduction of operation errors, and can quickly and effectively distinguish the different processing techniques of fresh bamboo sap, providing a material basis and technical support for the processing technique, quality standard improvement and efficacy guarantee of fresh bamboo sap; and solves the technical problems in the prior art that the sample pretreatment method of fresh bamboo sap is complicated, the specificity is poor, and the solid content between different batches of samples is large, and when only the sugar content is used as the quality control indicator, the difference in solid content caused by the different concentration degree in the preparation process of fresh bamboo sap needs to be eliminated.

[0041] (2) The present invention shortens the analysis time to 15 minutes through the combined effects of chromatographic column screening, mobile phase mass concentration ratio optimization, and ELSD detector condition optimization, which is significantly shorter than the current analysis time of 45 minutes for determining monosaccharides and disaccharides in fresh bamboo sap.

[0042] (3) The present invention adds triethylamine to the mobile phase, thereby eliminating the mutarotation isomerism of glucose molecules on the chromatographic column; the inventors improve the linear range of sugar detection by optimizing the setting parameters of the detector, such as the atomizer and drift tube temperature and carrier gas flow rate of the ELSD detector. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the HPLC-ELSD graph of sugar mixed reference substance and fresh bamboo sap stock solution prepared by different processing methods;

[0044] Figure 2 is a fructose standard curve according to Example 1 of the present invention;

[0045] Figure 3 is a glucose standard curve diagram according to Example 1 of the present invention;

[0046] Figure 4 is a sucrose standard curve according to Example 1 of the present invention;

[0047] Figure 5 This is a summary of the ratio of fructose and glucose in fresh bamboo sap liquid with different processing methods (GL: dry distillation; SZ: burning; ST: water extraction);

[0048] Figure 6 This is a summary of the ratio of sucrose and glucose in fresh bamboo sap liquid with different processing methods (GL: dry distillation; SZ: burning; ST: water extraction);

[0049] Figure 7 Optimization of sugar chromatography analysis conditions - comparison of different mobile phases. DETAILED DESCRIPTION

[0050] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0051] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0052] Instruments and reagents

[0053] 1. Instruments: 1260Ⅱ high performance liquid chromatograph (Agilent, USA), 1290Ⅱ evaporative light scattering detector (Agilent, USA), BT25S 1 / 100000 electronic balance (Sartorius, Germany).

[0054] 2. Test drug: 30 batches of fresh bamboo sap stock solution (including 10 batches of samples prepared by dry distillation, burning and water extraction), of which the dry distillation and burning samples were provided by a pharmaceutical company in Jiangxi, and 10 batches of water extraction samples were prepared in accordance with the "Fresh Bamboo Extract" item of the "Hunan Provincial Traditional Chinese Medicine Standard" (2009 edition). All fresh bamboo sap stock solutions are from Phyllostachys genus (Phyllostachys) of the Poaceae family (P. heterocycla (Carr.) Mitford cv. Pubescens).

[0055] D-anhydrous glucose reference (batch number: 110833-201707, ID: JGNY-QGLJ, purity: 99.9%), D-fructose reference (batch number: 100231-201807, ID: JEPK-W6GQ, purity: 99.4%) and sucrose reference (batch number: 111507-201704, ID: LCMX-DGTC, purity: 99.8%) were purchased from China Food and Drug Inspection Institutes.

[0056] The water was ultrapure water, and the other reagents were of analytical grade.

[0057] Example 1: Method for detecting the mass concentration ratio of sugar components based on HPLC-ELSD method to identify different processed fresh bamboo sap products (1) Preparation of test solution

[0058] Accurately measure 5 ml of fresh bamboo sap stock solution into a 10 ml volumetric flask, add an equal volume of 100% acetonitrile to the scale, shake well, pass through a 0.22 μM PVDF filter membrane, and take the filtrate to obtain the fresh bamboo sap test solution.

[0059] An equal volume of water was used to replace the fresh bamboo sap stock solution and the sample prepared according to the above method was used as a blank control.

[0060] (2) Preparation of sugar mixed reference solution

[0061] Take appropriate amount of fructose, glucose and sucrose reference substances, weigh them accurately, place them in a 10mL volumetric flask, add 50% acetonitrile-water solution to dilute to the mark, shake well, and prepare a 4.170mg·mL volumetric flask containing D-fructose, D-anhydrous glucose and sucrose, respectively. -1 , 4.102mg·mL -1 and 4.014 mg·mL -1 Sugar mixed reference solution①.

[0062] (3) Sugar chromatography analysis conditions

[0063] A Waters Amide Column (4.6 mm × 250 mm, 3.5 μm) was used, column temperature 35°C; 75% acetonitrile-water (containing 0.1% triethylamine), flow rate 1.0 mL min -1 , isocratic elution; ELSD parameters: evaporation temperature 50°C, drift tube temperature 50°C, carrier gas flow rate 1.6 L·min -1 , gain 1; injection volume 10μL.

[0064] Under the above chromatographic analysis conditions (3), other chromatographic peaks in the fresh bamboo sap sample had no interference with the fructose, glucose and sucrose peaks. Figure 1 .

[0065] (4) Linear relationship investigation

[0066] Accurately pipette 1 ml of the above-mentioned sugar mixed reference solution ① into a 2 mL volumetric flask, add 50% acetonitrile-water solution to dilute to the mark, and shake well to obtain mixed reference solution ②; dilute in half step by step according to the above method to obtain sugar mixed reference solutions ③ to ⑦ in turn; inject 10 μL of sugar mixed reference solutions ① to ⑦ in turn, and measure according to the above-mentioned sugar chromatographic analysis conditions (3).

[0067] The sample concentration (mg.mL -1 ) as the abscissa (x), and the logarithm of the peak area as the ordinate (y), and a standard curve is drawn to obtain a linear equation, see Figure 2-Figure 4 The results showed that fructose, glucose and sucrose had a good linear relationship within a certain range, as shown in Table 1.

[0068] Table 1 Results of linear relationship investigation of various components in fresh bamboo sap

[0069]

[0070] Note: a In the standard curve equation, y is the logarithm of the peak area, and x is the sample concentration (mg.mL -1 ) is the logarithm of .

[0071] (5) Precision test

[0072] Take the above-mentioned sugar mixed reference solution ④, pass it through a 0.22 μm PVDF filter membrane, and continuously inject it 6 times according to the above-mentioned chromatographic analysis conditions (3), record the peak area, and calculate the RSD of the fructose, glucose, and sucrose peak areas in the fresh bamboo sap mixed reference solution ④ to be 0.8%, 1.2%, and 1.1%, respectively (see Table 2), and the RSDs are all less than 5.0%, indicating that the instrument has good precision.

[0073] Table 2 Precision test results

[0074]

[0075] (6) Repeatability test

[0076] Take a batch of fresh bamboo sap stock solution prepared by dry distillation, burning method and water extraction method (dry distillation method: XZL-GL-01; burning method: XZL-SZ-01; water extraction method: XZL-ST-01) respectively, prepare 6 batches of fresh bamboo sap sample solutions in parallel according to the above-mentioned sample solution preparation method for each batch of samples, and perform the test according to the above-mentioned chromatographic analysis conditions (3). Calculate the content of fructose, glucose and sucrose in each batch of fresh bamboo sap samples, and the RSD of sugar content, and the results are shown in Table 3. The RSD of fructose, glucose and sucrose in fresh bamboo sap samples prepared by different processing methods are all less than 5.0%, indicating that the method has good repeatability.

[0077] Table 3 Repeatability test results

[0078]

[0079] (7) Stability test

[0080] Take an appropriate amount of each batch of fresh bamboo sap stock solution prepared by dry distillation, burning method and water extraction method (dry distillation method: XZL-GL-01; burning method: XZL-SZ-01; water extraction method: XZL-ST-01), prepare fresh bamboo sap test solution according to the above test solution preparation method, and inject and analyze according to the above chromatographic analysis conditions (3) at 0h, 2h, 4h, 8h, 12h and 24h, record the peak area respectively, and calculate the RSD of the peak area of ​​fructose, glucose and sucrose in each fresh bamboo sap stock solution, see Table 4.

[0081] The results showed that the RSDs of the fructose, glucose and sucrose peaks in the three processed fresh bamboo sap were all less than 5.0%, indicating that the three processed solutions had good stability within 24 hours.

[0082] Table 4 Stability test results

[0083]

[0084] (8) Sample recovery test

[0085] Take a batch of fresh bamboo sap prepared by different processing methods (dry distillation method: XZL-GL-01; burning method: XZL-SZ-01; water extraction method: XZL-ST-01), 3 portions per batch, a total of 9 portions, add equal amounts of fructose, glucose and sucrose reference substances, dilute to one volume. Prepare fresh bamboo sap test solution according to the above test solution preparation method, and measure according to the above chromatographic analysis conditions (3), calculate the sample recovery rate of each sample and the average sample recovery rate of each sugar in the 9 fresh bamboo sap samples and the RSD of the sample recovery rate, the results are shown in Table 5.

[0086] The results showed that the average recoveries of fructose, glucose and sucrose in 9 fresh bamboo sap samples were 101.0%, 103.8% and 100.6% respectively, and the average recoveries were between 95% and 105%, and the RSDs were 1.8%, 0.7% and 2.5% respectively, and the RSDs were all less than 5.0%. Accuracy refers to the degree of proximity between the results measured by this method and the true value or recognized reference value. The accuracy of the preparation detection method is generally verified by the sample recovery experiment. In Table 5, the sample recovery % = (measured amount × dilution factor - sample content) / added amount × 100, where the dilution factor is 2. Therefore, the above results show that the accuracy of this method is good.

[0087] Table 5 Sample recovery test results (n = 9)

[0088]

[0089]

[0090] Example 2: Content detection and analysis of different processed products of fresh bamboo sap

[0091] Ten batches of fresh bamboo sap prepared by dry distillation, burning and water extraction were taken, and fresh bamboo sap test solutions were prepared according to the above test solution preparation method. The samples were determined according to the above chromatographic analysis conditions (3). The contents of fructose, glucose and sucrose in each fresh bamboo sap sample were calculated by peak area according to the standard method. The results are shown in Table 6. Among them, the ratios of fructose: glucose: sucrose in fresh bamboo sap prepared by dry distillation, burning and water extraction were (0.0852-0.456): 1: (0-0.116); (0.753-1.04): 1: (0.297-4.73); (0.640-0.709): 1: (0.133-0.284).

[0092] Depend on Figure 5 and Figure 6It can be seen that the content ratio of fructose:glucose in the fresh bamboo sap prepared by three different processing methods is significantly different (P<0.001), among which the content ratio of sucrose:glucose in the burned fresh bamboo sap sample is significantly different from that in the samples prepared by the other two processing methods, and the proportion of sucrose in the fresh bamboo sap samples obtained by dry distillation and water extraction is low, so the difference is not obvious. It can be seen that the sugar ratio in fresh bamboo sap has a certain correlation with its processing method, and the processing method of fresh bamboo sap can be identified by the sugar ratio range.

[0093] Table 6 Sugar content test results of different processed products

[0094]

[0095]

[0096]

[0097] Note: Fru: fructose; Glu: glucose; Suc: sucrose

[0098] Example 3: Detection and analysis of commercially available fresh bamboo sap products

[0099] Thirteen batches of fresh bamboo sap products from different manufacturers were collected from the market. Fresh bamboo sap test solutions were prepared according to the above test solution preparation method. The samples were determined according to the above chromatographic analysis conditions (3) to calculate the contents of glucose, fructose and sucrose and the sugar ratios in the fresh bamboo sap. The preparation methods of the fresh bamboo sap samples were determined according to the ratio ranges of fructose, glucose and sucrose set in Example 2. The results are shown in Table 7.

[0100] The results showed that only 5 of the 13 batches of fresh bamboo sap samples had a fructose: glucose and sucrose: glucose ratio within the proposed range, and most samples only had one sugar component that met the ratio requirements, and it is possible that free sugars were added to the commercially available samples. Therefore, the processing method judgment results showed that most of the commercially available fresh bamboo sap products were prepared by burning and dry distillation, and no samples were prepared by water extraction.

[0101] Table 7 Sugar determination results and analysis of commercially available fresh bamboo sap products

[0102]

[0103]

[0104] Note: Fru: fructose; Glu: glucose; Suc: sucrose

[0105] Comparative Example 1: Comparison of the effects of different mobile phases on mutarotation

[0106] The sugar chromatography analysis conditions used in the comparative example are the same as those in Example 1 (3), with the only difference being that the mobile phase in Example 1 is 75% acetonitrile-water (containing 0.1% triethylamine), while the mobile phase in the comparative example does not contain 0.1% triethylamine, i.e., is only 75% acetonitrile-water.

[0107] Figure 7 This is a comparison chart of different mobile phases for optimization of sugar chromatography analysis conditions. In the figure, 1 is fructose, 2 is glucose, and 3 is sucrose. Figure 7 It can be seen that if triethylamine is not added, glucose will be separated into two peaks on the chromatographic column, affecting quantification.

[0108] Comparative Example 2: Comparison of the effects of different detection conditions on analysis time

[0109] The chromatographic column, mobile phase and ELSD detector conditions were optimized. Compared with the existing conditions of Comparative Example 2 shown in Table 8, a Waters Amide Column chromatographic column, 75% acetonitrile-water (containing 0.1% triethylamine) was used as the mobile phase, the drift tube temperature of the ELSD detector was 50°C, and the carrier gas flow rate was 1.6 L min -1 Under these conditions, the detection and analysis time of fresh bamboo sap was reduced to 15 minutes, reducing the experimental cost.

[0110] Table 8 Comparison of the impact of different detection conditions on analysis time

[0111]

[0112]

[0113] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for identifying different processed products of fresh bamboo sap by detecting the mass concentration ratio of sugar components based on HPLC-ELSD method, characterized in that: The following steps are involved: (1) Preparation of fresh bamboo sap test solution: Take the fresh bamboo sap stock solution obtained by the processing method, add acetonitrile as a solvent, and prepare the fresh bamboo sap test solution; (2) Preparation of mixed sugar reference solution: Take reference substances fructose, glucose and sucrose, add acetonitrile as solvent, and prepare mixed sugar reference solution; (3) HPLC-ELSD detection: The fresh bamboo sap test solution is injected into the HPLC-ELSD system to detect the sugar component content in the fresh bamboo sap test solution, wherein the sugar component is fructose, glucose and sucrose; (4) Calculation of the mass concentration ratio of sugar components: Calculate the mass concentration ratio of fructose: glucose: sucrose in the fresh bamboo sap stock solution. According to the mass concentration ratio range of sugar components in the fresh bamboo sap stock solution obtained by different processing methods, identify the processing technology of the fresh bamboo sap test solution, and thus determine the processing method of the fresh bamboo sap processed product.

2. The method according to claim 1, characterized in that The processing method is any one of dry distillation, burning or water extraction.

3. The method according to claim 1, characterized in that The specific preparation steps of the fresh bamboo sap test solution in step (1) include: measuring fresh bamboo sap stock solution into a volumetric flask, adding acetonitrile, filtering through a PVDF filter membrane, and taking a subsequent filtrate to obtain the fresh bamboo sap test solution, wherein the volume ratio of the fresh bamboo sap stock solution to acetonitrile is (0.5-1.5) : 1; preferably, the volume ratio of the fresh bamboo sap stock solution to acetonitrile is 1 : 1; Preferably, the acetonitrile is 100% acetonitrile by volume; In step (2), the acetonitrile has a volume percentage concentration of 50%.

4. The method according to claim 1, characterized in that: The chromatographic conditions in the HPLC-ELSD system in step (3) include: Chromatographic column: using triple-bonded amide groups based on ethylene bridge hybrid particles (BEH) as filler; preferably, the chromatographic column is a Waters Amide Column chromatographic column (4.6 mm×250 mm, 3.5 μm); Mobile phase: 75% acetonitrile-water; Isocratic elution: flow rate 1.0 mL / min; Detector: Evaporative Light Scattering Detector (ELSD).

5. The method according to claim 4, characterized in that The column temperature of the chromatographic column was 35° C., and the injection volume was 10 μL.

6. The method according to claim 4, characterized in that The 75% acetonitrile-water contains triethylamine with a volume percentage concentration of 0.1%.

7. The method according to claim 4, characterized in that The evaporation temperature of the evaporative light scattering detector is 50°C.

8. The method according to claim 4, characterized in that The drift tube temperature of the evaporative light scattering detector was 50° C., the carrier gas flow rate was 1.6 L / min, and the gain was 1.

9. The method according to claim 1, characterized in that: The mass concentration ratio range of the sugar components in the fresh bamboo sap stock solution obtained by the processing method is as follows: When the preparation method is dry distillation, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.0852-0.456) : 1 : (0-0.116); When the processing method is the burning method, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.753-1.04) : 1 : (0.297-4.73); When the preparation method is water extraction, the mass concentration ratio of fructose, glucose and sucrose in the fresh bamboo sap stock solution is (0.640-0.709):1:(0.133-0.284).

10. Use of the method according to any one of claims 1 to 9 in identifying different processed products of fresh bamboo sap.

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