CN113155993A - Method for detecting aroma components in milk or dairy products - Google Patents
Method for detecting aroma components in milk or dairy products Download PDFInfo
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- CN113155993A CN113155993A CN202110276155.8A CN202110276155A CN113155993A CN 113155993 A CN113155993 A CN 113155993A CN 202110276155 A CN202110276155 A CN 202110276155A CN 113155993 A CN113155993 A CN 113155993A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to a method for detecting aroma components in milk or dairy products, which comprises the steps of firstly extracting a to-be-detected product, and dissolving the to-be-detected product in a mixed solution of cyclohexane and ethyl acetate to obtain a mixed solution; injecting into gel permeation chromatographic column with ultraviolet-visible light luminosity detector, eluting with polystyrene microsphere gel, collecting eluate in 16.6-18.8min to obtain purified solution, evaporating to near dryness, and re-dissolving to obtain solution to be detected; then, detecting by using a gas chromatography-mass spectrometry system, analyzing a primary mass spectrogram and a secondary mass spectrogram to obtain the attribution category of each compound, and finally obtaining a corresponding structural formula and a corresponding molecular formula; and finally, comparing and extracting an ion flow diagram, and obtaining the concentration of the aroma component in the milk or the dairy product to be detected by a peak area method in combination with the concentration of the standard substance, the volume of the liquid to be detected and the quality of the product to be detected.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting aroma components in milk or dairy products.
Background
The means for analyzing the flavor components in the food mainly include gas chromatography (abbreviated as GC), ultra-high performance liquid chromatography (abbreviated as UPLC), phase-locked chromatography (abbreviated as CC), gas chromatography-mass spectrometry (abbreviated as GC-MS), ultra-high performance liquid chromatography-mass spectrometry (abbreviated as UPLC-MS), phase-locked chromatography-mass spectrometry (abbreviated as CC-MS), and electronic nose method (abbreviated as EN). Trapping of species is the first step in the study of volatile components in the matrix, and has a critical effect on the ratio of components in the study and on the quantitative limits.
The pretreatment modes for extracting and enriching volatile components in food mainly comprise liquid-liquid extraction (abbreviated as LLE), supercritical fluid extraction (abbreviated as SFE), solvent-assisted aroma component distillation (abbreviated as SAFE), headspace method (abbreviated as HS), dynamic headspace method (abbreviated as DHS), headspace solid-phase microextraction (abbreviated as HS-SPME) and the like. The method for extracting and enriching the volatile components of different food matrixes has limitations. In milk and dairy products, not only the composition of non-volatile components such as fats, proteins and saccharides is complicated, but also the composition of volatile components such as acids, esters, alcohols, ketones and aldehydes is also very complicated, and some of the volatile components have heat sensitivity and easy oxidation. For example, aliphatic hydroxy acid contains both hydroxyl and carboxyl, and its site has activity, when alpha-hydroxy acid is heated, two molecules of hydroxy acid can remove two molecules of water to form lactide structure, when beta-hydroxy acid is heated, one molecule of water can be removed to form unsaturated olefine acid, and when gamma-hydroxy acid is heated, one molecule of water can be removed from hydroxyl and carboxyl to form lactone.
In order to truly and comprehensively measure the composition of aroma components in milk and dairy products, a closed and low-temperature pretreatment mode needs to be selected. In addition, the milk and dairy products contain partial flavor componentsThe method has the advantages of low amount, low olfactive threshold concentration, great contribution to flavor composition, optimization of condition parameters of a determination method, and improvement of accuracy and precision of aroma component detection. The aroma components in milk and dairy products are classified according to functional group, including ketones (i.e. containing functional group-CO-), alkanes (i.e. containing functional group-CH)3or-CH2-), phenols (i.e., Ar-OH), carboxylic acids (i.e., containing a functional group-COOH), alcohols (i.e., containing a functional group-OH), amides (i.e., containing a functional group-NH)2) Thiols (i.e., containing the functional group-SH), aldehydes (i.e., containing the functional group-CHO), esters (i.e., containing the functional group-COOR), ethers (i.e., containing the functional group-O-CH)3or-O-CH2) And olefins (i.e., containing a functional group-CH ═ CH-), ketones, alkanes, phenols, and phenols are typical among the aroma components of milk and dairy products, and the results of a single analytical means do not fully reflect the compositions of these aroma components in milk and dairy products.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the method for detecting the aroma components in the milk or the dairy products, the pretreatment is simple, the method is quick and efficient, the reagent cost is low, the qualitative and quantitative result is accurate, the sensitivity is high, and the technical reference is provided for the quality safety monitoring of the milk and the dairy products.
The invention is realized by the following technical scheme:
a method for detecting aroma components in milk or milk products, when the aroma components are 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane, the method comprises the following steps:
step 1, extracting milk or dairy products to be detected by cyclohexane, and dissolving the milk or dairy products in a solvent with a volume ratio of 1:1 to obtain a mixed solution;
step 2, injecting the mixed solution into a gel permeation chromatographic column equipped with an ultraviolet-visible light photometric detector, wherein the filler of the chromatographic column is S-X3 polystyrene microsphere gel with the particle size of 200-400 mu m, eluting with the mixed solution of cyclohexane and ethyl acetate, collecting eluent within 16.6-18.8min to obtain a purified solution, and performing rotary evaporation on the purified solution until the purified solution is nearly dry and redissolving with n-hexane to obtain a solution to be detected;
step 3, detecting the liquid to be detected by using a gas chromatography-mass spectrometry system, obtaining a total ion flow diagram through gas chromatography, and obtaining a primary mass spectrogram and a secondary mass spectrogram through mass spectrometry;
and 5, comparing the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of the standard substance corresponding to each aroma component with the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio corresponding to each compound in the step 4 to obtain the mass-to-charge ratio corresponding to the aroma component to be detected, extracting an extracted ion flow diagram of each aroma component from a total ion flow diagram by using each mass-to-charge ratio, combining the peak area of the extracted ion flow diagram of each aroma component, the peak area of the extracted ion flow diagram of the standard substance and the concentration of the standard substance to obtain the concentration of each aroma component, and finally combining the concentration of each aroma component, the volume of the liquid to be detected and the mass of the milk or dairy product to be detected to obtain the concentration of the aroma component in the milk or dairy product to be detected, thereby completing the detection of the aroma component in the milk or dairy product.
Preferably, step 1, the uniformly mixed milk or dairy product sample to be detected is dissolved in cyclohexane, vortex mixing is carried out for 1-2min at 10000-12000r/min, then saturated sodium chloride water solution is added, oscillation is carried out for 15-20min, centrifugation is carried out for 3-5min at 8000-10000r/min, and finally a cyclohexane layer is taken and dissolved by mixed solution of cyclohexane and ethyl acetate with the volume of 1:1, so as to obtain mixed solution.
Further, the ratio of the sample to cyclohexane was (1-2) g: (10-15) mL.
Still further, the volume ratio of the saturated sodium chloride aqueous solution to cyclohexane is (3-5): (10-15).
Preferably, the purification conditions of the gel permeation chromatography column in step 2 are as follows:
the gel permeation chromatographic column is GPC purification column with specification of 20mm × 210mm, mobile phase is mixed solution of cyclohexane and ethyl acetate at volume ratio of 1:1, and flow rate is 3-4mLmin-1And the ultraviolet detection wavelength is 210 nm.
Preferably, in the step 2, the purified solution is subjected to rotary evaporation at the temperature of 20-25 ℃ until the purified solution is nearly dry, and then is redissolved by 3-5mL of n-hexane to obtain a solution to be detected.
Preferably, the gas chromatography conditions in step 3 are as follows:
the gas chromatographic column is an elastic quartz capillary chromatographic column with the specification of 30m multiplied by 0.25mm and 0.25 mu m, and adopts a non-flow-dividing constant-current mode with the flow rate of 1-1.5mLmin-1(ii) a The temperature rising procedure of the column oven is as follows: maintaining at 35-40 deg.C for 3-5min, and maintaining at 3-5 deg.C for min-1The temperature rise rate is increased from 35-40 ℃ to 150-170 ℃, and the temperature is kept for 3-5min, and finally the temperature is increased for 10-15 ℃ for min-1The temperature rise rate of (1) is increased from 150 ℃ to 170 ℃ to 220 ℃ and 230 ℃ and is kept at the temperature for 3 min.
Preferably, the mass spectrometry conditions in step 3 are as follows:
the ion source is an electron bombardment ion source, and the electron energy is 70-75 eV; the temperature of the sample inlet is 230-250 ℃; the temperature of the transmission line is 230-;
the ion source temperature is 220-230 ℃, and the solvent delay time is 3-5 min.
Further, step 3, a primary mass spectrogram is obtained through a full-scanning mode of the mass spectrum, and a secondary mass spectrogram is obtained through an ion scanning mode of the mass spectrum.
Preferably, the concentration of the aroma component in the milk or dairy product to be measured in step 5 is obtained by the following formula;
Compared with the prior art, the invention has the following beneficial technical effects:
the invention relates to a method for detecting aroma components in milk or dairy products, wherein 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane have strong polarity, according to the principle of similar compatibility, the milk or dairy products to be detected are firstly extracted by cyclohexane, so that the components can be extracted from the milk or dairy products, and meanwhile, the cyclohexane can compete for hydration water on the surface of protein in the milk or dairy products and destroy the hydration layer on the surface of colloid molecules to lead the protein to aggregate and precipitate, thereby achieving the purpose of enriching the target aroma components, an extracting solution is dissolved in the mixed solution of cyclohexane and ethyl acetate by a liquid-liquid extraction mode, macromolecular substances such as fat, glycan and the like can be removed, a primarily purified mixed solution is obtained, then the mixed solution is injected into a gel permeation chromatograph equipped with an ultraviolet-visible light photometric detector for purification, the neutral and 200-micron exclusion polystyrene microsphere gel and the porous polystyrene divinylbenzene medium contained in the gel can purify and separate target aroma components with small molecular weight and high polarity, the eluent is collected in a corresponding retention time range to obtain purified liquid containing the target aroma components, then the purified liquid is detected in a gas chromatography-mass spectrometry combined mode, a first-level mass spectrogram and a second-level mass spectrogram obtained by mass spectrometry are utilized to obtain a structural formula and a molecular formula of each compound, the mass charge ratio, the fragment abundance ratio and the isotope abundance ratio of a standard substance corresponding to each aroma component are combined to be compared with corresponding information of each previous compound, each mass charge ratio is utilized to extract an extracted ion flow diagram of each aroma component from total ion obtained by gas chromatography, and finally, the peak area and the like of each extracted ion flow diagram are utilized, The peak area of the standard substance and the concentration of the standard substance can obtain the concentration of each aroma component,and obtaining the concentration of the aroma components in the milk or the milk product to be detected by using the concentration of each aroma component, the volume of the liquid to be detected and the quality of the milk or the milk product to be detected. The method has accurate qualitative and quantitative result and high sensitivity, provides a systematic detection method for screening common aroma components in milk and dairy products, and results show that five types of aroma components have good linear relation in a linear range, correlation coefficients are all larger than 0.99, the standard recovery rate of the matrix reaches 81-90% under three addition levels of high (4 × CC beta), medium (2 × CC beta) and low (1 × CC beta), and the relative standard deviation is 1.8-5.9% after 6 times of repeated experiments. The determination limit (CC. alpha.) and the detection capacity (CC. beta.) were each 0.05. mu.g kg-1-0.33μg kg-1With 0.08. mu.g kg-1-0.52μg kg-1. The application of the gel permeation chromatography effectively makes up the problem that the traditional dispersed solid phase extraction purification method is not thorough in removing interfering substances, improves the sensitivity of the method, reduces the reagent cost, simplifies the pretreatment steps and improves the working efficiency; compared with conventional detection methods such as a liquid chromatography ultraviolet method, a liquid chromatography fluorescence method and the like, the gas chromatography-mass spectrometry combined method has strong matrix interference resistance and more accurate substance determination. In addition, compared with a liquid chromatography-mass spectrometry combined method, the method has the characteristics of low cost and high sensitivity and accuracy, and is suitable for large-scale batch detection of samples.
Drawings
FIG. 1a is a UV gel permeation chromatography outflow curve of a pasteurized milk sample according to the invention;
FIG. 1b is an ultraviolet gel permeation chromatography outflow curve of the ultra-high temperature instant sterilized milk of the present invention;
FIG. 1c is a UV gel permeation chromatography efflux curve for fermented milk according to the present invention;
fig. 1d is a uv gel permeation chromatography outflow curve of the infant formula of the present invention.
FIG. 2a is a graph of the corresponding response intensity of GPC effluent of the present invention over a collection time range of 16-16.8 min;
FIG. 2b is a graph of the corresponding response intensity of GPC effluent of the present invention over a collection time range of 18.2-19 min;
FIG. 3 is a GPC purge chromatogram of a target analyte in pasteurized milk.
Detailed Description
The principles and advantages of the present invention are explained and illustrated below with reference to specific embodiments so that those skilled in the art may better understand the present invention. The following description is exemplary only, and is not intended to limit the scope thereof.
The invention relates to a method for detecting aroma components in milk or dairy products by using gel permeation chromatography, wherein the aroma components belong to spices, and the method specifically comprises the following steps:
1) extracting target substances to be detected in milk and dairy products to be detected, namely 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane by using a liquid-liquid extraction treatment method;
weighing 1-2g (accurate to 0.01g) of the uniformly mixed sample in a 50mL centrifuge tube with a plug, adding 10-15mL of cyclohexane to dissolve the sample, mixing for 1-2min by high-speed vortex at 10000-12000r/min, adding 3-5mL of saturated sodium chloride aqueous solution, oscillating for 15-20min, and mixing for 8000 plus 10000rmin-1Centrifuging at low temperature for 3-5min, wherein the temperature is 4 deg.C, transferring cyclohexane layer to 10mL volumetric flask, and adding cyclohexane-ethyl acetate mixture (1:1, v/v) to constant volume of 10 mL;
2) based on a valve switching technology, purifying the extracted and concentrated target object to be detected by using gel permeation chromatography;
after milk and dairy product samples are extracted by cyclohexane, injecting the solution into a gel permeation chromatographic column by an automatic sampling device, eluting the samples by adopting cyclohexane-ethyl acetate mixed solution (1:1, v/v) through a high-pressure pump, and allowing the samples to flow out along gaps of polystyrene microsphere gel, wherein the samples have high relative molecular mass of protein and fat interfering in analysis of target compounds. The gel permeation chromatography system is provided with an ultraviolet-visible light photometric detector and is used for measuring the response intensity of the five aroma components, the detection wavelength is set to be 210nm, the peak emergence time is 4.0min-10.0min, the relative molecular mass of target analytes, namely 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane, is small, the target analytes can enter the interior of the polystyrene microsphere gel, and the peak emergence time is 16.6min-18.8min after the retention time is short;
gel permeation chromatography purification conditions: GPC purification column (specification is 20mm × 210mm), and packing is S-X3 polystyrene microsphere gel (particle size is 200 μm-400 μm); mobile phase cyclohexane-ethyl acetate with volume ratio of 1: 1; the flow rate is 3-4mLmin-1(ii) a The sample injection amount is 10.0 mL; the ultraviolet detection wavelength is 210 nm; rotatably evaporating the collected 16.6-18.8min purified solution at 20-25 deg.C to near dry, re-dissolving the near dry matter with 3-5mL n-hexane, and passing through 0.20 μm filter membrane for measurement;
3) detecting the processed sample by using a gas chromatography-mass spectrometry system, namely a 7890A-5975C gas chromatography-mass spectrometer;
the gas chromatography conditions were: capillary chromatographic column: DB-5MS (specification of 30m × 0.25mm, 0.25 μm) is specifically elastic quartz capillary chromatographic column, and adopts non-shunting constant flow mode with flow rate of 1-1.5mLmin-1(ii) a The temperature rising procedure of the column oven is as follows: maintaining at 35-40 deg.C for 3-5min, and maintaining at 3-5 deg.C for min-1Heating to 150--1Raising the temperature to 220 ℃ and 230 ℃ and keeping the temperature for 3 min; carrier gas: high purity helium (99.999%).
The mass spectrum conditions are as follows: electron impact (EI for short) ion source; the electron energy is: 70-75 eV; sample inlet temperature: 230 ℃ to 250 ℃; transmission line temperature: 230 ℃ to 250 ℃;
the ionization mode is as follows: ion source temperature: 220 ℃ and 230 ℃; solvent delay time: 3-5 min;
scanning time: 3min-60 min; and obtaining a primary mass spectrogram through the full scanning mode and obtaining a secondary mass spectrogram through the selective ion scanning mode by adopting the full scanning mode (SCAN for short) and the selective ion scanning mode (SIM for short).
4) Extracting the structure and element composition of target aroma components through an agent MSD chemstation workstation based on the obtained primary mass spectrogram and secondary mass spectrogram of the detected sample;
obtaining different molecular ion peaks by analyzing a primary mass spectrogram, obtaining different fragment ion peaks by analyzing a secondary mass spectrogram, obtaining accurate relative molecular mass by using the molecular ion peaks and the fragment ion peaks according to a mass loss mechanism, judging the attribution type of the substance, and deducing the structure and the element composition of the substance, namely the structural formula and the molecular formula of the substance by using the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of the substance in the secondary mass spectrogram;
5) and establishing aroma component information in a standard spectrum library by combining substances corresponding to the standard aroma components, wherein the aroma component information comprises the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of the five standard aroma components. Comparing the information of the five aroma components with the information of all compounds obtained by the tested object sample, and judging the aroma component structures in the milk and the dairy products within the error range of 0.05 Da. And then, using the mass-to-charge ratio of the aroma components, obtaining an extracted ion flow diagram of each substance from the total ion flow diagram obtained by the gas chromatography in the step 3, quantifying the concentration of the aroma components by using a peak area method, obtaining the concentration of each aroma component by combining the peak area of the extracted ion flow diagram, the peak area of a standard substance and the concentration of the standard substance, and finally obtaining the concentration of the aroma components in the milk or the dairy product to be detected by combining the concentration of each aroma component, the volume of the liquid to be detected and the quality of the milk or the dairy product to be detected, thereby completing the detection of the aroma components in the milk or the dairy product.
Obtained by the following formula;
The establishment of the standard spectrum library specifically comprises the following steps:
1a) preparing a standard solution of 5 types of aroma components (2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane) standard substance;
1b) screening the standard solution of the aroma components by using the gas chromatography-mass spectrometry system described in the step 3, and obtaining complete primary mass spectrograms and secondary mass spectrograms of the standard solution by adopting an electron bombardment ion source, a full scanning mode and a selective ion scanning mode, wherein gas chromatography-mass spectrometry parameters of 5 aroma component compounds are shown in a table 1;
TABLE 15 gas chromatography-Mass Spectrometry parameters for aroma components in milk and Dairy products
1c) And (4) processing the obtained primary mass spectrogram and secondary mass spectrogram through an agent MSD chemstation workstation according to the method described in the step 4 to obtain the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of each substance in the standard substance, and establishing a standard spectrum library of aroma components.
The preparation and specific analysis of the instruments, reagents and solutions used above are listed below:
1. instrument for measuring the position of a moving object
7890A-5975C gas chromatography-mass spectrometer (Agilent Technologies, USA), vortex mixer of vortex Gene2T type (Scientific Industries, USA), oscillator SA31 type (Yamata, Japan), ultrasonic cleaner (Kunshima ultrasonic instruments, Inc. of Jiangsu province), analytical balance of AL204-IC type (Mettler Toledo, Switzerland), rotary evaporation concentrator (Buchi, Switzerland), thermostatic drying cabinet of ED-115 type (Binder, Germany), SevenEasy S20K type pH meter (Toledo, Switzerland), gel permeation chromatograph (J2 Scientific, Germany), 20mm × 210mm GPC purification column (J2 Scientific), 200 + 400 μm S-X3 polystyrene microsphere gel packing (Bio-Beads, USA), Millil-Intrael polystyrene type (Millipore, USA).
2. Reagent
Milk and dairy products (Wal-Ma super, Jia le Fu supermarket), sodium chloride (GR) (Akzo Nobel, the Netherlands), 0.20 μm microporous membrane (Pall, USA), cyclohexane (HPLC) (Fisher, USA), ethyl acetate (HPLC) (Fisher, USA), n-Hexane (HPLC) (Fisher, USA), 5 aroma components and related chemical standard substances (Dr. Ehrentorfer GmbH, Germany, Sigma-Aldrich, Germany, Witega).
3. Preparation of the solution
2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid, 3-ethyl-5- (2-ethylbutyl) -octadecane mixed standard stock solution: 1000mgL-1Respectively and accurately weighing 0.1000g of 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane standard substance in a 100mL volumetric flask, dissolving with methanol, fixing the volume to the marked line, and uniformly oscillating. 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid, 3-ethyl-5- (2-ethylbutyl) -octadecane series mixed standard working solutions: accurately transferring appropriate amount of the above mixed standard stock solutions into 6 volumetric flasks of 100mL, diluting with methanol, diluting to constant volume to marked line, mixing, and making into 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid, and 3-ethyl-5- (2-ethylbutyl) -octadecane with mass concentration of 0.01mgL-1、0.02mgL-1、0.04mgL-1、0.10mgL-1、0.15mgL-1、0.20mgL-1The series was mixed with standard working solution.
4. Selection of gel permeation chromatography purification conditions
According to the retention time of a target analyte and impurities, fat and protein with relatively large molecular mass can be cut into a waste liquid flow path through a six-way valve communication position in the conversion gel permeation chromatography, a solution to be analyzed is switched into a trapping loop flow path, rotary evaporation and concentration are carried out, normal hexane is used for fixing the volume to 5mL, and then gas chromatography-mass spectrometry detection is carried out. Therefore, the design of the switching program of the communication position of the six-way valve is the key point of the gel permeation chromatography purification, and fig. 1a, fig. 1b, fig. 1c and fig. 1d are respectively the ultraviolet gel permeation chromatography outflow curves of the samples of pasteurized milk, ultrahigh temperature instant sterilized milk, fermented milk and infant formula milk powder after cyclohexane extraction.
Detecting pasteurized milk sample with gel permeation chromatography column ultraviolet 210nm, wherein fat and protein with relatively large molecular mass flow out in 4.00-9.10min in figure 1a, and solution to be analyzed flows out in 16.00-19.00 min; FIG. 1b shows the UV gel permeation chromatography outflow curve of the ultra-high temperature instant sterilized milk, wherein the fat and protein flow out at 4.00-9.00min and the solution to be analyzed flows out at 16.00-18.80 min; the UV gel permeation chromatography efflux curve of the fermented milk of FIG. 1c shows that fat and protein are shed at 4.70-8.90min and the solution to be analyzed is shed at 16.40-18.50 min; the uv gel permeation chromatography outflow curve of the infant formula powder of fig. 1d shows that fat and protein flow out at 4.70-8.80min and the solution to be analyzed flows out at 16.00-18.10min, thus determining that the optimal range of the collection time of the gel permeation chromatography effluent is 16.00-19.00 min.
The mass spectrum response (i.e. chromatographic peak area) of the 5 target substances is used as an index for evaluating the six-way valve communication position switching program.
The initial time of cutting the communication position of the six-way valve into the trapping ring flow path from the waste liquid flow path is initially set to 16.00min, the optimized step length is 0.20min, and the time of flowing the eluent into the trapping ring flow path is shortened; the time for switching the communication position of the fixed six-way valve to the waste liquid again is 19.0min, and 0.20min is an interval, so that the time for switching the communication position of the six-way valve is gradually advanced.
Figure 2 is the optimization results of the 5 compound efflux program. FIG. 2a shows the highest GPC effluent response intensity collected at 16.6min, and FIG. 2b shows the highest GPC effluent response intensity collected at 18.8min, which corresponds to the highest concentration of 5 compounds at the corresponding retention times, respectively. If the target analyte is cut into the trapping ring flow path too late by using the six-way valve, or the six-way valve communication flow path is cut into the waste liquid flow path when the target analyte compound is not completely eluted, the target substance to be analyzed is lost, and the recovery rate of the compound is reduced; if impurities such as fat and protein are cut into the trap ring by the six-way valve too early, impurities with relatively large molecular weight enter the trap ring, and if the target compound is eluted, the connecting flow path of the communicating valve is not cut into the waste liquid, and impurities with small molecular weight also enter the trap ring. Therefore, the collection time of the gel permeation chromatography is controlled to be 16.60min-18.80min, and the purification of the gel permeation chromatography on the sample is realized.
FIG. 3 shows that the response intensity of 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane is high in the collection time range, and the matrix interference is basically removed, which indicates that the optimized conditions can achieve good purification effect.
In addition, the influence of the elution speed on the separation effect of the target compound was investigated in the experiment, and 1.0mLmin was set-1，1.5mLmin-1，2.0mLmin-1，2.5mLmin-1，3.0mLmin-1，3.5mLmin-1，4.0mLmin-1，4.5mLmin-1，5.0mLmin-1When the flow rate is more than 4.0mLmin-1When the flow rate is less than 3.0mLmin, the separation effect of the target object from fat and protein is not good due to the high flow rate-1In time, too low flow rate widens the retention time zone of the sample, and diffusion is intensified to prolong the experimental time, so 3.5mLmin is adopted as the elution speed-1。
5. Analysis results
(1) Methodology validation
a standard curve, linear range and correlation coefficient of method
The ordinate y represents the peak area of the quantitative ion chromatogram peak of the standard aroma component, and x represents the concentration (mg L) of the standard component-1) And (5) drawing a standard substance calibration curve. The experimental result shows that the five types of aroma components in the detection method have good linear relation and correlation coefficient (r) in corresponding concentration ranges2) Are all greater than 0.99.
b quantitative lower limit, recovery and precision of the method
The detection limit and the quantitative lower limit take the determination limit (CC. alpha.) and the detection capacity (CC. beta.) as the examination criteria. CC α was determined using a calibration curve method, and its value was equal to 2.33 times the concentration value on the ordinate at which the response could be observed plus the corresponding standard deviation of reproducibility. The calculation method of CC beta is the sum of the value of CC alpha and 1.64 times of the standard deviation of reproducibility when the value of CC alpha is taken. The limit and detection capacity were determined to be 0.05. mu.g kg-1-0.33μg kg-1With 0.08. mu.g kg-1-0.52μg kg-1。
The accuracy and precision of the experiment were evaluated by the standard deviation of the recovery from the results of 6 replicates. Series mixed standard substance solutions (CC beta, 2 times of CC beta and 4 times of CC beta) of five types of aroma components at three concentration levels are added into a substrate, and the average recovery rate and the relative standard deviation of various aroma flavor substances are calculated by using 7890A-5975C gas chromatography-mass spectrometer for detection. The recovery rate and the relative standard deviation result are respectively 81% -90% and 1.8% -5.9%, and the established method has good accuracy and precision.
Actual sample detection
The invention, in its specific implementation, is practiced with the following specific parameters,
weighing 2g (accurate to 0.01g) of the uniformly mixed sample in a 50mL centrifuge tube with a plug, adding 10mL of cyclohexane to dissolve the sample, mixing for 1min by high-speed vortex at 12000r/min, adding 5mL of saturated sodium chloride aqueous solution, oscillating for 20min, and mixing with 10000rmin-1Centrifuging at low temperature for 5min, taking the cyclohexane layer, transferring to a 10mL volumetric flask, and fixing the volume to 10mL by using a cyclohexane-ethyl acetate mixed solution (1:1, v/v);
rotatably evaporating the collected purifying liquid at 25 ℃ until the purifying liquid is nearly dry, redissolving the nearly dry substance by using 5mL of normal hexane, and passing through a 0.20-micron filter membrane for measurement by an online machine;
the gas chromatography conditions were: flow rate of 1mLmin-1(ii) a The temperature rising procedure of the column oven is as follows: maintaining at 40 deg.C for 3min, and maintaining at 3 deg.C for 3min-1Heating to 170 deg.C and maintaining for 3min, and maintaining at 10 deg.C for min-1Raise to 230 ℃ and hold for 3 min.
The mass spectrum conditions are as follows: the electron energy is: 70 eV; sample inlet temperature: 250 ℃; transmission line temperature: 250 ℃;
the ionization mode is as follows: ion source temperature: 230 ℃; solvent delay time: 3 min;
applying the established liquid-liquid extraction-gel permeation chromatography-gas chromatography-mass spectrometry method to the commercial milk and dairy products: the results of the analytical tests on pasteurized milk (eighteen brands of twelve batches), ultra high temperature flash sterilized milk (twenty brands of twelve batches), fermented milk (thirteen brands of sixteen batches), and infant formula (sixteen brands of twelve batches) are shown in table 2. The experimental results show that2, 4-di-tert-butylphenol and 3-ethyl-5- (2-ethylbutyl) -octadecane are not present in commercially available milk and dairy products. The methodological verification of the three types of aroma components shows that the three types of aroma components have good linear relation in a linear range, the correlation coefficients are all larger than 0.99, the standard recovery rate of the matrix reaches 81-90% under three addition levels of high (4 × CC beta), medium (2 × CC beta) and low (1 × CC beta), the experiment is repeated for 6 times, and the relative standard deviation is 1.8-5.9%. The determination limit (CC. alpha.) and the detection capacity (CC. beta.) were each 0.05. mu.g kg-1-0.33μg kg-1With 0.08. mu.g kg-1-0.52μg kg-1. The method has accurate qualitative and quantitative result and high sensitivity, and provides a systematic detection method for screening common aroma components in milk and dairy products.
TABLE 2 typical milk and milk product ingredient assay results
The above-described embodiments of the present invention are examples, and any means having the same technical idea as the claims of the present invention and exhibiting the same effects are included in the present invention.
Claims (10)
1. A method for detecting aroma components in milk or dairy products is characterized by comprising the following steps when the aroma components are 2-nonanone, pentadecane, 2, 4-di-tert-butylphenol, lauric acid and 3-ethyl-5- (2-ethylbutyl) -octadecane:
step 1, extracting milk or dairy products to be detected by cyclohexane, and dissolving the milk or dairy products in a solvent with a volume ratio of 1:1 to obtain a mixed solution;
step 2, injecting the mixed solution into a gel permeation chromatographic column equipped with an ultraviolet-visible light photometric detector, wherein the filler of the chromatographic column is S-X3 polystyrene microsphere gel with the particle size of 200-400 mu m, eluting with the mixed solution of cyclohexane and ethyl acetate, collecting eluent within 16.6-18.8min to obtain a purified solution, and performing rotary evaporation on the purified solution until the purified solution is nearly dry and redissolving with n-hexane to obtain a solution to be detected;
step 3, detecting the liquid to be detected by using a gas chromatography-mass spectrometry system, obtaining a total ion flow diagram through gas chromatography, and obtaining a primary mass spectrogram and a secondary mass spectrogram through mass spectrometry;
step 4, analyzing the primary mass spectrogram and the secondary mass spectrogram to respectively obtain a plurality of molecular ion peaks and fragment ion peaks which correspond to each other one by one, obtaining the attribution type of each compound in the liquid to be detected through the molecular ion peaks and the fragment ion peaks, and finally obtaining the structural formula and the molecular formula of each compound respectively by utilizing the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of each compound in the secondary mass spectrogram;
and 5, comparing the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio of the standard substance corresponding to each aroma component with the mass-to-charge ratio, the fragment abundance ratio and the isotope abundance ratio corresponding to each compound in the step 4 to obtain the mass-to-charge ratio corresponding to the aroma component to be detected, extracting an extracted ion flow diagram of each aroma component from a total ion flow diagram by using each mass-to-charge ratio, combining the peak area of the extracted ion flow diagram of each aroma component, the peak area of the extracted ion flow diagram of the standard substance and the concentration of the standard substance to obtain the concentration of each aroma component, and finally combining the concentration of each aroma component, the volume of the liquid to be detected and the mass of the milk or dairy product to be detected to obtain the concentration of the aroma component in the milk or dairy product to be detected, thereby completing the detection of the aroma component in the milk or dairy product.
2. The method for detecting the aroma components in the milk or the dairy product according to claim 1, wherein the step 1 comprises the steps of dissolving the uniformly mixed sample of the milk or the dairy product to be detected in cyclohexane, mixing the sample in a vortex manner at 10000- & lt 12000 & gtr/min for 1-2min, adding a saturated sodium chloride aqueous solution, oscillating the mixture for 15-20min, centrifuging the mixture for 3-5min at 8000- & lt 10000 & gtr/min, and finally taking a cyclohexane layer and dissolving the cyclohexane layer with a mixed solution of cyclohexane and ethyl acetate with a volume of 1:1 to obtain a mixed solution.
3. The method for detecting the aroma components in milk or milk products according to claim 2, wherein the ratio of the sample to cyclohexane is (1-2) g: (10-15) mL.
4. The method for detecting the aroma components in the milk or the milk product according to claim 2, wherein the volume ratio of the saturated sodium chloride aqueous solution to the cyclohexane is (3-5): (10-15).
5. The method for detecting the aroma components in the milk or dairy product according to claim 1, wherein the purification conditions of the gel permeation chromatography column in the step 2 are as follows:
the gel permeation chromatographic column is GPC purification column with specification of 20mm × 210mm, mobile phase is mixed solution of cyclohexane and ethyl acetate at volume ratio of 1:1, and flow rate is 3-4mLmin-1And the ultraviolet detection wavelength is 210 nm.
6. The method for detecting the aroma components in the milk or the dairy product according to claim 1, wherein in the step 2, the purifying solution is subjected to rotary evaporation at the temperature of 20-25 ℃ until the purifying solution is nearly dry, and then 3-5mL of n-hexane is used for redissolving to obtain the liquid to be detected.
7. The method for detecting the aroma components in the milk or the milk product according to claim 1, wherein the gas chromatography conditions in the step 3 are as follows:
the gas chromatographic column is an elastic quartz capillary chromatographic column with the specification of 30m multiplied by 0.25mm and 0.25 mu m, and adopts a non-flow-dividing constant-current mode with the flow rate of 1-1.5mLmin-1(ii) a The temperature rising procedure of the column oven is as follows: maintaining at 35-40 deg.C for 3-5min, and maintaining at 3-5 deg.C for min-1The temperature rise rate is increased from 35-40 ℃ to 150-170 ℃, and the temperature is kept for 3-5min, and finally the temperature is increased for 10-15 ℃ for min-1The temperature rise rate of (1) is increased from 150 ℃ to 170 ℃ to 220 ℃ and 230 ℃ and is kept at the temperature for 3 min.
8. The method for detecting aroma components in milk or dairy products according to claim 1, wherein the mass spectrometric conditions in step 3 are as follows:
the ion source is an electron bombardment ion source, and the electron energy is 70-75 eV; the temperature of the sample inlet is 230-250 ℃; the temperature of the transmission line is 230-;
the ion source temperature is 220-230 ℃, and the solvent delay time is 3-5 min.
9. The method for detecting the aroma components in the milk or dairy product according to claim 8, wherein the step 3 is to obtain a primary mass spectrum by a full scan mode of mass spectrometry and obtain a secondary mass spectrum by an ion scan mode of mass spectrometry.
10. The method for detecting the aroma components in the milk or the milk product according to claim 1, wherein the concentration of the aroma components in the milk or the milk product to be detected in the step 5 is obtained by the following formula;
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