CN109765087B - Method for rapidly extracting and detecting malachite green in freshwater fish - Google Patents
Method for rapidly extracting and detecting malachite green in freshwater fish Download PDFInfo
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Abstract
The invention relates to a method for detecting malachite green, in particular to a method for quickly extracting and detecting malachite green in freshwater fish, which comprises the steps of firstly, quickly extracting the malachite green and recessive malachite green in the freshwater fish (raised in a pool containing the malachite green) by utilizing acetonitrile, dividing the malachite green and the recessive malachite green into two parts, adding potassium borohydride powder into one part to convert the malachite green into the recessive malachite green to obtain a sample background without the malachite green, acquiring an ultraviolet-visible spectrum of the sample to obtain background data N, directly acquiring the ultraviolet-visible spectrum of the sample to be detected as sample data c, secondly, establishing an ultraviolet-visible standard spectrum database V of the malachite green, and thirdly, calculating the content of the malachite green in the sample to be detected by adopting a vector-subspace included angle criterion method. The method solves the problems that the existing national standard extraction method of malachite green is complicated, the high performance liquid chromatography is long in time consumption for analyzing the malachite green, a large amount of solvents are used and the like, realizes the rapid analysis of the malachite green in the freshwater fish, and is convenient to popularize.
Description
Technical Field
The invention relates to a method for detecting Malachite Green (MG), in particular to a method for quickly extracting and detecting malachite green in freshwater fish.
Background
Malachite green (abbreviated as MG) belongs to triphenylmethane type dye, is toxic triphenylmethane chemical substance, and is often used for preventing and treating saprolegniasis, branchiomycosis, ichthyophthiriasis and the like of fish and for keeping fish fresh. In China 'NY 5071-2002 rules for use of pollution-free food and fish drugs', MG is listed as a forbidden drug. At present, the national standards and related literature methods for detecting aquatic products containing MG and LMG in China mainly comprise the following methods: high performance liquid chromatography, fluorescence method, Raman spectroscopy, enzyme linked immunosorbent assay, liquid-mass combined method, etc.
High performance liquid chromatography: the existing national standard and literature extraction method of MG and LMG in aquatic products comprises simultaneously or partially adding diethylene glycol, hydroxylamine hydrochloride, p-toluenesulfonic acid and ammonium acetate buffer solution for extraction, and adding KBH4And (3) performing solution reduction measurement on LMG or adding lead peroxide to perform oxidation measurement on MG, performing liquid-liquid extraction by adopting dichloromethane, and eluting and purifying by using a solid-phase extraction column such as neutral alumina, PRS and the like. The national standard recovery rate is 70-110%, and the detection limit is 0.5 mug/kg; the recovery rate of the related literature is over 82 percent after the national standard method is improved, and the accuracy is high.
Fluorescence method: the MG generates a fluorescence quenching phenomenon on the water-soluble Cd Te/Zn S quantum dots, a method for detecting aquatic products is established according to the property, and the fluorescence quenching intensity of the MG mass is in a linear relation between 0.0193-1.28MG/L and the Cd Te/Zn S quantum dot optimization system in a Tris-HCl buffer solution with the pH value of 8 and the concentration of 0.065mol/L and the concentration of Cd Te/Zn S quantum dot optimization system with the concentration of 1.60 multiplied by 10 < -4 > mol/L. The detection limit is 0.00543mg/L, the recovery rate is 90.4-100.3%, the RSD value is 0.37-1.01%, and the accuracy is high.
Raman spectroscopy: using MG sitesAt 432 and 437cm-1、1166-1170cm-1And 1613-1617cm-1The detection limit of MG in aquaculture water is 5MG/L, the precision RSD value is 0.51%, the method has good reproducibility, less time consumption, but poor sensitivity.
Enzyme linked immunosorbent assay: the detection rate of the residual MG and LMG content of different aquatic products in the market is 6.98% by applying one enzyme linked immunosorbent assay kit, and the enzyme linked immunosorbent assay kit can be used for rapidly screening a large number of aquatic products.
Liquid phase-mass spectrometry combination: deuterated LMG was used as internal standard. Adding hydroxylamine hydrochloride, p-toluenesulfonic acid and ammonium acetate, uniformly mixing, extracting a sample by using acetonitrile, centrifuging, purifying by using neutral alumina, adding dichloromethane for extraction, blowing nitrogen to be nearly dry, separating on a C18 column and detecting in an MRM mode after an initial mobile phase is constant, wherein the recovery rate is 86.5-91.8%, the detection limit is 0.1 mu g/L, the RSD is less than 7%, and the result has certain accuracy.
The chromatographic method has high sensitivity, but the time and the solvent consumption are high, and the spectrophotometry is widely applied in China, but has the defect of low sensitivity and has certain influence on low-concentration detection. Enzyme-linked immunoassay needs to eliminate protein interference, and the pretreatment process is complicated. The MG and LMG rapid detection technology comprises Raman spectroscopy and enzyme-linked immunoassay, but the sensitivity of the Raman spectroscopy is poor, the detection cost of the enzyme-linked immunoassay is high, and false negative and false positive conditions exist.
Disclosure of Invention
The invention aims to overcome the defects and provides a method for quickly extracting and detecting malachite green in freshwater fish.
In recent years, rapid analysis techniques are a development trend of food safety detection. The method adopts a reduction method, completely reduces Malachite Green (MG) into recessive malachite green (LMG) by using potassium borohydride powder, establishes a sample background without MG, and establishes a rapid detection method for MG in the freshwater fish based on a vector-subspace included angle criterion.
The technical scheme of the invention is as follows: a method for rapidly extracting and detecting malachite green in freshwater fish comprises the following steps:
step 1, putting freshwater fish into water containing malachite green for culturing for 24 hours, removing fish scales, fish skins and bones from an edible part, adding acetonitrile into fish meat, and quickly extracting to obtain a sample containing malachite green MG and recessive malachite green LMG.
Dividing the sample extracting solution into two parts, adding potassium borohydride powder into one part, completely converting malachite green into recessive malachite green, collecting the converted sample background ultraviolet-visible spectrum data N without the malachite green, and directly collecting the ultraviolet-visible spectrum of the sample as sample data c to be detected;
and 3, calculating the content of the malachite green of the sample by adopting a vector-subspace included angle criterion method.
Further, in step 1, the freshwater fish is a plurality of freshwater fish of different species.
The background spectrum data N (without the malachite green component) of the freshwater fish of different types/batches is completely consistent with other components in the sample to be detected, and a background database is not required to be established by liquid phase-spectrum combination.
Further, the vector-subspace angle criterion method includes:
(1) setting a deduction step number delta according to the quantitative precision;
(2) in the formula yi＝aix+biWith a larger x in1Value, get v1，yiTo representiAbsorbance value of malachite green at wavelength, ai、biDenotes a constant, x denotes the concentration of malachite green, v1I.e. with a concentration x1Multi-wavelength absorbance y of lower malachite green1，v1Is all of yiA moment consisting of the value ofArraying;
(3) deduction of v from spectral data c1Δ, the variable after subtraction is denoted dc(ii) a Background data N and variable dcMerging, marking as a comparison space R, and calculating the comparison spaces R and v1The included angle of (A);
(4) one-step subtraction of v from the spectral data c1Repeating the step (3) to calculate R and v1An included angle value;
(5) repeating the step (4), and comparing the R and v spaces when the malachite green is completely deducted from the spectral data c1Will have a maximum value thetamaxRecording of thetamaxNumber of deduction steps lambda corresponding to the occurrencemaxBy the concentration x of malachite green1Sum and subtract step number lambdamaxAnd calculating the content y of malachite green in the sample1(ii) a The calculation method is y1＝x1×λmaxA,/Δ, obtained y1The value is the content of malachite green in the sample; if the number of the deduction steps corresponding to the space included angle is 1 or the value of the space included angle is monotonically increased (decreased) along with the number of the deduction steps, the sample does not contain the component malachite green to be detected.
Furthermore, the detection limit of malachite green is 100 mug/kg and 0.002 mug/mL.
Further, the rapid extraction is that the sample is added with acetonitrile and then homogenized in a 10000r/min homogenizer, acid alumina is added for purification, then a rapid homogenizer is used for oscillation, the sample is centrifuged for 5min in a 4000r/min high-speed centrifuge, and the supernatant passes through a 0.45 mu m microporous filter membrane.
Further, in step 1, 1mL of 10.0 μ g/mL of malachite green can be completely converted into 6.5 μ g/mL of recessive malachite green, the addition amount of the potassium borohydride powder is 0.8mg, and the addition of excessive potassium borohydride does not affect the content of other background components.
Further, in the step 1, after adding potassium borohydride powder, performing high-speed centrifugation on the sample, and taking the supernatant for detection.
Further, in step 1, the fish meat sample is not less than 5g when the sample is taken.
The invention has the advantages that:
1. the invention firstly provides the rapid detection of MG in fish meat based on the vector-subspace included angle criterion, and the MG content in a sample is determined after MG is completely converted into LMG by potassium borohydride powder as the sample background, so that the problems of large amount of used solvent and long-time high performance liquid chromatography are solved, and the rapid detection and the field detection are greatly facilitated.
2. The method has the advantages of simple sample treatment process and short time consumption, the sample background is completely consistent with other components of the sample after the component to be detected is deducted from malachite green, a liquid phase-spectrum combined use is not needed for establishing background libraries of various fishes, and the method has a very optimistic prospect when being popularized to other fields.
3. The invention has accurate measuring result, the false negative and false positive rate is 0 percent in the concentration range higher than the detection limit, the detection limit is as follows: 100. mu.g/kg, 0.002. mu.g/mL.
4. The detection method of the invention has low cost and is convenient for popularization and utilization.
5. The invention uses acetonitrile to carry out rapid extraction, has high extraction efficiency and small influence of impurities.
Drawings
FIG. 1 is a study flow chart;
FIG. 2 is a graph illustrating an example of the calculation of the included angle for a low concentration sample;
FIG. 3 is a diagram illustrating an exemplary calculation of an included angle of a sample without an analyte
FIG. 4 is a high performance liquid chromatogram (610nm) of MG extracted from four fish by the fast extraction method;
FIG. 5 is a sample UV-Vis spectrum of four fish (watery MG);
FIG. 6 four fish (water-fed MG) plus KBH4The latter UV-Vis spectrum;
FIG. 7 is a malachite green standard spectrum;
FIG. 8 is a Malachite green standard operating curve;
FIG. 9 is a UV-Vis spectrum of four fish (aqueous MG) extracts plus MG standard solution.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various modifications and changes may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents may fall within the scope of the invention as defined in the claims appended hereto.
The invention discloses a calculation process demonstration:
a (830: 1000:); (the wavelength of MG is chosen to be 830:1000 rows, and all columns below this range)
b biaozhun (830: 1000:); (b is standard, biaozhun is the file name corresponding to the spectral data of MG standard)
c ═ U (830:1000,: r); (c denotes the sample to be tested, U is the file name corresponding to the sample database to be tested)
v ═ regmain (b, d); (least squares fit, d is concentration corresponding to MG standard)
V (: 1) + V (: 2); (least squares fit for standard UV-Vis spectral data and standard MG concentrations)
cn ═ findc (a, V, c (: n)); (calculating vector-subspace included angle, n is the number of columns of the sample to be measured)
[x,y]＝max(cn)
Two experimental methods
1, research process: see figure 1.
2 sample pretreatment method
Putting the living fishes of the Hehua fish, the yellow bee fish, the grass carp and the tilapia bought from the market into water with the concentration of 2.5 mu g/mL malachite green MG for 24 hours, removing fish scales, fish skins and bones from the edible part, taking 5g of fish meat, adding 10mL of acetonitrile, quickly extracting, namely homogenizing for 30s in a 10000r/min homogenizer, adding 5g of acidic alumina (200 plus 300 meshes) for purification, oscillating for 15s by a quick mixer, centrifuging for 5min by a 4000r/min high-speed centrifuge, and then passing the supernatant through a 0.45 mu m microfiltration membrane. Obtaining a sample extract containing MG: grass carp sample A, wasp fish sample B, grass flower fish sample C and tilapia sample D.
Removing fish scales, fish skin and fish bones from blank fish meat without MG in vivo, taking 5g of edible part of fish meat, adding 10mL of acetonitrile and 1mL of standard MG, quickly extracting, namely homogenizing in a homogenizer at 10000r/min for 30s, adding 5g of acidic alumina for purification, oscillating for 15s by a quick homogenizer, centrifuging at a high speed centrifuge of 4000r/min for 5min, and passing the supernatant through a 0.45-micrometer microporous filter membrane. Samples were obtained diluted 11 times the standard.
Respectively collecting data of the 220-one 1100nm ultraviolet visible spectrometer and data of the high performance liquid chromatography, and comparing results. And compared with the national standard method.
3-vector-subspace included angle criterion rapid analysis method
3.1 establishment of background data N and sample data c of freshwater fish sample
Dividing the sample extracting solution into two parts, adding potassium borohydride powder into one part to completely convert the malachite green into recessive malachite green to obtain a sample background without the malachite green, collecting ultraviolet-visible spectrum data N of the sample background, and directly collecting ultraviolet-visible spectrum data c of the sample on the other part.
3.2 establishment of MG Standard database V
Accurately weighing 0.0100gMG standard substance by using an analytical balance, dissolving the solid of the standard substance by using acetonitrile, transferring to a 100mL volumetric flask, carrying out constant volume to obtain 100 mu g/mL standard stock solution, preparing 10 mu g/mL standard stock solution, diluting the 10 mu g/mL standard stock solution into 0.08-6.0 mu g/mL standard substance respectively, collecting full-wavelength ultraviolet-visible spectrum data at 220-1100nm respectively, and obtaining a multi-wavelength standard MG ultraviolet spectrum database V by using a least square regression method.
3.3 data processing method
1) Acquiring background spectrum data N of a sample extracting solution without MG;
2) collecting spectral data c of the sample extracting solution;
3) collecting standard MG ultraviolet visible spectrum data, and establishing a standard ultraviolet spectrum database V;
4) determination of MG in the fish sample to be tested:
(1) the number of deduction steps Δ (set to 1000 in this example) is set according to the quantitative accuracy;
(2) in the formula yi＝aix+biWith a larger x in1Value, get v1；
yiTo representiAbsorbance value of malachite green at wavelength, ai、biDenotes a constant, x denotes the concentration of MG, v1I.e. with a concentration x1Multi-wavelength absorbance y of lower MG1，v1Is all of yiA matrix of values of (c);
(3) deduction of v from spectral data c1Δ, the variable after subtraction is denoted dc(ii) a A blank background database N and a variable dcMerging, marking as a comparison space R, and calculating the comparison spaces R and v1The included angle of (A);
(4) one-step subtraction of v from the spectral data c1Repeating the step (3) to calculate R and v1An included angle value;
(5) repeating the step (4), and comparing the spaces R and v when the MG in the spectrum number c is completely deducted1Will have a maximum value thetamaxRecording of thetamaxNumber of deduction steps lambda corresponding to the occurrencemaxBy concentration x of MG1Sum and subtract step number lambdamaxCalculating the content y of MG in the sample1(ii) a The calculation method is y1＝x1×λmaxA,/Δ, obtained y1The value is the content of MG in the sample; if the number of the deduction steps corresponding to the space included angle is 1 or the value of the space included angle is monotonically increased (decreased) along with the number of the deduction steps, the sample does not contain the component malachite green to be detected. The schematic diagrams of the calculation of the included angles of the samples to be measured with different concentrations are shown in fig. 2 and fig. 3.
5)KBH4Conversion test for MG
Due to KBH4The MG can be completely reduced to LMG, so that a certain amount of MG and LMG standard solution is added into 5g of blank Holdfish sample (not containing MG and LMG) fast extract, the added standard sample is divided into two parts, one part is added with a certain amount of potassium borohydride powder to be used as the background of MG determination, the other part is directly used for collecting the sample spectrum, and the dosage of potassium borohydride and the conversion equivalent weight of MG and LMG are inspected based on the vector-subspace angle criterion.
6) Precision test of MG
And (3) parallelly collecting 5 times of ultraviolet-visible spectrum data by taking a sample with known MG concentration and a standard MG with the concentration of 3 mu g/mL, calculating the content of the MG by using a vector-subspace angle criterion, and calculating the RSD value.
7) Experiment of recovery rate of MG with standard
And (3) adding 10mL of acetonitrile into 5g of blank fish meat without MG, uniformly mixing, and extracting according to a quick extraction method to obtain a blank sample. Adding a certain amount of standard MG solution into the sample, collecting ultraviolet spectrum data of the sample under the conditions of 220-1100nm, determining the standard adding content of the sample by adopting a vector-subspace included angle criterion method, and calculating the standard adding recovery rate.
8) Limit of detection experiment for MG
Taking 5g of the edible part of the plecoglossus altivelis which does not contain MG in 3 parts, respectively adding 10mL of acetonitrile and 1mL of standard solutions of 0.5, 2 and 4 mu g/mL, and quickly extracting. And obtaining a sample of the 11-time diluted standard substance, collecting data of the 220-fold 1100nm ultraviolet-visible spectrometer, and calculating the content of MG by using a vector-subspace included angle criterion method, wherein the corresponding detection limit concentration unit is mu g/kg.
And sequentially diluting 0.5 mu g/mL malachite green standard solution, and calculating the content of MG by adopting a vector-subspace angle criterion method until the deduction step number corresponding to the maximum angle is more than 20 and the corresponding detection limit concentration unit is mu g/mL.
Results of four experiments
1. Results of different pretreatment methods of samples
Respectively taking 5g of the edible parts of the blank grass carp and tilapia flesh which do not contain MG, chopping, adding 1mL of standard MG solution with the concentration of 10 mug/mL or 4 mug/mL, and adding 10mL of acetonitrile for quick extraction. A sample was obtained that was theoretically diluted 11 times the standard. And extracting by adopting a national standard method to obtain a series of samples. The data of the 220-plus 1100nm ultraviolet-visible spectrometer are respectively collected and calculated by adopting a vector-subspace included angle criterion, and the results are shown in tables 3-4. And compared with the high performance liquid phase data result.
The HPLC for extracting MG from four fish by fast extraction is shown in FIG. 4.
The extraction rate of the sample by the rapid extraction method is 47-52%, and the extraction rate of the sample by the national standard method is 38-44%, so that the extraction rate of the rapid extraction method is superior to that of the national standard method. The RSD of the extraction rate of the fast extraction method is 4.62 percent, the repeatability is good, the reliability is strong, and the method can be used for fast extracting the content of MG in fish meat.
TABLE 3 fast extraction method for MG extraction rate experiment in fish meat
TABLE 4 experiment of extraction rate of MG from fish meat by national standard method
2. Establishment and calculation result of vector-subspace data model
2.1 acquisition of sample background spectral data N and sample spectral data c, the ultraviolet-visible spectrum of the sample of four fish (MG water culture) is shown in figure 5, and KBH is added to the sample of four fish (MG water culture)4The resulting UV-Vis spectrum is shown in FIG. 6.
2.2 establishment of MG Standard database V
Diluting 100 mu g/mL MG with acetonitrile into standard MG solutions of 0.08 mu g/mL, 0.60 mu g/mL, 1.00 mu g/mL, 2.00 mu g/mL, 3.00 mu g/mL, 4.00 mu g/mL and 6.00 mu g/mL respectively, collecting the data of the ultraviolet-visible spectrometer under the wavelength of 220 nm-1100 nm respectively, establishing a standard substance spectrum database and naming the database as: biaozhun. The linear equation, the correlation coefficient and the linear range of the standard working curve are respectively as follows: 0.1054 x-0.00433; r20.9994; 0.08-6.0 μ g/mL, as shown in figures 7-8.
2.3 calculation results of MG content in Fish meat sample
Taking 5g of the above samples (four live fishes are put into water containing MG for feeding) and 5g of commercially available four fish meat edible parts, adding 10mL of acetonitrile, quickly extracting, collecting the ultraviolet-visible spectra of the four fish (water-borne MG) extracting solutions and the MG standard solution as shown in FIG. 9, calculating the MG content of the samples by adopting a vector-subspace included angle criterion method, and comparing the content with the high performance liquid phase measurement result as shown in the following Table 5. According to the same method, after other commercial samples are quickly extracted, 220-1100nm spectral data can be directly collected for quantitative analysis.
TABLE 5 comparison of results of MG content calculation for samples by two methods
The results are shown in table 5, and the results of the two calculation methods are close, so that the content of the sample can be quickly calculated by directly collecting the spectrum of the sample based on the vector-subspace included angle criterion, the calculation is accurate, and the time is greatly shortened compared with the chromatographic analysis.
3. Experiment of conversion rate of potassium borohydride to MG
A certain amount of malachite green and recessive malachite green standard solution is added into the extracting solution of a blank Holdfish sample (not containing malachite green and recessive malachite green), a certain amount of potassium borohydride powder is added into 1mL of the extracting solution, and the dosage of the potassium borohydride and the conversion equivalent of the malachite green (MW:463.5) and the recessive malachite (MW:330.5) are considered as shown in Table 6. The result shows that 0.8mg of potassium borohydride powder is added, the malachite green can be completely converted into recessive malachite green, and the conversion equivalent is 0.65.
TABLE 6 conversion equivalents of Malachite Green to recessive Malachite Green
Add malachite green and the green standard solution of recessive malachite of known concentration in blank fish meat extract, get 1mL solution and add 2.0mg potassium borohydride powder conversion back, survey malachite green and the green content of recessive malachite, calculate the green total content of recessive malachite according to 0.65's conversion equivalent, as shown in Table 7, the green rate of recovery of recessive malachite is greater than 90%, the calculated result is accurate, it is 0.65 to further verify that the conversion equivalent that malachite green turns into the green of recessive malachite completely.
TABLE 7 conversion results of malachite green in fish meat
As shown in tables 6-7, the potassium borohydride powder can completely reduce MG, so that the sample background does not contain the component MG to be detected, and excessive KBH is added4The powder does not change the background other component content of the sample.
Precision test of MG
Taking grass carp 2 with known liquid phase concentration (actual value)#(commercially available) and wasp 2#(commercial) samples and 4 mu g/mL standard MG solution are parallelly collected for 5 times of ultraviolet-visible spectrum data, the content of MG is calculated by using a vector-subspace angle criterion, the result is shown in Table 8, RSD is less than or equal to 4.67%, and the method is credible and has good precision.
TABLE 8MG precision experiments
Test for recovery of MG by adding standard
Respectively taking grass carp No. 1 (sold in the market) and wasp fish No. 1 (sold in the market) in the table 4, adding MG standard solution with the same volume into the blank sample extracting solution to obtain a sample of a diluted 2-time standard substance, collecting spectral data of the sample under the condition of 220 plus one ion at 1100nm, calculating the sample standard addition recovery rate by using a vector-subspace included angle criterion method, wherein the MG standard addition recovery rate is 94-115% as shown in the table 8, and the method is proved to have high calculation result recovery rate, high accuracy and strong reliability.
TABLE 9 MG spiking recovery test
6. Limit of detection experiment
Taking 5.0g of the edible part of the plecoglossus altivelis without MG 3 parts, respectively adding 10mL of acetonitrile and 1mL of standard MG solution with 0.5, 2 and 4 mu g/mL, and quickly extracting. Obtaining a sample of a standard substance diluted by 11 times, collecting data of 220-1100nm ultraviolet-visible spectrometer of the sample, calculating the content of MG by using a vector-subspace included angle criterion, wherein the calculation result is shown in table 10, the result shows that 5.0g of fish meat sample is added with 1mL of 0.5 mu g/mL malachite green standard solution, and then acetonitrile is used for rapid extraction, and the content of MG can be detected based on the vector-subspace included angle criterion, so the detection limit is 100 mu g/kg.
And taking 0.5 mu g/mL malachite green standard solution, and sequentially diluting until the deduction step number corresponding to the maximum included angle value is more than 20, wherein the detection limit concentration is 0.002 mu g/mL.
TABLE 10 detection limit test for MG
Five conclusions and discussion
The invention optimizes the extraction method of malachite green in freshwater fish (meat), and adopts a vector-subspace included angle criterion to establish a rapid detection method of malachite green. Firstly, acetonitrile is used for quickly extracting malachite green in freshwater fish (raised in a pool containing the malachite green), an extracting solution is divided into two parts, potassium borohydride powder is added into one part of the extracting solution to completely convert the malachite green into recessive malachite green, a sample background without the malachite green is obtained, ultraviolet-visible spectrum data N of the sample background are collected, ultraviolet-visible spectrum data c of the sample are directly collected from the other part of the extracting solution, secondly, an ultraviolet-visible standard spectrum database V of the malachite green is established, and thirdly, the malachite green content in the sample to be detected is quickly calculated by adopting a vector-subspace included angle criterion algorithm. The result of the method is close to the result calculated by the high performance liquid chromatography, and the recovery rate of the added standard is between 94 and 115 percent; the precision RSD is less than or equal to 4.67 percent; the detection limit was 100. mu.g/kg.
The extraction rate of the acetonitrile on the malachite green is 47 to 52 percent, which is superior to the national standard method; the optimal chromatographic conditions are acetonitrile: 0.02mol/L acetic acid-ammonium acetate buffer solution (pH4.6) ═ 75: 25.
Micro KBH4Powder 0.8MG can completely convert 1mL10 μ g/mL MG into LMG and excess KBH4The content of other components in the sample is not changed. Thus the handle sampleDividing the extractive solution into two parts, adding KBH into one part4The powder is used as the background of the sample to be detected, the other part is used as the sample to be detected, a background database is not required to be established by liquid phase-spectrum combination, the false negative and false positive rates of detecting the MG content in the freshwater fish are 0 percent in a linear range and a range higher than a detection limit, the quantification is accurate, and the operation is quicker and simpler. The problems that the existing extraction method of malachite green in national standard is complicated, the operation is complex, the extraction rate is low, and the problems that the high performance liquid chromatography is long in time consumption and large in solvent consumption in malachite green analysis are solved, so that the method is convenient to popularize.
The experiment of the invention should be noted with the following 2 points:
1. in the process of sample dilution and standard recovery rate adding experiments, because the acetonitrile content is increased, the solubility of water-soluble impurities in a fish sample is reduced, and a turbidity phenomenon can occur, so that the problem that the baseline does not return to zero when data of an ultraviolet-visible spectrometer are collected is caused, and calculation errors are caused. The method adopted in the experiment is to centrifuge for 5min in a high-speed centrifugal machine with the speed of 4000r/min, and to collect the data of the ultraviolet-visible spectrometer by taking the supernatant. High speed centrifugation is also required to zero the baseline after conversion by addition of potassium borohydride powder.
2. Experiments prove that when the amount of the fish sample exceeds 5g, the interference of impurities in the range of 580-655nm in a visible light region is not large, the detection limit is favorably reduced, and at the moment, the deviation is not large when the result calculated by adopting a vector-subspace included angle criterion method is compared with the calculation result of the high performance liquid chromatography, and the calculation result has high accuracy.
Claims (5)
1. A method for rapidly extracting and detecting malachite green in freshwater fish is characterized by comprising the following steps:
step 1, putting freshwater fish into water containing malachite green for culturing for 24 hours, removing fish scales, fish skins and bones from an edible part, adding acetonitrile into fish meat, and quickly extracting to obtain a sample containing malachite green MG and recessive malachite green LMG;
dividing the sample extracting solution into two parts, adding potassium borohydride powder into one part, completely converting malachite green into recessive malachite green, collecting the converted sample background ultraviolet-visible spectrum data N without the malachite green, and directly collecting the ultraviolet-visible spectrum of the sample as sample data c to be detected;
step 2, establishing a malachite green standard substance database V, accurately weighing the malachite green standard substance by using an analytical balance, dissolving the solid of the standard substance by using acetonitrile to prepare a standard stock solution, respectively diluting the standard stock solution into standard substances with different concentrations, collecting ultraviolet-visible spectrum data under the full wavelength, and obtaining the ultraviolet-visible spectrum database V of the malachite green standard substance by using a least square regression method;
step 3, calculating the content of malachite green of the sample by adopting a vector-subspace included angle criterion method;
the vector-subspace included angle criterion method comprises the following steps:
(1) setting a deduction step number delta according to the quantitative precision;
(2) in the formula yi＝aix+biWith a larger x in1Value, get v1，yiDenotes the absorbance value of malachite green at the i wavelength, ai、biDenotes a constant, x denotes the concentration of malachite green, v1I.e. with a concentration x1Multi-wavelength absorbance y of lower malachite green1，v1Is all of yiA matrix of values of (c);
(3) deduction of v from spectral data c1Δ, the variable after subtraction is denoted dc(ii) a Background data N and variable dcMerging, marking as a comparison space R, and calculating the comparison spaces R and v1The included angle of (A);
(4) one-step subtraction of v from the spectral data c1Repeating the step (3) to calculate R and v1An included angle value;
(5) repeating the step (4), and comparing the R and v spaces when the malachite green is completely deducted from the spectral data c1Will have a maximum value thetamaxRecording of thetamaxNumber of deduction steps lambda corresponding to the occurrencemaxBy the concentration x of malachite green1Sum and subtract step number lambdamaxAnd calculating the content y of malachite green in the sample1(ii) a The calculation method is y1＝x1×λmaxA,/Δ, obtained y1The value is the content of malachite green in the sample; if the number of the deduction steps corresponding to the space included angle is 1 or the space included angle value is monotonically increased/decreased along with the number of the deduction steps, the sample does not contain the component to be detected, namely malachite green;
the malachite green detection limit is 100 mug/kg and 0.002 mug/mL; in the step 1, 1mL of 10.0 mu g/mL malachite green can be completely converted into 6.5 mu g/mL recessive malachite green, the addition amount of potassium borohydride powder is 0.8mg, and the content of other background components is not influenced by adding excessive potassium borohydride.
2. The rapid extraction and detection method of claim 1, wherein: in the step 1, the freshwater fish is a plurality of freshwater fish of different species.
3. The rapid extraction and detection method of claim 1, wherein: the rapid extraction is specifically that a sample is added with acetonitrile and then is homogenized in a 10000r/min homogenizer, 5g of acidic alumina is added, 200-mesh and 300-mesh purification is carried out, then a rapid mixer is used for oscillation, a high-speed centrifuge at 4000r/min is used for centrifugation for 5min, and the supernatant passes through a 0.45 mu m microporous membrane.
4. The rapid extraction and detection method of claim 1, wherein: in the step 1, after adding potassium borohydride powder, carrying out high-speed centrifugation on a sample, and taking supernatant for detection.
5. The rapid extraction and detection method of claim 1, wherein: in the step 1, when the fish is sampled, the fish sample is not less than 5 g.
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