阅读说明：本技术 一种水果水分氢氧同位素比值的测定方法和应用 (Method for measuring hydrogen-oxygen isotope ratio of fruit moisture and application thereof ) 是由 李安 潘立刚 靳欣欣 于 2020-04-30 设计创作，主要内容包括：本发明涉及一种水果水分氢氧同位素比值的测定方法和应用,将水果榨汁后获得的果汁样品过固相萃取柱去除杂质,将经净化的水分样品还原为H<Sub>2</Sub>和CO,然后进行氢氧同位素比值的测定。本发明提供的水果水分氢氧同位素分析方法,与现有方法相比,操作简便快速、测定精度和准确度好,且能实现氢氧同位素的同时测定。本发明方法对于推动氢氧同位素技术进行水果产地溯源、果汁掺伪鉴别等领域的更广泛应用具有重要意义。(The invention relates to a method for measuring hydrogen-oxygen isotope ratio of fruit moisture and application thereof, which comprises the steps of removing impurities in a fruit juice sample obtained after fruit juicing through a solid-phase extraction column, and reducing a purified moisture sample into H 2 And CO, and then the determination of the hydrogen-oxygen isotope ratio is performed. Compared with the existing method, the method for analyzing the hydrogen and oxygen isotopes of the fruit moisture provided by the invention is simple, convenient and quick to operate, has good determination precision and accuracy, and can realize simultaneous determination of the hydrogen and oxygen isotopes. The method of the invention is used for promoting the hydrogen-oxygen isotope technology to carry out the fields of fruit producing area tracing, fruit juice adulteration identification and the likeHas important significance for wider application.)

1. A method for measuring the hydrogen-oxygen isotope ratio of fruit water is characterized in that a fruit juice sample obtained after fruit juicing is processed by a solid-phase extraction column to remove impurities, and the purified water sample is reduced to H₂And CO, and then the determination of the hydrogen-oxygen isotope ratio is performed.

2. The method of claim 1, comprising:

step 1), purifying: juicing fruits and centrifuging the juice sample to remove impurities by a solid-phase extraction column;

step 2), filtering: filtering the juice sample subjected to impurity removal in the step 1) through a microporous filter membrane to obtain a further purified water sample;

step 3), determination: reducing the moisture sample in step 2) to H₂And CO, and then the hydrogen-oxygen isotope ratio is determined.

3. The method of claim 2, further comprising:

step 4), correcting: and (3) measuring the hydrogen and oxygen isotopes of the standard water sample by adopting the methods of the steps 1) to 3), establishing a correction curve, and correcting the measured value of the sample in the step 3).

4. The method according to claim 2, wherein in step 1), the solid phase extraction column is an activated carbon solid phase extraction column, preferably a 500mg/6mL activated carbon solid phase extraction column.

5. The method according to claim 3, wherein in step 1), the sample loading amount is 2-3 mL, and the collected sample volume is 0.25-0.5 mL, preferably 0.25 mL.

6. The method as set forth in any one of claims 2 to 5, wherein in the step 2), the microfiltration membrane is a 0.45 μm aqueous membrane.

7. The method according to any one of claims 2 to 6, wherein in step 3), simultaneous determination of hydrogen and oxygen isotopes is performed in step 2) using elemental analyzer-stable isotope ratio mass spectrometry; preferably, the instrument test parameters are: the flow rate of the helium carrier gas is 90-110 mL/min, preferably 100 mL/min; the temperature of the pyrolysis tube is 1350-1410 ℃, and 1380 ℃ is preferable; the temperature of the molecular sieve chromatographic column is 65-85 ℃, preferably 75 ℃; the amount of the sample was 0.1. mu.L.

8. The method according to any one of claims 2 to 7, wherein the calibration curve in step 4) is a linear fit curve with the measured values of the standard water sample as abscissa and the real values of the standard water sample as ordinate.

9. The method according to any one of claims 2 to 8, wherein in step 1), the impurities comprise organic substances, preferably the organic substances are sugars, organic acids, pigments; and/or, the centrifugation conditions are: centrifuging at 8000-12000 rpm, preferably 10000rpm, for 3-10 min, preferably 5 min.

10. Use of the assay according to any one of claims 1 to 9 for the identification of fruit origin or adulteration of fruit juice.

Technical Field

The invention relates to the field of stable isotope analysis, in particular to a method for determining hydrogen-oxygen isotope ratio of fruit moisture and application thereof.

Background

The stable hydrogen and oxygen isotopes in the fruit moisture are important basis for tracing the fruit producing area and identifying adulteration of fruit juice. The composition of water-stable hydrogen-oxygen isotopes in the fruit juice is influenced by geographical, climatic and environmental factors such as altitude, temperature, latitude, and coast distance of fruit planting areaThus becoming a source tracing index for origin identification; the water stability heavy isotope(s) in the fruit due to transpiration during the fruit growth process²H and¹⁸o) is relatively enriched compared with source water such as underground water, atmospheric precipitation and the like, so that the hydrogen-oxygen isotope composition of the water in the fruit is obviously different from the doped exogenous water, and the stable hydrogen-oxygen isotope composition is the basis for distinguishing fresh fruit juice from blended fruit juice.

As the application of the hydrogen-oxygen isotope technology is more and more extensive, the detection method is also continuously promoted and improved. According to the literature report, the hydrogen and oxygen isotope ratio analysis of fruit moisture stability mainly comprises two types, namely, the method comprises the steps of collecting the water of a squeezed sample in a distillation mode, and measuring by using an element analyzer-stable isotope mass spectrometer; and secondly, after the water vapor and the external source gas are subjected to balance exchange by adopting an off-line balance method or an on-line balance method, the stable isotope mass spectrometer is used for determination. The former has the disadvantages that the distillation process of water is easy to cause the fractionation of heavy isotopes and light isotopes, which affects the accuracy of the result, and in addition, the distillation extraction requires a special vacuum extraction device of water and takes a long time (generally, the complete distillation requires more than 2 h). The latter is a method which is currently applied more because the operation is more automatic, but has the following disadvantages: firstly, the balancing process takes long time, and balancing is needed for 6 to 15 hours if the oxygen isotope is measured; secondly, the sample is easy to ferment and deteriorate due to long balance time, so that the accuracy of the result is influenced; respectively balancing and not simultaneously processing the hydrogen isotope and the oxygen isotope during the determination; and volatile components in the sample matrix such as ethanol and the like are easy to perform isotope exchange with the equilibrium reaction gas, and the measurement result is interfered.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art and realize simple, convenient and quick operation, high measurement precision and accuracy and simultaneous measurement of hydrogen and oxygen isotopes, the invention provides a method for measuring the hydrogen and oxygen isotope ratio of fruit moisture and application thereof.

The invention provides a method for measuring the hydrogen-oxygen isotope ratio of fruit moisture, which comprises the steps of passing a fruit juice sample obtained after fruit juicing through a solid-phase extraction columnRemoving impurities and reducing the purified water sample to H₂And CO, and then the determination of the hydrogen-oxygen isotope ratio is performed.

According to some preferred embodiments of the invention, comprising:

step 1), purifying: juicing fruits and centrifuging the juice sample to remove impurities by a solid-phase extraction column; the solid phase extraction column can effectively remove impurities such as sugar and the like;

step 2), filtering: filtering the juice sample subjected to impurity removal in the step 1) through a microporous filter membrane to obtain a further purified water sample; the microporous filter membrane is used for filtering particulate matters, bacteria and the like;

step 3), determination: reducing the moisture sample in step 2) to H₂And CO, and then the hydrogen-oxygen isotope ratio is determined.

According to some preferred embodiments of the present invention, further comprising:

step 4), correcting: and (3) measuring the hydrogen and oxygen isotopes of the standard water sample by adopting the methods of the steps 1) to 3), establishing a correction curve, and correcting the measured value of the sample in the step 3).

According to some preferred embodiments of the present invention, in step 1), the solid phase extraction column is an activated carbon solid phase extraction column, preferably 500mg/6mL activated carbon solid phase extraction column.

According to some preferred embodiments of the present invention, in step 1), the sample loading amount is 2-3 mL, and the collected sample volume is 0.25-0.5 mL, preferably 0.25 mL.

According to some preferred embodiments of the invention, in step 2), the microfiltration membrane is a 0.45 μm aqueous membrane.

According to some preferred embodiments of the present invention, in step 3), the hydrogen and oxygen isotopes are simultaneously determined in step 2) by using an element analyzer-stable isotope ratio mass spectrometer; preferably, the instrument test parameters are: the flow rate of the helium carrier gas is 90-110 mL/min, preferably 100 mL/min; the temperature of the pyrolysis tube is 1350-1410 ℃, and 1380 ℃ is preferable; the temperature of the molecular sieve chromatographic column is 65-85 ℃, preferably 75 ℃; the amount of the sample was 0.1. mu.L.

According to some preferred embodiments of the inventionIn an implementation mode, the moisture sample enters a pyrolysis tube of an element analyzer to be converted into H₂And CO, separated in the chromatographic column of molecular sieve under the drive of carrier gas, and enter the mass spectrometer of stable isotope ratio, the charged ion of different mass-to-charge ratios is separated under the influence of magnetic field, and receive and change into the electrical signal by the Faraday cup, the system calculates the isotope ratio of hydrogen and oxygen separately, fig. 1 is H that the instrument obtains after gathering the relevant signal₂And CO isotopic isomer ion mass spectra. Preference is given to H in mass spectrometers₂Is ionized by the ion source into isotopic isomer ions having mass to charge ratios of 2 and 3, and CO is ionized by the ion source into isotopic isomer ions having mass to charge ratios of 28,29 and 30.

According to some preferred embodiments of the present invention, the calibration curve in step 4) is a linear fitting curve with the measured value of the standard water sample as abscissa and the real value of the standard water sample as ordinate.

According to some preferred embodiments of the present invention, in step 1), the impurities include organic matters, preferably, the organic matters are sugar, organic acid, pigment; and/or, the centrifugation conditions are: centrifuging at 8000-12000 rpm, preferably 10000rpm, for 3-10 min, preferably 5 min.

The invention also provides the application of the determination method in tracing the fruit producing area or identifying the adulteration of the fruit juice.

The invention has the beneficial effects that: compared with the prior art, the method for analyzing the hydrogen and oxygen isotopes of the fruit moisture provided by the invention at least has the following advantages: the method is simple, convenient and quick to operate, and does not need time-consuming water distillation extraction operation and water vapor equilibrium reaction; secondly, interference factors in the pretreatment process are few, and the measurement precision and accuracy are good; and the hydrogen and oxygen isotopes can be simultaneously measured, so that the detection cost is further saved. The method has important significance for promoting the hydrogen-oxygen isotope technology to carry out the wider application in the fields of fruit producing area tracing, fruit juice adulteration identification and the like

Drawings

FIG. 1 is the isotope mass spectrum of the determination of the water stability hydrogen-oxygen isotope ratio of the fruit juice.

FIG. 2 shows the total sugar content variation in different volumes of effluent.

FIG. 3 is a calibration curve of hydrogen-oxygen isotope ratio (left: hydrogen isotope; right: oxygen isotope).

FIG. 4 shows the distribution range of hydrogen-oxygen isotope ratio of peach fruits in different producing areas.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The embodiments or examples of the present invention do not indicate any particular technique or condition, and the embodiments or examples are performed according to the technique or condition described in the literature in the art or according to the product specification. In the present invention, the instruments and the like used are conventional products which are purchased from regular vendors, not indicated by manufacturers. The raw materials used in the invention can be conveniently bought in domestic product markets.

According to some embodiments of the invention, the method specifically comprises the following steps:

(1) centrifuging: the juice sample obtained from the squeezed juice was centrifuged at 10000rpm for 5 min.

(2) Purifying: using a 500mg/6mL activated carbon solid-phase extraction column as an adsorption purification column to adsorb sugar in the fruit juice, taking 2-3 mL of centrifuged fruit juice supernatant, pressurizing by using an injector to enable the sample to slowly pass through the solid-phase extraction column, controlling the flow rate to be 0.5-1mL/min, and collecting 0.25mL of purified water flowing out initially.

(3) And (3) filtering: and (3) passing the collected water through a 0.45-micron water system microporous filter membrane, filtering out particulate impurities and bacteria eluted when the collected water passes through a solid-phase extraction column, transferring the sample into a sample injection small bottle with an inner lining pipe, and waiting for computer analysis.

(4) And (3) determination: determination of Stable Hydrogen-oxygen isotope ratio of purified Water Using an element Analyzer-Stable isotope Mass spectrometer²H and¹⁸o). After the moisture sample enters a high-temperature cracking tube of an element analyzer, hydrogen and oxygen elements of the moisture are respectively converted into H₂And CO, the converted gas is separated in the molecular sieve chromatographic column under the drive of the carrier gas and then enters a stable isotope ratio mass spectrometer, and H in the mass spectrometer₂Ionized by the ion source to mass-to-charge ratios of 2 and 3Isotopic isomer ions, CO, are ionized by an ion source into isotopic isomer ions with mass-to-charge ratios of 28,29 and 30, charged ions with different mass-to-charge ratios are separated under the action of a magnetic field and received by a Faraday cup to be converted into electric signals, and the system respectively calculates the isotopic ratio of hydrogen to oxygen. The instrument test parameters are as follows: the flow rate of the helium carrier gas is 100 mL/min; the temperature of the pyrolysis tube is 1380 ℃; the temperature of the molecular sieve chromatographic column is 75 ℃; the amount of the sample was 0.1. mu.L.

(5) And (3) correction: and (3) treating the same batch of active carbon solid phase extraction columns by using a standard water sample according to the fruit juice water purification method and analyzing and determining the same instrument parameters to obtain the stable isotope ratio of the standard water sample, establishing a correction curve, and correcting the hydrogen-oxygen isotope ratio of the sample.