A fish muscle pretreatment method for detecting odorous substances and an on-machine sample detection method

Abstract

The present application belongs to the technical field of sample pretreatment and substance detection, and provides a fish muscle pretreatment method for off-flavor substance detection and a sample detection method after machine loading. The pretreatment method of the present application is simple to operate, can meet batch processing of multiple samples, and has strong practicability for detection of off-flavor substances in actual batch fish muscle samples. Moreover, the extraction method of the present application can realize simultaneous extraction of multiple off-flavor substances (n-hexanal, n-hexanol, 1-octen-3-ol, 2-methylisoborneol and geosmin) and has high recovery rate. In addition, the pretreatment method of the present application does not require expensive instruments and equipment, and the required consumables are cheap and low in cost. The present application also provides a detection method of the sample after machine loading obtained by the pretreatment method, and the detection method of the present application has a sample loading time of only 18 min for a single sample, is short in sample analysis time, and can simultaneously detect five off-flavor substances.

Description

Technical Field

[0001] The present invention relates to the technical field of sample pretreatment and substance detection, and in particular to a fish muscle pretreatment method for detecting odorous substances and an on-machine sample detection method. Background Art

[0002] Eutrophication of water bodies and industrial and agricultural wastewater pollution can lead to excessive growth of algae and bacterial microorganisms, which in turn continuously secrete odorous secondary metabolites, known as odorants. These odorants include n-hexaldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol, and geosmin. These odorants can penetrate through the gills and skin, easily accumulating in the tissues of aquatic products and producing odor. Freshwater fish are a major aquatic product. Their muscle tissue is nutritious, delicious, and contains essential amino acids. The accumulation of odorants in muscle can extend the harvest period, increase disease incidence, increase aquaculture costs, and even severely impact the market value of aquatic products. However, research on the odor problem in freshwater aquaculture fish in my country is very limited.

[0003] Establishing analytical methods for odor-producing substances in fish is a crucial step in studying their biological effects. Currently, methods for analyzing odor-producing substances in fish muscle tissue primarily involve converting these substances into a liquid phase through steam distillation and microwave distillation, followed by enrichment through purge-and-trap and solid-phase microextraction (SPM), followed by quantitative analysis using gas chromatography (GC) or gas chromatography-mass spectrometry (GC / MS). However, the reported distillation-SPM extraction methods are complex and require high operational requirements; in addition, the extraction heads used in these methods are very expensive. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a fish muscle pretreatment method and an on-machine sample detection method for detecting odor substances. The fish muscle pretreatment method provided by the present invention is simple to operate and low in cost.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a fish muscle pretreatment method for detecting odorous substances, comprising the following steps:

[0007] Homogenize the muscle sample of the fish to be tested to obtain a homogenate;

[0008] mixing the homogenate and the extractant, performing extraction, and combining the extracts;

[0009] The extract is concentrated and enriched in sequence to obtain a sample for use in an apparatus;

[0010] The extractant includes ethyl acetate and a salting-out agent;

[0011] The extraction comprises sequentially performing vortex extraction and ultrasonic extraction.

[0012] Preferably, the tissue homogenization comprises: mixing the muscle sample of the fish to be tested, water and quartz sand, and grinding them.

[0013] Preferably, the ratio of the fish muscle sample to be tested to water is 5 g:5 mL.

[0014] Preferably, the ratio of the fish muscle sample to be tested to ethyl acetate is 5 g:20 mL.

[0015] Preferably, the concentration of the salting-out agent in the extractant is 0.2 g / mL.

[0016] Preferably, the salting-out agent comprises sodium chloride.

[0017] Preferably, the rotation speed of the vortex extraction is 2000-3000 rpm, and the time is 10 minutes.

[0018] Preferably, the power of the ultrasonic extraction is 100-150W, and the time is 5 minutes.

[0019] Preferably, the enrichment is adsorbent enrichment, and the adsorbent of the adsorbent enrichment includes PSA.

[0020] The present invention also provides a method for detecting a sample obtained by the fish muscle pretreatment method described in the above technical solution, comprising the following steps:

[0021] Performing gas chromatography-mass spectrometry on the sample on the machine;

[0022] The gas chromatography-mass spectrometry detection includes gas chromatography detection and mass spectrometry detection;

[0023] The conditions for the gas chromatography detection include:

[0024] Chromatographic column model: SH-Rsi-5sil-MS capillary column, 30m×250μm×0.25μm;

[0025] Gas chromatography parameters: 40°C for 2 min, increase to 180°C at 20°C / min, hold for 5 min, increase to 220°C at 20°C / min, hold for 2 min;

[0026] The injection port temperature was 200°C; the carrier gas was He, and the carrier gas flow rate was 1 mL / min; splitless injection;

[0027] The mass spectrometry detection conditions include: a transfer line temperature of 260°C; an ion source temperature of 230°C; and an electron energy of 70 eV.

[0028] The present invention provides a fish muscle pretreatment method for detecting odorous substances, comprising the following steps: homogenizing a fish muscle sample to be tested to obtain a homogenate; mixing the homogenate with an extractant, extracting, and combining the extracts; concentrating and enriching the extracts in sequence to obtain a sample for use on an apparatus; the extractant comprises ethyl acetate and a salting-out agent; and the extraction comprises sequentially performing vortex extraction and ultrasonic extraction. The pretreatment method of the present invention is simple to operate, meets the requirements for batch processing of multiple samples, and has strong practicality for detecting odorous substances in actual batches of fish muscle samples. Moreover, the extraction method of the present invention can achieve simultaneous extraction of multiple odorous substances (n-hexylaldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol, and geosmin) with a high recovery rate. Furthermore, the pretreatment method of the present invention does not require expensive instruments and equipment, and the required consumables are inexpensive and low in cost.

[0029] The present invention also provides a method for detecting a sample obtained by the fish muscle pretreatment method described in the above technical solution, comprising the following steps: subjecting the sample to gas chromatography-mass spectrometry; the gas chromatography-mass spectrometry detection includes gas chromatography and mass spectrometry; the gas chromatography detection conditions include: chromatographic column model: SH-Rsi-5sil-MS capillary column, 30m×250μm×0.25μm; gas chromatography parameters: 40°C for 2 minutes, ramped to 180°C at 20°C / min, held for 5 minutes, ramped to 220°C at 20°C / min, held for 2 minutes; inlet temperature: 200°C; carrier gas: He at a carrier gas flow rate of 1 mL / min; splitless injection; mass spectrometry detection conditions include: transfer line temperature: 260°C; ion source temperature: 230°C; electron energy: 70 eV. The detection method provided by the present invention has an injection time of only 18 minutes for a single sample, shortening the sample analysis time; and can simultaneously detect five odor substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the standard chromatogram of each odorant in SIM mode (50 μg / L);

[0031] Figure 2 This is the effect of different extractants on the recovery rate of five odor substances;

[0032] Figure 3 This is the effect of different ionic strengths on the recovery rates of five odorous substances;

[0033] Figure 4 This is a graph showing the effect of different vortex times on the recovery rates of five odorous substances;

[0034] Figure 5 This is a graph showing the effect of different ultrasonic times on the recovery rates of five odorous substances. DETAILED DESCRIPTION

[0035] The present invention provides a fish muscle pretreatment method for detecting odorous substances, comprising the following steps:

[0036] Homogenize the muscle sample of the fish to be tested to obtain a homogenate;

[0037] mixing the homogenate and the extractant, performing extraction, and combining the extracts;

[0038] The extract is concentrated and enriched in sequence to obtain a sample for use in an apparatus;

[0039] The extractant includes ethyl acetate and a salting-out agent;

[0040] The extraction comprises sequentially performing vortex extraction and ultrasonic extraction.

[0041] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0042] The present invention homogenizes the muscle sample of the fish to be tested to obtain a homogenate.

[0043] In the present invention, the tissue homogenization preferably comprises: mixing and grinding a fish muscle sample to be tested, water, and quartz sand. In the present invention, the ratio of the fish muscle sample to be tested to water is preferably 5g:5mL. In the present invention, the ratio of the fish muscle sample to be tested to quartz sand is preferably 5g:1g. In the present invention, the grinding time is preferably 2 to 5 minutes.

[0044] After obtaining the homogenate, the present invention mixes the homogenate with an extractant, performs extraction, and combines the extracts.

[0045] In the present invention, the extractant comprises ethyl acetate and a salting-out agent. In the present invention, the concentration of the salting-out agent in the extractant is preferably 0.2 g / mL. In the present invention, the salting-out agent preferably comprises sodium chloride. In the present invention, the ratio of the fish muscle sample to be tested to ethyl acetate is preferably 5 g:20 mL.

[0046] In the present invention, the extraction includes vortex extraction and ultrasonic extraction in sequence. In the present invention, the rotation speed of the vortex extraction is preferably 2000-3000 rpm, more preferably 2500 rpm; the time of the vortex extraction is preferably 10 minutes. In the present invention, the power of the ultrasonic extraction is preferably 100-150W, more preferably 120W; the time of the ultrasonic extraction is preferably 5 minutes. After the ultrasonic extraction, the present invention preferably also includes centrifugation and drying. In the present invention, the rotation speed of the centrifugation is preferably 3000-5000 r / min, more preferably 4000 r / min; the time is preferably 2-10 minutes, more preferably 5 minutes. In the present invention, the drying is preferably drying with a desiccant, and the desiccant of the desiccant drying preferably includes anhydrous sodium sulfate. In the present invention, the drying preferably includes: passing the liquid phase obtained by centrifugation through a funnel filled with anhydrous sodium sulfate to a chicken heart bottle.

[0047] In the present invention, the extraction is preferably performed three times.

[0048] After obtaining the extract, the present invention sequentially concentrates and enriches the extract to obtain an on-machine sample.

[0049] In the present invention, the concentration temperature is preferably 30-40°C; the concentration time is preferably 8-15 minutes. After the concentration, the present invention preferably further comprises a volume adjustment, and the reagent for the volume adjustment preferably comprises ethyl acetate.

[0050] In the present invention, the enrichment is preferably adsorbent enrichment, and the adsorbent for the adsorbent enrichment preferably includes PSA. In the present invention, the enrichment preferably includes: transferring the concentrated material to a centrifuge tube containing PSA, and sequentially performing vortexing and centrifugation. In the present invention, the vortexing speed is preferably 2000-3000 rpm, and the vortexing time is preferably 5 minutes; the centrifugation speed is preferably 10,000 rpm, and the time is preferably 3 minutes.

[0051] After the enrichment, the present invention preferably further comprises filtration, and the filtration membrane is preferably a 0.22 μm filter membrane.

[0052] The present invention also provides a method for detecting a sample obtained by the fish muscle pretreatment method described in the above technical solution, comprising the following steps:

[0053] The samples were tested by gas chromatography-mass spectrometry;

[0054] The gas chromatography-mass spectrometry detection includes gas chromatography detection and mass spectrometry detection;

[0055] The conditions for the gas chromatography detection include:

[0056] Chromatographic column model: SH-Rsi-5sil-MS capillary column, 30m×250μm×0.25μm;

[0057] Gas chromatography parameters: 40°C for 2 min, increase to 180°C at 20°C / min, hold for 5 min, increase to 220°C at 20°C / min, hold for 2 min;

[0058] The injection port temperature was 200°C; the carrier gas was He, and the carrier gas flow rate was 1 mL / min; splitless injection;

[0059] The mass spectrometry detection conditions include: a transfer line temperature of 260°C; an ion source temperature of 230°C; and an electron energy of 70 eV.

[0060] In the present invention, the gas chromatography-mass spectrometry detection is preferably performed on a gas chromatography-mass spectrometer, and the model of the gas chromatography-mass spectrometer is preferably GCMS-QP2010 Ultra.

[0061] In the present invention, the odor substances include n-hexyl aldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol and geosmin.

[0062] In the present invention, the characteristic ion m / z of n-hexyl aldehyde is 44; the characteristic ion m / z of n-hexanol is 56; the characteristic ion m / z of 1-octen-3-ol is 57; the characteristic ion m / z of 2-methylisoborneol is 95; and the characteristic ion m / z of geosmin is 112.

[0063] The fish muscle pretreatment method for detecting odorous substances and the on-machine sample detection method provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0064] Experimental instrument: gas chromatography-mass spectrometry (GCMS-QP2010 Ultra), chromatographic column model: SH-Rsi-5sil-MS capillary column (30m×250μm×0.25μm).

[0065] Experimental reagents: Standard substances: n-hexaldehyde (purity > 99.0%, Aladdin, Shanghai); n-hexanol (purity > 99.8%, Aladdin, Shanghai); 1-octen-3-ol (purity > 98.0%, Yuanye, Shanghai); 2-methylisoborneol (purity > 98.0%, Sigma, Shanghai); geosmin (purity > 97.0%, Sigma, Shanghai); ethyl acetate, dichloromethane, acetonitrile (analytical grade, National Pharmaceutical Chemical Reagent Co., Ltd.); ethyl acetate (chromatographic grade, National Pharmaceutical Chemical Reagent Co., Ltd.); PSA adsorbent (Shanghai Yuexu Technology Co., Ltd.); sodium chloride, anhydrous sodium sulfate (National Pharmaceutical Chemical Reagent Co., Ltd.).

[0066] Preparation of mixed standard solution

[0067] Standard samples of n-hexaldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol and geosmin were dissolved in chromatographically pure ethyl acetate, and mixed standard solutions of five odor substances at concentrations of 0.5, 1, 5, 10 and 50 μg / L were prepared and stored at -20°C.

[0068] Sample collection and storage:

[0069] Grass carp samples were collected from the fish ponds of the Green Agriculture Technology Company in Yangzhong, Zhenjiang, Jiangsu Province. Fish samples were dissected and muscle removed, wrapped in tinfoil, and stored at −80°C.

[0070] Determination of optimal process parameters

[0071] (1) Selection of gas chromatography-mass spectrometry detection conditions

[0072] Gas chromatography parameters: 40°C for 2 min, increase to 180°C at 20°C / min, maintain for 5 min, increase to 220°C at 20°C / min, maintain for 2 min; injection port temperature 200°C; carrier gas He, carrier gas flow rate 1 mL / min; splitless injection.

[0073] Mass spectrometry parameters: transfer line temperature 260°C; ion source temperature 230°C; electron energy 70 eV.

[0074] Qualitative analysis was performed in full scan mode, and the retention times of n-hexaldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol and geosmin were determined to be 4.406, 5.048, 6.138, 8.046 and 9.807 min, respectively.

[0075] Quantitative analysis was performed in the selected ion mode. The characteristic ions m / z of hexanal, 1-hexanol, 1-octene-3-alcohol (1-octene-3-ol), 2-methylisocamphenyl alcohol (2-MIB) and geosmin (GSM) were 44, 56, 57, 95 and 112, respectively (e.g. Figure 1 The standard chromatograms of each odorant under SIM mode (50 μg / L) are shown.

[0076] (2) Optimization of pre-treatment process parameters

[0077] The recovery rates of five odorous substances under different test conditions were compared at a spike level of 10 μg / kg to determine the optimal process parameters. The specific implementation steps are as follows:

[0078] 2.1. Selection of extraction agent

[0079] The physical and chemical properties of different extractants will directly affect the extraction efficiency of the target analytes. Considering the principle of like dissolves like, the recovery rates of three organic solvents with different polarities, dichloromethane, ethyl acetate and acetonitrile, on odorous substances in muscle samples were compared to determine an extractant with high recovery rate and low impurity content. The results are as follows Figure 2 As shown. Figure 2 It can be seen that when ethyl acetate is used as the extraction solvent, the average recovery rate of the five odorous substances is between 74% and 80%. Dichloromethane has low polarity and is effective in separating from the aqueous layer during extraction, but the average recovery rate of odorous substances is low. Acetonitrile has good compatibility with animal tissues, strong penetration, and can precipitate proteins and degrease, but the average recovery rate of odorous substances is less than 20%. Therefore, ethyl acetate is the optimal extraction solvent.

[0080] 2.2. Selection of ionic strength

[0081] Adding an appropriate amount of sodium chloride during the organic solvent extraction process can promote salting out, increase the solubility of the target analyte in the organic solvent, and improve the sample recovery rate. The effect of different concentrations of salting out agent (i.e., ionic strength) in the extractant on the recovery rate of the five odor substances is shown in Figure 2. Figure 3 As shown, from Figure 3 As can be seen, the average recovery rates of the five odor compounds ranged from only 42% to 66% at NaCl concentrations of 0 and 0.4 g / mL. Adding NaCl at a concentration of 0.2 g / mL effectively improved the extraction efficiency of odor compounds in muscle samples. Therefore, 0.2 g / mL of NaCl was selected as the optimal ionic strength.

[0082] 2.3. Selection of vortex time

[0083] Vortex extraction is an important step in the extraction process of pretreatment analysis. In the vortex environment, the extractant can fully contact with the target analyte. The effects of different vortex times on the recovery rates of the five odorous substances are shown in the figure below. Figure 4 As shown. Figure 4 As can be seen, the average recovery rate of odorous substances in muscle ranged from 33% to 65% after vortexing for 5 minutes. After vortexing for 10 minutes, the average recovery rate reached over 74%. Increasing the vortexing time to 15 minutes showed no significant change in the average recovery rate of each odorous substance. Based on these considerations, the final vortexing time of 10 minutes was selected.

[0084] 2.4. Selection of ultrasound time

[0085] Ultrasonic extraction can use ultrasound to improve the physical and chemical properties of substances such as dissolution and distribution, thereby improving the separation and extraction efficiency of target analytes. Figure 5 As shown. Figure 5 It can be seen that the average recovery rate of odor substances in muscle was above 72% under 5, 10 and 15 min ultrasonic extraction. Taking all factors into consideration, the best ultrasonic extraction time was 5 min.

[0086] (3) Validation of the method

[0087] 3.1 Linearity and detection limit

[0088] The linearity of the method was evaluated by testing a mixed standard solution of five odorous substances (0.5, 1, 5, 10, and 50 μg / L) under the above conditions. The results are shown in Table 1.

[0089] Table 1 Standard curve of odor substances

[0090]

[0091] It can be seen from Table 1 that the peak areas and concentrations of the five odor substances have a good linear relationship between 0.5 and 50 μg / L.

[0092] The detection limit was set at a signal three times the noise level. This method has a low detection limit, as shown in Table 2.

[0093] Table 2 Method detection limit

[0094]

[0095] 3.2 Accuracy and Precision

[0096] In order to evaluate the accuracy and precision of the pretreatment method and detection method of the present invention, three addition levels of 0.5, 1 and 10 μg / kg were set, and the recovery rates of odorous substances in grass carp muscle samples were calculated. The results are shown in Table 3.

[0097] Table 3 Recovery rates of five odorous substances added to muscle tissue

[0098]

[0099]

[0100] As shown in Table 3, within the range of 0.5 to 10 μg / kg spiked into muscle tissue, the average recoveries of n-hexaldehyde, n-hexanol, 1-octen-3-ol, 2-methylisoborneol, and geosmin were 64.8% to 75.9%, 69.0% to 75.8%, 62.8% to 74.3%, 67.7% to 77.1%, and 70.2% to 80.1%, respectively. Their relative standard deviations (RSDs) were 1.38% to 6.39%, 3.73% to 6.07%, 3.90% to 7.48%, 4.73% to 5.43%, and 3.63% to 7.65%, respectively. This method demonstrates high accuracy and good precision, meeting the requirements for detecting odorous substances in muscle tissue of fish in fish ponds.

[0101] Example 1

[0102] To validate the practicality of this method, grass carp muscle samples were collected from three fish ponds owned by Tongwei Co., Ltd. in Yangzhong, Zhenjiang City, Jiangsu Province. The contents of five odor compounds in the muscle were analyzed using the pretreatment and detection methods described above. The results are shown in Table 4.

[0103] Table 4 Contents of odorous substances in grass carp muscle tissues from three fish ponds

[0104]

[0105]

[0106] Three sampling points were set up in each fish pond.

[0107] “-” means that the substance was not detected under the conditions of this method.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting odorous substances in fish muscle, characterized in that: The following steps are involved: Homogenize the muscle sample of the fish to be tested to obtain a homogenate; mixing the homogenate and the extractant, performing extraction, and combining the extracts; The extract is concentrated and enriched in sequence to obtain a sample for use in an apparatus; Performing gas chromatography-mass spectrometry on the sample on the machine; The extractant includes ethyl acetate and a salting-out agent, and the salting-out agent includes sodium chloride; The extraction includes sequentially performing vortex extraction and ultrasonic extraction; the rotation speed of the vortex extraction is 2000-3000 rpm, and the time is 10 minutes; the power of the ultrasonic extraction is 100-150W, and the time is 5 minutes; The enrichment is adsorbent enrichment, and the adsorbent of the adsorbent enrichment is PSA; The gas chromatography-mass spectrometry detection includes gas chromatography detection and mass spectrometry detection; The conditions for the gas chromatography detection include: Chromatographic column model: SH-Rsi-5sil-MS capillary column, 30m×250μm×0.25μm; Gas chromatography parameters: 40°C for 2 min, increase to 180°C at 20°C / min, hold for 5 min, increase to 220°C at 20°C / min, hold for 2 min; The injection port temperature was 200°C; the carrier gas was He, and the carrier gas flow rate was 1 mL / min; splitless injection; Mass spectrometry detection conditions included: transfer line temperature of 260°C; ion source temperature of 230°C; electron energy of 70 eV; The odor substances include n-hexanal, n-hexanol, 1-octen-3-ol, 2-methylisoborneol and geosmin.

2. The detection method according to claim 1, wherein The tissue homogenization comprises: mixing the muscle sample of the fish to be tested, water and quartz sand, and grinding the mixture.

3. The detection method according to claim 2, characterized in that The ratio of the fish muscle sample to be tested to water is 5 g:5 mL.

4. The detection method according to claim 1, wherein The ratio of the fish muscle sample to be tested to ethyl acetate is 5 g:20 mL.

5. The detection method according to claim 1, wherein The concentration of the salting-out agent in the extractant is 0.2 g / mL.

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