CN111175408A

CN111175408A - High performance liquid chromatography method for determining soluble sugar in tomato fruits

Info

Publication number: CN111175408A
Application number: CN202010088657.3A
Authority: CN
Inventors: 黄腾波; 邱琦芸; 李紫薇
Original assignee: Longhua Bio-Industry Innovation Research Institute Of Shenzhen University; Shenzhen University
Current assignee: Longhua Bio-Industry Innovation Research Institute Of Shenzhen University; Shenzhen University
Priority date: 2020-02-12
Filing date: 2020-02-12
Publication date: 2020-05-19

Abstract

The invention provides a high performance liquid chromatography method for measuring soluble sugar in tomato fruits, which utilizes a high performance liquid chromatography to be connected with an RID-20A differential refraction detector in series to detect the soluble sugar, can distinguish glucose and fructose which are all pentoses, achieves the effect of accurately detecting three soluble sugars of glucose, fructose and sucrose at the same time, requires less samples, and can carry out accurate detection only by 0.1g or even less samples; the method has the advantages of few detection steps, simplicity, rapidness and good reproducibility, reduces experimental errors, improves the accuracy of data, can perform qualitative and quantitative analysis on three soluble sugars, namely glucose, fructose and sucrose, and provides scientific basis for accurate judgment and rapid detection of the mixture of the soluble sugars in the tomatoes.

Description

High performance liquid chromatography method for determining soluble sugar in tomato fruits

Technical Field

The invention relates to the field of analytical chemistry, in particular to a high performance liquid chromatography method for determining soluble sugar in tomato fruits.

Background

The plant of the genus Lycopersicon, the family Solanaceae (Solanaceae), the family tubular florae, the genus Lycopersicon, is a vegetable economic crop with high nutritive value, and is widely planted in the south and north of China. The fruit is sour and sweet in taste and rich in nutrition, so that the fruit is very popular with consumers.

The taste of tomatoes is determined by many factors, the taste of tomatoes is influenced by many factors, and soluble sugar is one of the most important determinants. The main soluble sugar components in tomato are glucose, fructose and sucrose, with higher levels of glucose and fructose. As the tomato fruit ripens, the amount of soluble sugars in the tomato changes, which in turn affects the flavor of the tomato.

Currently, soluble sugars are analyzed by anthrone-sulfuric acid colorimetry, phenol method, fibration method, thin layer chromatography, gas chromatography, liquid chromatography, and the like. Wherein, the anthrone-sulfuric acid colorimetric method, the phenol method and the fibhlin method can only detect the total amount of soluble sugar, can not detect the content of different soluble sugar components, has larger error and high cost, and is easy to cause danger and pollution by using chemical reagents, and has complex operation. The gas chromatography requires derivatization treatment of a test sample, and the operation process is complex. Because glucose and fructose are both pentose and are difficult to separate characteristic peaks in high performance liquid chromatography, a mixture of various soluble sugars cannot be detected and analyzed simultaneously, and accurate detection is difficult.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography method for measuring soluble sugar in tomato fruits, and aims to solve the problem that a plurality of soluble sugars cannot be simultaneously detected by adopting the high performance liquid chromatography in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a high performance liquid chromatography method for determining soluble sugar in tomato fruits comprises the following steps:

preparing a mixed standard solution of a glucose standard, a fructose standard and a sucrose standard, carrying out high performance liquid chromatography and RID-20A differential refraction detector series connection on the mixed standard solution to obtain a standard map, and making a standard curve to obtain a regression equation of each standard;

preparing a tomato product to be tested; and carrying out high performance liquid chromatography-tandem RID-20A differential refraction detector detection on the tomato to-be-detected product under the same detection condition as the mixed standard solution to obtain a chromatogram of the tomato to-be-detected product, determining the types of glucose, fructose and sucrose according to the retention time of the chromatogram of the tomato to-be-detected product, and calculating according to the chromatographic peak areas of the types to obtain the contents of the glucose, the fructose and the sucrose.

The invention provides a method for simultaneously detecting three soluble sugars of glucose, fructose and sucrose in tomatoes by connecting a High Performance Liquid Chromatography (HPLC) with a RID-20A refractive index detector in series, which comprises the steps of firstly analyzing a mixed standard solution of a glucose standard substance, a fructose standard substance and a sucrose standard substance to obtain a standard map and making a standard curve; and then pretreating the tomato to-be-detected product, carrying out high performance liquid chromatography-tandem RID-20A differential refraction detector detection on the solution obtained by pretreating a small amount of sample under the same detection condition as the mixed standard solution, determining the type of each soluble sugar according to the retention time of the chromatogram of the tomato to-be-detected product, and analyzing according to the chromatographic peak area and the standard curve to obtain the content of each soluble sugar. The detection method utilizes a high performance liquid chromatography to be connected with an RID-20A differential refraction detector in series to detect the soluble sugar, can distinguish glucose and fructose which are all pentoses, achieves the effect of accurately detecting three soluble sugars, namely glucose, fructose and sucrose at the same time, requires less samples, and can carry out accurate detection only by 0.1g or even less samples; the method has the advantages of few detection steps, simplicity, rapidness and good reproducibility, reduces experimental errors, improves the accuracy of data, can perform qualitative and quantitative analysis on three soluble sugars, namely glucose, fructose and sucrose, and provides scientific basis for accurate judgment and rapid detection of the mixture of the soluble sugars in the tomatoes.

Drawings

FIG. 1 is a chromatogram of a standard solution of fructose, glucose and sucrose provided by the embodiment of the invention.

Fig. 2 is a standard curve diagram of a fructose standard provided by the embodiment of the invention.

FIG. 3 is a standard curve diagram of a glucose standard provided by an embodiment of the invention.

Fig. 4 is a standard curve diagram of a sucrose standard provided by an embodiment of the present invention.

Fig. 5 is a chromatogram of a liquid to be tested of tomatoes provided by an embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the description of the present invention, the description of the various instruments and reagents involved is as follows:

the models of the instruments are as follows: the high performance liquid chromatograph is Shimadzu LC-16; the electronic balance is sartorius; the constant-temperature water bath kettle is Jiangsu Jinyi; vacuum freeze-drying to obtain Ningbo Xinzhi; the centrifuge is eppendorf; the combined solvent filter is a Shanghai worker.

Each reagent was produced: Standard-D- (+) -glucose and Standard-sucrose were purchased from Sigma-Aldrich; the standard product-D (-) -fructose is purchased from a national standard product net; the methanol and the acetonitrile are purchased from sigma company, and the purity is chromatographic purity; the pure water is Wahaha pure water.

The embodiment of the invention provides a high performance liquid chromatography method for measuring soluble sugar in tomato fruits, which comprises the following steps:

s01, preparing a mixed standard solution of a glucose standard, a fructose standard and a sucrose standard, carrying out high performance liquid chromatography and RID-20A differential refraction detector serial connection on the mixed standard solution to obtain a standard map, and making a standard curve to obtain a regression equation of each standard;

s02, preparing a tomato product to be tested; and carrying out high performance liquid chromatography-tandem RID-20A differential refraction detector detection on the tomato to-be-detected product under the same detection condition as the mixed standard solution to obtain a chromatogram of the tomato to-be-detected product, determining the types of glucose, fructose and sucrose according to the retention time of the chromatogram of the tomato to-be-detected product, and calculating according to the chromatographic peak areas of the types to obtain the contents of the glucose, the fructose and the sucrose.

The invention provides a method for detecting soluble sugar by using a High Performance Liquid Chromatography (HPLC) series RID-20A differential refraction detector, which can distinguish glucose and fructose which are all pentoses, so as to achieve the effect of simultaneously and accurately detecting three soluble sugars, namely glucose, fructose and sucrose, and the required sample amount is less, and the accurate detection can be carried out only by using 0.1g or even less samples; the method has the advantages of few detection steps, simplicity, rapidness and good reproducibility, reduces experimental errors, improves the accuracy of data, can perform qualitative and quantitative analysis on three soluble sugars, namely glucose, fructose and sucrose, and provides scientific basis for accurate judgment and rapid detection of the mixture of the soluble sugars in the tomatoes.

Specifically, in step S01, a mixed standard solution of a glucose standard, a fructose standard and a sucrose standard is prepared, wherein the standard-D- (+) -glucose and the standard-sucrose are purchased from sigma-Aldrich; the standard product-D (-) -fructose is purchased from a national standard product net; providing a standard substance mixed standard solution, carrying out high performance liquid chromatography detection on the standard substance mixed standard solution, analyzing to obtain a standard map, and further determining retention time of different types of standard substances and determining a standard curve.

Preferably, in the mixed standard solution, the concentration of the glucose standard substance is 50 mg/mL; the concentration of the fructose standard substance is 50 mg/mL; the concentration of sucrose standard was 50 mg/mL. In a preferred embodiment of the present invention, the preparation method of the mixed standard solution is as follows: respectively and precisely weighing a proper amount of glucose standard substance, fructose standard substance and sucrose standard substance, and dissolving the standard substances with ultrapure water to prepare the mixed standard solution.

Preferably, the mixed standard solution is diluted step by step to prepare at least 5 mixed standard diluents with different concentrations, and the mixed standard diluents with different concentrations are subjected to high performance liquid chromatography and RID-20A refractive index detector series connection detection to obtain a standard map. At least 5 mixed standard diluents with different concentrations are provided, and a certain number of samples with different concentrations are detected, so that a standard curve can be drawn to carry out subsequent experimental analysis.

Preferably, the step of diluting the mixed standard solution step by step to prepare at least 5 mixed standard dilutions with different concentrations is to prepare 6 mixed standard dilutions with different concentrations. Further preferably, the 6 mixed standard dilutions with different concentrations comprise 30mg/mL mixed standard dilution, 20mg/mL mixed standard dilution, 10mg/mL mixed standard dilution, 5mg/mL mixed standard dilution, 2.5mg/mL mixed standard dilution and 1.25mg/mL mixed standard dilution.

Further preferably, 6 mixed standard dilutions prepared with different concentrations are filtered to remove impurities in the mixed standard dilutions, so as to ensure high purity in the mixed standard dilutions. In the preferred embodiment of the invention, in the step of filtering treatment, a filter membrane with the diameter of 0.45 μm is used for filtering, so that a good impurity removal effect is ensured.

Further preferably, 6 mixed standard diluents with different concentrations obtained by filtering are degassed, so that redundant air is not contained in the solution, the detection result is not affected, and the chromatographic column is protected. In a preferred embodiment of the invention, the degassing treatment is selected from, but not limited to, methods of ultrasonic degassing.

Further, the mixed standard diluent with different concentrations in the 6 are respectively subjected to high performance liquid chromatography and RID-20A differential refractometer detection, and a standard map can be obtained.

Preferably, the high performance liquid chromatography detection adopts an Shimadzu LC-16 liquid chromatograph detector, the high performance liquid chromatography is connected with an RID-20A differential refraction detector in series, and the RID-20A differential refraction detector has a 4-partition photodiode technology and shows excellent linearity in a wide concentration range. The RID-20A shows the technical parameters of the differential refraction detector: the method comprises the following steps: a deflection type; refractive index range: 1.00-1.75 RIU; measurement range: 0.01-500 x 10-6 RIU; noise <0.25x10-8 RIU; linearity: 5x 10-4; temperature control: 30-60 ℃; maximum usage flow rate: 20 ml/min; polarity switching: comprises the following steps of; size: w260 XD 420 XH 140mm,12 kg. The RID-20A differential refraction detector can ensure higher detection sensitivity, and can separate the peaks of glucose and fructose which are both pentose, thereby realizing simultaneous detection and improving the accuracy of detection results.

Preferably, the conditions of the high performance liquid chromatography detection are as follows: the chromatographic column is NH₂A chromatographic column; NH (NH)₂The chromatographic column is an amino chromatographic column, and can separate hydrophilic and polar compounds such as carbohydrate and monosaccharide, oligosaccharide, sugar alcohol, etc. in reversed phase mode. Further preferably, the NH is₂The column size was 3 μm, 4.6X 250 mm. NH with a particle size of 3 μm and a size of 4.6X 250mm was selected₂The chromatographic column ensures that the chromatographic column has better analysis speed, separation capability and detection capability.

Preferably, the column temperature is 30-50 ℃, the column temperature under the conditions is kept, the qualitative and quantitative analysis of glucose, sucrose and fructose can be better carried out, and the accuracy of the detection result is improved. In the detection process, the change of the column temperature can influence the peak emergence time and the peak type of the sample, if the column temperature is too high, the peak emergence is faster, the retention time is shortened, and the reproducibility of the retention time is poor, so that the qualitative result of the components of the sample is influenced, and meanwhile, the over-high column temperature can cause the half-peak width to be narrowed, the peak height to be heightened, and the quantification of the components of the sample is influenced; if the column temperature is too low, the peak is slower, the retention time is longer, and the reproducibility of the retention time is poor, so that the qualitative result of the sample components is influenced, and meanwhile, if the column temperature is too low, the half-peak width is widened, the peak height is lowered, and the quantification of the sample components is influenced. In the preferred embodiment of the invention, the column temperature is 40 ℃, and under the column temperature condition of 40 ℃, the stability of the peak-out time and the moderate peak area size of the 3 kinds of soluble sugars can be ensured, and the accuracy of the experiment is ensured.

Preferably, the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile. Due to the use of NH2 chromatography columns, the columns separate hydrophilic and polar compounds, such as carbohydrates and monosaccharides, oligosaccharides, sugar alcohols, etc. in reversed phase mode. Therefore, the mobile phase needs to provide a polar solvent for elution. Further preferably, the elution mode is isocratic elution; when isocratic elution is carried out, the volume percentage of the mobile phase A is 15-20% and the percentage of the mobile phase B is 80-85% within 15-20 min. And controlling the volumes of the flowing solution A and the flowing phase solution B, so that the peaks of all substances can be controlled to be separated.

Preferably, the elution time is 15-20 min; the elution treatment is carried out by adopting the elution time, so that each component can be detected, and the accuracy of the time is ensured. If the elution time is too short, two samples will be detected simultaneously during the elution process, which further affects the accuracy of the experiment. In a preferred embodiment of the invention, the elution time is 16 min; and the elution treatment is carried out at the time, so that the better elution effect can be ensured, and the detection result is credible.

Preferably, the sample amount is 10-30 μ L; the sample amount of the conditions is kept, glucose, sucrose and fructose can be separated accurately, the accuracy of the experiment is guaranteed, and the accuracy of the detection result is improved. If the sample amount is too large, the peak area of each substance is too large, so that two sample peaks can not be separated, and subsequent analysis is influenced; if the sample amount is too small, the peak area of each substance is too small, and the too small peak area cannot be accurately and quantitatively analyzed, so that the accuracy of the experiment is affected. In a preferred embodiment of the present invention, the sample volume is 20 μ L; the sample amount under the conditions is kept, the 3 kinds of soluble sugar can be accurately separated, the accuracy of an experiment is ensured, and the accuracy of a detection result is improved.

Preferably, the elution flow rate is 1.0-1.5 mL/min; the elution flow rate affects the retention time of the sample, and thus the accuracy of the experiment. If the elution flow rate is too high, the retention time is shortened, the peak emergence time is accelerated, the distance between peaks is shortened, the peaks can not be completely separated, and the detection is influenced; if the elution flow rate is too slow, the retention time increases, which all results in inaccurate experimental results. In the preferred embodiment of the invention, the elution flow rate is 1.2mL/min, and the elution treatment is carried out according to the flow rate, so that the accurate retention time can be ensured, and the detection result is credible.

The method has the advantages that the chromatographic conditions are adopted for high performance liquid chromatography detection, the qualitative and quantitative detection can be carried out on glucose, sucrose and fructose at the same time, the detection efficiency is improved, the detection steps are few, the method is simple and rapid, the reproducibility is good, the experimental error is reduced, and the accuracy of data is improved.

Preferably, the type of each standard substance is determined by the retention time in the standard map, a standard curve is prepared by using the peak area of each standard substance and the concentration of each corresponding standard substance, and a regression equation of each standard substance is obtained. Further preferably, the mixed standard diluents with different concentrations in the 6 are respectively subjected to high performance liquid chromatography detection to obtain standard maps, the types of the standard substances are determined according to the retention time in the standard maps, standard curves are made according to the peak areas of the standard substances and the corresponding concentrations of the standard substances, and regression equations of the standard substances are obtained; preferably, the standard curve is plotted with the peak area of each standard as the ordinate (Y) and the concentration of the corresponding standard as the abscissa (X, mg/mL).

Specifically, in step S02, a tomato sample is prepared; and carrying out high performance liquid chromatography-tandem RID-20A differential refraction detector detection on the tomato to-be-detected product under the same detection condition as the mixed standard solution to obtain a chromatogram of the tomato to-be-detected product, determining the types of glucose, fructose and sucrose according to the retention time of the chromatogram of the tomato to-be-detected product, and calculating according to the chromatographic peak areas of the types to obtain the contents of the glucose, the fructose and the sucrose.

Preferably, the step of preparing the tomato test sample comprises the following steps:

s021, providing a tomato sample, and collecting the pulp of the tomato sample;

s022, adding an organic solvent into the pulp to extract to obtain a first mixture, heating the first mixture, centrifuging, and collecting supernatant of the first mixture;

s023, concentrating the supernatant of the first mixture to obtain a first extract;

s024, adding ultrapure water into the first extract, mixing, and filtering to obtain the tomato product to be tested.

In the above step S021, a tomato sample is provided, and the tomato sample may be any one of tomato varieties. In a preferred embodiment of the invention, the tomato sample is a laboratory-grown tomato of the Micro-Tom variety.

Preferably, in the step of collecting the pulp of the tomato sample, the tomato sample is treated, the peel and the pulp are separated, the seeds in the pulp are removed, and the pulp of the tomato sample is collected and ground into powder in liquid nitrogen.

In step S022, the organic solvent is added to the pulp to extract the pulp to obtain a first mixture, the first mixture is heated and centrifuged, and a first mixture supernatant is collected.

Preferably, in the step of extracting the pulp with an organic solvent to obtain a first mixture, the concentration of the first mixture is 50-100 mg/mL. Further preferably, the organic solvent is selected from 80% ethanol solution.

Preferably, in the step of heating the first mixture, the temperature of the heating is 35 to 40 ℃, and the time of the heating is 25 to 30 minutes. In a preferred embodiment of the present invention, in the step of subjecting the first mixture to a heat treatment, the temperature of the heat treatment is 35 ℃ and the time of the heat treatment is 30 minutes.

Preferably, in the step of subjecting the first mixture to a centrifugal treatment after the heating treatment, the centrifugal treatment is performed under the following conditions: the rotation speed of the centrifugal treatment is 10000 rcf-12000 rcf; the time of the centrifugal treatment is 20-25 minutes. In a preferred embodiment of the present invention, in the step of subjecting the first mixture to a centrifugal treatment after the heating treatment, the centrifugal treatment is performed under the following conditions: the rotation speed of the centrifugal treatment is 12000 rcf; the time for the centrifugation treatment was 20 minutes.

In the step S023, concentrating the supernatant of the first mixture to obtain a first extract; in a preferred embodiment of the present invention, the supernatant of the first mixture is concentrated by a vacuum centrifugal concentrator until all the solvent is evaporated and the bottom yellow non-volatile material is the extracted soluble sugar.

In the step S024, the first extract is added to ultrapure water, mixed, and filtered to obtain the tomato sample. Preferably, the filtration treatment is performed by adopting a 0.45 μm filter membrane to obtain the tomato product to be tested.

Specifically, the tomato to-be-detected product is subjected to detection by a high performance liquid chromatography-tandem RID-20A differential refraction detector under the same detection condition as the mixed standard solution, preferably, the chromatographic detection experimental conditions are as follows: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; the chromatographic column is NH₂A chromatographic column; the column temperature is 30-50 ℃; the sample injection amount is 20 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; the elution mode is isocratic elution, and when the isocratic elution is carried out, the volume percentage of the mobile phase A is 15-20% and the percentage of the mobile phase B is 80-85% within 15-20 min; the elution time is 15-20 min; the elution flow rate is 1.0-1.5 mL/min. Under the conditions, the high performance liquid chromatography-series RID-20A refractive index detector detection is carried out on the tomato solution to be detected, so that the separation and the accuracy of glucose, sucrose and fructose can be ensuredAnd (6) detecting.

In a preferred embodiment of the present invention, the chromatographic assay is performed under the following experimental conditions: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; the chromatographic column adopts 3 mu m, 4.6 multiplied by 250mm NH₂A chromatographic column; the column temperature was 40 ℃; the sample injection amount is 20 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; the elution mode is isocratic elution, and when the isocratic elution is carried out, the volume ratio of the mobile phase A solution to the mobile phase B solution is 15: 85 parts by weight; the elution time is 16 min; the elution flow rate was 1.2 mL/min. And carrying out high performance liquid chromatography-series RID-20A refractive index detector detection on the tomato solution to be detected under the conditions, so that separation and accurate detection on glucose, sucrose and fructose can be ensured.

Specifically, after high performance liquid chromatography is carried out and RID-20A refractive index detector is used for detection, a chromatogram of the tomato to-be-detected product is obtained, the types of glucose, sucrose and fructose are determined according to the retention time of the chromatogram of the tomato to-be-detected product, and the concentrations of the glucose, the sucrose and the fructose in the tomato to-be-detected product are calculated according to the peak areas of various chromatograms and the standard curves and regression equations of various prepared substances.

The content of each soluble sugar in the fresh weight per gram of the sample is known by substituting the obtained concentration into the following formula.

Wherein, X represents the content of soluble sugar in the sample, and the unit is mg/g;

c, obtaining the mass concentration of soluble sugar in the sample solution by a standard curve, wherein the unit is mg/mL;

v1-final volume fixing of the sample liquid,

m is the mass of the sample in g.

Preferably, the high performance liquid chromatography method for determining soluble sugar in tomato fruit described herein comprises the following steps:

preparing glucose standard substance, fructose standard substance and caneDiluting the mixed standard solution of sugar standard products step by step to prepare 6 mixed standard diluents with different concentrations, carrying out high performance liquid chromatography tandem RID-20A differential refraction detector detection on the mixed standard diluents with different concentrations to obtain a standard map, determining the types of the standard products according to the retention time in the standard map, making a standard curve according to the peak area of each standard product and the concentration of each corresponding standard product, and obtaining a regression equation of each standard product; wherein the chromatographic conditions are as follows: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; NH is selected as chromatographic column₂A chromatographic column; the column temperature is 30-50 ℃; the sample injection amount is 10-30 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; the elution mode is isocratic elution, and when the isocratic elution is carried out, the volume percentage of the mobile phase A is 15-20% and the percentage of the mobile phase B is 80-85% within 15-20 min; the elution time is 15-20 min; the elution flow rate is 1.0-1.5 mL/min;

providing a tomato sample, collecting the pulp of the tomato sample; adding an organic solvent into the pulp to extract to obtain a first mixture, heating the first mixture, centrifuging, and collecting the supernatant of the first mixture; concentrating the supernatant of the first mixture to obtain a first extract; adding ultrapure water into the first extract, mixing and filtering to obtain the tomato product to be detected;

and carrying out high performance liquid chromatography-tandem RID-20A differential refraction detector detection on the tomato to-be-detected product under the same detection condition as the mixed standard solution to obtain a chromatogram of the tomato to-be-detected product, determining the types of glucose, fructose and sucrose according to the retention time of the chromatogram of the tomato to-be-detected product, and calculating according to the peak areas of the chromatograms of the types to obtain the contents of the glucose, the fructose and the sucrose.

The description is further described in the context of specific embodiments.

Example 1

Standard Curve for preparing standard substance

Preparing a mixed standard solution of a glucose standard substance, a sucrose standard substance and a fructose standard substance: respectively and precisely weighing a proper amount of glucose, sucrose and fructose standard products, preparing a mixed standard solution containing 50mg of glucose, sucrose and fructose in each 1mL of Wahaha purified water, and filtering the mixed standard solution by using a 0.45-micrometer filter membrane.

Preparing a mixed standard diluent: diluting the mixed standard solution step by step to prepare 6 mixed standard diluents with different concentrations, wherein the 6 mixed standard diluents with different concentrations comprise 30mg/mL mixed standard diluents, 20mg/mL mixed standard diluents, 10mg/mL mixed standard diluents, 5mg/mL mixed standard diluents, 2.5mg/mL mixed standard diluents and 1.25mg/mL mixed standard diluents.

High performance liquid chromatography tandem RID-20A differential refractometer analysis: detecting the 6 mixed standard diluents with different concentrations to obtain standard maps, wherein the chromatographic conditions are as follows: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; the chromatographic column adopts 3 mu m, 4.6 multiplied by 250mm NH₂A chromatographic column; the column temperature was 40 ℃; the sample injection amount is 20 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; when isocratic elution is carried out, the volume ratio of the mobile phase A solution to the mobile phase B solution is 15: 85 parts by weight; the elution time is 16 min; the elution flow rate was 1.2 mL/min. Determining the types of the standard products through the retention time in the standard map, making a standard curve according to the peak area of each standard product and the concentration of each corresponding standard product, and obtaining the regression equation of each standard product.

Precision analysis: precisely absorbing the 6 mixed standard diluents with different concentrations, repeatedly injecting samples for 3 times according to the chromatographic conditions, measuring the peak areas of the substances, calculating the RSD value of the peak areas, and analyzing the precision of the instrument.

Example 2

Determination of soluble sugar content of tomatoes in different development stages

Preparing a tomato sample: taking 4 tomato samples in different development stages, separating peels and pulps, removing seeds in the pulps, and grinding the pulps without the seeds in the pulps into powder by adopting liquid nitrogen; adding 0.1g of sample into a 15mL centrifuge tube, accurately weighing in the test tube, recording the fresh weight of the sample and 1mL of 80% ethanol, uniformly mixing, performing heat preservation extraction in a 35 ℃ water bath for 30min, centrifuging at the normal temperature of 12000rcf for 20min, and taking the supernatant in another centrifuge tube. Repeatedly extracting the fruit sample with 80% ethanol for 2 times, collecting the extractive solution, centrifuging and concentrating to dry, dissolving the residue with 200 μ L Wahaha purified water, filtering with 0.45 μm filter membrane, and ultrasonic degassing to obtain 4 tomato products to be tested at different development stages; 3 different samples were taken at each time period and 3 sets of parallel experiments were performed.

High performance liquid chromatography tandem RID-20A differential refractometer analysis: detecting tomato products to be detected in 4 different development stagesTo a standard spectrum, the chromatographic conditions were as follows: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; the chromatographic column adopts 3 mu m, 4.6 multiplied by 250mm NH₂A chromatographic column; the column temperature was 40 ℃; the sample injection amount is 20 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; the elution mode is isocratic elution, and when the isocratic elution is carried out, the volume ratio of the mobile phase A solution to the mobile phase B solution is 15: 85 parts by weight; the elution time is 16 min; the elution flow rate was 1.2 mL/min.

And obtaining a chromatogram of the tomato to-be-detected product, determining the types of glucose, sucrose and fructose according to the retention time of the chromatogram of the tomato to-be-detected product, and calculating the concentrations of the glucose, the sucrose and the fructose in the tomato to-be-detected product according to the chromatographic peak areas of the types.

v1-final volume of sample solution, 0.2mL in this example;

m is the mass of the sample in g.

And (4) analyzing results:

in example 1, the mixed standard solution is subjected to high performance liquid chromatography and RID-20A differential refractometer detection to obtain a standard spectrum as shown in figure 1, and can be obtained from figure 1, and 3 soluble sugars can be separated by performing high performance liquid chromatography and RID-20A differential refractometer analysis on the mixed standard solution under the chromatographic conditions, wherein the retention time of fructose is 7.109 min; the retention time of glucose is 8.343 min; the retention time of sucrose was 11.178 min.

High performance liquid chromatography tandem RID-20A difference is carried out on 6 mixed standard diluents with different concentrationsAnd detecting by a refraction detector, analyzing one by one to obtain standard curves of fructose, glucose and sucrose, and drawing the standard curves by taking the concentration (mg/mL) of the standard substance as a horizontal coordinate (X) and taking the peak area as a vertical coordinate (Y). As shown in fig. 2 to 4, fig. 2 is a standard curve of fructose standard, and the regression equation of fructose is that y is 167245x +26093.6, and R is calculated²0.9990, and a linear range of 0.125-30 (mg/mL); FIG. 3 is a standard curve of glucose standard, which is calculated to have a regression equation of y 139061x +43769.3, R²0.9995, linear range of 0.125-30 (mg/mL); fig. 4 is a standard curve of sucrose standard, and the regression equation of sucrose is 142109x +54142.7, R2 is 0.9997, and the linear range is 0.125-30 (mg/mL). The results show that the linear ranges of the glucose, fructose and sucrose standards are good.

Precisely absorbing the 6 mixed standard diluents with different concentrations, repeatedly injecting samples for 3 times according to the chromatographic conditions, measuring the peak areas of all substances, calculating the RSD values of the peak areas, and analyzing to obtain the RSD ranges of the peak areas of glucose, fructose and sucrose, wherein the RSD ranges are 0.17% -1.44%, and the instrument precision is good.

In example 2, the tomato sample is subjected to high performance liquid chromatography-RID-20A differential refractive index detector detection, the chromatogram thereof is shown in FIG. 5 and can be obtained from FIG. 5, and the mixed standard solution is subjected to high performance liquid chromatography-RID-20A differential refractive index detector analysis through the chromatographic conditions, so that 3 soluble sugars can be separated, wherein the retention time of fructose is 6.952 min; the retention time of glucose is 8.050 min; the retention time of sucrose was 10.967 min. The variety to be detected of the tomato also has other impurity substances which have certain influence on the high performance liquid chromatography detection, so the retention time of each substance has certain influence.

And measuring the peak areas of all components in the tomato samples at different times, and substituting the peak areas into a regression equation to calculate the concentration of all soluble sugar components.

Furthermore, the content of each soluble sugar in each gram of fresh weight of the sample can be known by substituting the concentration into the following formula.

v1-final volume of sample solution, 0.2mL in this example;

m is the mass of the sample in g.

The average of 3 results in each group was found, and the results are shown in Table 1, wherein in the tomato green stage (MG), the fructose content was 6.522MG/g, the glucose content was 5.303MG/g, and the sucrose content was 0.348 MG/g; the total amount of the three soluble sugars is 12.173 mg/g; in the tomato color breaking period (BR), the fructose content is 7.738mg/g, the glucose content is 4.132mg/g, and the sucrose content is 0.668 mg/g; the total amount of the three soluble sugars is 12.528 mg/g; in five days (BR +5) of tomato color breaking, the fructose content is 17.860mg/g, the glucose content is 3.342mg/g, and the sucrose content is 0.707 mg/g; the total amount of the three soluble sugars is 21.909 mg/g; in the tomato mature period (RM), the fructose content is 15.450mg/g, the glucose content is 4.855mg/g, and the sucrose content is 0.480 mg/g; the total amount of the three soluble sugars is 20.785 mg/g; as can be seen from Table 1, the tomato fruits at different periods all have different contents of glucose, sucrose and fructose.

The high performance liquid chromatography detection method for the soluble sugar of the tomato fruits adopts the high performance liquid chromatography to be connected with the RID-20A differential refraction detector in series for detection, can accurately detect 3 soluble sugars of glucose, sucrose and fructose in the tomato fruits, has high sensitivity, good specificity, accuracy and simplicity, can effectively evaluate the content of the soluble sugar of the tomato, and lays a foundation for the research of the flavor of the tomato.

TABLE 1

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A high performance liquid chromatography method for measuring soluble sugar in tomato fruits is characterized by comprising the following steps:

2. The HPLC method for determining soluble sugars in tomato fruit according to claim 1, wherein the HPLC assay conditions are as follows:

the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector;

the chromatographic column is NH₂A chromatographic column;

the column temperature is 30-50 ℃;

the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile;

the elution mode is isocratic elution;

the elution time is 15-20 min.

3. The HPLC method for determining soluble sugars in tomato fruits as claimed in claim 2, wherein the volume percentage of mobile phase A is 15-20% and the volume percentage of mobile phase B is 80-85% when isocratic elution is performed.

4. The HPLC method for determining soluble sugars in tomato fruits according to claim 3, wherein the HPLC assay conditions are as follows:

the NH₂The specification of the chromatographic column is 3 μm and 4.6 × 250 mm;

the sample amount is 10-30 μ L;

the elution flow rate is 1.0-1.5 mL/min.

5. The HPLC method for determining soluble sugar in tomato fruit according to any one of claims 1 to 4, wherein the step of preparing a tomato sample comprises the following steps:

providing a tomato sample, collecting the pulp of the tomato sample;

adding an organic solvent into the pulp to extract to obtain a first mixture, heating the first mixture, centrifuging, and collecting the supernatant of the first mixture;

concentrating the supernatant of the first mixture to obtain a first extract;

and adding ultrapure water into the first extract, mixing and filtering to obtain the tomato product to be detected.

6. The HPLC method for determining soluble sugars in tomato fruits as claimed in claim 5, wherein the step of extracting the pulp with organic solvent to obtain the first mixture has a concentration of 50-100 mg/L.

7. The HPLC method according to claim 5, wherein in the step of subjecting the first mixture to heat treatment, the temperature of the heat treatment is 35 to 40 ℃ and the time of the heat treatment is 25 to 30 minutes.

8. The HPLC method for determining soluble sugars in tomato fruits as claimed in claim 5, wherein in the step of subjecting said first mixture to a centrifugation process after heating, the conditions of said centrifugation process are as follows: the rotation speed of the centrifugal treatment is 10000 rcf-12000 rcf; the time of the centrifugal treatment is 20-25 minutes.

9. The HPLC method according to any one of claims 1 to 4, wherein the step of subjecting the mixed standard solution to HPLC-RID-20A differential refraction detector detection to obtain a standard map, and making a standard curve to obtain a regression equation for each standard, the step of diluting the mixed standard solution step by step to prepare at least 5 mixed standard dilutions with different concentrations, subjecting the mixed standard dilutions with different concentrations to HPLC-RID-20A differential refraction detector detection to obtain a standard map, determining the type of each standard by the retention time in the standard map, and making a standard curve by using the peak area of each standard and the concentration of each corresponding standard to obtain a regression equation for each standard.

10. The high performance liquid chromatography method for the determination of soluble sugars in tomato fruits as claimed in claim 1, characterized in that it comprises the following steps:

preparing a mixed standard solution of a glucose standard, a fructose standard and a sucrose standard, diluting the mixed standard solution step by step, preparing 30mg/mL, 20mg/mL, 10mg/mL, 5mg/mL, 2.5mg/mL and 1.25mg/mL6 mixed standard diluents with different concentrations, detecting the mixed standard diluents with different concentrations by high performance liquid chromatography-tandem RID-20A differential refraction detector to obtain a standard map, determining the type of each standard through the retention time in the standard map, making a standard curve by using the peak area of each standard and the concentration of each corresponding standard, and obtaining a regression equation of each standard; wherein the chromatographic conditions are as follows: the detector is Shimadzu LC-16 liquid chromatograph matched with RID-20A refractive index detector; NH is selected as chromatographic column₂A chromatographic column; the column temperature is 30-50 ℃; the sample injection amount is 10-30 mu L; the mobile phase A solution is pure water, and the mobile phase B solution is acetonitrile; the elution mode is isocratic elution, and when the isocratic elution is carried out, the volume percentage of the mobile phase A is 15-20%, and the percentage of the mobile phase B is 80-85%; the elution time is 15-20 min; the elution flow rate is 1.0-1.5 mL/min;