CN108918725B - Analysis and detection method for effectively identifying different types of Chinese softshell turtles - Google Patents
Analysis and detection method for effectively identifying different types of Chinese softshell turtles Download PDFInfo
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Abstract
The invention discloses an analysis and detection method for effectively identifying different types of Chinese softshell turtles, which comprises the following steps: uniformly grinding muscle tissues of the Chinese softshell turtle to be detected, and adding dichloromethane-methanol mixed extraction solution as an extracting solution; carrying out ultrasonic-assisted extraction on the extracting solution, adding ultrapure water, freezing and centrifuging; adding dichloromethane into the primary water phase obtained by freezing and centrifuging for secondary extraction, and combining the primary organic phase obtained by freezing and centrifuging and the secondary organic phase obtained by secondary extraction to obtain an extract; drying the extract under a nitrogen blowing instrument, fixing the volume with acetonitrile, and filtering with 0.22 μm organic filter membrane to obtain a solution to be detected; and (3) analyzing and detecting the liquid to be detected by a liquid chromatography-mass spectrometry method, and using the phospholipid ions of m/z790,816,818 and 866 as potential markers for species identification of the Chinese softshell turtles. The method can effectively distinguish three kinds of Chinese soft-shelled turtles, namely Zhejiang black-bone turtle, Qingxi black-bone turtle and Zhejiang new-flower turtle.
Description
Technical Field
The invention relates to a lipidomics method for analyzing phospholipids in turtles, in particular to a method for effectively identifying different types of Chinese softshell turtles and whether the Chinese softshell turtles are adulterated or not.
Background
Chinese soft-shelled turtle is a high-value commercial species, and is often eaten as a tonic and food in Asian countries such as China, Japan, Korea, and the like. The soft-shelled turtle contains nutritional factors such as protein, essential amino acids, polyunsaturated fatty acids, trace elements and other active substances, and has effects of improving immunity, promoting metabolism, and preventing diabetes and anemia. In recent years, several new strains of trionyx sinensis have been obtained by cross breeding, and are expected to replace the currently unmodified varieties. However, in the market, these high value soft-shelled turtles and their processed products are replaced by cheap and even inferior meat to gain economic benefit to the merchant. Because of their similar shapes, such deceptive means are difficult to identify through the external and anatomical features of soft-shelled turtles, and further increase the difficulty of identification when they are processed. Therefore, an effective and reliable method for analyzing and detecting the class of the Chinese soft-shelled turtle is needed by related departments to guarantee the food safety of consumers.
At present, most of methods for identifying the species of the Chinese softshell turtles are methods for genotypes such as Polymerase Chain Reaction (PCR), restriction fragment length polymorphism (CAPs), multifunctional targeted proteomics and the like. However, these methods are time consuming and expensive and are subject to major limitations in analytical testing when the analyte is complex or DNA contamination is present. Furthermore, when the species under investigation is not present in the database, specific probe sequences and associated DNA sequencing are required, which makes the genotyping method for species identification technically demanding.
As the complexity of samples increases, the identification of species of trionyx sinensis requires powerful analytical instruments with high sensitivity, high selectivity and a large detection range.
The three kinds of Chinese soft-shelled turtle, namely Zhejiang black-bone turtle, Qingxi black-bone turtle and Zhejiang new-flower turtle, have similar strains and similar appearances and are difficult to distinguish by naked eyes, so that a method for effectively distinguishing the Chinese soft-shelled turtles is urgently needed to be developed.
Disclosure of Invention
The invention aims to provide an analysis and detection method for effectively identifying different types of Chinese soft-shelled turtles. The method has the characteristics of high resolution, high sensitivity, rapidness, reliability and economy; can effectively identify three Chinese softshell turtle strains of Zhejiang black turtle No. two, Qingxi black turtle and Zhejiang new flowered turtle.
In order to solve the technical problems, the invention provides an analysis and detection method for effectively identifying different types of Chinese softshell turtles, which comprises the following steps:
1) taking 0.2g of muscle tissue of the Chinese softshell turtle to be detected, grinding uniformly, adding 1.2 +/-0.1 mL of dichloromethane-methanol mixed extraction solution, and oscillating uniformly (oscillation time is about 5 +/-1 min) at 30 +/-5 ℃ to obtain an extracting solution (A product);
in the dichloromethane-methanol mixed extraction solution, the ratio of dichloromethane: 1.9-2.1% of methanol: a volume ratio (v/v) of 1 (preferably 2: 1);
2) after the ultrasonic-assisted extraction is carried out on the extracting solution (product A), 0.8 plus or minus 0.1mL of ultrapure water is added, and the extracting solution is frozen and then centrifuged;
3) adding 2 +/-0.2 mL of dichloromethane into the primary water phase (upper layer) obtained by freezing and centrifuging in the step 2) for secondary extraction to obtain a secondary water phase at the upper layer and a secondary organic phase layer at the lower layer;
4) combining the primary organic phase (lower layer) obtained in the step 2) through freezing and centrifuging and the secondary organic phase obtained in the step 3) through secondary extraction to obtain extract liquor;
drying the extract under a nitrogen blowing instrument, fixing the volume with 1mL of acetonitrile, and filtering the extract through a 0.22-micron organic filter membrane to be used as a solution to be detected;
5) and (3) analyzing and detecting the liquid to be detected by a liquid chromatography-mass spectrometry (a method combining liquid chromatography and mass spectrometry), and using the phospholipid ions of m/z790,816,818 and 866 as potential markers for species identification of the Chinese softshell turtles.
The improvement of the analysis and detection method for effectively identifying different types of Chinese softshell turtles is as follows: in the step 5), the step of mixing the raw materials,
the liquid chromatogram is: the liquid chromatographic column is YMC Triart diol liquid chromatographic column, 4.6 x 250mm,3 μm; the temperature of the chromatographic column is 40 ℃, and the flow rate is 0.2 mL/min-1The sampling amount is 2 mL;
the mobile phase A is: firstly, preparing an acetic acid acetonitrile solution with acetic acid concentration of 53 +/-5 mM, and then adjusting the pH value to 4.0-4.5;
the mobile phase B is as follows: adding pure water into 60 +/-5 mmol ammonium acetate and 53 +/-5 mmol acetic acid to reach a constant volume of 1L, and adjusting the pH value to 3.0 +/-0.2;
the gradient elution was: the mobile phase A is kept at 95 percent for 0 to 3 minutes; after 3-13 minutes, the mobile phase A is uniformly reduced from 95% to 70%; after 13-18 minutes, the mobile phase A is uniformly reduced to 50 percent; in the gradient elution, the balance is mobile phase B, and the above% is volume% for each time period.
Remarks explanation: washing the column with pure water and acetonitrile in the volume ratio of 1:1 for the next 18-25 min; the washed column can be reused.
The analysis and detection method for effectively identifying different types of Chinese softshell turtles is further improved as follows: in the step 5), the step of mixing the raw materials,
mass spectrometry mode: using a needle pump syringe at 200. mu.L.min-1Injecting the liquid to be detected (as a sample) obtained in the step 4) into an atmospheric pressure ion chemical source at the flow rate of 2 mu L, and ionizing in a negative ion mode;
the mass spectrum conditions are as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; ion source gases, including dry gas (GS1), nebulizer gas (GS2), and curtain gas, set at 50psi, 60psi, and 25psi, respectively; the mass spectrum measuring range is m/z450-950, and the scanning speed is 1s per spectrum;
instrument control and data acquisition, processing and analysis were performed using analyst1.5.1 software.
The analysis and detection method for effectively identifying different types of Chinese softshell turtles is further improved as follows: in the step 5), the step of mixing the raw materials,
when the relative range of the abundance of the phospholipid ions of m/z816 is 15% -21%, the relative range of the abundance of the phospholipid ions of m/z790 is 2% -4%, and the relative range of the abundance of the phospholipid ions of m/z818 is 10% -12%, the Chinese softshell turtle to be detected is Zhewu II;
when the relative range of the abundance of the phospholipid ions of the m/z816 is 6-10%, the Chinese softshell turtle to be detected is Qingxi soft-shelled turtle.
When the relative range of the abundance of the phospholipid ions of the m/z816 is 11% -14%, the Chinese softshell turtle to be detected is a new Hua softshell turtle.
The analysis and detection method for effectively identifying different types of Chinese softshell turtles is further improved as follows: in the step 4): the extract was dried at ambient temperature of 4. + -. 1 ℃ under nitrogen flow (40. + -. 5 ℃ C.), and then diluted with 1mL of acetonitrile to a constant volume, and passed through a 0.22 μm organic filter to obtain a test solution.
Note: the ambient temperature is controlled in order to minimize the risk of lipid oxidation.
As a further improvement of the analysis and detection method for effectively identifying different types of Chinese soft-shelled turtles, in the step 2): extracting the extractive solution (product A) with ultrasonic wave at 50 deg.C under 53KHz for 30 + -5 min, adding 0.8 + -0.1 mL ultrapure water, and centrifuging at-20 + -2 deg.C for 15 + -2 min.
As a further improvement of the analysis and detection method for effectively identifying different types of Chinese soft-shelled turtles, in the step 3): adding 2 +/-0.2 mL of dichloromethane into the primary water phase (upper layer) obtained by centrifuging in the step 2) for secondary extraction to obtain a secondary water phase at the upper layer and a secondary organic phase layer at the lower layer; replacing the primary water phase obtained by centrifuging in the step 2) with the secondary water phase, and repeating the secondary extraction for 1-2 times;
and 2) combining the primary organic phase (lower layer) obtained by centrifuging in the step 2) and all the secondary organic phases obtained by extraction in the step 3) to obtain an extract.
Compared with the prior identification technology, the method utilizes a lipidomics method to distinguish the trionyx sinensis of different strains through higher resolution and sensitivity and high-quality and accurate phospholipid analysis and determination. Advances in analytical methods such as Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR), Gas Chromatography (GC), and High Performance Liquid Chromatography (HPLC) have accelerated the development of lipidomics. The technology combining high performance liquid chromatography and mass spectrometry is the most commonly used method for separating and detecting lipid, and because the technology has satisfactory resolution, sensitivity and repeatability, the technology can be used for detecting and analyzing the lipid in a complex sample; according to the invention, the detection conditions of the liquid chromatography and the mass spectrometry are respectively set according to the characteristics of Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidylinositol (PI), so that the high performance liquid chromatography and the mass spectrometry have a good effect when used for detecting a sample. And it is known from a large number of experiments that phospholipids are easily ionized in negative ion mode, and that PE and PI molecular species are ionized to [ M-H ]]-And PC molecular species are easily ionized to [ M + CH3COO]-(this is because acetic acid is added to the mobile phase), an optimum ionization signal can be obtained by the ionization method of negative ions, and the detection accuracy and effect are improved.
The present invention optimizes various factors affecting the extraction process, such as extraction temperature, solvent amount and extraction time, in order to obtain the best extraction performance at the minimum cost. In terms of extraction temperature, when the temperature is lower, the viscosity of the sample is reduced along with the increase of the extraction temperature, so that the sample is well dispersed in the organic phase, but when the temperature is increased to a certain degree, the sample is possibly degraded by oxidation, so that 50 ℃ is selected as the optimal extraction temperature in the ultrasonic extraction of the step 2); in terms of the amount of the solvent (the sum of the amounts of the dichloromethane-methanol mixed extract solution in step 1) and the ultrapure water in step 2)), the recovery rate of the phospholipid sample gradually increases as it increases from 0.5mL to 2mL, and remains unchanged as it continues to increase, so that the amount of the solvent is selected to be 2 mL; in the aspect of the ultrasonic extraction time in the step 2), when the extraction time is 10-30 minutes, the extraction efficiency is remarkably improved, and as the extraction time is prolonged, the standard substance is more sufficiently contacted with the extraction medium, so that the frequency of migration of the standard substance between two phases is higher, and the extraction efficiency is kept unchanged, therefore, the extraction time is selected to be 30 minutes.
In addition, the acetonitrile is used as a proper elution solution in the invention, because the acetonitrile can effectively retain the target analyte to a great extent and remove the interference of the interfering matrix on the detection result, thereby generating larger abundance and clearer spectrum and having better elution effect. While other solutions such as chloroform and methanol result in severe loss of phospholipids. Meanwhile, in order to improve the sensitivity of mass spectrometry, reduce the matrix effect and realize the optimal separation of lipids, the pH value of the mobile phase is optimized before mass spectrometry. Ionic strength plays a key role in hydrophilic chromatographic separations, while low pH mobile phases are weaker in ionic strength, and in addition, PC and PE exist in cationic or neutral form due to negatively charged phosphate groups and positively charged head groups, while pH may influence the ratio of these two forms. Therefore, the invention compares the spectra with and without acetic acid, and finds that the spectrum peak shape is better after adding 0.1% acetic acid, the retention time is relatively shorter, and the spectrum peak width of the mobile phase without acidification is in a tail shape and has poor symmetry. Therefore, the invention selects the mobile phase A (acetic acid acetonitrile solution with acetic acid concentration of 53 +/-5 mM, and adjusts the pH value to 4.0-4.5) to carry out liquid chromatography separation.
In conclusion, the method can effectively identify the Chinese softshell turtles of different strains through phospholipid analysis and determination with higher resolution and sensitivity and high quality accuracy, and can also detect and analyze the lipid in complex samples. The method also sets corresponding detection conditions of the liquid chromatography and the mass spectrometry according to the characteristics of Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidylinositol (PI), so that the high performance liquid chromatography and the mass spectrometry have good effect when used for detecting the sample.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a graph of recovery of phospholipid standards as a function of (A) extraction temperature, (B) elution solvent volume, and (C) extraction time;
FIG. 2 is a mass spectrum of Qingxi black turtle (A) and phospholipid standard (B) PC, (C) PE and (D) PI;
FIG. 3 is a HILIC-MS/MS chromatogram of (A) PC, (B) PE and (C) PI of a Neodyma thunbergii sample;
FIG. 4 is a diagram showing the principal component analysis of phospholipid molecular species of different species of Trionyx sinensis.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
in the following case, the rotation speed of the centrifuge is 8000 g.
1) and taking 0.2g of muscle tissue of the Chinese softshell turtle to be detected, uniformly grinding the muscle tissue into paste, adding 1.2mL of dichloromethane-methanol mixed extraction solution, and uniformly oscillating the mixture at the temperature of 30 ℃ for 5 minutes to obtain an extraction solution (A product).
Dichloromethane-methanol mixed extraction solution, dichloromethane: methanol 2: a volume ratio of 1 (v/v);
2) and performing ultrasonic assisted extraction on the extract (product A) at 50 deg.C under 53KHz for 30 min, adding 0.8mL ultrapure water, and rapidly freezing and centrifuging at-20 deg.C for 15 min.
3) Adding 2mL of dichloromethane into the primary water phase (upper layer) obtained by the step 2) through freezing and centrifuging for secondary extraction to respectively obtain a secondary water phase at the upper layer and a secondary organic phase layer at the lower layer;
the secondary extraction is repeated for 2 times by replacing the primary water phase obtained by the centrifugation in the step 2) with the secondary water phase,
4) combining the primary organic phase (lower layer) obtained by the step 2) freezing and centrifuging and the secondary organic phase obtained by the step 3)3 times of extraction to obtain an extraction liquid;
drying the extract at 4 + -1 deg.C under nitrogen blowing instrument with nitrogen flow (40 + -5 deg.C), diluting with 1mL acetonitrile to constant volume, and filtering with 0.22 μm organic filter membrane to obtain solution to be detected;
note: the ambient temperature is controlled in order to minimize the risk of lipid oxidation.
5) Analyzing and detecting the liquid to be detected by a liquid chromatography-mass spectrometry (a method combining liquid chromatography and mass spectrometry), and using phospholipid ions of m/z790,816,818 and 866 as potential markers for species identification of the Chinese soft-shelled turtles to obtain a detection result; the method comprises the following specific steps:
the liquid chromatographic column is YMC Triart diol liquid chromatographic column, 4.6 x 250mm,3 μm;
the mobile phase A is: firstly, preparing an acetic acid acetonitrile solution with acetic acid concentration of 53mM, and then adjusting the pH value to 4.0-4.5;
the mobile phase B is as follows: adding pure water into 60mmol ammonium acetate and 53mmol acetic acid to reach 1L, and adjusting pH to 3.0 +/-0.2;
the temperature of the chromatographic column is 40 ℃, and the flow rate is 0.2 mL/min-1The sample volume was 2 mL.
The gradient elution was: the mobile phase A is kept at 95 percent for 0 to 3 minutes; after 3-13 minutes, the mobile phase A is uniformly reduced from 95% to 70%; after 13-18 minutes, the mobile phase A is uniformly reduced to 50 percent; in the gradient elution, the balance is mobile phase B, and the above% is volume% for each time period.
Remarks explanation: washing the column with pure water and acetonitrile in the volume ratio of 1:1 for the next 18-25 min; the washed column can be reused.
Mass spectrometry mode in liquid chromatography-mass spectrometry:
using a needle pump syringe at 200. mu.L.min-1Injecting a sample (the liquid to be detected obtained in the step 4) into an atmospheric pressure ion chemical source at a sample injection amount of 2 mu L, and ionizing in a negative ion mode;
the mass spectrum conditions are as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; ion source gases, including dry gas (GS1), nebulizer gas (GS2), and curtain gas, set at 50psi, 60psi, and 25psi, respectively; the mass spectrum measuring range is m/z450-950, and the scanning speed is 1s per spectrum;
instrument control and data acquisition, processing and analysis were performed using analyst1.5.1 software.
Selecting soft-shelled turtles of the varieties known as Zhejiang black-bone turtle, Qingxi black-shelled turtle and Zhejiang new-flowered turtle as samples respectively, and detecting according to the method, wherein the obtained results are as follows:
total Ion Chromatography (TIC) and extracted ion chromatography (XIC) obtained from qingxi wu turtle are shown in fig. 2A, PC 14: 0/14: 0, PE 15: 0/15: 0 and PI 16: 0/16: the external standard of 0 separates well and baseline separation is achieved between groups. Elution of PC14 first: 0/14: 0(9.33 min), re-elution of PE 15: 0/15: 0(11.15 min) and PI 16: 0/16: 0(16.17 min). The retention capacity of the target on the HILIC column is related to a complex relationship among the pH of the mobile phase, the ionization degree of the compound, the surface of the stationary phase and the adsorbed water layer. Since different phospholipid molecular species within the same class have the same polarity head, their retention times are very close, so all phospholipid molecular species can be found by correlation mass spectrometry on phospholipid standards. Neodychium japonicum, as shown in FIG. 3, shows peaks corresponding to different phospholipid molecular species, which are caused by different fatty acyl substitutions on the glycerol backbone. A total of 22 PC molecules were detected, with m/z816 ions being most abundant. As described in table 1; the spectra of PE show diversity, with 23 PE molecular species being co-detected, such as m/z772([ PE 38: 1-H)]-)、m/z742([PE 36：2-H]-)、m/z746([O-PE 38：7-H]-) And the like. The spectrum of PI is simple, the ions are easy to clarify, and the spectrum is expressed in m/z885([ PI 38: 4-H)]-) Ion dominated, highly unsaturated [ PI 40: 6-H]-It is also abundant. A total of 10 PI molecules were identified, less than PC and PE. Then, the peak area of each phospholipid molecule species is obtained by using the single ion current response of the XIC function, the relative content of each phospholipid molecule species is normalized, and the phospholipid molecule species are identified by using the obvious characteristics of different Chinese softshell turtle species after treatment.
TABLE 1 characteristics and quantities of phospholipid molecular species in three turtles
According to the table, the following judgment rules for three kinds of Chinese soft-shelled turtles, namely Qingxi black-shelled turtle, Zhejiang black-shaped turtle No. two and Zhejiang new-flowered turtle, can be known;
when the relative range of the abundance of the phospholipid ions of m/z816 is 15% -21%, the relative range of the abundance of the phospholipid ions of m/z790 is 2% -4%, and the relative range of the abundance of the phospholipid ions of m/z818 is 10% -12%, the Chinese softshell turtle to be detected is Zhewu II;
when the relative range of the abundance of the phospholipid ions of the m/z816 is 6-10%, the Chinese softshell turtle to be detected is Qingxi soft-shelled turtle.
When the relative range of the abundance of the phospholipid ions of the m/z816 is 11% -14%, the Chinese softshell turtle to be detected is a new Hua softshell turtle.
Example 2, the ultrasonic extraction temperature parameters in step 2 of example 1 are respectively set to 40 ℃, 50 ℃ and 60 ℃; the rest is equivalent to embodiment 1. A comparison of the results obtained for recovery is shown in FIG. 1A.
From FIG. 1A, it is understood that the optimum extraction temperature is 50 ℃.
Examples 3,
The sum of the amounts of the dichloromethane-methanol mixed extract solution in step 1) and the ultrapure water in step 2) in example 1 was set to 0.5mL, 1mL, 2mL, and 3mL, respectively (dichloromethane-methanol mixed extract solution: ultrapure water in a volume ratio of 1.5: 1); the rest is equivalent to embodiment 1. A comparison of the results obtained for recovery is shown in FIG. 1B.
From FIG. 1B, it is found that the optimum volume of the extraction solvent is 2 mL.
Example 4, the ultrasonic extraction time parameters in step 2) of example 1 were set to 10, 20, 30, 40 and 50 minutes, respectively; the rest is equivalent to embodiment 1. A comparison of the results obtained for recovery is shown in FIG. 1C.
From FIG. 1C, it is understood that the optimum extraction time is 30 minutes.
Experiment 1: the 3 phospholipid standards of PC, PE and PI were set to 5 concentration levels (0.1, 1, 10, 100, 200. mu.g.mL)-1) On the basis, an intercept linear regression and a 1/x weighting factor linear regression are adopted to establish a calibration curve of the phospholipid standard. As shown in Table 2, the LOQ value is about 200. mu.g/mL-1The linear relationship is good within the range, the correlation coefficient R2 is between 0.9978 and 0.9985, and the peak area (Y) and the concentration (x, mu g & mL) of the tested compound-1) With a good linear relationship.
LOD and LOQ can accurately identify and determine the minimum concentration of an analyte. These two parameters were used to evaluate the sensitivity of the proposed HILIC-based lipidomics approach. The LOD and LOQ values are calculated by using signal-to-noise ratios (ratio of noise to peak intensity) of 3 and 10, respectively. As shown in Table 2, the LOD and LOQ values were less than 0.48. mu.g.mL-1And 1.36. mu.g.mL-1The method has higher sensitivity under optimized chromatographic and spectral parameters.
TABLE 2 calibration curves, correlation coefficients, limits of detection (LOD) and limits of quantitation (LOQ) for the three phospholipid standards
The precision of the method was evaluated in terms of the corresponding day precision and day precision. At 50. mu.g/mL-1The phospholipid standard of (2) was added to the trionyx sinensis sample. The fortified sample was then extracted using the method described previously. For in-day precision, six replicates of the sample solution were performed in three consecutive tests; to pairResults were obtained at day precision by 6 day calculations. As shown in table 3, the variation within and between days was less than 5.63% and 7.29%, respectively, indicating that the method had better precision.
Adding 50 μ g/mL of the extract before extraction-1The recovery experiment is carried out on the trionyx sinensis sample with the phospholipid standard, the recovery rate is compared with that of a sample without actual sample extract with the same concentration, and the result shows that the content of phospholipid in the sample is 50 mu mol/mL-1The recovery of the sample was 100%. PC 14: 0/14: 0, PE 15: 0/15: 0, PI 16: 0/16: the relative recovery of 0 is between 86.4% and 93.6%, and the relative standard deviation is less than 5.89%, indicating that the method has good recovery.
TABLE 3 precision and recovery obtained by HILIC-MS/MS method for three different phospholipid standards
Experiment 2: the detection method established by the invention is adopted to detect 18 Chinese soft-shelled turtle samples (6 parallel samples of each Chinese soft-shelled turtle), the three types of Chinese soft-shelled turtle samples are Zhejiang new Hua turtle (ZXH), Qingxi black soft-shelled turtle (QXW) and Zhejiang black second number (ZWE), and the results are subjected to mathematical statistics analysis. The top panel of fig. 4 shows the first two principal components of a trionyx sinensis sample (D1 and D2), accounting for the 50.8 cumulative percent (cum%) value and the data lumped variance of 49.2 cum%, respectively. These trionyx sinensis samples were well clustered into three statistically significant groups (ST-ZXH, ST-QXW and ST-ZWE) and distributed at different positions in the graph. To reveal the most influential phospholipid molecular species that influence the separation between these three clusters, coefficients were calculated to determine the weights of the original variables by constructing loading maps. The lower graph of fig. 4 shows the loading of the variables in D1 and D2. Most of the identified phospholipid molecular species cluster near the central zero line, while other ions that have a significant effect on the difference are far from the blue circle. For example, in D1 with a load value of 0.71, m/z816 is the dominant contributor, while in D2 with load values of 0.46, 0.42, and 0.51, respectively, m/z790, 818, and 866 are the dominant eigenvalues. These ions can be used as potential markers for identifying the strain of the Chinese softshell turtle.
Six samples are respectively arranged for each kind of soft-shelled turtle, eighteen samples are detected, the figure 4 is obtained according to the obtained data of each sample, and the data of the samples of the same kind can be well gathered together, and three circles (representing three kinds of soft-shelled turtles) are not overlapped, so that the method can well distinguish different kinds of soft-shelled turtles.
A well-established HILIC-based lipidomics approach was applied to six randomly collected blind samples. The proposed phospholipid ions of the potential markers m/z790,816,818 and 866 were used for the differentiation of turtle strains. The 4 samples were tentatively identified as ZWEs because they had relatively high m/z816 abundance and low levels of m/z790 and 818. The other two blind samples were classified as QXW due to their low levels of m/z816 and 866. For validation and confirmation, the results were imported into the PCA plot, indicating that four ZWE samples and two QXW samples were well-homopolymerized. The lipidomics method based on the combination of high performance liquid chromatography and mass spectrometry has great advantages in the rapid identification of three types of Chinese softshell turtles, and can provide relevant basis and reference for effectively identifying different types of Chinese softshell turtles and identifying whether the Chinese softshell turtles are adulterated, thereby ensuring the food safety of consumers.
Verification experiment, the detection of the fresh brook black turtle, Zhe black second and Zhe new flowered turtle, Huanghe turtle and Jiangxi turtle according to the method of example 1 (each kind of Chinese turtle is provided with 3 groups), the results are respectively shown in Table 4:
TABLE 4
In comparative experiment 1, the mobile phase A in example 1 is changed from "preparing an acetic acid acetonitrile solution with acetic acid concentration of 53 +/-5 mM, and then adjusting the pH to 4.0-4.5" to "acetonitrile", and the rest is the same as example 1. The test was carried out using a turtle identical to that used in the validation experiment, according to the method described in this comparative experiment 1. The results obtained are compared with the validation experiments as described in table 5 below.
TABLE 5 phospholipid ion abundance (%)
TABLE 6
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. The analysis and detection method for effectively identifying different types of Chinese softshell turtles is characterized by comprising the following steps:
1) taking 0.2g of muscle tissue of the Chinese softshell turtle to be detected, grinding uniformly, adding 1.2 +/-0.1 mL of dichloromethane-methanol mixed extraction solution, and oscillating uniformly at 30 +/-5 ℃ to obtain an extraction solution;
in the dichloromethane-methanol mixed extraction solution, the ratio of dichloromethane: 1.9-2.1% of methanol: 1 in a volume ratio;
2) after the ultrasonic-assisted extraction is carried out on the extracting solution, 0.8 plus or minus 0.1mL of ultrapure water is added, and then the mixture is refrigerated and centrifuged;
3) adding 2 +/-0.2 mL of dichloromethane into the primary water phase obtained by the step 2) through freezing and centrifuging for secondary extraction to obtain a secondary water phase at the upper layer and a secondary organic phase layer at the lower layer;
4) combining the primary organic phase obtained by the step 2) freezing and centrifuging and the secondary organic phase obtained by the step 3) secondary extraction to obtain an extraction liquid;
drying the extract under a nitrogen blowing instrument, fixing the volume with 1mL of acetonitrile, and filtering the extract through a 0.22-micron organic filter membrane to be used as a solution to be detected;
5) analyzing and detecting the liquid to be detected by a liquid chromatography-mass spectrometry method, and using phospholipid ions of m/z790,816,818 and 866 as potential markers for species identification of the Chinese softshell turtles;
the liquid chromatogram is: the liquid chromatographic column is YMC Triart diol liquid chromatographic column, 4.6 x 250mm,3 μm; the temperature of the chromatographic column is 40 ℃, and the flow rate is 0.2 mL/min-1The sampling amount is 2 mL;
the mobile phase A is: firstly, preparing an acetic acid acetonitrile solution with acetic acid concentration of 53 +/-5 mM, and then adjusting the pH value to 4.0-4.5;
the mobile phase B is as follows: adding pure water into 60 +/-5 mmol ammonium acetate and 53 +/-5 mmol acetic acid to reach a constant volume of 1L, and adjusting the pH value to 3.0 +/-0.2;
the gradient elution was: the mobile phase A is kept at 95 percent for 0 to 3 minutes; after 3-13 minutes, the mobile phase A is uniformly reduced from 95% to 70%; after 13-18 minutes, the mobile phase A is uniformly reduced to 50 percent; in the gradient elution, the balance is mobile phase B in each time period, and the percentage is volume percent;
the mass spectrum conditions are as follows: setting the ion spray voltage at 4500V in the negative ion mode, the ion source temperature was maintained at 500 ℃; ion source gases, including dry gas (GS1), nebulizer gas (GS2), and curtain gas, set at 50psi, 60psi, and 25psi, respectively; the mass spectrum measuring range is m/z450-950, and the scanning speed is 1s per spectrum;
instrument control and data acquisition, processing and analysis are carried out by using Analyst1.5.1 software;
when the relative range of the abundance of the phospholipid ions of m/z816 is 15% -21%, the relative range of the abundance of the phospholipid ions of m/z790 is 2% -4%, and the relative range of the abundance of the phospholipid ions of m/z818 is 10% -12%, the Chinese softshell turtle to be detected is Zhewu II;
when the relative range of the abundance of the phospholipid ions of the m/z816 is 6-10%, the Chinese softshell turtle to be detected is Qingxi soft-shelled turtle;
when the relative range of the abundance of the phospholipid ions of the m/z816 is 11% -14%, the Chinese softshell turtle to be detected is a new Hua softshell turtle.
2. The assay and detection method for effectively identifying different species of trionyx sinensis according to claim 1, wherein in the step 5),
mass spectrometry mode: using a needle pump syringe at 200. mu.L.min-1Injecting the solution to be detected obtained in the step 4) into an atmospheric pressure ion chemical source at the flow rate of 2 mu L, and ionizing in a negative ion mode.
3. The assay and detection method for effectively identifying different species of trionyx sinensis according to claim 1 or 2, wherein in the step 4): blowing the extract liquid with nitrogen flow at the ambient temperature of 4 +/-1 ℃, then fixing the volume with 1mL of acetonitrile, and passing through a 0.22 mu m organic filter membrane to be used as a liquid to be detected.
4. The assay and detection method for effectively identifying different species of trionyx sinensis according to claim 1 or 2, wherein:
in the step 2): extracting the extractive solution with ultrasonic wave at 50 deg.C and 53KHz for 30 + -5 min, adding 0.8 + -0.1 mL ultrapure water, and centrifuging at-20 + -2 deg.C for 15 + -2 min.
5. The assay detection method for effectively identifying different species of trionyx sinensis according to claim 1 or 2, wherein in the step 3): adding 2 +/-0.2 mL of dichloromethane into the primary water phase obtained by centrifuging in the step 2) for secondary extraction to obtain a secondary water phase at the upper layer and a secondary organic phase layer at the lower layer; replacing the primary water phase obtained by centrifuging in the step 2) with the secondary water phase, and repeating the secondary extraction for 1-2 times;
and 2) combining the primary organic phase obtained by centrifugation in the step 2) and the secondary organic phase obtained by all the extractions in the step 3) to obtain an extraction liquid.
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