CN113447581A - Method for detecting microcystin in algae food and raw materials for producing algae food - Google Patents
Method for detecting microcystin in algae food and raw materials for producing algae food Download PDFInfo
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- CN113447581A CN113447581A CN202110696858.6A CN202110696858A CN113447581A CN 113447581 A CN113447581 A CN 113447581A CN 202110696858 A CN202110696858 A CN 202110696858A CN 113447581 A CN113447581 A CN 113447581A
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- 239000012498 ultrapure water Substances 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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Abstract
The invention provides a method for detecting microcystin in algae food and raw materials for producing the same. Mixing a sample to be detected with an extraction solvent, extracting by vortex oscillation, centrifuging and collecting a sample extracting solution; purifying the sample extracting solution by using a purifying tube, and filtering to obtain a sample detection solution to be detected; wherein the purifying tube is filled with magnesium sulfate, N-propyl ethylenediamine adsorbent and C18And neutral alumina; and detecting the detection liquid of the sample to be detected by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer to realize the qualitative and quantitative analysis of 8 kinds of microcystins. The method has the characteristics of short analysis time, stable and reliable qualitative and quantitative detection results and the like.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and relates to a method for detecting microcystins in algae food and production raw materials thereof.
Background
The algae food and its raw materials contain abundant proteins, amino acids, polysaccharides, dietary fibers, unsaturated fatty acids, mineral elements and various physiologically active substances. In recent years, algal foods typified by spirulina have been attracting attention and favored by consumers as novel foods having health-care functions. At present, the raw materials of the algae health food approved by China comprise spirulina, chlorella, dunaliella salina, haematococcus pluvialis and the like. Spirulina including Spirulina platensis (Arthrospira platensis) and Spirulina maxima (Arthrospira maxima) is one of the most important raw materials for algal food. In addition, Chlorella pyrenoidosa (Chlorella pyrenoidosa) was approved as a new resource food in 2012, becoming an emerging popular food ingredient.
Other symbiotic algae such as microcystis, anabaena and the like may exist in the water area for cultivating the spirulina and can generate microcystin harmful to human bodies. The microcystins have obvious hepatotoxicity, embryonic development and reproductive toxicity, and can cause embryo malformation, developmental retardation and even death; in addition, the medicine has certain negative effects on the nervous system and the immune system, including: induce apoptosis, oxidative stress, cause alteration of mitochondrial function, and the like. The occurrence of water bloom due to the eutrophication of the culture fresh water can cause the pollution of the spirulina by the microcystin. Because of the good chemical stability of microcystins, if the quality control is not strict during the processing and production process of spirulina, the microcystins are likely to be transferred to the final product. The consumption of spirulina food contaminated with microcystins by consumers can result in potential harm to the body. As early as 2002, relevant researchers of Chinese disease prevention and control center investigate the pollution condition of microcystin in raw materials and products for producing spirulina health food and find that: the spirulina production process and the products sold in the market have microcystin pollution to different degrees. The research considers that the intake of microcystins by taking spirulina possibly affects the health of human beings, and proposes to set a limit standard of microcystins in algae health food which accords with the characteristics of China. Until now, the limit standard of the microcystins of related products is not provided in China. In the aspect of detection standards, the national standard GB/T16919-. GB 5009.273-2016 determination of microcystins in aquatic products of national food safety Standard is suitable for determination of microcystins in aquatic products such as fish, shrimp, freshwater mussel, etc., algae food and raw materials thereof are not included in the application range, and the standard only relates to detection of microcystins-LR, -RR and-YR, and other types of microcystins which can cause pollution cannot be covered. In the technical requirements of the health food raw material catalog spirulina filed products published by the general administration of market supervision (No. 4 in 2021) about the dosage form and the technical requirements of five health food raw material filed products such as coenzyme Q10 and the like (the technical requirements of the health food raw material catalog spirulina filed products), two indexes of 'beta-carotene' and 'phycocyanin' are added as mark components besides physical and chemical indexes and microbial indexes, and the microcystins are not included in the detection range and the limit of the microcystins is not specified.
At present, methods for determining microcystins include High Performance Liquid Chromatography (HPLC), Capillary Electrophoresis (CE), liquid chromatography-mass spectrometry (LC-MS) and the like, and mainly focus on the environmental industrial fields of water quality monitoring and the like and the detection of microcystins in drinking water and aquatic products. The research on the pollution condition of the microcystins in the algae food and raw materials thereof is less, the coverage of toxin types is narrower, most methods adopt the traditional solid phase extraction column to purify the sample, and the sample preparation efficiency is lower. In order to effectively evaluate the safety risk in the algae food and the raw materials thereof and accurately identify the types and the pollution degree of the microcystin pollutants, a method for measuring the content of the microcystin in the algae food and the raw materials thereof needs to be established.
Disclosure of Invention
Based on the defects in the prior art, the invention aims to provide a method for detecting microcystins in algae food and production raw materials thereof, the detection method realizes the detection of 8 microcystins in algae food such as algae powder, algae tablets, algae granules, algae capsules and the like and the production raw materials thereof, and has the characteristics of short analysis time, stable and reliable qualitative and quantitative detection results and the like.
The purpose of the invention is realized by the following technical means:
the invention provides a method for detecting microcystin in algae food and raw materials for producing the same, which comprises the following steps:
mixing the sample to be detected (such as algae food such as algae powder, algae tablet, algae granule, algae capsule, etc. and raw materials for producing the same) with extraction solvent, extracting by vortex oscillation, centrifuging, and collecting sample extractive solution;
purifying the sample extracting solution by using a purifying tube, and filtering to obtain a sample detection solution to be detected; wherein the purification pipe is filled with magnesium sulfate, Primary Secondary Amine (PSA) adsorbent, and C18And neutral alumina;
the invention adopts ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) to qualitatively and quantitatively detect the content of microcystins in algae food and raw materials thereof.
In the detection method of the present invention, magnesium sulfate, N-Propylethylenediamine (PSA) adsorbent, and C are packed18And a neutral alumina (Al-N) purification tube, wherein C18The adsorbent can be used for removing fat and high-content chlorophyll and PSA adsorbent, and can be used for removing sugar and organic acid, and Al-N can be used for removing fat, thereby avoiding instrument pollution, improving detection sensitivity, and ensuring that the recovery rate of 8 microcystins can meet the detection requirement. The purified extracting solution can be filtered and then can be put into a machine for detection, thereby omitting the steps of solid phase extraction column activation, sample loading, elution and the like required by the traditional sample purification and also reducing a vacuum device required by solid phase extraction. After the purification tube is adopted for treatment, 8 kinds of microcystins can obtain better mass spectrum response. The pretreatment process is simple and quick, the complex operation steps in the traditional purification mode are avoided, and the blank of the detection technology of the microcystins in the algae food and raw materials thereof is made up.
In the above method, preferably, the microcystin includes one or more of microcystin-lr (mclr), microcystin-rr (mcrr), microcystin-yr (mcyr), microcystin-lw (mclw), microcystin-lf (mclf), microcystin-la (mcla), microcystin-ly (mcly), and microcystin-wr (mcwr); the method of the invention can realize the quantitative and qualitative detection of the 8 kinds of microcystins simultaneously.
In the above method, preferably, the extraction solvent comprises 70% aqueous methanol.
In the above process, when methanol: when the volume percentage of water is 70:30, the overall extraction efficiency of 8 microcystins is better.
In the above method, preferably, the ratio of the sample to be detected to the extraction solvent is 1 g: (5-20) mL.
In the above method, preferably, the ratio of the sample to be detected to the extraction solvent is 1 g: 10 mL.
In the method, the vortex oscillation time is preferably 30-60 s; preferably, the time of vortex oscillation is 60 s.
In the method, the temperature for centrifuging after vortex oscillation is preferably 0-8 ℃, the rotating speed is 5000-10000 rpm, and the centrifuging time is preferably 5-10 min.
In the above method, preferably, the temperature for centrifugation after vortex oscillation is 4 ℃, the rotation speed is 10000rpm, and the centrifugation time is 5 min.
In the above process, preferably, the magnesium sulfate, the N-Propylethylenediamine (PSA) adsorbent, and the C are mixed18And the mass ratio of the neutral alumina is (6-27): (2-9): (1-3): (1-3).
In the above process, preferably, the magnesium sulfate, the N-Propylethylenediamine (PSA) adsorbent, and the C are mixed18And the mass ratio of the neutral alumina is 15: 5: 3: 3.
in the above method, preferably, the filtration after purification is performed using a microporous organic filtration membrane.
In the above method, preferably, the microporous organic filter membrane is a 0.22 μm polytetrafluoroethylene filter membrane.
In the above method, preferably, the chromatographic conditions of the hplc-triple quadrupole mass spectrometer include:
a chromatographic column: SHIMADZU
AQ-C18 chromatographic column with length of 100mm, inner diameter of 2.1mm and particle diameter of 1.9 μm;
mobile phase: 0.01% formic acid in water and acetonitrile; gradient elution is adopted; the column temperature is 35-45 ℃, preferably 45 ℃, and the flow rate is 0.2-0.4 mL/min, preferably 0.3 mL/min; the sample injection amount is 2-10 mu L, preferably 5 mu L; the elution procedure is as follows:
| time/min | 0.01% formic acid in water/%) | Acetonitrile/%) |
| 0 | 75 | 25 |
| 3 | 50 | 50 |
| 4 | 35 | 65 |
| 6 | 10 | 90 |
| 8 | 10 | 90 |
| 8.1 | 75 | 25 |
| 10 | 75 | 25 |
In the above method, when "water (0.01% formic acid) + acetonitrile" is used as the mobile phase, the mass spectrum response of 8 microcystins is better.
In the above method, preferably, the mass spectrometer conditions of the hplc-triple quadrupole mass spectrometer include:
the ionization mode is an electrospray ion source positive ion mode, a multi-reaction monitoring mode signal is acquired, and the flow rate of atomized gas is 2.5-3.5L/min, preferably 3L/min; the flow rate of the drying gas is 8-12L/min, preferably 10L/min; the flow rate of the heating gas is 8-12L/min, preferably 10L/min; the interface temperature is 250-350 ℃, and preferably 300 ℃; the interface voltage is 3.0-5.0 kV, preferably 4.0 kV.
In the above method, preferably, the qualitative analysis of microcystins by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry comprises:
acquiring an ion pair comprising a precursor ion and a product ion by adopting a positive ion scanning mode;
selecting two or more groups of ion pairs, wherein when the retention time of chromatographic peaks of the selected ion pairs in the sample is consistent with that of corresponding standard chromatographic peaks; and meanwhile, when the relative abundance ratio of the product ions in the sample is compared with the relative abundance ratio of the product ions in the standard solution with the same concentration to meet the requirement of allowable deviation, the qualitative requirement is met. The maximum allowable deviation from ion abundance when confirmed by qualitative analysis is as follows:
| relative ion abundance/%) | k＞50 | 50≥k＞20 | 20≥k＞10 | k≤10 |
| Allowable relative deviation/%) | ±20 | ±25 | ±30 | ±50 |
In the above method, preferably, the quantitative analysis of microcystins by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry comprises:
analyzing the microcystin standard series solution by adopting ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and injecting a sample by using a matrix matching standard solution; obtaining the corresponding relation between the quantitative ion peak area and the solution concentration, and drawing a matrix matching standard curve;
and (3) feeding a sample detection solution to be detected, detecting the peak area of the quantitative ion, and obtaining the content of the microcystin in the sample detection solution to be detected according to the matrix matching standard curve.
The invention has the beneficial effects that:
(1) the detection method provided by the invention has the advantages of simple and rapid pretreatment of the algae food and the raw materials thereof, few operation steps, high working efficiency, reduced workload, relatively low requirement on the operation technical level of inspectors and the like, and can be used for risk assessment and screening monitoring of microcystins in the algae food and the raw materials thereof.
(2) The method adopts the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to detect 8 microcystins in algae food and raw materials thereof, has the advantages of few operation steps, short analysis time, high sensitivity and stable and reliable qualitative and quantitative detection results, can be used for risk assessment and screening monitoring of the microcystins in the samples, and has wide application prospect. Fills the blank of the method standard of the project, and provides scientific basis and technical support for the quality safety supervision of the algae food. The detection method has important significance for identifying the type of the microcystins in the algae food and raw materials thereof and accurately evaluating the intake risk caused by pollution.
Drawings
FIG. 1 is a total ion flow diagram of MCLR, MCRR, MCYR, MCLW, MCLF, MCLA, MCLY and MCWR in example 1 of the present invention.
FIG. 2 shows the relative response intensity of 8 microcystins according to the present invention measured in comparative example 1 with different mobile phase types and different formic acid contents (in the figure, A: water (0.01% formic acid) + acetonitrile; B: water (0.025% formic acid) + acetonitrile; C: water (0.05% formic acid) + acetonitrile; D: water (0.1% formic acid) + acetonitrile; E: water (0.01% formic acid) + methanol; F: water (0.025% formic acid) + acetonitrile (0.025% formic acid); G: water (0.01% formic acid) + acetonitrile (0.01% formic acid)).
Fig. 3 shows the relative extraction efficiencies of 8 microcystins measured with different extraction solvents in comparative example 2 of the present invention (in the figure, 60% methanol: water 60:40 (volume%), 70% methanol: water 70:30 (volume%), 80% methanol: water 80:20 (volume%), and 90% methanol: water 90:10 (volume%).
FIG. 4 shows the relative response intensity of 8 microcystins measured in comparative example 3 by different extraction methods and different purification methods (in the figure, SS: SHIMESN QuEChERS dSPE column; WS: inventive purification tube; CM: LUMTECH MPFC-QuEChERS (Complex matrix) ultrafiltration type purification column; HL: LUMTECH M)PFC-QuEChERS (high lipid) ultrafiltration type purification column; WP Waters
PRIME HLB Extraction Cartridges; VT: vortex extraction is adopted, and the extraction time is 60 s; 10: ultrasonic extraction is adopted, and the extraction time is 10 min; 20: ultrasonic extraction is adopted, and the extraction time is 20 min; 30: ultrasonic extraction is adopted, and the extraction time is 30 min).
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. The skilled person can use the content of the present invention to properly modify the sample preparation method, select the equivalent chromatographic column, and properly adjust the parameters of the apparatus. It is expressly intended that all such modifications, equivalents and adaptations which are apparent to those skilled in the art are intended to be included within the present invention. The raw materials tested in the following examples were all commercially available and commonly used unless otherwise specified.
The reagents and instruments used in the method for detecting the components of 8 microcystins in the algae food and the raw materials thereof can be purchased from the market. The CAS numbers, names, molecular formulas, molecular weights and molecular structural formulas of MCLR, MCRR, MCYR, MCLW, MCLF, MCLA, MCLY and MCWR are shown in Table 1, and the CAS numbers, names, molecular formulas, molecular weights and molecular structural formulas of 8 microcystins are shown in Table 1.
Table 1:
the instrument used was as follows: LCMS-8060 model ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometer (Shimadzu corporation, Japan)) (ii) a Analytical balance model AL 204 (Mettler Toledo, switzerland); vortex Genie 2 Vortex mixer (Scientific Industries, USA); GTR21-1B medical centrifuge (Beijing New times Beili medical instruments Co., Ltd.);
pro VF ultrapure water purification system (Sartorius, Germany).
The invention is further illustrated by the following examples:
example 1:
the embodiment provides a method for detecting microcystins in algae food and production raw materials thereof, which adopts an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer and comprises the following specific processes:
(1) sample extraction:
accurately weighing 0.5g (accurate to 0.001g) of sample (algae powder of certain algae food), placing in a 15mL centrifuge tube, adding 70% methanol-containing aqueous solution to 5mL, and shaking at high speed for 60s in a vortex mixer to mix the sample and the extraction solvent thoroughly.
(2) Sample preparation:
centrifuging the sample extractive solution at 4 deg.C and 10000rpm for 5min, purifying the centrifuged extractive solution with a purifying tube, filtering with 0.22 μm polytetrafluoroethylene microporous membrane, and collecting the filtrate as the sample detection solution.
The purification tube of this example was packed with 150mg of magnesium sulfate, 50mg of N-Propylethylenediamine (PSA) adsorbent, and 30mg of C18And 30mg of neutral alumina (Al-N).
(3) Sample detection:
the purified sample was subjected to ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry using LCMS-8060 ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer (shimadzu corporation, japan).
The chromatographic conditions were as follows:
a chromatographic column: SHIMADZU
AQ-C18 chromatographic column with length of 100mm, inner diameter of 2.1mm and particle size of 1.9 μm; mobile phase: 0.01% formic acid in water and acetonitrile; gradient elution is adopted; the column temperature was 45 ℃; the flow rate is 0.3 mL/min; the amount of sample was 5. mu.L. The mobile phase and gradient elution procedure are shown in table 2, and table 2 shows the mobile phase composition and gradient elution procedure when the ultra performance liquid chromatography-triple quadrupole mass spectrometer is adopted.
Table 2:
the mass spectrometry conditions were as follows:
an ion source: electrospray ion source positive ion mode (ESI)+) (ii) a Detection mode: in the Multiple Reaction Monitoring (MRM) mode, the atomization gas flow rate is 3L/min, the heating gas flow rate is 10L/min, the interface temperature is 300 ℃, the Desolvation Line (DL) temperature is 250 ℃, the drying gas flow rate is 10L/min, and the interface voltage is 4.0 kV.
The liquid mass analysis of 8 kinds of microcystins adopts single charge [ M + H ] in positive ion scanning mode]+Or double charge [ M +2H]2+Mode(s). When the single charge mode is adopted, the responses of other 7 kinds of microcystins except microcystin-RR (MCRR) are better, so that the single charge mode is adopted for the other 7 kinds of microcystins except MCRR, and the double charge mode is adopted for MCRR. Performing first-order mass spectrum full scan on the target compound to obtain single charge [ M + H ] of the target compound]+Precursor ions or double charges [ M +2H]2+Precursor ions are subjected to secondary scanning, 2 sub-ions with strongest response are selected by optimizing voltage, and collision energy when the sub-ions respond to the ions in an optimal mode is optimized in a multi-reaction monitoring mode (MRM), so that optimal mass spectrum conditions are obtained. The total ion flow diagram of 8 kinds of microcystins is shown in figure 1; the mass spectrum parameters, parent ions and ion ions of 8 kinds of microcystins are shown in Table 3, and Table 3 shows 8 kinds of microcystins-LR (M)CLR, microcystin-RR (MCRR), microcystin-YR (MCYR), microcystin-LW (MCLW), microcystin-LF (MCLF), microcystin-LA (MCLA), microcystin-LY (MCLY) and microcystin-WR (MCWR).
Table 3:
note: quantification of ions.
When mass spectrometry is adopted, the co-extract in the matrix can inhibit or enhance the response value of the microcystin instrument to be detected, so that the content measurement is higher or lower, and the accuracy and reliability of the result are influenced. The invention uses standard curve measurement to examine the substrate effect by matching the substrate with the slope (k) of the standard curveM) Slope (k) of the standard curve with solventS) And comparing to quantitatively evaluate the influence of the matrix of the sample to be tested on the components to be tested. When (k)M/kS-1) x 100% is a negative value, indicating that the matrix has an inhibitory effect on the component to be tested; when (k)M/kS-1) x 100% is a positive value, indicating that the matrix has an enhancing effect on the component to be measured; when (k)M/kS-1). times.100% is zero, this indicates that no matrix effect is present. In the present invention, it is considered that when (k)M/kSWhen the-1) x 100% is less than or equal to 20%, the influence of the matrix effect on the component to be detected is small, and the interference on the mass spectrum response can be ignored; when (k)M/kS-1)×100％|>At 20%, the matrix effect has a large influence on the component to be detected, and the interference on mass spectrum response is obvious, and the inhibition effect or the enhancement effect generated by the matrix effect is considered at the moment. The matrix effect of 8 microcystins in mass spectrometry was evaluated, and the experimental results are shown in table 4, where table 4 shows the effect of matrix effect on mass spectrometry of 8 microcystins.
Table 4:
| microcystin species | kM | kS | (kM/kS-1)×100％ |
| MCLF | 4090 | 12543 | -67 |
| MCLW | 4584 | 10637 | -57 |
| MCLY | 10522 | 11653 | -10 |
| MCLA | 17521 | 15949 | 10 |
| MCWR | 4303 | 2813 | 53 |
| MCLR | 16954 | 9222 | 84 |
| MCYR | 14900 | 6861 | 117 |
| MCRR | 47254 | 53537 | -12 |
From table 4, it can be seen that: the sample matrix has small influence on MCLY, MCRR and MCLA, and the interference effect is negligible; the method has strong inhibition effect on the determination of MCLF and MCLW; has strong enhancement effect on the measurement of MCWR, MCLR and MCYR. Based on the experimental results, the aim of synchronously measuring 8 kinds of microcystins is comprehensively considered, and the matrix matching standard curve method is adopted for quantitative analysis so as to eliminate or reduce matrix effect influence.
Example 2:
by adopting the extraction method and the detection method of the sample in the embodiment 1, considering that the detection sensitivities of different kinds of microcystins are different, the influence difference possibly generated by the matrix effect is larger when the liquid quality is detected, and the potential influence is possibly generated on the measurement result of the component to be measured in the sample, so that the recovery rate is measured by setting 3 sample adding concentrations to examine the analysis and measurement effect of the component to be measured in the sample under different microcystin content conditions. The specific process is as follows:
18 parts of blank matrix sample are weighed, each part is 0.5g (accurate to 0.0001g) and placed in a 15mL centrifuge tube, and 8 microcystin standard solutions with low, medium and high concentrations are respectively added, each concentration is 6 parts in parallel (n is 6).
And adding an extraction solvent methanol: water (70:30, V: V) to 5mL was shaken at high speed on a vortex mixer for 60s to mix the sample thoroughly with the extraction solvent. Centrifuging the sample extract at 4 deg.C, 10000rpm for 5min, purifying the centrifuged extract with the purification tube of the invention, filtering with 0.22 μm polytetrafluoroethylene microporous membrane to obtain the liquid to be tested, and performing ultra performance liquid chromatography-triple quadrupole mass spectrometry under the same conditions. Taking the blank matrix extracting solution as a diluent, preparing a matrix matching standard solution of the microcystin to be detected, and drawing a matrix matching standard curve by taking the peak area of the quantitative ions of the target compound as a vertical coordinate and the concentration of the matrix matching standard solution as a horizontal coordinate after ultra performance liquid chromatography-triple quadrupole mass spectrometry. And (3) injecting a sample to be detected into the solution, determining the qualitative property according to the retention time, measuring the peak area of the quantitative ions, and obtaining the content of the microcystins in the solution to be detected according to a standard curve. The recovery rates and standard deviations were calculated and the recovery rates under 3 different spiking concentrations are shown in table 5.
Table 5:
table 5 the results of the experiments show that: the extraction method and the detection method adopted by the invention can realize better analysis and detection on samples with different microcystin contents, and the recovery rates of 3 added standard concentrations meet the experimental requirements and can be used as a preferred method.
Comparative example 1:
because the microcystins are a group of cyclic heptapeptides, the general structure is: cyclo (-D-Ala-L-X-D-Masp-L-Z-Adda-D-Glu-Mdha), wherein: l is levorotatory; d is dextrorotation. Masp (sometimes written as MeAsp) is D-erythro-beta-methylaspartic acid; adda is (2s,3s,8s,9s) -3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyl-4, 6-dienoic acid; mdha is N-methyl dehydroalanine. X and Z are two variable L-amino acids, and due to the difference in XZ and the differences in methylation or demethylation of Masp and Adda, a variety of different microcystin isomers can be formed.
When the mobile phase composition is optimized, the sensitivity of polypeptide formed by different amino acid combinations to pH value change and the protonation degree in the mass spectrometry process are fully considered, 2 different types of organic phases of acetonitrile and methanol are respectively selected and combined with water, formic acid with different contents is added to adjust the pH value of the mobile phase, so that the influence of the pH value change of the mobile phase on the mass spectrum response intensity of 8 types of microcystins is considered, the respective response values of the same type of microcystins under different types of mobile phase conditions are taken as molecules, the average value of the response values of the same type of microcystins under different types of mobile phase conditions is taken as denominator, and the respective relative response values of the 8 types of microcystins under each type of mobile phase conditions are respectively calculated according to the ratio of the two types of the microcystins.
The mass spectrum response of 8 microcystins under the following 7 mobile phase compositions is examined, the mobile phase compositions are shown in Table 6, and the relative response intensity of 8 microcystins under different mobile phase types and different formic acid contents is shown in FIG. 2.
Table 6:
figure 2 results show that: the mass spectrum response of 8 microcystins was overall better when water (0.01% formic acid) + acetonitrile was used as the mobile phase, so water (0.01% formic acid) + acetonitrile was chosen as the mobile phase.
Comparative example 2:
by adopting the method for detecting 8 kinds of microcystins in example 1, considering the complexity of the algae food and the raw material matrix thereof, when different extraction solvents are used for extracting the component to be detected in the sample, the component to be detected is not fully extracted due to the difference of the types and polarities of the extraction solvents, thereby affecting the analysis accuracy. Therefore, this comparative example selects 4 different composition extraction solvents to extract 8 microcystins in order to investigate the effect of methanol: the extraction efficiency of 8 microcystins is respectively 60:40, 70:30, 80:20 and 90:10 in the volume percentage of water.
Taking the respective response values of the same microcystin under different types of extraction solvents as the numerator, taking the average value of the response values of the same microcystin under different types of extraction solvents as the denominator, respectively calculating the respective relative response values of 8 types of microcystin under each type of extraction solvent according to the proportion of the two, and measuring the extraction efficiency according to the calculation result. The experimental results are shown in FIG. 3, and FIG. 3 shows the relative extraction efficiencies of 8 kinds of microcystins under different extraction solvents.
The results of FIG. 3 are compared to find that: when the ratio of methanol: when the volume percentage of water is 70:30, the overall extraction efficiency of 8 microcystins is better. When other extraction solvents are adopted, higher extraction efficiency can be obtained only for part of kinds of microcystins, and the extraction efficiency is too low for other kinds of microcystins, so that the accuracy of qualitative and quantitative analysis is difficult to ensure.
Comparative example 3:
because the accuracy of the measurement result is directly influenced by the selection of the sample pretreatment method, the sample is pretreated by adopting different purification means, so that the purification effect of the combination of different types of purification means is studied by combining with an extraction mode, and a suitable sample pretreatment method is selected.
By adopting the 8 microcystin detection methods and extraction solvents in example 1, considering that the matrix of the algae food and raw materials thereof is complex, and the extraction and purification effects have direct influence on the matrix effect of the component to be detected in the mass spectrometry, different extraction methods and purification methods are used for extracting and purifying the sample so as to reduce the influence of matrix interference on the mass spectrometry of the component to be detected.
Blank matrix samples were weighed out into 15mL centrifuge tubes at 0.5g each (to the nearest 0.0001g), and 8 microcystin standard solutions of the same concentration were added, and methanol: extracting solvent with water (70:30, V: V) to 5mL, respectively extracting with 3 different ultrasonic time extraction modes such as 10min, 20min and 30min and vortex extraction for 60s, centrifuging at 4 deg.C, 10000rpm and 5min, combining each extraction mode with 5 purification devices in a crossing way, purifying with the purification devices to obtain sample extractive solution, filtering with 0.22 μm polytetrafluoroethylene microporous membrane to obtain solution to be tested, and analyzing with ultra high performance liquid chromatography-triple quadrupole mass spectrometer to determine mass spectrum response of 8 microcystins under different extraction modes and purification methods.
5 the purification device comprises: SHIMESN QuEChERS dSPE column, purification tube of the invention, LUMTECH MPFC-QuEChERS (complex matrix) ultrafiltration type purification column, LUMTECH MPFC-QuEChERS (high lipid) ultrafiltration type purification column and Waters
PRIME HLB Extraction Cartridges. Taking the respective response values of the same microcystin under different extraction conditions and purification modes as molecules, taking the average value of the response values of the same microcystin under different extraction conditions and purification modes as denominators, respectively calculating the respective relative response values of 8 microcystins under the same extraction conditions and purification modes according to the proportion of the two values, and comprehensively measuring the extraction and purification effects. The experimental results are shown in fig. 4, and fig. 4 is a graph showing the relative response intensity of 8 kinds of microcystins measured under different extraction modes and different purification modes.
Figure 4 results show that: when the sample is extracted by vortex oscillation for 60s, and is centrifugally separated under the conditions that the temperature is 4 ℃, the rotating speed is 10000rpm and the time is 5min, and the sample is purified by the purifying tube, 8 microcystins can obtain better mass spectrum response. When the component to be detected is extracted by ultrasonic, the 8 microcystins are difficult to obtain better mass spectrum response under the purification of different purification devices, probably because: (1) the ultrasonic process causes partial degradation or decomposition of the microcystins; (2) the interaction of the microcystins and the sample matrix in the ultrasonic process can cause the microcystins and components of lipids, proteins or metals in the matrix to form substances with more stable structures, and the substances are difficult to effectively strip microcystins molecules to be detected through the absorption, combination, exchange and other action mechanisms of the purification material in the purification process, so that the microcystins cannot enter a solution to be detected after being filtered by the filter membrane.
In summary, when methanol is used: water (70:30, V: V) is used as an extraction solvent, a sample is subjected to centrifugal separation at the temperature of 4 ℃ and the rotation speed of 10000rpm for 5min after being swirled for 60s, and the sample is purified by the purification tube of the invention and then the sample extraction solution is filtered by a polytetrafluoroethylene microporous filter membrane of 0.22 mu m to prepare the sample, so that the quantitative and qualitative detection of 8 microcystins in algae food and production raw materials thereof can be effectively realized, and the method has the advantages of short required working time and high efficiency.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A method for detecting microcystins in algae food and raw materials for producing the same comprises the following steps:
mixing a sample to be detected with an extraction solvent, extracting by vortex oscillation, centrifuging and collecting a sample extracting solution;
purifying the sample extracting solution by using a purifying tube, and filtering to obtain a sample detection solution to be detected; wherein the purification pipe is filled with magnesium sulfate, N-propyl ethylenediamine adsorbent and C18And neutral alumina;
and detecting the detection liquid of the sample to be detected by adopting an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer to realize qualitative and quantitative analysis of the microcystins.
2. The method of claim 1, wherein the microcystins comprise one or more of microcystin-LR, microcystin-RR, microcystin-YR, microcystin-LW, microcystin-LF, microcystin-LA, microcystin-LY, and microcystin-WR.
3. The method of claim 1, wherein the extraction solvent comprises 70% aqueous methanol;
preferably, the ratio of the amount of the sample to be detected to the amount of the extraction solvent is 1 g: (5-20) mL;
preferably, the ratio of the amount of the sample to be detected to the amount of the extraction solvent is 1 g: 10 mL.
4. The method according to claim 1, wherein the time of vortex oscillation is 30-60 s; preferably, the time of vortex oscillation is 60 s;
preferably, the temperature for centrifuging after vortex oscillation is 0-8 ℃, the rotating speed is 5000-10000 rpm, and the centrifuging time is 5-10 min;
preferably, the temperature for centrifugation after vortex oscillation is 4 ℃, the rotation speed is 10000rpm, and the centrifugation time is 5 min.
5. The method of claim 1, wherein the magnesium sulfate, the N-propylethylenediamine adsorbent, the C18And the mass ratio of the neutral alumina is (6-27): (2-9): (1-3): (1-3);
preferably, said magnesium sulfate, said N-propylethylenediamine adsorbent, said C18And the mass ratio of the neutral alumina is 15: 5: 3: 3.
6. the method of claim 1, wherein the purified filtration is a microporous organic filtration membrane;
preferably, the microporous organic filter membrane is a 0.22 μm polytetrafluoroethylene filter membrane.
7. The method of claim 1, wherein the chromatographic conditions of the ultra performance liquid chromatography-triple quadrupole mass spectrometer comprise:
a chromatographic column: SHIMADZU
AQ-C18A chromatographic column with the length of 100mm, the inner diameter of 2.1mm and the particle size of 1.9 mu m;
mobile phase: 0.01% formic acid in water and acetonitrile; gradient elution is adopted; the column temperature is 35-45 ℃, preferably 45 ℃, and the flow rate is 0.2-0.4 mL/min, preferably 0.3 mL/min; the sample injection amount is 2-10 mu L, preferably 5 mu L; the elution procedure is as follows:
。
8. The method of claim 1, wherein the mass spectrometer conditions of ultra performance liquid chromatography-triple quadrupole mass spectrometry comprise:
the ionization mode is an electrospray ion source positive ion mode, a multi-reaction monitoring mode signal is acquired, and the flow rate of atomized gas is 2.5-3.5L/min, preferably 3L/min; the flow rate of the drying gas is 8-12L/min, preferably 10L/min; the flow rate of the heating gas is 8-12L/min, preferably 10L/min; the interface temperature is 250-350 ℃, and preferably 300 ℃; the interface voltage is 3.0-5.0 kV, preferably 4.0 kV.
9. The method of claim 1, wherein performing qualitative analysis of the microcystins using ultra high performance liquid chromatography-triple quadrupole mass spectrometry comprises:
acquiring an ion pair comprising a precursor ion and a product ion by adopting a positive ion scanning mode;
selecting two or more groups of ion pairs, wherein when the retention time of chromatographic peaks of the selected ion pairs in the sample is consistent with that of corresponding standard chromatographic peaks; and meanwhile, when the relative abundance ratio of the product ions in the sample is compared with the relative abundance ratio of the product ions in the standard solution with the same concentration to meet the requirement of allowable deviation, the qualitative requirement is met.
10. The method of claim 1, wherein the quantitative analysis of microcystins using ultra performance liquid chromatography-triple quadrupole mass spectrometry comprises:
analyzing the microcystin standard series solution by adopting ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and injecting a sample by using a matrix matching standard solution; obtaining the corresponding relation between the quantitative ion peak area and the solution concentration, and drawing a matrix matching standard curve;
and (3) feeding a sample detection solution to be detected, detecting the peak area of the quantitative ion, and obtaining the content of the microcystin in the sample detection solution to be detected according to the matrix matching standard curve.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103076420A (en) * | 2012-12-31 | 2013-05-01 | 江苏省环境监测中心 | Method for detecting multicomponent microcystins through ultra-high performance liquid chromatography/triple quadrupole tandem mass spectrometry |
| CN103940921A (en) * | 2014-01-09 | 2014-07-23 | 山东出入境检验检疫局检验检疫技术中心 | Microcystic toxins liquid chromatogram-tandem mass spectrum detection method |
| CN104977383A (en) * | 2015-06-18 | 2015-10-14 | 中山鼎晟生物科技有限公司 | Method for rapidly and quantitatively detecting microcystins in spirulina food |
| CN106324123A (en) * | 2016-08-09 | 2017-01-11 | 国家烟草质量监督检验中心 | Determination method of pesticide residual quantity in tobacco and tobacco products |
| US20180321237A1 (en) * | 2016-10-21 | 2018-11-08 | The University Of Toledo | Protocol for Preconcentration and Quantification of Microcystins Using LC-MS |
| CN109142607A (en) * | 2018-10-15 | 2019-01-04 | 浙江省检验检疫科学技术研究院 | The method of Amitraz and metabolite residual quantity in QuEChERS- liquid chromatography-mass spectrography/mass spectrometric determination honey |
| CN113009002A (en) * | 2019-12-20 | 2021-06-22 | 中粮营养健康研究院有限公司 | Sample pretreatment method containing furfural substances and method for detecting content of furfural substances |
-
2021
- 2021-06-23 CN CN202110696858.6A patent/CN113447581A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103076420A (en) * | 2012-12-31 | 2013-05-01 | 江苏省环境监测中心 | Method for detecting multicomponent microcystins through ultra-high performance liquid chromatography/triple quadrupole tandem mass spectrometry |
| CN103940921A (en) * | 2014-01-09 | 2014-07-23 | 山东出入境检验检疫局检验检疫技术中心 | Microcystic toxins liquid chromatogram-tandem mass spectrum detection method |
| CN104977383A (en) * | 2015-06-18 | 2015-10-14 | 中山鼎晟生物科技有限公司 | Method for rapidly and quantitatively detecting microcystins in spirulina food |
| CN106324123A (en) * | 2016-08-09 | 2017-01-11 | 国家烟草质量监督检验中心 | Determination method of pesticide residual quantity in tobacco and tobacco products |
| US20180321237A1 (en) * | 2016-10-21 | 2018-11-08 | The University Of Toledo | Protocol for Preconcentration and Quantification of Microcystins Using LC-MS |
| CN109142607A (en) * | 2018-10-15 | 2019-01-04 | 浙江省检验检疫科学技术研究院 | The method of Amitraz and metabolite residual quantity in QuEChERS- liquid chromatography-mass spectrography/mass spectrometric determination honey |
| CN113009002A (en) * | 2019-12-20 | 2021-06-22 | 中粮营养健康研究院有限公司 | Sample pretreatment method containing furfural substances and method for detecting content of furfural substances |
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