CN115166088A - Method for identifying blended fresh bamboo juice based on linear relation of sugar content - Google Patents
Method for identifying blended fresh bamboo juice based on linear relation of sugar content Download PDFInfo
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Abstract
The method for identifying and blending fresh bamboo juice based on the linear relation of sugar content comprises the following steps of firstly constructing a chromatographic condition and system applicability test, then preparing a solution of the fresh bamboo juice to be identified, then accurately absorbing a proper amount of the solution of the fresh bamboo juice to be identified for a plurality of times, respectively injecting the solution of the fresh bamboo juice to be identified into a liquid chromatograph, determining the quantitative limits of fructose, glucose and sucrose in the solution of the fresh bamboo juice to be identified under the constructed chromatographic condition and system applicability test, and finally determining the proportion of the fructose, the glucose and the sucrose in the fresh bamboo juice prepared by an ancient method direct firing according to the linear relation of sugar content extracted by screening to be 1.7-1.3; when the ratio of fructose, glucose and sucrose in the fresh bamboo juice to be identified is not in the range, the blended fresh bamboo juice is obtained; compared with the prior art, the method has the advantages that the operation steps are effectively simplified, qualitative and quantitative analysis on the sugar content in the fresh bamboo juice can be simply and quickly realized, the accuracy rate of identifying and blending the fresh bamboo juice is high, and the method has good application prospects.
Description
Technical Field
The invention relates to the technical field of fresh bamboo juice detection, in particular to a method for identifying blended fresh bamboo juice based on a linear relation of sugar content.
Background
Draining fresh bamboo, cutting off fresh bamboo, standing, burning the middle part of the bamboo, allowing light yellow juice at two ends of the bamboo to flow out, and holding in a container to obtain succus Bambusae with bamboo fragrance, sweet and cold nature and taste, and wind spasm; according to the recipe, the medicine can dispel wind, reduce pathogenic fire, moisten dryness, remove phlegm, nourish blood, benefit yin, benefit orifices, improve eyesight, treat vomiting due to apoplexy, phlegm-induced coma, mania due to wind spasm, vexation, diabetes and blood deficiency spontaneous perspiration; therefore, fresh succus Bambusae has effects of clearing heat, eliminating phlegm, arresting convulsion and benefiting orifices, and can be used for treating severe infection, lung heat expectoration, asthma, chest distress, apoplexy, tongue stiffness, excessive phlegm and saliva, infantile phlegm heat convulsion, etc.
Fresh succus Bambusae contains sugar, amino acids, phenol, etc., and the sugar can be used as main energy substance for cell life activity, and has obvious immunity promoting effect. The investigation of fresh bamboo juice sold in the market shows that part of fresh bamboo juice is directly blended and processed by glucose, fructose or sucrose, which seriously affects the quality of fresh bamboo juice and its preparation.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for identifying blended fresh bamboo juice based on a linear relation of sugar content, so as to solve the problems in the background technology.
The technical problem solved by the invention is realized by adopting the following technical scheme:
the method for identifying blended fresh bamboo juice based on the linear relation of sugar content comprises the following steps:
1) Test for establishing chromatographic conditions and system applicability
2) Preparing the fresh bamboo juice solution to be identified
Precisely measuring a proper amount of fresh bamboo juice to be identified, placing the fresh bamboo juice in a measuring flask, adding water to dilute the fresh bamboo juice to a scale, and shaking up to obtain a fresh bamboo juice solution to be identified;
3) Measuring fresh bamboo juice solution to be identified
Precisely absorbing a proper amount of fresh bamboo juice solution to be identified for several times, respectively injecting into a liquid chromatograph, and determining the quantitative limits of fructose, glucose and sucrose in the fresh bamboo juice solution to be identified under the chromatographic conditions and the system applicability test constructed in the step 1);
4) Judging fresh bamboo juice to be identified
Linear relation of sugar content is selected and extracted: the regression equation of the fructose is y =1.2071x +5.1752 2 =0.9994, fructose is in good linearity between 0.813552 and 4.067760 mu g; the regression equation of glucose is y =1.1898x +4.9832 2 =0.9996, glucose is in good linearity between 1.056942 and 5.284710 mug; the regression equation for sucrose is y =1.1887x +5.0956 2 =0.9993, sucrose presents good linearity between 1.093808-5.469040 μ g, the ratio of fructose, glucose and sucrose in the fresh bamboo juice prepared by the ancient method direct firing is 1.7-1.3 according to the linear relation of sugar content; when the ratio of fructose, glucose and sucrose in the fresh bamboo juice to be identified, which is measured in the step 3), is not between 1.0 and 1.3.
In the present invention, the liquid chromatograph is an LC 20AT high performance liquid chromatograph.
In the invention, the acetonitrile is chromatographically pure, the water is the drinking pure water of Waohaha, and other reagents are analytically pure.
Has the advantages that: the method extracts the linear relation of sugar content, measures the proportion of fructose, glucose and sucrose in the fresh bamboo juice prepared by direct firing through an ancient method according to the linear relation of sugar content, and further establishes the method for quickly identifying the true and false fresh bamboo juice based on the linear relation of sugar content.
Drawings
FIG. 1 is a HPLC-ELSD chromatogram of a mixed control solution in a preferred embodiment of the invention.
FIG. 2 is a HPLC-ELSD chromatogram of a test solution in a preferred embodiment of the present invention.
FIG. 3 is a negative control solution HPLC-ELSD chromatogram of a preferred embodiment of the invention.
FIG. 4 is a schematic diagram of a fructose standard curve in a preferred embodiment of the present invention.
FIG. 5 is a schematic diagram of a glucose calibration curve according to a preferred embodiment of the present invention.
FIG. 6 is a schematic diagram of a sucrose calibration curve in accordance with a preferred embodiment of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.
The method for identifying blended fresh bamboo juice based on the linear relation of sugar content comprises the following steps:
1. instruments and reagents
An LC 20AT high performance liquid chromatograph, an Alltech3300 evaporative light scattering detector, and a one-hundred-thousandth electronic balance of Sartorius BT 25S type;
d-fructose reference substances (batch number: 100231-201305, purity: 99.4%), D-anhydrous glucose reference substances (batch number: 110833-201205, purity: 99.5%), sucrose reference substances (batch number: 111507-201303, purity: 99.8%) and all the D-fructose reference substances are purchased from China food and drug testing research institute; baking the obtained fresh bamboo juice sample (batch number: 20180822);
acetonitrile is chromatographically pure, water is the pure drinking water of Wahaha, and other reagents are analytically pure;
2. preparation of reagents
2.1 chromatographic conditions and System suitability test
2.2 preparation of control solutions
Respectively taking appropriate amount of fructose, anhydrous glucose and sucrose as reference substances, adding water to obtain solutions containing 0.2mg of fructose per 1ml as mixed reference substance solutions;
2.3 preparation of test solutions
Precisely measuring 2ml of the fire-baked fresh succus Bambusae sample (lot number: 20180822), placing in a 50ml measuring flask, adding water to dilute to scale, and shaking to obtain sample solution;
2.4 preparation of negative control solution
Preparing negative sample from other medicinal materials without succus Bambusae according to process, and preparing into negative control solution according to the preparation method of test solution;
2.5 assay method
Precisely sucking 10 μ l of each of the reference solution, the sample solution and the negative reference solution, injecting into a liquid chromatograph, and measuring to obtain chromatograms shown in FIGS. 1-3;
3. linear relation of screened and extracted sugar content
Respectively injecting 5 μ l, 10 μ l, 15 μ l, 20 μ l and 25 μ l of the reference solution into liquid chromatograph, and measuring peak area according to text chromatogram conditionTaking the common logarithm of peak area as the ordinate and the common logarithm of sample amount of reference substance as the abscissa, drawing a standard curve, wherein the regression equation of fructose is y =1.2071x +5.1752 2 Results are shown in table 1 and fig. 4, indicating that fructose is in good linearity between 0.813552 and 4.067760 μ g; the regression equation of glucose is y =1.1898x +4.9832 2 Results are shown in table 2 and fig. 5, indicating that glucose is in good linearity between 1.056942 and 5.284710 μ g; the regression equation for sucrose is y =1.1887x +5.0956 2 =0.9993, and as a result, as shown in table 3 and fig. 6, sucrose showed good linearity between 1.093808 and 5.469040 μ g;
TABLE 1 measurement of fructose linearity Table
| Sample volume (ug) | Lg Sample size of reference | Measuring the peak area | Lg Measuring the peak area |
| 0.813552 | -0.0896 | 114657 | 5.0594 |
| 0.813552 | -0.0896 | 115327 | 5.0619 |
| 1.627104 | 0.2114 | 275012 | 5.4394 |
| 1.627104 | 0.2114 | 278648 | 5.4451 |
| 2.440656 | 0.3875 | 440992 | 5.6444 |
| 2.440656 | 0.3875 | 443226 | 5.6466 |
| 3.254208 | 0.5124 | 604979 | 5.7817 |
| 3.254208 | 0.5124 | 616339 | 5.7898 |
| 4.067760 | 0.6094 | 813895 | 5.9106 |
| 4.067760 | 0.6094 | 815123 | 5.9112 |
TABLE 2 table of results of glucose linearity measurement
TABLE 3 sucrose Linear relationship measurement results Table
| Sample volume (ug) | Lg Sample size of reference | Measuring the peak area | Lg Measuring the peak area |
| 1.093808 | 0.0389 | 135293 | 5.1313 |
| 1.093808 | 0.0389 | 138001 | 5.1399 |
| 2.187616 | 0.3400 | 323549 | 5.5099 |
| 2.187616 | 0.3400 | 323068 | 5.5093 |
| 3.281424 | 0.5161 | 515982 | 5.7126 |
| 3.281424 | 0.5161 | 523050 | 5.7185 |
| 4.375232 | 0.6410 | 700702 | 5.8455 |
| 4.375232 | 0.6410 | 707211 | 5.8495 |
| 5.46904 | 0.7379 | 941205 | 5.9737 |
| 5.46904 | 0.7379 | 937123 | 5.9718 |
4. Sugar content precision test
Precisely sucking 10 μ l of 2.3 prepared sample solution, continuously feeding sample for 6 times under the above chromatographic conditions, and measuring peak area, the results are shown in tables 4-6:
table 4 table of fructose precision test results
| Precision 1 | Precision 2 | Precision 3 | Precision 4 | Precision 5 | Precision 6 | Mean value of | RSD(％) |
| 373771 | 380175 | 385994 | 380021 | 373428 | 383379 | 379461 | 1.3 |
TABLE 5 table of results of saccharin density tests
| Precision 1 | |
Precision 3 | Precision 4 | |
Precision 6 | Mean value of | RSD(％) |
| 297880 | 294416 | 308792 | 297445 | 300761 | 295513 | 299135 | 1.7 |
Table 6 sucrose precision test results table
| Precision 1 | |
Precision 3 | Precision 4 | |
Precision 6 | Mean value of | RSD(％) |
| 404504 | 390777 | 406866 | 392234 | 396126 | 399077 | 398264 | 1.6 |
5. Sugar content repeatability test
Precisely weighing 5 parts of fresh succus Bambusae sample of the same lot (lot number: 20180822), preparing test solution according to the above method, respectively, analyzing and determining under the above chromatographic conditions to obtain fructose, glucose and sucrose with average content of 5.4036 mg/mL -1 、6.3912mg·mL -1 And 6.2366 mg. ML -1 RSD is 1.3%, 0.9% and 1.7% respectively, which shows that the test reproducibility is good; the results are shown in tables 7 to 9The following steps:
TABLE 7 table of fructose reproducibility test results
TABLE 8 glucose reproducibility test results Table
TABLE 9 sucrose reproducibility test results Table
6. Sugar content stability test
Precisely sucking 10 μ l of a sample solution (fresh bamboo juice sample batch number: 20180822) prepared by 2.3, respectively carrying out sample injection measurement for 0h, 6h, 12h, 18h and 24h, and determining that the peak area average values of fructose, glucose and sucrose are 369903, 290412, 376556 and RSD are 1.2%, 1.8% and 1.6% respectively, which indicates that the sample solution is stable in 24h, and the results are shown in tables 10-12:
TABLE 10 fructose stability test results table
| 0h | 6h | 12h | 18h | 24h | Mean value of | RSD％ |
| 373379 | 365712 | 365098 | 370322 | 375003 | 369903 | 1.2 |
TABLE 11 glucose stability test results Table
| 0h | 6h | 12h | 18h | 24h | Mean value of | RSD％ |
| 295513 | 286322 | 283668 | 293579 | 292979 | 2904126 | 1.8 |
Table 12 table of sucrose stability test results
| 0h | 6h | 12h | 18h | 24h | Mean value of | RSD％ |
| 379077 | 382075 | 377772 | 377411 | 366446 | 3765566 | 1.6 |
7. Sugar content sample recovery test
Adopting a sample adding and recovering test, precisely absorbing 200 mu l of the fresh bamboo juice sample (batch number: 20180822) with the measured sugar content, respectively placing 6 parts of the fresh bamboo juice sample into 5ml measuring bottles, precisely adding 3ml of a mixed reference substance solution of D-fructose, D-anhydrous glucose and sucrose, adding water to dilute the solution to a scale, shaking the solution uniformly, precisely absorbing 10 mu l of the solution, injecting the solution into a high performance liquid chromatograph, recording a chromatogram, calculating the average sample adding and recovering rate, and measuring the peak area and the sample adding and recovering rate, wherein the results are shown in tables 13-18:
TABLE 13 peak area table for fructose assay
| Numbering | Peak area 1 | |
Average peak area | Lg Average peak area | Lg Sample volume | Sample size (g) |
| 1 | 351079 | 350991 | 351035.0 | 5.5454 | 0.3067 | 2.0263 |
| 2 | 352004 | 352009 | 352006.5 | 5.5466 | 0.3077 | 2.0310 |
| 3 | 356911 | 356280 | 356595.5 | 5.5522 | 0.3123 | 2.0526 |
| 4 | 351085 | 351215 | 351150.0 | 5.5455 | 0.3068 | 2.0267 |
| 5 | 356090 | 355680 | 355885.0 | 5.5513 | 0.3116 | 2.0493 |
| 6 | 355125 | 357737 | 356431.0 | 5.5520 | 0.3122 | 2.0521 |
TABLE 14 fructose sample application recovery rate test results Table
TABLE 15 area table of glucose measurement peaks
| Numbering | Peak area 1 | |
Average peak area | Lg Average peak area | Lg Sample volume | Sample volume (g) |
| 1 | 289427 | 289298 | 289362.5 | 5.4614 | 0.4019 | 2.5229 |
| 2 | 286383 | 287014 | 286698.5 | 5.4574 | 0.3986 | 2.5038 |
| 3 | 286779 | 286998 | 286888.5 | 5.4577 | 0.3988 | 2.5050 |
| 4 | 290562 | 290828 | 290695.0 | 5.4634 | 0.4036 | 2.5328 |
| 5 | 286230 | 288023 | 287126.5 | 5.4581 | 0.3991 | 2.5067 |
| 6 | 288586 | 288463 | 288524.5 | 5.4602 | 0.4009 | 2.5171 |
TABLE 16 test results of glucose loading recovery
TABLE 17 sucrose measurement Peak area Table
| Numbering | Peak area 1 | |
Average peak area | Lg Average peak area | Lg Sample volume | Sample volume (g) |
| 1 | 381386 | 380868 | 381127.0 | 5.5811 | 0.4084 | 1.2805 |
| 2 | 375548 | 377536 | 376542.0 | 5.5758 | 0.4040 | 1.2676 |
| 3 | 376094 | 378517 | 377305.5 | 5.5767 | 0.4047 | 1.2696 |
| 4 | 380821 | 380779 | 380800.0 | 5.5807 | 0.4081 | 1.2796 |
| 5 | 380936 | 381921 | 381428.5 | 5.5814 | 0.4087 | 1.2814 |
| 6 | 376168 | 374867 | 375517.5 | 5.5746 | 0.4030 | 1.2647 |
TABLE 18 sucrose sample application recovery test results Table
8. Detection limit and quantification limit
Precisely weighing 7.01mg of fructose reference substance, 10.15mg of glucose reference substance and 9.70mg of sucrose reference substance, respectively placing in a 50ml measuring flask, adding water to dissolve and dilute to scale, shaking up, precisely weighing 3ml of each of the three reference substance solutions, placing in the same 10ml measuring flask, adding water to dilute to scale, shaking up to serve as a mixed reference substance solution, precisely sucking 2 mul and 3 mul of the mixed reference substance solution, respectively injecting into a liquid chromatograph, and measuring to obtain detection limits of fructose, glucose and sucrose of 0.10mg/ml, 0.30mg/ml and 0.15mg/ml respectively; precisely sucking 13 mul and 15 mul of the mixed reference substance solution, respectively injecting into a liquid chromatograph, and measuring to obtain the quantitative limits of fructose, glucose and sucrose of 0.73mg/ml, 1.36mg/ml and 1.31mg/ml respectively;
9. establishment of limits
Taking 33 batches of the ancient method direct firing process and 10 batches of the fresh bamboo juice samples simulating the dry distillation process (more than or equal to 120 ℃) of the ancient method, carrying out determination according to the method, wherein the results are shown in table 19, the determination results can be obtained, the fresh bamboo juice of 43 batches in total contains three sugars of fructose, glucose, sucrose and the like, and the proportion relation among the three is as follows: fructose: glucose: sucrose = 1.7-1.3, 1.0-6.0, and if not, adding and blending;
TABLE 19 Table of the results of the proportional relationship among fructose, glucose and sucrose in fresh bamboo juice
10. Determination of samples
After detecting 10 batches of commercial fresh bamboo juice samples and 19 batches of collected fresh bamboo juice raw materials, only 3 batches of the samples accord with the proposed proportion, the qualification rate is only 10.3 percent, which indicates that the commercial fresh bamboo juice is seriously blended, and the results are shown in a table 20 which shows the total table of the sugar measurement results in the fresh bamboo juice:
TABLE 20 overview of fresh bamboo juice preparation and sugar determination results in raw materials
The sugar content precision test, the sugar content repeatability test and the sugar content sample adding recovery test are carried out to obtain the sugar content, the ratio of fructose, glucose and sucrose in the fresh bamboo juice sample prepared by the ancient method direct firing is 1.7-1.3, 1.0-6.0, the fresh bamboo juice sample which is not in the range is added and blended, the sugar content in the fresh bamboo juice is in a linear relation, the fructose regression equation is y =1.2071x +5.1752, R 2 =0.9994, fructose is in good linearity between 0.813552 and 4.067760 μ g; the regression equation of glucose is y =1.1898x +4.9832 2 =0.9996, glucose is in good linearity between 1.056942 and 5.284710 mu g; the regression equation for sucrose is y =1.1887x +5.0956 2 =0.9993, sucrose presents good linearity between 1.093808-5.469040 mug, and the method identifies and blends fresh bamboo juice, and has convenient operation, fast speed and accurate result.
Claims (4)
1. The method for identifying blended fresh bamboo juice based on the linear relation of sugar content is characterized by comprising the following specific steps of:
1) Test for establishing chromatographic conditions and system applicability
Establishing a chromatographic column and a mobile phase, and determining the test conditions of the system applicability;
2) Preparing the fresh bamboo juice solution to be identified
Precisely measuring a proper amount of fresh bamboo juice to be identified, placing the fresh bamboo juice in a measuring flask, adding water to dilute the fresh bamboo juice to a scale, and shaking up to obtain a fresh bamboo juice solution to be identified;
3) Measuring fresh bamboo juice solution to be identified
Precisely absorbing a proper amount of fresh bamboo juice solution to be identified for several times, respectively injecting into a liquid chromatograph, and determining the quantitative limits of fructose, glucose and sucrose in the fresh bamboo juice solution to be identified under the chromatographic conditions and the system applicability test constructed in the step 1);
4) Judging fresh bamboo juice to be identified
Linear relation of sugar content is selected and extracted: the regression equation for fructose is y =1.2071x +5.1752 2 =0.9994, fructose is in good linearity between 0.813552 and 4.067760 μ g; the regression equation for glucose is y =1.1898x +4.9832 2 =0.9996, glucose is in good linearity between 1.056942 and 5.284710 mug; the regression equation for sucrose is y =1.1887x +5.0956 2 =0.9993, sucrose presents good linearity between 1.093808-5.469040 μ g, the ratio of fructose, glucose and sucrose in the fresh bamboo juice prepared by the ancient method direct firing is 1.7-1.3 according to the linear relation of sugar content; when the ratio of fructose, glucose and sucrose in the fresh bamboo juice to be identified, which is measured in the step 3), is not between 1.0 and 1.3.
2. The method for identifying fresh blended bamboo juice based on linear relationship of sugar content as claimed in claim 1, wherein in step 1), the chromatographic column is luna ® Omega SUGAR 100A chromatography column (250 mm. Times.4.6 mm,3 μm), mobile phase: acetonitrile-Water (85: 15) flow rate 1.0mL min -1 (ii) a The column temperature is 35 ℃, and the ELSD drift tube temperature is 70 ℃.
3. The method for identifying and blending fresh bamboo juice based on the linear relation of sugar contents as claimed in claim 1, wherein in the step 1), the acetonitrile is chromatographically pure, the water is the purified drinking water of Waoha, and other reagents are analytically pure.
4. The method for identifying blended fresh bamboo juice based on linear relationship of sugar content as claimed in claim 1, wherein in step 3), the liquid chromatograph is LC 20AT high performance liquid chromatograph.
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