CN113960236A

CN113960236A - Method for determining geosmin and dimethyl isoborneol in fish body based on rapid pretreatment technology

Info

Publication number: CN113960236A
Application number: CN202111180121.5A
Authority: CN
Inventors: 曲宝成; 王侯宇; 谢晴; 赵洪霞; 刘鹰; 岳扬; 王炫凯; 张敬波
Original assignee: Dalian University of Technology; Dalian Ocean University
Current assignee: Dalian University of Technology; Dalian Ocean University
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-01-21
Anticipated expiration: 2041-10-11
Also published as: CN113960236B

Abstract

The invention belongs to the technical field of aquaculture and food monitoring, discloses a method for determining geosmin and dimethyl isoborneol in fish bodies based on a rapid pretreatment technology, and establishes a detection method for determining GSM and 2MIB in fish tissues by using a dispersive solid-phase extraction-gas chromatography-mass spectrometry method. In the method, a sample is extracted by acetonitrile solution, salting out is assisted for extraction, and after purification, under the condition of gradient temperature rise, a target analyte is qualitatively analyzed by comparing retention time and ion pair information in a selected ion monitoring mode, and is quantitatively determined by an internal standard method. The method has the advantages that: the method has the advantages of less required sample amount and reagent amount, high analysis speed, high sensitivity and good reproducibility, can meet the requirement of mass detection of GSM and 2MIB in the fish tissue sample, and provides a rapid detection technical guarantee for evaluating the occurrence condition of the earthy smell substances in the fish tissue and exploring the reduction and removal of the typical earthy smell substances in the fish body.

Description

Method for determining geosmin and dimethyl isoborneol in fish body based on rapid pretreatment technology

Technical Field

The invention relates to a method for determining earthy smell substances in fish bodies based on rapid pretreatment, belongs to the technical field of aquaculture and food monitoring, and mainly relates to freshwater fish, seawater fish and the like. The method is mainly based on a dispersed solid phase extraction technology, effective extraction of the geosmin and the dimethyl isoborneol in a sample is completed by adopting an improved dispersed extraction and purification method, and then GC/MS quantitative analysis is carried out, so that the rapid extraction and detection of the geosmin and the dimethyl isoborneol in cultured fishes and wild fishes can be realized, the analysis time can be effectively saved, and the analysis cost can be saved.

Background

The problem of earthy taste in aquatic products is long-standing, and earthy taste substances in the aquatic products have a profound influence on links of cultivation, processing, sale, consumption and the like of the aquatic products, so that serious economic loss can be caused to related industries. The earthy smell is a general term for various chemical substances and has a complex composition, and among them, Geosmin (GSM) and dimethyl isoborneol (2-MIB) are considered as two main substances causing earthy smell, and the related research is also the most. The production of GSM and 2-MIB is mostly caused by environmental factors, and also comprises related biochemical reactions in aquatic animals, such as the oxidation of fatty acid, the enzyme-catalyzed conversion of sulfur-containing and nitrogen-containing precursor substances, the degradation of trimethylamine in fish bodies under the action of microorganisms and enzymes, and the like.

Research shows that the contents of GSM and 2-MIB in the water environment reach 9ng/L and 4ng/L respectively, so that the aquatic products have earthy taste. The content of GSM or 2-MIB in fish meat exceeds 0.7 μ g/kg, and the fish has earthy smell. At present, a great deal of research is focused on content analysis of GSM and 2-MIB in water, China also promulgates a standard test method GB/T32470-2016 for GSM and 2-MIB of drinking water in 2016, and relatively few researches on effective analysis of GSM and 2-MIB in fish bodies are carried out, so that the currently developed methods mainly comprise a sensory analysis method and an instrument analysis method. Sensory analysis is considered as the most direct and simple method, but cannot achieve the purpose of quantitative detection, and the experimental result cannot be repeated for many times, so that the method has great limitation; and the instrumental analysis method can achieve accurate, qualitative and quantitative determination. At present, Gas Chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) is commonly adopted to carry out instrument analysis on GSM and 2-MIB, but as the earthy smell substances in the fish body are all in trace amount, a series of pre-treatments such as enrichment, concentration and the like on the GSM and 2-MIB in a sample are particularly important. The existing pretreatment methods aiming at fish tissue samples mainly comprise a microwave distillation extraction method (MAD-SE), a stirring rod adsorption method (SBSE), a solid phase microextraction method (SPME), a headspace solid phase microextraction method (HSPME), a purging and trapping method (P & T) and the like, and the development of the pretreatment technologies directly influences the application and popularization of the detection of the earthy flavor substances in the fish tissues.

Among the pretreatment technologies at home and abroad, microwave distillation and solid-phase microextraction are the main pretreatment methods currently adopted. The microwave distillation has relatively simple operation steps, but has poor enrichment effect on organic matters with strong volatility, such as earthy flavor substances and the like. The microwave distillation-solid phase microextraction can fully extract volatile components in fish bodies, does not need organic solvents or concentration, but has longer extraction time, expensive extraction head and shorter service life, and is not suitable for large-scale detection and analysis. Microwave distillation-purging and trapping do not need organic solvent, so that time is saved, but the extraction solvent sodium chloride with too high concentration can cause blockage to the instrument, and the technology is not completely mature. The stirring rod adsorbs without an organic solvent, but the adsorption balance can be achieved only in a longer time, and according to the sample amount, the adsorption balance can be achieved only in 1h when the sample amount is short and in 3-4 h when the sample amount is long. Dynamic headspace extraction can be used for continuous gas phase extraction, but the sample matrix can interfere with the analysis result, and the absorption and desorption processes can cause loss of sample components and have poor reproducibility. In conclusion, the pretreatment methods have the disadvantages of large dependence on matched instruments, long detection time, high detection cost and low applicability to large-scale sampling analysis.

Also for the reasons mentioned above, there is an interest in actively developing rapid, inexpensive, and effective pretreatment techniques for GSM and 2-MIB in fish tissues to satisfy large-scale detection and analysis. Among them, the liquid-liquid extraction and the dispersive solid-phase extraction methods are attracting attention again because of their simple and rapid operation and high repeatability. In recent process studies, researchers have developed pretreatment techniques using n-hexane extraction. However, when n-hexane is extracted, in addition to the target substances GSM and 2-MIB, its lipophilicity can cause the extraction of fat and lipophilic compounds in fish tissues, so the operation needs to increase the SPE column purification treatment process, which results in the increase of pretreatment extraction time, process increase and detection cost. The research technology is based on the principle of dispersive solid-phase extraction, acetonitrile and a water system are adopted, the target objects GSM and 2-MIB can be quickly extracted at normal temperature and normal pressure, instrument analysis is carried out after quick purification, the detection cost can be obviously reduced, the detection time is saved, the repeatability is high, and the method is suitable for large-scale quick detection of GSM and 2-MIB in fish tissues.

The fish is one of the main food sources consumed by human, the earthy smell substances in the fish body always influence the whole process from cultivation (or fishing) to dining tables for a long time, and the objective evaluation of the earthy smell substances in the fish body and the development of a reduction technology are seriously dependent on the development of related detection technologies. In order to reduce the detection cost and save the detection time, it is necessary to develop a rapid, simple and convenient pretreatment technology suitable for mass detection aiming at typical earthy smell substances GSM and 2-MIB.

Disclosure of Invention

The object of the present invention is to provide a method for quickly, easily and efficiently determining GSM and 2-MIB in fish based on the above-mentioned state of the art.

The technical scheme of the invention is as follows:

a method for determining geosmin and dimethyl isoborneol in fish bodies based on a rapid pretreatment technology comprises the following steps:

(1) liquid-solid extraction: adding acetonitrile into the homogenized fish tissue sample for extraction, and performing vortex oscillation for 30-60 s;

(2) salting out and back extraction: adding sodium chloride into the sample obtained in the step (1), adding 1-2 g of sodium chloride into every 2g of fish tissue sample, performing vortex oscillation for 30-60 s, and centrifuging for 3-5 min at 8000-12000 r/min;

(3) dewatering and purifying: adding anhydrous sodium sulfate into the liquid to be purified obtained in the step (2), adding 4-6 g of anhydrous sodium sulfate into every 2g of fish tissue samples, carrying out vortex oscillation for 30-60 s, centrifuging at 8000-12000 r/min for 3-5 min, and transferring supernatant;

(4) adsorption and purification: adding an adsorption purifying agent into the supernatant obtained in the step (3), carrying out vortex oscillation for 30-60 s, centrifuging for 3-5 min at 8000-12000 r/min, and transferring the supernatant; the adsorption purificant is PSA and activated carbon, and 0.1g of the adsorption purificant is added into each 2g of fish tissue samples;

(5) membrane-passing purification: filtering the supernatant obtained in the step (4) by a membrane, purifying and then loading the supernatant on a machine;

(6) preparing a standard solution: firstly, 10mg of 2-MIB is weighed to be 10mL by methanol to prepare a standard stock solution of 1mg/mL, and 10mg of GSM is weighed to be 10mL by methanol to prepare a standard stock solution of 1 mg/mL. Respectively measuring appropriate amount of GSM and 2-MIB, and preparing into mixed standard intermediate solution with concentration of 1mg/L with methanol. Sucking a proper amount of mixed standard intermediate solution, diluting the mixed standard intermediate solution into standard series working solutions containing 1ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 40ng/mL of GSM and 2-MIB respectively by using methanol, wherein the content of the internal standard 2-isobutyl-3-methoxypyrazine is 4 ng/mL;

(7) and (3) qualitative and quantitative analysis: and (3) putting the liquid to be analyzed into a gas chromatography-mass spectrometer, monitoring by adopting an SIM scanning mode, and quantifying by adopting an internal standard method.

a) And (3) qualitative analysis: measuring a sample by using a GC-MS instrument and establishing a standard working curve, wherein if the retention time of a chromatographic peak detected in the sample is consistent with that of a chromatographic peak of a corresponding standard substance, the allowable deviation is less than +/-2.5%; and comparing the relative abundance of each component qualitative ion in the sample with the relative abundance of the corresponding qualitative ion in the mixed standard working solution with the approximate concentration, and judging that the corresponding substance to be detected exists in the sample if the deviation does not exceed the range specified in the table 1.

TABLE 1 maximum permissible deviation of relative ion abundance for qualitative confirmation

Relative ion abundance	＞50％	More than 20% to 50%	More than 10% to 20%	≦10％
					Maximum deviation allowed	±20％	±25％	±30％	±50％

b) Quantitative analysis: measuring the mixed standard series working solution by using a GC-MS method to obtain the chromatographic peak area of the corresponding standard solution, and drawing a standard curve by taking the concentration of the mixed standard working solution as a horizontal coordinate and the peak area of the chromatographic peak as a vertical coordinate; and then, measuring the sample to be measured under the same conditions to obtain the chromatographic peak area of the sample to be measured, and obtaining the concentration of each component in the liquid to be measured according to the standard curve. The response value of each component to be measured in the sample should be within the linear response range of the standard curve.

(7) And (4) calculating a result:

calculating the formula:

in formula (1):

x is the content of the component to be detected in the sample, and the unit is microgram per kilogram (mu g/kg);

c-the concentration of each component to be measured in the measurement solution read from the standard curve, in units of micrograms per liter (μ g/L);

m is the weighed mass of the sample, and the unit is gram (g);

v-volumetric volume of sample solution in milliliters (mL).

The standard stock solution can be stored for 3 months at-18 ℃ in the dark.

The mixed standard series working solution is prepared at the time of use.

The gas chromatography-mass spectrometry test conditions are as follows:

the chromatographic conditions of the GC-MS are as follows:

a) a chromatographic column: DB-35MS quartz capillary column, 30m × 0.32mm (i.d), film thickness of 0.25 μm, or equivalent.

b) Temperature of the column: starting at 100 deg.C, it is raised to 220 deg.C at 12 deg.C/min, and then raised to 280 deg.C at 30 deg.C/min (5 min hold).

c) Sample inlet temperature: 230 ℃ to 230 ℃.

d) Interface temperature of chromatography-mass spectrometry: 280 ℃.

e) Carrier gas: helium with purity of more than or equal to 99.999 percent and 1.5 mL/min.

f) Sample introduction amount: 1 μ L.

g) And (3) sample introduction mode: the sample is injected without split flow, and the valve is opened after 0.75 min.

The mass spectrum conditions of the GC-MS are as follows:

k) an ionization mode: EI.

l) ionization energy: 70 eV.

m) detection mode: an ion monitoring mode (SIM) is selected.

n) solvent retardation: 3.0 min.

The method has the advantages that after a sample is subjected to solvent extraction by acetonitrile, salting-out back extraction is assisted, and after an extracting solution is adsorbed and purified by a purifying agent, gas quality analysis is performed under the condition of temperature programming. The average recovery rate of the compound is 87.2-115.7%, and the Relative Standard Deviation (RSD) is 0.6-6.2%. When the sample weighing amount is 2g and the constant volume is 5mL, the detection limit of GSM is 0.37 mug/kg, and the detection limit of 2-MIB is 0.51 mug/kg; the limit of quantitation for GSM is 1.23. mu.g/kg, and for 2-MIB 1.69. mu.g/kg.

The method has the advantages of simple pretreatment, small usage amount of samples and solvents, small matrix interference, high analysis speed, high sensitivity, good reproducibility and the like, can be suitable for rapid detection of a large number of samples, and meets the requirement of daily detection work.

Drawings

FIG. 1(a) is a mass spectrum (qualitative and quantitative ion selection) of GSM according to an embodiment of the present invention;

FIG. 1(b) is a mass spectrum (qualitative and quantitative ion selection) of MIB of example 2 of the present invention;

FIG. 2 is a Selected Ion Monitoring (SIM) chromatogram of GSM and 2-MIB in fish tissues according to an embodiment of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions of the present invention are further explained below with reference to the drawings and the following examples, but the technical solutions of the present invention are not limited to the scope of the present invention.

Example 1

(1) Liquid-solid dispersion extraction: weighing 2.0g of sample, placing the sample in a 20mL polypropylene centrifuge tube, adding 2mL of water, carrying out vortex oscillation for 30s, dispersing the tissue sample, adding 5mL of acetonitrile extraction solution, carrying out vortex oscillation for 30s, adding 1g of sodium chloride for back extraction, carrying out vortex oscillation for 30s, adding 4g of anhydrous sodium sulfate, centrifuging at 8000r/min for 5min, transferring the supernatant into the 20mL polypropylene centrifuge tube, and purifying.

(2) Liquid-solid adsorption purification: adding 0.1g PSA powder and 0.1g C18 powder into the supernatant, vortex oscillating for 30s, centrifuging at 8000r/min for 1min, collecting supernatant, and filtering with microporous membrane for analysis.

(3) Preparing a standard solution: first, 0.01g (to the nearest 0.0001g) of GSM and 2-MIB were weighed out separately and made up to 10mL with methanol to make 1mg/mL stock solutions. Respectively measuring appropriate amount of GSM and 2-MIB, and preparing into mixed standard intermediate solution with concentration of 1mg/L with methanol. Sucking a proper amount of mixed standard intermediate solution, diluting the mixed standard intermediate solution into standard series working solutions containing 1ng/mL, 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 40ng/mL of GSM and 2-MIB respectively by using methanol, wherein the content of the internal standard 2-isobutyl-3-methoxypyrazine is 4ng/mL

(4) And (3) qualitative and quantitative analysis: and (3) introducing the solution to be analyzed into a gas chromatography-mass spectrometer (GC-MS), monitoring by adopting a Selective Ion Monitoring (SIM) mode, and quantifying by adopting an internal standard method. The specific test conditions for qualitative and quantitative analysis are as follows:

gas chromatography conditions: DB-35MS quartz capillary column chromatographic column (30m multiplied by 0.32mm, the film thickness is 0.25 mu m, or equivalent) is selected, and the temperature programming condition of the chromatographic column is as follows: starting at 100 deg.C, it is raised to 220 deg.C at 12 deg.C/min, and then raised to 280 deg.C at 30 deg.C/min (5 min hold). Sample inlet temperature: 230 ℃ to 230 ℃. Interface temperature of chromatography-mass spectrometry: 280 ℃. Carrier gas: helium with purity of more than or equal to 99.999 percent and 1.5 mL/min. And (3) sample introduction mode: the sample is injected without split flow, and the valve is opened after 0.75 min. Sample introduction amount: 1 μ L. Mass spectrum parameter conditions: ionization mode is Electrospray Ionization (EI), ionization energy: 70 eV. The detection mode is a selective ion monitoring mode (SIM). Solvent retardation: 3.0 min.

When the chromatographic column is selected, three different chromatographic column packings, namely an HP-5 capillary column chromatographic column (30m multiplied by 0.25mm, the film thickness is 0.25 mu m '), a DB-35MS quartz capillary column chromatographic column (30m multiplied by 0.32mm, the film thickness is 0.25 mu m '), and a DB-35MS quartz capillary column chromatographic column (30m multiplied by 0.53mm, the film thickness is 3.0 mu m ') are respectively selected. The results show that GSM and 2-MIB have good chromatographic retention behaviors on three chromatographic columns, and DB-35MS quartz capillary column chromatographic columns (30m multiplied by 0.32mm, the film thickness is 0.25 mu m) are finally selected by comprehensively considering the peak shape, the response, the separation degree and the commodity price of each compound to be detected.

In the invention, when the extraction solvent is selected, the related compounds are all easily dissolved in organic reagents such as methanol, acetonitrile, n-hexane and the like, so that methanol, acetonitrile, n-hexane and ethyl acetate which are commonly used in laboratories are selected as the extraction solvent in the sample extraction process. The experimental result shows that the extraction efficiency of methanol, acetonitrile normal hexane and ethyl acetate on GSM and 2-MIB can reach more than 80%, and in order to save the use amount of an organic solvent and consider that the acetonitrile has obvious precipitation effect on free protein containing samples, the invention determines the acetonitrile as the extraction solvent in order to achieve a better purification effect.

In the selection of sample purification, the invention respectively considers 100mg PSA/100mg C₁₈100mg PSA/100mg neutral alumina, 100mg C₁₈Impact of/100 mg neutral alumina, 3 combinations on sample purification efficacy. PSA is a commonly used dispersed solid phase extraction cleanser which can remove fatty acids, organic acids, polar substances, etc. from tissues by weak anion exchange or polar action. C₁₈Effectively adsorb compounds such as fat and fatty acid. Neutral alumina is a strong polar adsorbent, has neutral surface, and is easy to retain heterocyclic and organic amine and other electron-rich compounds. By aiming at different C₁₈Combinations of neutral alumina and PSA, 100mg PSA/100mg C₁₈When the combination is the purifying agent, the recovery rate of the GSM and the 2-MIB is 80 to 120 percent. Thus, 100mg PSA/100mg C was finally used₁₈And carrying out sample purification treatment in combination.

In the invention, the influence of anhydrous sodium sulfate and anhydrous magnesium sulfate is considered during the selection of the dehydrating agent, the influence of the anhydrous sodium sulfate and the anhydrous magnesium sulfate has no significant influence on the recovery rates of GSM and 2-MIB, and the anhydrous sodium sulfate is adopted as the dehydrating agent in the invention in view of stronger water absorption capacity and weaker adsorption effect of the anhydrous sodium sulfate.

In the embodiment of the invention, the GSM is purchased from Shanghai Michelin Biotechnology limited, and the purity is more than or equal to 97 percent; 2-MIB was purchased from Chem Service, USA, and the purity was more than or equal to 99%.

Results and analysis

a) Under the selected chromatographic and mass spectrum conditions, according to the standard solution with the determined series of concentrations, the concentration of the target substance is used as an abscissa (x), the peak area of a chromatographic peak is used as an ordinate (y), the result of regression analysis is shown in table 2, and as can be seen from the table 2, GSM and 2-MIB have good linearity within the range of 0.5-500 mug/L, and the correlation coefficient r is more than 0.997. According to the definition of detection limit by International Union of Pure and Applied Chemistry (IUPAC), serial mixed standard solutions are diluted and tested, and the signal-to-noise ratio (S/N) is calculated, with S/N being 3 as the detection Limit (LOD) and S/N being 10 as the quantification Limit (LOQ). The mixed standard solution was quantitatively added to a blank sample containing no target substance and subjected to detection after treatment, LOD of GSM and 2-MIB were classified into 0.37. mu.g/kg and 0.51. mu.g/kg, and LOQ of the method was 1.23. mu.g/kg and 1.69. mu.g/kg.

TABLE 2 Linear equations, correlation coefficients, Linear Range (n 6), detection limits and quantitation limits for GSM and 2-MIB

Analyte	Linear equation of equations	r	Linear range/. mu.g/L	LOD(μg/kg)	LOQ(μg/kg)
						GSM	y＝79.037x-78.481	0.999	0.5-500	0.37	1.23
2-MIB	y＝80.022x+19.192	0.999	0.5-500	0.51	1.69

b) Fish tissue samples without GSM and 2-MIB were selected, tested for additive recovery and precision according to three concentration levels, 6 levels each, measured according to the procedure specified in the method, and the average recovery of the method was examined, see table 3. The results show that the standard addition average recovery rate of GSM and 2-MIB is between 87.2 and 115.7 percent, and the Relative Standard Deviation (RSD) is between 0.6 and 6.2 percent, which both meet the requirement of trace analysis of samples.

TABLE 3 recovery and precision of GSM and 2-MIB spiked for different fish tissue samples (n ═ 6)

Claims

1. A method for measuring geosmin and dimethyl isoborneol in fish bodies based on a rapid pretreatment technology is characterized by comprising the following steps:

(6) preparation of a standard series of solutions: preparing standard series of working solutions on a computer, wherein the working solutions contain geosmin and dimethylisoborneol which are respectively 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL and 20ng/mL by using a methanol solution;

2. The method of claim 1, wherein the membrane-passing purification material in step (5) is a 0.22 μm filter membrane.

3. The method according to claim 1 or 2, wherein the chromatographic conditions of the GC-MS in step (7) are as follows:

a) a chromatographic column: DB-35MS quartz capillary column, 30m × 0.32mm (i.d), film thickness 0.25 μm;

b) temperature of the column: starting at 100 deg.C, heating to 220 deg.C at 12 deg.C/min, heating to 280 deg.C at 30 deg.C/min, and maintaining for 5 min;

c) sample inlet temperature: 230 ℃;

d) interface temperature of chromatography-mass spectrometry: 280 ℃;

e) carrier gas: helium, 1.5 mL/min;

f) sample introduction amount: 1 mu L of the solution;

4. The method of claim 1 or 2, wherein the mass spectrometry conditions of the GC-MS in step (7) are as follows:

k) an ionization mode: EI;

l) ionization energy: 70 eV;

m) detection mode: selecting an ion monitoring mode;

n) solvent retardation: 3.0 min.

5. The method of claim 3, wherein the mass spectrometry conditions of GC-MS in step (7) are as follows:

k) an ionization mode: EI;

l) ionization energy: 70 eV;

m) detection mode: selecting an ion monitoring mode;

n) solvent retardation: 3.0 min.