CN114518419A

CN114518419A - Method for determining earthy smell substances in freshwater fish

Info

Publication number: CN114518419A
Application number: CN202210070848.6A
Authority: CN
Inventors: 田良良; 胥亚夫; 孔聪; 蔡友琼; 方长玲; 沈晓盛
Original assignee: East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences
Current assignee: East China Sea Fishery Research Institute Chinese Academy of Fishery Sciences
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2022-05-20

Abstract

The invention discloses a method for measuring earthy smell substances in freshwater fish, which comprises the following steps: firstly, extracting earthy substances in the minced freshwater fish by using normal hexane twice to obtain an extracting solution; secondly, adding C18 powder, graphitized carbon black and anhydrous magnesium sulfate into the extracting solution obtained in the first step, fully oscillating and centrifuging to achieve the purification effect; thirdly, purifying and concentrating the sample extracting solution by adopting a silica gel solid phase extraction column to obtain an eluent; and fourthly, determining the content of the earthy flavor substances in the eluent obtained in the third step by using a gas chromatography-mass spectrometry method. The method for measuring the earthy taste substances in the freshwater fish is simple and easy to operate, and has higher accuracy and better stability.

Description

Method for determining earthy smell substances in freshwater fish

Technical Field

The invention belongs to the technical field of food detection, and relates to a method for determining earthy-smell substances, namely Geosmin (GSM) and 2-methylisoborneol (2-MIB) in freshwater fish.

Background

The freshwater fish yield in China is very high, accounts for 65% of the world, and plays a key role in effective supply of aquatic products in the market. However, the fresh water fish generally has unpleasant earthy taste, the taste and the economic value of the fresh water fish and the processed product thereof are seriously influenced, the healthy development of the fresh water fish culture and the processing industry is restricted, and substances which generate the earthy taste mainly comprise geosmin and 2-methyl isoborneol. In view of the situation, an effective method for detecting the earthy taste substances in the freshwater fish is urgently needed to be established, and technical support is provided for further research.

In the last 30 years, a plurality of methods for measuring GSM and 2-MIB in water environment have been developed, but the research on the detection method of the earthy smell substances in the freshwater fish is less at home and abroad, although the human nose can detect the earthy smell substances and the 2-methylisoborneol in the range of parts per billion, the human nose can only provide semi-quantitative data, and the problem of sensory overload is easy to occur; the electronic nose adopting a special sensor can detect the geosmin and the 2-methylisoborneol, but the sensitivity and the selectivity in a complex substrate such as a fish body cannot meet the requirements; the microwave distillation-solid phase microextraction method has the disadvantages of complicated device, low recovery rate and unsatisfactory effect.

Disclosure of Invention

The invention aims to provide an effective method for measuring the earthy smell substances in the freshwater fish, which can be conveniently popularized and used, and can measure the earthy smell substances, namely the geosmin and the 2-methylisoborneol in the freshwater fish.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a method for measuring earthy smell substances in freshwater fish, which comprises the following steps:

firstly, extracting 2-10g of earthy flavor substances in minced freshwater fish by adopting 10-50mL of normal hexane twice to obtain an extracting solution;

secondly, adding C18 powder, graphitized carbon black and anhydrous magnesium sulfate into the extracting solution obtained in the first step, wherein the mass ratio of the C18 powder to the graphitized carbon black to the anhydrous magnesium sulfate is 1:1 (3-5), and centrifuging after fully oscillating to achieve a purification effect;

thirdly, purifying and concentrating the sample extracting solution by adopting a silica gel solid phase extraction column to obtain an eluent;

and fourthly, determining the content of the earthy flavor substances in the eluent obtained in the third step by using a gas chromatography-mass spectrometry method.

The freshwater fish is selected from crucian and grass carp.

The first step comprises the steps of: adding normal hexane into the freshwater fish meat paste, fully oscillating, performing first ultrasonic treatment and first centrifugation, and taking supernatant to a centrifugal tube; adding n-hexane into the fish meat, fully shaking, performing ultrasonic treatment for the second time, centrifuging for the second time, taking the supernatant, and adding the supernatant into a centrifugal tube.

The time of the first ultrasonic treatment in the first step is 5min-30 min.

The time of the second ultrasonic in the first step is 5min-30 min.

The rotating speed of the first centrifugation in the first step is 4000r/min-10000r/min, and the time is 5min-30 min.

The rotation speed of the second centrifugation in the first step is 4000r/min-10000r/min, and the time is 5min-30 min.

The rotation speed of the centrifugation in the second step is 2000r/min-10000r/min, and the time is 3min-10 min.

The third step includes the steps of: activating a silica gel column (500mg/3mL) by using 3-10 mL of n-hexane, passing the supernatant obtained after centrifugation in the second step through the column, leaching by using 1-10 mL of n-hexane, drying the solid phase extraction column, eluting by using 1-5 mL of n-hexane/ethyl acetate with the volume ratio of (1-3): 1, drying the solid phase extraction column, receiving eluent, mixing in a vortex mode, and putting into a sample injection vial.

The gas chromatography conditions in the fourth step: HP-5MS capillary column: 30 m.times.0.25 mm.times.0.5 μm; 99.999 percent of high-purity helium, adopting a constant flow mode, and enabling the flow rate of carrier gas to be 1.0 mL/min; controlling temperature programming, wherein the initial temperature is 60 ℃, keeping for 1min, increasing to 120 ℃ at 5 ℃/min, keeping for 3min, increasing to 170 ℃ at 10 ℃/min, keeping for 1min, increasing to 280 ℃ at 20 ℃/min, keeping for 5 min; the injection port temperature is 250 ℃, and split-flow injection is not carried out.

The mass spectrum condition in the fourth step is as follows: and (3) bombarding an ion source EI by electrons, wherein the ion source temperature is 250 ℃, the transmission line temperature is 280 ℃, the electron energy is 70eV, and data are acquired by selecting an ion scanning SIM mode.

The earthy substance is geosmin and 2-methylisoborneol.

The content of geosmin is 0.4-100 μ g/kg.

The content of the 2-methylisoborneol is 0.4-100 mu g/kg.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the invention adopts the normal hexane which is easy to dissolve in GSM and 2-MIB for extraction, can improve the extraction efficiency and is beneficial to the realization of the purification and concentration steps. And the QuChERS method is adopted for purification, and the silica gel column is adopted for purification and concentration, so that the impurity interference can be avoided, the detection sensitivity is improved, and the gas chromatography-mass spectrometry determination is facilitated. The method can be realized only by a centrifuge and a semi-automatic solid phase extractor which are commonly used for chemical analysis, and compared with the solid phase microextraction method reported in the prior art, the method needs complex microwave and airflow devices, is simpler to operate, has higher accuracy and better stability, and is easier to realize simultaneous determination of a large number of samples.

Drawings

FIG. 1 is a schematic diagram showing the effect of different sample inlet temperatures on the signal-to-noise ratio of 2-MIB and GSM chromatographic peaks.

FIG. 2 is a schematic representation of the effect of different column initial temperatures on peak heights of 2-MIB and GSM chromatograms.

FIG. 3 is a graph showing the effect of different column initial temperatures on the signal-to-noise ratio of the 2-MIB and GSM chromatographic peaks.

FIG. 4 is a schematic diagram of a change rule of 2-MIB and GSM peak high at different temperature rise rates.

FIG. 5 is a graph showing the recovery of the target substances in the reagents after adsorption of 2-MIB and GSM onto C18 powder in the different reagents.

FIG. 6 is a graph showing the recovery of the target substances in the reagents after the graphitized carbon black powder in different reagents adsorbs 2-MIB and GSM.

FIG. 7 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM by PSA in different reagents.

FIG. 8 is a graph showing the recovery of the target substances from the reagents after adsorption of 2-MIB and GSM on anhydrous magnesium sulfate in the different reagents.

FIG. 9 is a graph showing the recovery of the target from the reagents after adsorption of 2-MIB and GSM onto silica powder in the different reagents.

FIG. 10 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM on neutral alumina powder in different reagents.

FIG. 11 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM on basic alumina in different reagents.

FIG. 12 is a graph showing the recovery of the target in the reagent after adsorption of 2-MIB and GSM onto Florisil in the different reagents.

FIG. 13 is a graph showing the recovery of the target from the reagent after adsorption of 2-MIB and GSM onto diatomaceous earth in different reagents.

FIG. 14 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM to chitosan in different reagents.

FIG. 15 is a graph illustrating the effect of different ultrasound times on 2-MIB and GSM.

FIG. 16 is a graph showing elution efficiency of n-hexane and acetone, which are elution reagents in different ratios.

FIG. 17 is a graph showing elution efficiency of n-hexane and ethyl acetate as elution reagents in different ratios.

FIG. 18 is a graphical representation of the effect of different capacity silica gel solid phase extraction columns on recovery of 2-MIB and GSM.

FIG. 19 is a graphical representation of the matrix effect of different sample matrices on 2-MIB and GSM assays.

FIG. 20 is a schematic diagram of an apparatus used in the microwave distillation-solid phase microextraction process.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1

The method for determining the earthy taste substances including geosmin and 2-methylisoborneol in the freshwater fish by using n-hexane extraction-solid phase extraction concentration-gas chromatography-mass spectrometry comprises the following steps:

removing scales, viscera and skin of crucian, taking out muscle along the back, homogenizing, and mixing.

The first step, extraction: weighing 5g of crucian carp minced meat, adding 15mL of n-hexane, fully shaking, carrying out ultrasonic treatment for 15min, centrifuging at 4500r/min for 8min, and taking supernatant to a 50mL centrifuge tube; and adding 10mL of n-hexane into the fish, fully shaking, carrying out ultrasonic treatment for 15min, centrifuging at 4500r/min for 8min, taking the supernatant, and adding the supernatant into a 50mL centrifuge tube.

And step two, purification: 0.5g of C18 powder, 0.5g of graphitized carbon black and 2g of anhydrous magnesium sulfate are added into the centrifuge tube in the first step, the mixture is shaken for 7min, and centrifuged for 5min at 2500 r/min.

Step three, silica gel column purification and concentration: activating a silica gel column (500mg/3mL) by using 10mL of n-hexane, passing the supernatant obtained after the second step of centrifugation through the column, leaching by using 3mL of n-hexane, drying the solid phase extraction column, eluting by using 2mL of n-hexane/ethyl acetate with the volume ratio of 3:1, drying the solid phase extraction column, receiving the eluent by using a 5mL glass tube, mixing in a vortex mode, and placing into a sample injection vial.

Fourthly, gas chromatography-mass spectrometry determination: and (4) carrying out gas chromatography-mass spectrometry on the sample in the sample injection vial in the third step to determine the content of the earthy taste substances.

Gas chromatography conditions: HP-5MS capillary column (30 m.times.0.25 mm. times.0.5 μm); high-purity helium (99.999%) is adopted, and the flow rate of carrier gas is 1.0mL/min in a constant flow mode; controlling temperature programming, wherein the initial temperature is 60 ℃, keeping for 1min, increasing to 120 ℃ at 5 ℃/min, keeping for 3min, increasing to 170 ℃ at 10 ℃/min, keeping for 1min, increasing to 280 ℃ at 20 ℃/min, keeping for 5 min; the injection port temperature is 250 ℃, and split-flow injection is not carried out.

Mass spectrum conditions: electron impact ion source (EI), ion source temperature 250 ℃, transmission line temperature 280 ℃ and electron energy 70 eV. Data is acquired in a selected ion Scan (SIM) mode. 2-MIB quantitative ions are m/z94.9, and qualitative ions are m/z94.9, 107.9 and 134.9; the GSM quantitative ions are m/z111.9, and the qualitative ions are m/z111.9, 124.9 and 96.9.

1. And (4) injecting according to the gas chromatography and mass spectrum conditions, and determining the peak area. And (4) drawing a linear curve by taking the peak area as a vertical coordinate (Y) and the analyte concentration as a horizontal coordinate (X) to obtain a linear regression equation and a linear range of each component.

Linear curve: the linear equation of the 2-methylisoborneol is Y ═ 609.962+13239.1X, the linear range is 2-500ng/mL, and R is²＝0.9997。

Linear equation of geosmin is 960.238+15714.5X, linear range is 2-500ng/mL, R²＝0.9997。

The detection limit of the method is 0.6 mu g/kg.

2. Precision test

Precisely absorbing the same mixed reference substance solution, continuously feeding samples for 6 times respectively according to chromatographic and mass spectrum conditions, recording the peak area of the component to be detected, calculating the RSD value, and inspecting the precision in the day; the analysis was continued for 3 days, and the RSD values of the peak areas of the 2 components were calculated to examine the precision during the day. The precision RSD of the instrument in the day is 1.3% -5.1%, the precision RSD of the instrument in the day is 1.6% -7.8%, and the precision RSD is less than 15%, which indicates that the precision of the instrument is good.

3. Method condition optimization

3.1 Mass Spectrometry Condition optimization

Respectively preparing 2ug/mL standard solutions of 2-methylisoborneol and geosmin, respectively performing full scan measurement, searching mass spectrum database and comparing characteristic ions in literature to determine the peak of the target, determining the characteristic ions of 2-methylisoborneol as 94.9/107.9/134.9 and the characteristic ions of geosmin as 111.9/124.9/96.9 according to the abundance and specificity of ions in the full scan.

3.2 chromatographic Condition optimization

3.2.1 Effect of injection Port temperature

Experiments compare the influence of the sample inlet temperature of 200 ℃, 250 ℃ and 280 ℃ on the detection sensitivity of the 2-MIB and the GSM, and find that the peak area and the peak height change are not obvious, but the change of the signal-to-noise ratio (S/N) is obvious, as shown in figure 1, and figure 1 is a schematic diagram of the influence of different sample inlet temperatures on the signal-to-noise ratios of the 2-MIB and the GSM chromatographic peaks. When the temperature of the sample inlet is 250 ℃, the signal-to-noise ratio is highest, so that the temperature of the sample inlet is selected to be 250 ℃.

3.2.2 Effect of initial temperature of temperature program on 2-MIB and GSM sensitivity

Experiments compare the influence of initial column temperatures of 60 ℃ and 100 ℃ on the detection sensitivity of a target object, and find that the peak area changes little, but the peak height and the signal-to-noise ratio are obviously different, as shown in fig. 2 and fig. 3, fig. 2 is a schematic diagram of the influence of different column initial temperatures on the peak heights of 2-MIB and GSM chromatograms, and fig. 3 is a schematic diagram of the influence of different column initial temperatures on the signal-to-noise ratios of 2-MIB and GSM chromatograms. As can be seen from the figure, the peak height at the initial temperature of the column at 60 ℃ is higher, the signal-to-noise ratio is better, and the peak height at the initial temperature of the column at 100 ℃ is lower, the peak width is wider, so the signal-to-noise ratio is smaller. Therefore, an initial temperature of 60 ℃ is selected.

3.2.3 Effect of ramp Rate on 2-MIB and GSM sensitivity

Measuring the 100ng/mL mixed standard solution of the 2-MIB and the GSM by adopting different heating rates of 2 ℃/min, 5 ℃/min, 8 ℃/min, 9 ℃/min, 10 ℃/min, 11 ℃/min, 12 ℃/min, 13 ℃/min, 14 ℃/min and 15 ℃/min, and as shown in figure 4, figure 4 is a schematic diagram of the variation rule of the peak high of the 2-MIB and the GSM under different heating rates. It can be seen from the figure that as the temperature rise rate increases, the peak areas of 2-MIB and GSM do not change significantly, the peak height gradually increases, and the corresponding signal-to-noise ratio also increases, which is likely to be higher because the faster the temperature rise rate, the smaller the peak width, and so the higher the peak height. Therefore, when the sample is measured, the detection sensitivity is improved by adopting a larger temperature rise rate under the condition of ensuring no interference of an impurity peak.

3.3 adsorption rates of different purification materials in different reagents on 2-MIB and GSM

At present, the determination method for 2-MIB and GSM mainly adopts a solid phase microextraction method, so that reference data for extracting and purifying reagents of the method is lacked, and in order to design an extraction and purification scheme for 2-MIB and GSM, the adsorption rates of different materials in different reagents on the 2-MIB and the GSM are tested. Respectively preparing 5mL of standard solutions of 50ng/mL of 2-MIB and GSM by adopting four reagents of normal hexane, ethyl acetate, acetone and acetonitrile, respectively adding 0.5g of graphitized carbon black, C18, PSA, anhydrous magnesium sulfate, silica gel, neutral alumina, basic alumina, Florisil, diatomite and chitosan, fully oscillating for adsorption, standing, taking supernatant for determination, and performing two parallel experiments. The results are shown in FIGS. 5 to 14. FIG. 5 is a graph showing the recovery of the target substance from the reagents after adsorption of 2-MIB and GSM onto C18 powder in the different reagents. FIG. 6 is a graph showing the recovery rates of the target substances in the reagents after the graphitized carbon black powder in different reagents adsorbs 2-MIB and GSM. FIG. 7 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM by PSA in different reagents. FIG. 8 is a graph showing the recovery of the target substances from the reagents after adsorption of 2-MIB and GSM on anhydrous magnesium sulfate in the different reagents. FIG. 9 is a graph showing the recovery of the target from the reagents after adsorption of 2-MIB and GSM onto silica powder in the different reagents. FIG. 10 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM on neutral alumina powder in different reagents. FIG. 11 is a graph showing the recovery of the target in the reagents after adsorption of 2-MIB and GSM onto basic alumina in the different reagents. FIG. 12 is a graph showing the recovery of the target in the reagent after adsorption of 2-MIB and GSM onto Florisil in the different reagents. FIG. 13 is a graph showing the recovery of the target from the reagent after adsorption of 2-MIB and GSM onto diatomaceous earth in different reagents. FIG. 14 is a graph showing the recovery of target from reagents after adsorption of 2-MIB and GSM to chitosan in different reagents.

As can be seen from the figure, the adsorption of C18 powder, graphitized carbon black, N-Propyl Silane (PSA) to 2-MIB and GSM increases with the increasing polarity of the reagent, and the adsorption in N-hexane is minimal; the adsorption of the anhydrous magnesium sulfate to the 2-MIB and the GSM in different reagents is small; silica gel powder and neutral alumina powder completely adsorb 2-MIB and GSM in normal hexane, but do not adsorb in ethyl acetate and acetone basically, and partially adsorb in acetonitrile; the basic alumina and the florisil can adsorb more than 70 percent of 2-MIB and GSM in normal hexane, and the adsorption in ethyl acetate and acetone is less than 10 percent. By combining the adsorption characteristics of the materials on 2-MIB and GSM in different reagents, C18 powder, graphitized carbon black, PSA, anhydrous magnesium sulfate and silica gel powder are selected for the next experiment.

3.4 optimization of extraction reagents

Acetonitrile and n-hexane were used for sample extraction experiments, respectively. Weighing 5g of crucian carp, adding 750ng of 2-MIB and GSM standard substance, respectively extracting by 15mL of acetonitrile and n-hexane, purifying by QuChERS (0.5g of graphitized carbon black, 0.5g of C18,0.5g of PSA, 2g of anhydrous magnesium sulfate), and measuring after passing through a 0.2 mu m organic filter membrane. The results showed that acetonitrile extracted less impurities but the recovery rate was 37% to 76%, while n-hexane extracted more impurities but the recovery rate was more than 90%. The low recovery rate of the target in the acetonitrile is probably related to the high adsorption rate of the QuChERS material to the 2-MIB and the GSM in the acetonitrile, and no material capable of completely adsorbing the target exists in the acetonitrile, so that the possibility of concentration is limited, and the QuChERS material cannot be used as an extraction reagent; although the extraction of n-hexane has certain interference, the n-hexane is finally selected as the extracting solution because a purifying material can further purify and concentrate.

3.5 selection of extraction ultrasound time

Because the literature reports that the ultrasonic can cause the degradation of 2-MIB and GSM, the ultrasonic time during the extraction is tested, 100ng/mL standard solution is subpackaged into 2mL sample injection vials, each sample injection vial is respectively filled with 0.7mL, and the ultrasonic time is respectively 15min, 30min, 1.0h, 1.5h, 2.0h, 2.5h and 3.0 h. FIG. 15 is a schematic diagram showing the effect of different ultrasound times on 2-MIB and GSM, as shown in FIG. 15. The results show that the recovery rate is relatively increased with the prolonged ultrasound time, which is probably caused by the fact that the normal hexane reagent is volatilized due to the temperature increase of the liquid caused by the ultrasound, the volume of the solution is reduced, and the concentration of the target substance is increased. Within 1.0h of ultrasonic treatment, the recovery rate difference between 2-MIB and GSM is not obvious, but along with the prolonging of ultrasonic time, the recovery rate difference between the 2-MIB and the GSM is larger and larger, the 2-MIB is increased more quickly, the GSM basically tends to be stable, probably because the degradation of geosmin caused by the ultrasonic treatment is related, the ultrasonic time is long, although the reagent is volatilized to cause the volume reduction, the degradation of the reagent causes the concentration reduction, a certain balance is achieved, and the degradation speed of the 2-methylisoborneol is slower, so the difference between the 2-MIB and the GSM is caused. This is consistent with the first order kinetics of degradation of 2-MIB and GSM reported in the literature with rate constants of 0.07 and 0.12min-1, respectively. Therefore, when ultrasonic extraction is adopted, under the condition of ensuring complete extraction, the ultrasonic time is not too long to cause degradation, and finally ultrasonic treatment is selected for 15 min.

3.6QuChERS purification Condition optimization

The QuChERS purification commonly uses C18, graphitized carbon black, PSA and anhydrous magnesium sulfate, so the combination of the two is tested, 80ng/mL of 2-MIB and 10mL of GSM standard solution are prepared by normal hexane, 0.5g of C18,0.5g of graphitized carbon black, 0.5g of PSA and 2g of anhydrous magnesium sulfate are respectively added, the mixture is fully vibrated, centrifuged, and the supernatant is taken for determination. As a result, it was found that the recovery rates of 2-MIB were 74.4 and 76.4% and the recovery rates of GSM were 87.6 and 89.5%. As can be seen from the adsorption results of different purifying materials in different reagents on a target object, the recovery rate is low mainly due to the relatively high adsorption rate of PSA on the target object, so that graphitized carbon black, C18 and anhydrous magnesium sulfate are selected as the purifying materials, and the use of PSA is abandoned.

3.7 selection and optimization of concentration conditions

Experiments of nitrogen blowing and rotary evaporation by adopting a 200ng/mL standard solution show that the nitrogen blowing concentration is only 0-3.3% in the recovery rate of heating nitrogen blowing, and 2.2-32.6% in the recovery rate of non-heating small air flow blowing; when rotary evaporation concentration is adopted, the recovery rate is 0-22.1% when the vacuum degree is large, and the recovery rate is 1.04-33.5% when the vacuum degree is small. Since 2-MIB and GSM are semi-volatile substances, the nitrogen-blowing and rotary evaporation concentration recovery rate is low, and the stability is poor, the concentration cannot be carried out by adopting the method.

Because the adsorption rate of the silica gel to the 2-MIB and the GSM in the normal hexane can reach 99 percent, the adsorption rate of the silica gel is less than 5 percent in an ethyl acetate and acetone system, and the purification and the concentration of the extracting solution can be realized according to the characteristic.

Elution experiments were performed using a silica gel solid phase extraction column (500mg/3 mL). 1mL of standard solution dissolved by 100ng/mL of n-hexane passes through a solid phase extraction column activated by n-hexane, the column is dried, and elution is carried out by using 2mL of n-hexane/acetone (4/1, 3/1, 2/1 and 1/1) and n-hexane/ethyl acetate (0/1, 1/1, 3/1, 5/1 and 7/1), and the results are shown in FIGS. 16 and 17, and FIG. 16 is a schematic diagram of elution efficiency of n-hexane and acetone which are elution reagents in different proportions. FIG. 17 is a graph showing elution efficiency of n-hexane and ethyl acetate as elution reagents in different ratios. It can be seen from the figure that the deviation of the elution efficiency of the n-hexane/acetone is relatively large, the deviation of the n-hexane and the ethyl acetate is relatively small, and the n-hexane/ethyl acetate (v/v, 3/1) is selected as the eluent by comprehensively considering the elution efficiency and the elution effect.

3.8 selection of silica gel columns of different capacities

When the column packing is less, the volume of the reagent used for elution is small, and the concentration multiple is large; when the column packing is large, the volume of the reagent used for elution is large, and the concentration multiple is small. Therefore, the solid phase extraction column with less filler should be selected under the condition of meeting the experimental requirements. The effect of 200mg/3mL and 500mg/3mL silica gel solid phase extraction columns were compared.

Weighing grass carp and crucian carp samples, extracting and purifying, respectively adding 100ng2-MIB and GSM, mixing, respectively passing through 200mg/3mL and 500mg/3mL silica gel solid phase extraction columns, collecting eluent, and determining. As shown in FIG. 18, FIG. 18 is a schematic diagram showing the effect of different capacity silica gel solid phase extraction columns on the recovery of 2-MIB and GSM.

As can be seen from FIG. 18, the recovery rates for 2-MIB of the two columns are not very different, but for GSM, the recovery rate is about 60% when the amount of the filler is 200mg, and about 110% when the amount of the filler is 500mg/mL, which is probably because GSM is difficult to retain relative to 2-MIB, and when the amount of the column filler is small, the column capacity is close to saturation, so that 2-MIB is preferentially retained, and a part of GSM is not retained on the column, thereby causing the recovery rate to be low. Therefore, the solid phase extraction column of 500mg/3mL is more suitable.

3.9 matrix Effect

Preparing a blank matrix solution of a sample according to a determined experimental scheme by adopting blank grass carp and crucian carp samples, adding 50 mu L of 1.0 mu g/mL 2-MIB and GSM mixed standard solution into the blank matrix solution of 450 mu L to prepare a matrix standard solution of 100ng/mL, and measuring and calculating the recovery rate of the matrix standard solution to the reagent standard solution, wherein the result is shown in figure 19, and figure 19 is a schematic diagram of the influence of different sample matrixes on the matrix effect of 2-MIB and GSM measurement. For 2-MIB, the matrix effect of the three sample matrixes is less than 5%, for GSM, the matrix effect of the crucian matrix is small, and the matrix effect of the grass carp matrix is maximum and about 11%, so that the basic requirement of quantification is met, and therefore, a reagent standard curve is adopted for quantification during determination.

3.10 method accuracy and precision

Taking blank crucian carp and grass carp as test objects, carrying out 3 addition recovery experiments with concentration levels of 2, 10 and 20 mug/kg, repeating the experiments for 6 times, and calculating the recovery rate and the RSD value, wherein the results are shown in tables 1 and 2. Under the addition level, the average recovery rate of the 2-MIB and the GSM is 79.9-89.3%, and the relative standard deviation is 3.19-6.63%, which shows that the method has good accuracy and precision.

TABLE 1.2 MIB and GSM recovery and precision at different levels of spiking in crucian carp

TABLE 2.2 MIB and GSM recovery and precision at different spiking levels in grass carp

3.11 determination of actual Positive samples

Purchasing a live grass carp of 1kg weight, pouring 2-MIB and GSM standard substances through a mouth, after 1 hour of cultivation, removing scales, viscera and skin, taking out muscles along the back, homogenizing and mixing evenly. 5g of minced grass carp meat is weighed and treated according to the method, and then the result is determined, the content of 2-MIB is 11.9 mug/kg, the content of GSM is 3.26 mug/kg, and the RSD is less than 5%, which shows that the method has good stability when the method is used for measuring actual samples.

TABLE 3 content of 2-MIB and GSM in positive grass carp samples

Comparative example 1

Geosmin and 2-methylisoborneol are semi-volatile substances, and many different auxiliary distillation and extraction techniques are usually adopted when the reported detection method is extracted from a matrix, and the captured residue is usually enriched before GC-MS analysis, such as solid phase micro-extraction. When large scale assays of samples are involved, the purge and trap process involves the assembly of complex equipment, requiring more workspace to perform the parallel operations. Furthermore, due to the semi-volatility of GSM and 2-MIB, headspace sampling requires additional time to re-clean and the headspace sampling or trap purging process may result in poor reproducibility of repeated analyses, thus these methods currently require more consumables, equipment and space for large scale sample analysis.

The determination method of the earthy taste substances in the freshwater fish is characterized in that a detection method is established by utilizing the characteristic of difficult volatilization of the substances for the first time, the substances are extracted by utilizing the normal hexane which is easily dissolved by GSM and 2-MIB and are not easy to lose after extraction, and then the substances are purified by a QuCHERS method and purified and concentrated by a silica gel column so as to be conveniently determined by a gas chromatography-mass spectrometry method, so that the method is simpler and easy to operate, higher in accuracy and stability, small in occupied space and higher in efficiency when a large number of samples are simultaneously determined.

Comparative example 2

The microwave distillation-solid phase microextraction method has complicated equipment, as shown in FIG. 20, and FIG. 20 is a schematic view of the equipment used in the microwave distillation-solid phase microextraction method. The occupied space of the equipment is large, and only one set of equipment is generally used, so that only one sample can be measured during use, and the batch measurement of the samples cannot be realized.

The method disclosed by the invention is used for extracting in a 50mL centrifuge tube, is flexible and convenient to operate, and can be used for simultaneously operating dozens of or even hundreds of samples. 2-MIB and GSM are semi-volatile substances, and in an actual fish sample, the earthy smell substances are tightly combined with fish, so that the earthy smell substances cannot be completely volatilized although the volatilization of the earthy smell substances is promoted by adopting a microwave distillation mode, and the recovery rate is basically between 30 and 70 percent; the method is characterized in that the normal hexane is used for extraction, the earthy taste substances are easy to dissolve in the normal hexane, the normal hexane is assisted with ultrasound during extraction, the normal hexane can be fully contacted with minced fillet, the earthy taste substances can be completely extracted, and the recovery rate is 70-100%. When the earthy taste substance is extracted by using a microwave distillation-solid phase microextraction method, the earthy taste substance is in a gas state for a period of time, so that the requirement on the sealing property of equipment in the experimental process is particularly high, the cost of the equipment is higher, and the popularization of the experimental method is limited to a certain extent; the method of the invention has simple equipment, and the used materials are all universal in laboratories, and do not need special condition restriction, thus being beneficial to the popularization and application of the method.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for measuring earthy smell substances in freshwater fish is characterized by comprising the following steps:

thirdly, purifying and concentrating the sample extracting solution by adopting a silica gel solid phase extraction column to obtain eluent;

2. The method for measuring the earthy smell substances in the freshwater fish as claimed in claim 1, wherein the freshwater fish is selected from crucian carp and grass carp.

3. The method of claim 1, wherein the first step comprises the steps of: adding normal hexane into the freshwater fish meat paste, fully oscillating, performing first ultrasonic treatment and first centrifugation, and taking supernatant to a centrifugal tube; adding n-hexane into the fish meat, fully shaking, performing ultrasonic treatment for the second time, centrifuging for the second time, taking the supernatant, and adding the supernatant into a centrifugal tube.

4. The method for measuring the earthy taste substances in the freshwater fish as claimed in claim 3, wherein the time of the first ultrasonic treatment in the first step is 5min to 30 min;

the time of the second ultrasonic in the first step is 5min-30 min.

5. The method for measuring the earthy smell substances in the freshwater fish as claimed in claim 3, wherein the rotating speed of the first centrifugation in the first step is 4000r/min-10000r/min, and the time is 5min-30 min;

6. The method for measuring the earthy smell substances in the freshwater fish as claimed in claim 1, wherein the rotation speed of the centrifugation in the second step is 2000r/min-10000r/min, and the time is 3min-10 min.

7. The method for measuring earthy smell substances in fresh water fish according to claim 1, wherein said third step comprises the steps of: activating a silica gel column by using 3-10 mL of n-hexane, passing the supernatant after centrifugation in the second step through the column, leaching by using 1-10 mL of n-hexane, blow-drying a solid phase extraction column, eluting by using 1-5 mL of n-hexane/ethyl acetate with the volume ratio of (1-3): 1, blow-drying the solid phase extraction column, receiving eluent, mixing in a vortex mode, and putting into a sample injection vial.

8. The method for measuring the earthy smell substances in the freshwater fish according to claim 1, wherein the gas chromatography conditions in the fourth step are as follows: HP-5MS capillary column: 30 m.times.0.25 mm.times.0.5 μm; 99.999 percent of high-purity helium, adopting a constant flow mode, and enabling the flow rate of carrier gas to be 1.0 mL/min; controlling temperature programming, wherein the initial temperature is 60 ℃, keeping for 1min, increasing the temperature to 120 ℃ at 5 ℃/min, keeping for 3min, increasing the temperature to 170 ℃ at 10 ℃/min, keeping for 1min, increasing the temperature to 280 ℃ at 20 ℃/min, and keeping for 5 min; the injection port temperature is 250 ℃, and split-flow injection is not carried out.

9. The method for measuring the earthy taste substances in the freshwater fish according to claim 1, wherein the mass spectrum conditions in the fourth step are as follows: and (3) bombarding an ion source EI by electrons, wherein the temperature of the ion source is 250 ℃, the temperature of a transmission line is 280 ℃, and the electron energy is 70eV, and selecting an ion scanning SIM mode to acquire data.

10. The method for measuring the earthy smell substances in the freshwater fish according to claim 1, wherein the earthy smell substances are geosmin and 2-methylisoborneol;

the content of the geosmin is 0.4-100 mug/kg;

the content of the 2-methylisoborneol is 0.4-100 mu g/kg.