CN111122721B - Simple determination of citric acid delta in fruit juice 13 Method of C value - Google Patents

Simple determination of citric acid delta in fruit juice 13 Method of C value Download PDF

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CN111122721B
CN111122721B CN201911299288.6A CN201911299288A CN111122721B CN 111122721 B CN111122721 B CN 111122721B CN 201911299288 A CN201911299288 A CN 201911299288A CN 111122721 B CN111122721 B CN 111122721B
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citric acid
juice
acid
sample
delta
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CN111122721A (en
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钟其顶
王道兵
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China National Research Institute of Food and Fermentation Industries
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

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Abstract

The invention relates to a method for simply measuring citric acid delta in fruit juice 13 A method of C value comprising the steps of: a. sample dilution: diluting a fruit juice sample by 15-25 times with purified water to obtain a sample diluent; b. hydrolysis of sucrose: adding an acid to the sample dilution to completely hydrolyze sucrose present in the sample dilution, thereby obtaining a hydrolysis solution; c. diluting and filtering hydrolysate: diluting the hydrolysate to ensure that the concentration of citric acid in the diluted solution is 150-250 times of the concentration of citric acid in the juice sample, and then filtering to obtain filtrate; d. analysis of citric acid delta by liquid chromatography-stable isotope ratio mass spectrometry 13 A C value, wherein the column used in the liquid chromatography-stable isotope ratio mass spectrometry is a hydrogen ion exchange liquid chromatography column. The method avoids the influence of sucrose on the determination of citric acid by a hydrolysis method, has simple operation and low cost, and is delta-citrate 13 The analysis efficiency of C is high, and the application of the stable isotope technology in the true and false identification of the fruit juice, especially in the detection of the adulteration of the citric acid can be promoted.

Description

Simple determination of citric acid delta in fruit juice 13 Method of C value
Technical Field
The invention relates to a method for simply measuring citric acid delta in fruit juice 13 The invention relates to a method for measuring the C value, in particular to a method for measuring the citric acid stable carbon isotope ratio (marked as delta) in fruit juice 13 C) Belonging to the technical field of stable isotope analysis.
Background
The fruit juice has the natural flavor of the fruit and is rich in various nutritional ingredients, so the fruit juice is deeply loved by consumers, but the problem of identifying the truth of the fruit juice at present, particularly the problem of adulteration detection, is a difficult problem faced by the current quality supervision department. Citric acid is an important organic acid contained in various fruits, and the citric acid content in the fruit juice can provide an important basis for identifying adulterated fruit juice. However, it is not limited toThe citric acid content in the fruit is different due to different production places, varieties and varieties, but has a certain fluctuation range, and the adulteration molecules can be adjusted by adding the citric acid when the citric acid content in the fake and counterfeit products is lower than the normal value, so that the difficulty is brought to the identification of the adulteration of the fruit juice. The carbon isotope stabilization technology has an important role in distinguishing compounds from different sources (see non-patent document 1), and has potential application value in detecting externally added citric acid in fruit juice, so that the citric acid delta in the fruit juice 13 Accurate determination of C-values is a hotspot of current research. Citric acid delta according to the requirement of stable isotope ratio mass spectrometry 13 The C value is measured while eliminating the interference of other organic matters, and the fruit juice is a mixture containing a large amount of organic matters such as saccharides and organic acids, so that the separation and purification of the citric acid become delta 13 The key to the C assay.
From non-patent documents 2 to 8, it is known that citric acid delta is contained in fruit juice 13 The assay for C goes through three stages of development: (1) adjusting acidity of fruit juice by Doner (to pH 8.5), heating to 60 deg.C, centrifuging to obtain citrate, cleaning, drying, and performing citric acid delta by two-way sampling-stable isotope ratio mass spectrometry 13 C analysis, but this technique does not completely separate citric acid from other organic acids and will contain some of the carbohydrate; (2) jamin first separates saccharides and organic acid by ion exchange resin, then uses preparative liquid chromatography to separate malic acid and citric acid, and then uses elemental analysis-stable isotope ratio mass spectrometry to carry out transformation and analysis; (3) with the development of the technology, the carbon isotope composition of macromolecular organic matters can be analyzed on line by the liquid chromatography-stable isotope ratio mass spectrometry technology, but the mobile phase of the liquid chromatography cannot contain organic matters under the technology, so that the technology is matched with certain hydrophilic chromatographic columns to realize alcohol substances, carbohydrate compounds and amino acid delta 13 On-line analysis of C, however, direct determination of citric acid delta without pretreatment has not been found yet 13 C report: guyon established the determination of glucose, fructose, glycerol and ethanol δ using an Alltech 700cH Carbohydrate column liquid chromatography 13 Method of C, but in any case optimizing the colorUnder the spectrum condition, the citric acid can not be accurately measured under the system, so that the average delta of the organic acid in the lemon juice can be directly measured 13 C value; the application of the pegamun was tried by the belvevenus, but the calcium ion exchange column can not effectively separate citric acid from malic acid, and the hydrogen ion exchange column can separate citric acid from malic acid, but the influence of sucrose exists, so that the pegamun adopts a mode that a liquid phase is prepared, the calcium ion exchange column is used for firstly separating sucrose from citric acid, fractions are collected, and then the fractions are measured on a liquid chromatogram-stable isotope ratio mass spectrum which is prepared with the hydrogen column, although the stability of the result is good, two liquid chromatographs are required to work at the same time, the analysis cost is increased, and the sample measuring time is long. At present, a method for rapidly and economically determining the citric acid delta 13C value in fruit juice is urgently needed, and the research realizes the citric acid delta 13C value in the fruit juice by establishing a simple pretreatment technology according to the advancement of a liquid chromatograph-stable isotope ratio mass spectrometer, the analysis requirement of citric acid and the physicochemical characteristics of cane sugar 13 The rapid and accurate determination of the C value provides technical support for the source analysis of the citric acid in the fruit juice.
Cited non-patent documents
Non-patent document 1: rossmann A.determination of stable isoborne rates in Food analysis [ J ]. Food reviews International, 2001, 17 (3): 347-381.
Non-patent document 2: doner L W.carbon isotope ratios in natural and synthetic citric acids as indicators of lemon juice addition [ J ]. Journal of Agricultural and Food Chemistry, 1985, 33 (4): 770-772.
Non-patent document 3: jamin E, Gonzalez J, Remaud G, et al.detection of exogenous substrates or organic acids addition in pine juice and concentrations by 13C IRMS analysis [ J ]. Journal of Agricultural and Food Chemistry, 1997, 45 (10): 3961-3967.
Non-patent document 4: gonzalez J, Jamin E, Remaud G, et al, authentication of lemon threads and centres by a combined multi-isotope approach using SNIF-NMR and IRMS [ J ]. Journal of Agricultural and Food Chemistry, 1998, 46 (6): 2200-2205.
Non-patent document 5: houerou G, Kelly S D, Dennis M J.determination of the oxygen-18/oxygen-16 isoborne rates of sugar, citric acid and water from single strand organic juice j.Rapid communications in mass spectrometry, 1999, 13 (13): 1257-1262.
Non-patent document 6: jamin E, Martin F, Santamaria-Fernandez R, et al.detection of exogenous citric acid in front of juice J by table isotope ratio analysis [ J ]. Journal of aggregative and food chemistry, 2005, 53 (13): 5130-5133.
Non-patent document 7: guyon F, Auberger P, Gaill L, et al.13C/12C isocopate rates of organic acids, glucose and free determined by HPLC-co-IRMS for lemon juics automation [ J ]. Food chemistry, 2014, 146: 36-40.
Non-patent document 8: (Zhuweixia, Yangji, Yanna, et al. liquid chromatography-isotope ratio Mass Spectrometry method for studying carbon isotope ratio of citric acid in orange juice [ J ]. analytical laboratory, 2014, 33 (12): 1402-1407.)
Disclosure of Invention
In view of the above problems of the prior art, it is an object of the present invention to provide a method for measuring the citric acid δ in fruit juice easily and rapidly 13 Novel method for C value.
Means for solving the problems
The present inventors have conducted extensive studies to solve the above problems, and as a result, have found that removal of sucrose by acidolysis (particularly, using sulfuric acid) of a diluted sample does not affect the stability of citric acid [ delta ] by liquid chromatography-stable isotope ratio mass spectrometry using a hydrogen ion exchange column 13 And C value measurement. Based on the above findings, the present invention has been completed.
Briefly, the citric acid delta in the current juice is aimed at 13 C, establishing a rapid, accurate and economic analysis method for the citric acid delta in the fruit juice 13 Analysis of C and its application provide a technical approach.
The invention is based on the ingredient characteristics of the fruit juice product, according to the physical and chemical characteristics and the formula of the cane sugarThe analytical performance of a liquid chromatogram-stable isotope ratio mass spectrum system of a hydrogen ion exchange column is solved, and the problem of the prior fruit juice citric acid delta is solved 13 And C, off-line purification is required during determination to influence the analysis efficiency. The invention relates to citric acid delta of fruit juice 13 The rapid determination of C provides an effective solution that will promote the citric acid delta of fruit juice 13 And C, research and application provide a technical method for identifying the authenticity of the juice.
Specifically, the invention provides a method for simply measuring citric acid delta in fruit juice 13 A method of C value comprising the steps of:
a. sample dilution: diluting a fruit juice sample by 15-25 times with purified water to obtain a sample diluent;
b. hydrolysis of sucrose: adding a non-carbonaceous inorganic acid to the sample dilution to completely hydrolyze sucrose present in the sample dilution, thereby obtaining a hydrolysis solution;
c. diluting and filtering hydrolysate: diluting the hydrolysate to ensure that the concentration of citric acid in the diluted solution is 150-250 times of the concentration of citric acid in the juice sample, and then filtering to obtain filtrate;
d. analysis of citric acid delta by liquid chromatography-stable isotope ratio mass spectrometry 13 And C value.
Wherein the chromatographic column used in the liquid chromatography-stable isotope ratio mass spectrometry is a hydrogen ion exchange liquid chromatographic column.
In one embodiment, the solvent used for dilution in steps a and c is water, preferably ultrapure water.
In another embodiment, in step b, the acid is a dilute hydrochloric acid or dilute sulfuric acid, preferably, the concentration of the dilute sulfuric acid is 0.1mol/L to 2mol/L, preferably 1mol/L, the addition ratio of the dilute sulfuric acid is 10% to 30% relative to the weight of the sample diluent, and the sucrose hydrolysis is performed at 60 ℃ to 80 ℃ for 20 min to 150 min.
In a further embodiment, the filtration in step c is performed using a filter membrane having a pore size of 0.22 μm or 0.45 μm, preferably 0.22 μm.
In one embodiment, the hydrogen ion exchange liquid chromatography column is a HyperREZ XP carbonhydate H + model chromatography column.
In one embodiment, the mobile phase of the liquid chromatography is a sulfuric acid solution with pH 2, and the flow rate is 0.20-0.6 mL/min, preferably 0.25 mL/min; the temperature of the chromatographic column is 25-70 ℃, and preferably 30 ℃.
In one embodiment, the liquid chromatography-stable isotope ratio mass spectrometry is equipped with an IsoLink oxidation interface that uses phosphoric acid and sodium peroxodisulfate solution as reaction aids.
In one embodiment, the concentration and flow rate of the phosphoric acid and sodium peroxodisulfate solution are such that the ion current signal intensity at m/z 32 is 4000 to 18000mV, preferably 8000 mV.
In one embodiment, the fruit juice is from a fruit having citric acid as an important flavor profile component.
In a preferred embodiment, the fruit juice is orange juice, lemon juice, apple juice, peach juice, pear juice, grape juice, mango juice, pineapple juice, or papaya juice. In a further preferred embodiment, the fruit juice is orange, orange or lemon juice.
Advantageous results of the invention
The method avoids the influence of sucrose on the determination of citric acid by a hydrolysis method, has simple operation and low cost, and is delta to citric acid 13 The analysis efficiency of C is high, and the application of the stable isotope technology in the true and false identification of the fruit juice, especially in the detection of the adulteration of the citric acid can be promoted.
According to the characteristics of the fruit juice sample, the citric acid is purified and separated without complex sample pretreatment technology, the citric acid is prepared without adding one more liquid chromatogram, and the citric acid delta of the fruit juice is simplified by combining the technical characteristics of the liquid chromatogram-stable isotope ratio mass spectrum and the physical and chemical characteristics of the sucrose 13 C value determination step, the citric acid delta of the fruit juice is realized 13 The C value is quickly and accurately measured, and a new method is provided for the adulteration detection research and the production process control technology research of the fruit juice product.
In particular, the present invention provides an excellent effect as compared with the method of the great congratulatory clouds of non-patent document 8. Specifically, the method of great green requires two liquid chromatographs to work simultaneously, which increases the analysis cost and also requires a long sample measurement time: as can be seen from the article by pegleg, sucrose and citric acid cannot be effectively distinguished by using the hydrogen type ion column alone, so that a method for preparing a liquid phase is used, i.e., sucrose and citric acid are separated by using the zomax Ca type column on another liquid chromatography, the collected effluent contains citric acid and other organic matters (only sucrose is not available), and then citric acid and other organic matters are separated by using the hydrogen ion column, which requires 2 liquid chromatographs working simultaneously, 2 chromatographic columns are equipped, 2 detectors are used, the cost is high, and the analysis time is long because only one sample can be processed at a time when the liquid phase is prepared. The method combines a chemical hydrolysis method with an isotope determination method before isotope determination, can process n samples at one time during hydrolysis, only needs water and dilute acid, and reduces the material cost and time cost.
Drawings
FIG. 1 LC-IRMS measurement of sucrose and glucose delta 13 C, ion flow diagram;
FIG. 2 LC-IRMS determination of citric acid delta 13 C, ion flow diagram;
FIG. 3 LC-IRMS measurement of the delta of the solution after acid hydrolysis 13 C, ion flow diagram;
FIG. 4 LC-IRMS determination of citric acid delta in orange juice after acid hydrolysis 13 And C, ion flow diagram.
Detailed Description
The present invention removes sucrose by acid hydrolysis (particularly with sulfuric acid) of diluted samples, then converts and determines citric acid delta using liquid chromatography-stable isotope ratio mass spectrometry equipped with a hydrogen ion exchange column 13 C。
More specifically, the invention determines the citric acid delta of the fruit juice 13 A method of C value comprising the steps of:
a. diluting a sample;
b. hydrolyzing sucrose;
c. diluting and filtering the hydrolysate;
d. and (3) measuring the ratio of liquid chromatography to stable isotope by mass spectrometry.
The steps are described in detail below.
a. Sample dilution
In the measurement, the juice sample needs to be diluted by 15 to 25 times. The solvent used for sample dilution is water, preferably ultrapure water. The purpose of sample dilution is to reduce the content of organic substances such as saccharides and organic acids in a sample to an appropriate range, and particularly to hydrolyze the organic substances with acid as described below in a case where the sugar degree is appropriate, to improve the hydrolysis efficiency.
b. Hydrolysis of sucrose
According to literature analysis, the citric acid delta in the juice is influenced 13 The factor in the C assay is mainly sugar interference, especially sucrose. Therefore, the separation and purification of citric acid becomes delta 13 The key to the C assay. In the prior art, complex pretreatment of juice samples is required to eliminate the influence of sugars (especially sucrose) and other organic substances in juice.
The inventors have surprisingly found that the removal of sucrose by acid hydrolysis (particularly with sulphuric acid) of a diluted sample, rather than a complex sample pretreatment technique to purify and separate citric acid, does not affect the performance of liquid chromatography-stable isotope ratio mass spectrometry on citric acid delta by using a hydrogen ion exchange column 13 And C value determination.
In step b, adding a non-carbon-containing inorganic acid into the sample diluent, and completely hydrolyzing sucrose in the sample diluent under appropriate reaction conditions to obtain a hydrolysate.
The acid added during hydrolysis can be dilute hydrochloric acid or dilute sulfuric acid, preferably dilute sulfuric acid, for example, dilute sulfuric acid with the concentration of 0.1-2 mol/1, preferably 1mol/L, the adding proportion of the dilute sulfuric acid is 10-30% relative to the weight of the sample diluent, and the sucrose hydrolysis is carried out at 60-80 ℃ for 20-150 min. The hydrolysis is preferably carried out under water bath conditions. It should be noted that since the carbon isotope of citric acid is measured in the present invention, other carbon elements cannot be introduced, and therefore, carbon-containing inorganic acids such as carbonic acid and organic acids are not suitable.
Of course, other acids, other addition ratios, reaction temperatures, reaction times, and the like may be used as long as sucrose can be completely hydrolyzed. One skilled in the art can determine the reaction conditions required for complete hydrolysis of sucrose.
c. Diluting and filtering the hydrolysate;
and (3) diluting the hydrolysate to ensure that the concentration of citric acid in the diluted solution is 150-250 times of that in the juice sample, and then filtering to obtain filtrate.
For example, after the juice sample is diluted 15 to 25 times in step a, the hydrolyzed solution may be further diluted 10 times. The solvent used for sample dilution is still water, preferably ultrapure water.
Filtration is carried out using a filter membrane, for example, an aqueous microporous filter membrane, having a pore size of 0.45 μm or 0.22. mu.m, preferably 0.22. mu.m. The purpose of filtration is to prevent the macromolecular substances from blocking the liquid chromatographic column because water-insoluble impurities are to be removed during the liquid chromatographic analysis.
The sample loading volume after dilution is preferably 50. mu.L.
The reason why the sucrose hydrolysate needs to be further diluted in this step is that the acid needs to reach a certain concentration to be effective in hydrolyzing sucrose, but the concentration of the acid entering the liquid chromatography column causes damage to the instrument. Therefore, the step is mainly used for reducing the concentration of the acid, and simultaneously, the content of the citric acid is further diluted and is suitable for measurement.
d. Liquid chromatography-stable isotope ratio mass spectrometry
The liquid chromatography-stable isotope ratio mass spectrometry refers to that a hydrogen ion exchange column (preferably HyperREZ XP carbonhydate H +) is matched with an instrument, and various parameters are adjusted to be in a working state: the carrier gas for stabilizing the isotope ratio mass spectrum is helium; the mobile phase of the liquid chromatographic column is a sulfuric acid solution with pH 2; the flow rate is 0.25-0.60 mL/min; the column temperature is 25-70 ℃; adjusting the concentration and the flow rate of the reaction auxiliary agent until the background signal intensity of m/z which is 32 is 4000-18000 mv; the injection volume was 50. mu.L.
Specifically, citric acid separated by liquid chromatography needs to be oxidized to generate carbon dioxide detected by an instrument, so that the liquid chromatography-stable isotope ratio mass spectrum is provided with an IsoLink oxidation interface, phosphoric acid and a sodium peroxydisulfate solution are used as reaction aids in the IsoLink oxidation interface, wherein sodium peroxydisulfate is used as an oxidizing agent, and phosphoric acid is used as an acidifying agent. Too high a concentration of sodium peroxodisulfate tends to clog the capillary system, while too low a concentration tends to result in insufficient oxidizing power. In addition, the flow rate of the oxidizer and acidulant also affects the oxidizing ability. Through research, the ion current signal intensity of the ion current signal for adjusting the concentration and the flow rate of the reaction auxiliary agent to m/z of 32 is preferably 4000-18000 mV, preferably 8000 mV. In this case, the oxidation capacity meets the analytical requirements of the instrument.
Optionally, confirming that working environment, airtightness and vacuum degree of ion chamber of the stable isotope ratio mass spectrometer meet requirements, and then testing CO by an instrument 2 Middle delta 13 Precision and stability of C, adjusting ion source parameter values as necessary.
The invention is particularly suitable for juices from fruits having citric acid as an important flavour attribute, for example orange, lemon, apple, peach, pear, grape, mango, pineapple or papaya, and particularly for citric acid delta in orange, orange or lemon juice 13 And C value is measured, so that the adulterated fruit juice can be identified.
Examples
The invention will be explained in more detail below by means of the following examples. The following examples are illustrative only, and it should be understood that the present invention is not limited by the following examples.
Example 1:
1.1 instruments
Liquid chromatography-stable isotope ratio mass spectrometry (LC-IRMS, equipped with oxidation interface Isolink), all purchased from seimer feishell science and technology (china) ltd;
1.2 materials and reagents
Sucrose (analytical grade), glucose (analytical grade), citric acid (analytical grade); ultrapure water (Milli-Q system preparation); orthophosphoric acid (purity more than or equal to 99%) and sodium peroxodisulfate (purity more than or equal to 99%); helium (purity is more than or equal to 99.999%); 0.22 μm filter (aqueous system); sulfuric acid (purity is more than or equal to 99%).
1.3 chromatographic conditions
HyperRez XP Carbohydrate H + chromatography column (7.7X 300mm/8 μm); the mobile phase is 0.005mol/L sulfuric acid solution, and the flow rate is 0.25 mL/min; the column temperature was 30 ℃.
1.4 Oxidation interface operating conditions
The oxidation interface (IsoLink) used phosphoric acid and sodium peroxodisulfate solutions as reaction aids, and the ion current signal intensities at m/z 32, 33 and 34 were monitored simultaneously by IRMS, and the concentrations and flow rates of phosphoric acid and sodium peroxodisulfate solutions were adjusted to ensure an ion current signal intensity at m/z 32 of 8000 mV.
1.5 sample preparation
Purified water is used as a solvent, 0.6g/L sugar solution (sucrose: glucose is 1: 1) and 0.3g/L citric acid solution are prepared, and the mixture is filtered by a 0.22 mu m filter membrane for later use.
1.6 sample determination
Adjusting the liquid chromatogram-stable isotope ratio mass spectrometer to working state, and respectively taking sugar solution and citric acid solution for sample injection determination (sample injection volume is 50 μ L), sucrose/glucose and citric acid delta 13 The ion flow diagrams of C are shown in fig. 1 and 2, respectively.
1.7 analysis of results
As can be seen from FIGS. 1 and 2, the starting and stopping time of sucrose ion flow on the liquid chromatography-stable isotope ratio mass spectrum is 1347s to 1489s, the starting and stopping time of glucose is 1522 to 1674s, and the starting and stopping time of citric acid is 1324s to 1504s, and sucrose and citric acid have obvious overlap, which is the same as the research result of the mozzarella, and the separation degree of sucrose and citric acid of any hydrogen-type ion exchange column is lower.
Example 2
2.1 instruments
Liquid chromatography-stable isotope ratio mass spectrometry (LC-IRMS, equipped with oxidation interface Isolink), all purchased from seimer feishell science and technology (china) ltd;
2.2 materials and reagents
Sucrose (analytical grade), glucose (analytical grade), citric acid (analytical grade); ultrapure water (produced by Milli-Q systems); orthophosphoric acid (purity is more than or equal to 99 percent) and sodium peroxodisulfate (purity is more than or equal to 99 percent); helium (purity is more than or equal to 99.999%); 0.22 μm filter (aqueous); sulfuric acid (purity is more than or equal to 99%).
2.3 chromatographic conditions
HyperRez XP Carbohydrate H + chromatography column (7.7X 300mm/8 μm); the mobile phase is 0.005mol/L sulfuric acid solution, and the flow rate is 0.25 mL/min; the column temperature was 30 ℃.
2.4 Oxidation interface operating conditions
The oxidation interface (IsoLink) used phosphoric acid and sodium peroxodisulfate solutions as reaction aids, and the ion flux signal intensities of m/z 32, 33 and 34 were monitored simultaneously by IRMS, and the concentrations and flow rates of the phosphoric acid and sodium peroxodisulfate solutions were adjusted to ensure that the ion flux signal intensity of m/z 32 was 8000 mV.
2.5 sample preparation
Using purified water as a solvent, preparing 6g/L of sugar solution A (sucrose: glucose is 1: 1), 3g/L of citric acid solution B and sugar acid mixed solution D (sucrose, glucose and citric acid are all 3 g/L).
2.6 sample pretreatment
2.6.1 respectively diluting the sugar solution, the citric acid solution and the sugar acid mixed solution by 10 times and filtering the diluted solutions by using a 0.22 mu m filter membrane to obtain samples A1 and B1;
2.6.2 preparing 1mo/L sulfuric acid solution;
2.6.3 taking 1mL of each of the sugar solution A, the citric acid solution B and the sugar acid mixed solution, and adding 10% sulfuric acid solution to obtain samples A2, B2 and D2;
2.6.4 samples A2 and B2 were placed in a 70 ℃ water bath for 90min, diluted 10 fold after cooling and filtered through a 0.22 μm filter to give samples A3, B3 and D3.
2.7 determination
Adjusting the liquid chromatogram-stable isotope ratio mass spectrometer to be in a working state, and respectively measuring the delta of organic matters in the samples A1, B1, A3, B3 and D3 13 C values, results are shown in Table 1. The ion flow diagram of sample A1 is the same as in FIG. 1, the ion flow diagrams of samples B1 and B3 are the same as in FIG. 2, and the ion flow diagram of sample A3 is shown in FIG. 23。
TABLE 1 organic. delta 13 C measurement results
Figure BDA0002319448240000091
Figure BDA0002319448240000101
As can be seen from Table 1, the delta values of sucrose and glucose in sample A1 13 The C values were-18.63% and-12.02% respectively, sucrose was not detected in sample A3, and since A1 was a control sample in which A3 was not subjected to acid hydrolysis, it was considered that acid hydrolysis treatment completely hydrolyzed sucrose, while delta of glucose was 13 The C value is changed to-14.07 per mill because sucrose is hydrolyzed into glucose and fructose. Comparison of B1 and B3 revealed that citric acid delta before and after acidolysis 13 The difference of the C value is only 0.05 per mill (within 0.2 per mill of the determination error), namely the acidolysis treatment does not influence the delta of the citric acid 13 And C value measurement.
Sample D contained sucrose, glucose and citric acid together, due to the delta of sucrose and citric acid used in the test 13 C values are respectively-18.63 per mill and-11.66 per mill, while the first example shows that the starting and stopping time of cane sugar and citric acid flowing out of the chromatographic column is 1347 s-1489 s and 1324 s-1504 s, respectively, so that if the acidolysis process can not completely hydrolyze cane sugar, the residual cane sugar and citric acid can flow out of the chromatographic column at the same time, and delta of ion flow appears from 13024 s-1504 s 13 The value of C will necessarily be between that of sucrose and citric acid. The data in Table 1 show that the assay in sample D3 was-11.58% o, delta to citric acid 13 The C values are the same (-11.66 per mill and-11.73 per mill), which indicates that the sugar acid mixed solution completely hydrolyzes the sucrose after the acid hydrolysis treatment, so the organic matter in the chromatogram from 1320s to 1504s is citric acid.
Example 3
3.1 Instrument
Liquid chromatography-stable isotope ratio mass spectrometry (LC-IRMS, equipped with oxidation interface Isolink), all purchased from seimer feishell science and technology (china) ltd;
3.2 materials and reagents
Sucrose (analytical grade), glucose (analytical grade), citric acid (analytical grade); ultrapure water (Milli-Q system preparation); orthophosphoric acid (purity is more than or equal to 99 percent) and sodium peroxodisulfate (purity is more than or equal to 99 percent); helium (purity is more than or equal to 99.999%); 0.22 μm filter (aqueous system); sulfuric acid (purity is more than or equal to 99%).
3.3 chromatographic conditions
HyperRez XP Carbohydrate H + chromatography column (7.7X 300mm/8 μm); the mobile phase is 0.005mol/L sulfuric acid solution, and the flow rate is 0.25 mL/min; the column temperature was 30 ℃.
3.4 Oxidation interface operating conditions
The oxidation interface (IsoLink) used phosphoric acid and sodium peroxodisulfate solutions as reaction aids, and the ion current signal intensities at m/z 32, 33 and 34 were monitored simultaneously by IRMS, and the concentrations and flow rates of phosphoric acid and sodium peroxodisulfate solutions were adjusted to ensure an ion current signal intensity at m/z 32 of 8000 mV.
3.5 sample preparation
3.5.1 preparing 1mo/L sulfuric acid solution;
3.5.2 orange juice, lemon juice and apple juice are used as test raw materials, diluted by 15 times respectively, then 10% sulfuric acid solution is added, then the mixture is placed in a water bath at 70 ℃ for 90min, cooled, diluted by 10 times and filtered by a 0.22 mu m filter membrane, and the filtrate is collected to be tested (each sample is treated for 2 times respectively).
3.5.3 orange juice is used as test raw material, 20%, 40%, 60% and 80% citric acid (analytically pure) is added into the orange juice according to the content of citric acid in the orange juice, diluted 15-25 times respectively, then 10% sulfuric acid solution is added, then the orange juice is placed in a 70 ℃ water bath for 90min, after cooling, diluted 10 times and filtered by a 0.22 mu m filter membrane, and the filtrate is collected to be tested (each sample is treated 2 times respectively).
3.6 determination
Adjusting the liquid chromatogram-stable isotope ratio mass spectrometer to a working state, and respectively measuring the citric acid delta in the juice 13 C values, results are shown in Table 2. Delta of citric acid in fruit juice 13 The C ion flow diagram is shown in fig. 4.
TABLE 2 citric acid delta in juice samples 13 Measurement of C valueDetermine the result
Figure BDA0002319448240000111
As can be seen from table 2, the maximum deviation was only 0.18% o, which is determined in duplicate for each sample, indicating that the reproducibility of the method is good.
Delta of citric acid in orange, apple and lemon juices 13 C value is less than-26.53 per mill, which is equal to citric acid in the second embodiment (analytically pure, delta) 13 C-11.66 ‰) because analytically pure citric acid is produced by fermentation of microorganisms starting from corn (C4 plants), while orange, apple and lemon juices are C3 plants. While the blending test shows that the orange juice is added with citric acid to obtain delta 13 C shows a gradual positive trend and is in obvious positive correlation with the addition amount of citric acid (R) 2 0.999), which shows that the method can accurately determine the citric acid delta 13C value in the fruit juice, and on the other hand, the technology and theory can be used for detecting whether the fruit juice contains exogenously added citric acid.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (18)

1. Simple determination of citric acid delta in fruit juice 13 A method of C value comprising the steps of:
a. sample dilution: diluting a fruit juice sample by 15-25 times with purified water to obtain a sample diluent;
b. hydrolysis of sucrose: adding a non-carbonaceous mineral acid to the sample dilution to completely hydrolyze sucrose present in the sample dilution to obtain a hydrolysate;
c. diluting and filtering hydrolysate: diluting the hydrolysate to ensure that the concentration of citric acid in the diluted solution is 150-250 times of the concentration of citric acid in the juice sample, and then filtering to obtain filtrate;
d. analysis of citric acid delta by liquid chromatography-stable isotope ratio mass spectrometry 13 The value of C is the sum of the values of,
wherein the chromatographic column used in the liquid chromatography-stable isotope ratio mass spectrometry is a hydrogen ion exchange liquid chromatographic column.
2. The method of claim 1, wherein the solvent used for dilution in steps a and c is water.
3. The method according to claim 1, wherein in the step b, the inorganic acid is diluted hydrochloric acid or diluted sulfuric acid, the diluted sulfuric acid is added in a proportion of 10-30% relative to the weight of the sample diluent, and the sucrose hydrolysis is performed at 60-80 ℃ for 20-150 min.
4. The method of claim 1, wherein in step c the filtration is performed using a filter membrane having a pore size of 0.22 μm or 0.45 μm.
5. The method of claim 1, wherein the hydrogen ion exchange liquid chromatography column is a HyperREZ XP carbohydrate H + model chromatography column.
6. The method according to claim 1 or 5, wherein the mobile phase of the liquid chromatography is a sulfuric acid solution with pH 2, and the flow rate is 0.20-0.6 mL/min; the temperature of the chromatographic column is 25-70 ℃.
7. The method of claim 1, wherein the liquid chromatography-stable isotope ratio mass spectrometry is equipped with an IsoLink oxidation interface that uses phosphoric acid and sodium peroxodisulfate solution as reaction aids.
8. The method of claim 7, wherein the concentration and flow rate of the phosphoric acid and sodium peroxodisulfate solution are such that the ion current signal intensity at m/z-32 is 4000 to 18000 mv.
9. The method of claim 1, wherein the fruit juice is derived from fruit having citric acid as an important flavor profile component.
10. The method of claim 1, wherein the fruit juice is orange juice, lemon juice, apple juice, peach juice, pear juice, grape juice, mango juice, pineapple juice, or papaya juice.
11. The method of claim 2, wherein the water is ultrapure water.
12. The method of claim 3, wherein the dilute sulfuric acid has a concentration of 0.1 to 2 mol/L.
13. The method of claim 12, wherein the dilute sulfuric acid has a concentration of 1 mol/L.
14. The method of claim 4, wherein the pore size of the filter is 0.22 μm.
15. The method of claim 6, wherein the mobile phase of the liquid chromatography is a sulfuric acid solution with a pH of 2 and a flow rate of 0.25 mL/min.
16. The method of claim 6, wherein the chromatography column temperature is 30 ℃.
17. The method of claim 8, wherein the ion current signal intensity is 8000 mV.
18. The method of claim 10, wherein the fruit juice is orange, orange or lemon juice.
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