CN112198254A - Method for measuring existence form and content of semicarbazide in shellfish tissue - Google Patents
Method for measuring existence form and content of semicarbazide in shellfish tissue Download PDFInfo
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Abstract
The invention relates to a method for measuring the existence form and content of semicarbazide in shellfish tissues, belongs to the technical field of aquatic product detection, and confirms that semicarbazide exists in shellfish tissues in a combined state and a free state simultaneously, and provides a method for detecting the content of semicarbazide in two forms. The detection limit of the aminourea in the mantle, the gonad, the adductor muscle and the visceral tissues of the shellfish is 0.5 mug/kg. The semicarbazide has a recovery rate of 70-120% in the added concentration range of 1.0-10 mug/kg, and the relative standard deviation in batches and the relative standard deviation between batches of the method are both less than or equal to 15%. The invention establishes the method for measuring the existence form and the content of the semicarbazide as the environmental pollutant in the shellfish for the first time, has more accurate and reliable measurement result, high sensitivity, good result reproducibility and accurate quantification, and defines the existence form of the semicarbazide as the environmental pollutant in the shellfish for the first time.
Description
Technical Field
The invention belongs to the technical field of aquatic product detection, and relates to a method for detecting the existence form and content of an environmental pollutant semicarbazide in shellfish tissues by using high performance liquid chromatography-tandem mass spectrometry (LC/-MS/MS).
Background
Semicarbazide (SEM) has been recognized as a characteristic metabolite of nitrofurazone drugs and is often used as a marker for monitoring illegal use of nitrofurazone drugs in foods. Furacilin and its metabolites are specified in the european union, japan and china on a sequential basis and cannot be detected in animal-derived foods. However, researches in recent years show that the metabolic degradation of nitrofurazone is not the only source of residual semicarbazide in aquatic products, and semicarbazide pollution can be introduced by a plurality of ways. The main sources of semicarbazides include: (1) the decomposition of furacilin medicine is a synthetic broad-spectrum antibacterial medicine, has antibacterial and insecticidal effects on most gram-positive bacteria and negative bacteria, some fungi and protozoa, and has been widely used in aquaculture industry. Furacilin can be rapidly catabolized in an animal body, and finally, a metabolite-semicarbazide with high stability, long residual time and stronger toxic effect is formed, which is one of the main sources of semicarbazide in the environment. (2) Semicarbazide, an intermediate in chemical raw material and medical synthesis, is often used as a reagent for measuring and synthesizing aldehyde ketone and cyanate. Semicarbazides are used in a number of pharmaceutical processes, particularly in the organic synthesis of metal semicarbazide derivatives. (3) Decomposition of azodicarbonamide is commonly used as a foaming agent for food-contact plastics, and decomposition at high temperatures can produce semicarbazides, which are derived from the thermal decomposition of azodicarbonamide in metal-backed glass-packaged food products, including infant and baby food, fruit juices, jams, honey, and the like. In addition, azodicarbonamide, as a flour blowing agent, can be decomposed at high temperature to produce semicarbazide. (4) The food can generate semicarbazide through hypochlorite treatment, and the semicarbazide is detected in samples of northern sea shrimps, chicken, milk, protein powder, soybean flakes, red algae, carrageenan and the like which are treated by hypochlorite solution, which indicates that the semicarbazide is a product after the hypochlorite reacts with amino acid in the food. (5) Semicarbazides occur naturally in some animals and plants. The semicarbazide produced by the various ways can be discharged into the environment, becomes a novel pollutant in the environment, pollutes the environment in which aquatic organisms depend to live, and finally accumulates in the body of the aquatic organisms. At present, there are reports in literature that part of sea water and submarine sediments in sea areas are polluted to different degrees, and semicarbazide residues are detected in marine organisms. However, whether the semicarbazide in the environment exists in a bound state or a free state after entering the shellfish body is not clear at present.
Disclosure of Invention
The invention aims to solve the technical problem of establishing a method for detecting the form and the content of semicarbazide, an environmental pollutant, in shellfish tissues. The method of the invention proves that the semicarbazide in the shellfish tissue exists in a combined state and a free state simultaneously, and provides a detection method for the semicarbazide content in two forms: and respectively measuring the total amount of semicarbazide and the bound semicarbazide, and calculating the content of free semicarbazide. Method for measuring total amount of semicarbazide: hydrolyzing the sample with hydrochloric acid, derivatizing with 2-nitrobenzaldehyde, quantitatively adding dipotassium hydrogen phosphate solution, adding ethyl acetate solution for liquid-liquid extraction, concentrating and purifying the supernatant, measuring by a liquid chromatography-tandem mass spectrometer, and quantifying by an internal standard method. The method for determining the bound semicarbazide comprises the steps of respectively washing a sample with methanol/water, methanol and water to obtain a free semicarbazide, adding hydrochloric acid for hydrolysis, and determining the content of the bound semicarbazide according to the steps. The content of the free semicarbazide is obtained by subtracting the bonded semicarbazide from the total semicarbazide.
The detection limit of the aminourea in the mantle, the gonad, the adductor muscle and the visceral tissues of the shellfish is 0.5 mug/kg. The semicarbazide has a recovery rate of 70-120% in the added concentration range of 1.0-10 mug/kg, and the relative standard deviation in batches and the relative standard deviation between batches of the method are both less than or equal to 15%.
The invention is completed according to the following operation method:
a method for measuring the existing form and content of semicarbazide in shellfish tissues comprises the following steps of measuring the total amount of semicarbazide, measuring the combined form of semicarbazide, drawing a standard curve, and using instrument conditions, qualitative determination and quantitative determination:
1) measurement of Total amount of semicarbazide
(1) Sample hydrolysis and derivatization
Adding hydrochloric acid, a derivatization reagent and an isotope internal standard into a sample, hydrolyzing and derivatizing for 16h at 37 ℃, taking out and placing to room temperature;
further, the derivatization reagent in the step (1) is 2-nitrobenzaldehyde with the concentration of 100mmol/L, and the ratio of the added volume to the mass of the sample is 3:40(mL: g).
Further, the concentration of hydrochloric acid in the step (1) is 0.5mol/L, and the mass ratio of the added hydrochloric acid volume to the sample is 5:2(mL: g).
(2) Extraction and purification
Adding a dipotassium phosphate solution into the sample treated in the step (1) to ensure that the pH value of the sample solution is 7.0-7.5, adding ethyl acetate liquid-liquid extraction, carrying out vortex mixing, centrifuging, taking supernate, transferring the supernate into nitrogen for drying at 45 ℃, adding a 5% methanol aqueous solution, carrying out vortex oscillation to dissolve residues, transferring the supernate into an ultrafiltration centrifugal tube, centrifuging at 14000r/min, and determining the supernate by using a high performance liquid chromatography-tandem mass spectrometer;
further, the concentration of the dipotassium hydrogen phosphate solution added in the step (2) is 1.0mol/L, and the mass ratio of the added volume to the sample is 3:1(mL: g).
2) Determination of bound semicarbazide
(1) Washing the sample with methanol/water, methanol and water respectively to obtain free semicarbazide;
(2) then, measuring according to the step 1);
3) drawing of standard curve
Respectively transferring semicarbazide standard solutions with certain concentrations, carrying out sample hydrolysis and derivatization, extraction and purification according to the operation steps of (1) and (2) in the step 1) except that no sample is added, then carrying out LC-MS/MS analysis, and drawing a standard curve;
4) conditions of the apparatus used
The chromatographic conditions were as follows:
a) a chromatographic column: c18A reversed phase chromatographic column with the diameter of 2.1mm multiplied by 100mm and the diameter of 2.6-5 mu m;
b) column temperature: room temperature;
c) flow rate: 0.3 mL/min;
d) sample introduction amount: 10 mu L of the solution;
e) mobile phase: a: methanol, B: 2mmol/L aqueous ammonium acetate solution, gradient elution program is shown in Table 1.
TABLE 1 mobile phase gradient elution conditions
The mass spectrometry conditions were as follows:
an electrospray ion source; spraying voltage: 5500V, positive ion scan; ion source temperature: 550 ℃, collision gas: medium; air curtain air: 30 psi; atomizing: 35 psi; auxiliary heating gas: 35 psi; ion mode: multiple reaction selective monitoring, wherein parameters of selected reaction monitoring parent ions, ionic ions, collision energy and cone hole voltage mass spectra are shown in a table 2;
table 2 selective reaction monitoring of parent ions, daughter ions and collision energy
Note: represents quantitative fragment ions
5) Qualitative and quantitative
(1) Qualitative determination
Under the same test conditions, the deviation of the retention time of the target compound in the sample solution and the retention time of the target compound in the standard working solution is within +/-5 percent, and the relative abundance of the detected qualitative ions is consistent with the relative abundance of the qualitative ions in the standard working solution with similar concentration, and the deviation is in accordance with the requirement of table 3;
TABLE 3 base peak to sub-intense fragment ion abundance ratio requirements
(2) Quantitative determination
Taking a sample solution and a corresponding standard working solution, performing multi-point calibration, and quantifying according to an internal standard method; the response values of the target compounds in the standard solution and the sample solution are within the linear range detected by the instrument; the semicarbazide is quantified by taking an isotope thereof as an internal standard;
(3) blank experiment
The measurement is carried out according to the measurement conditions and steps except that no sample is added;
(4) result calculation and presentation
The residual amount of the target compound in the sample was calculated according to the formula (a):
in the formula:
x is the residual amount of the target compound in the sample, μ g/kg;
c-concentration of target compound in sample solution, ng/mL;
v, the volume of the final sample liquid is determined to be mL;
f, dilution multiple;
m-sample size, g;
note: blank values need to be deducted from the calculation result;
the total amount of semicarbazide and the bound semicarbazide content were calculated according to the formula (a), and the free semicarbazide content was the total amount of semicarbazides-bound semicarbazides content.
Sensitivity, accuracy and precision of the method of the invention
Sensitivity: the detection limits of the semicarbazide in the adductor muscle, mantle, gill, viscera and gonad are all 0.5 mug/kg, and the quantification limit is 1.0 mug/kg.
Accuracy: the method has the semicarbazide recovery rate of 70-120% within the adding concentration range of 1.0-10 mug/kg.
Precision: the relative standard deviation in batches and the relative standard deviation between batches of the method are both less than or equal to 15 percent.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes the existence form and the distribution characteristic of the semicarbazide in the shellfish product for the first time, and applies the method to determine the existence form and the distribution characteristic of the semicarbazide in different tissues of the shellfish for the first time. The method has very important significance for developing research on environmental pollutants in shellfish products and ensuring the quality safety of aquatic products.
The existing detection technology mainly aims at detecting in-vivo metabolites of nitrofuran drugs, and the drugs mainly exist in a protein binding state after entering the body. Semicarbazide is a novel environmental pollutant and exists in a free state in a natural state. As a novel environmental pollutant, whether the pollutant exists in a bound state or a free state after entering a scallop body is unknown, and no detection method for the existence state of the semicarbazide in different tissues of the shellfish exists at present. The method fills the blank that no detection method for detecting the existence form of the semicarbazide, which is a novel environmental pollutant, in the bodies of the shellfish is available in China.
The invention calculates free semicarbazide by methods of measuring total semicarbazide amount and bound semicarbazide respectively, adopts liquid chromatography-tandem mass spectrometry to measure, can eliminate interference and accurately determine the nature, and simultaneously can accurately determine the quantity.
The method optimizes the concentration and the dosage of hydrochloric acid and dipotassium hydrogen phosphate, the concentration of the hydrochloric acid is 0.5mol/L, 6mL of 1.0mol/L dipotassium hydrogen phosphate is directly and quantitatively added after derivatization, the pH value of the solution is 7.0-7.5, and sodium oxide with different concentrations is not needed to adjust the pH value. According to the method, 5mL of hydrochloric acid and 6mL of dipotassium hydrogen phosphate solution are quantitatively added, the pH value of the solution can be 7.0-7.5, particularly, during batch operation, the complicated operation process of adjusting the pH value one by one is omitted, and the extraction efficiency is greatly improved.
Drawings
FIG. 1: characteristic ion mass chromatograms of SEM in adductor muscle and its internal standard;
FIG. 2: a characteristic ion mass chromatogram of SEM and internal standard in the mantle;
FIG. 3: SEM in gill and characteristic ion mass chromatogram of internal standard thereof;
FIG. 4: characteristic ion mass chromatograms of SEM and internal standards in the viscera;
FIG. 5: characteristic ion mass chromatograms of SEM and its internal standard in gonads.
Detailed Description
The technical contents of the invention are described in detail below by way of examples with reference to the accompanying drawings:
the invention relates to a detection method for analyzing the existence form and the content of semicarbazide, which is an environmental pollutant, in shellfish tissues by utilizing a high performance liquid chromatography-tandem mass spectrometry method.
The shells are various, such as scallops, clams, oysters, mussels and the like, and are remained in the bodies of the shells due to pollution of novel environmental pollutants in the growth process of the shells, and the scallops with the most representative shells are selected as verification examples.
Example 1 measurement of the morphology of semicarbazide in scallop
1. The instrument and equipment selected for this embodiment: LC-MS/MS liquid chromatogram-tandem mass spectrometer
(1) HPLC part (Shimadzu, model LC-20A) using Kinetex XB C18Reverse phase chromatography column, 100mm × 2.1mm,2.6 μm.
(2) AB Qtrap 5500 mass spectrometer (AB corporation, usa): the system is provided with an ESI ion source, a triple quadrupole rod, a collision pool, a vacuum system and a gas path control system;
(3) an ultrasonic cleaning instrument (Kunshan ultrasonic instruments Co., Ltd., model KQ-600 DE);
(4) a high-speed centrifuge: 8000r/min (Thermo Fisher Co.);
(5) vortex mixers (Talboys, usa);
(6) nitrogen blowing instrument (U.S. organization, model N-EVAP 112);
(7) a micro high-speed centrifuge: 14000r/min (Sigma, Germany);
(8) analytical balance: sensory quantity 0.00001g (Siderelis, Germany, model CP 225D);
(9) balance: sensory quantity 0.01g (Siderelis, Germany, model CPA 1003P);
(10)10K ultrafiltration centrifuge tubes (Millipore);
(11) a constant temperature oscillator (model IS-RDS3, manufactured by TIQi Co., Ltd.);
(12) Milli-Q ultra pure water instruments (Millipore, USA).
2. Preparing standard solution and reagent
(1) Acetonitrile, methanol: pure chromatography (Merck, Germany).
(2) Ammonium acetate (Merck, Germany).
(3) 2-nitrobenzaldehyde (Sigma, USA)
(4) Hydrochloric acid, dipotassium hydrogen phosphate: super grade pure (national drug group chemical reagents limited).
(5)2mmol/L ammonium acetate solution: accurately weighing 0.154g of ammonium acetate, dissolving with water, metering to 1L, and mixing uniformly for later use.
(9) SEM standard stock solution: weighing SEM standard substance about 10.0mg, dissolving with methanol, diluting to 10mL, preparing standard stock solutions with concentration of 1.0mg/mL respectively, and freezing at-20 deg.C for storage.
(10)SEM-13C,15N2Isotope internal standard stock solution: weighing SEM-13C,15N2Dissolving the standard substance about 10.0mg with methanol, diluting to 10mL, preparing internal standard stock solutions with concentration of 1.0mg/mL respectively, and freezing and storing at-20 deg.C.
(11) SEM standard working solution: accurately transferring appropriate amount of standard stock solution, diluting with methanol, diluting to constant volume, preparing into standard working solution with concentration of 100 μ g/L and 10 μ g/L, and refrigerating at 2-8 deg.C.
(12)SEM-13C,15N2Isotope internal standard working solution: accurate transfer of appropriate amount of SEM-13C,15N2Diluting the isotope internal standard stock solution with methanol and fixing the volume to prepare an internal standard working solution with the concentration of 100 mug/L, and refrigerating and storing at 2-8 ℃.
The water used in the whole experiment process is ultrapure water.
3. Step of sample treatment
(1) Sample pretreatment of semicarbazide Total
(ii) hydrolysis and derivatization
Weighing 2g of amenorrhea muscle, mantle, gill, viscera and gonad homogenate tissues in 50mL of centrifugation, adding 50 mu L of isotope internal standard, standing for 5min, adding 5mL of 0.5mol/L HCl, adding 150 mu L of 100 mmol/L2-nitrobenzaldehyde, vortexing for 1min, and placing at 37 ℃ for constant temperature oscillation in dark for 16 h.
② extraction and purification
Taking out the centrifuge tube, cooling to room temperature, quantitatively adding 6mL (pH value is 7.0-7.5) of 1.0mol/L dipotassium phosphate solution, adding 8mL of ethyl acetate, carrying out vortex oscillation for 1min, centrifuging for 5min at 8000r/min, taking supernatant, transferring the supernatant into a10 mL centrifuge tube, and drying at 40 ℃ with nitrogen. Accurately adding 1.0mL of 5% methanol solution, fully vortexing and shaking to dissolve residues, transferring the solution into an ultrafiltration centrifugal tube, centrifuging for 10min at 14000r/min, and taking the supernatant for liquid chromatography-tandem mass spectrometry.
(2) Sample pretreatment of bound semicarbazide
The amenorrhea, mantle, gill, viscera and gonad homogenates were weighed at 2g each, washed once with 6mL of methanol/water solution (50:50, v/v), once with 6mL of methanol/water solution (75:25, v/v), once with 6mL of methanol, and once with 6mL of water.
50 μ L of internal standard was added and the following procedure was followed as in (1).
(3) Drawing of standard curve
Accurately transferring a proper amount of standard working solution into a 50mL centrifuge tube, and performing the steps of (1) and (II) except for no sample, so that the final solution concentration of the semicarbazide is respectively 0.0005. mu.g/mL, 0.001. mu.g/mL, 0.002. mu.g/mL, 0.005. mu.g/mL, 0.010. mu.g/mL and 0.020. mu.g/mL, and performing LC-MS/MS analysis to draw a standard curve.
4. Conditions of the instruments used for the experiment
The chromatographic conditions were as follows:
a) a chromatographic column: kinetex XB C18Reversed phase chromatographic column, 2.1mm × 100mm, 2.6 μm;
b) column temperature: room temperature;
c) flow rate: 0.3 mL/min;
d) sample introduction amount: 10 mu L of the solution;
e) mobile phase: a: methanol, B: 2mmol/L aqueous ammonium acetate solution, gradient elution program is shown in Table 1.
The mass spectrometry conditions were as follows:
a) ionization mode: electrospray ion source (ESI);
b) the scanning mode is as follows: scanning positive ions;
c) ion source temperature: 550 ℃;
d) collision gas (CAD): medium;
e) air Curtain gas (curtaingas): 30 psi;
f) atomizing Gas (Gas 1): 35 psi;
g) auxiliary heating Gas (Gas 2): 35 psi;
h) scanning mode: selective Reaction Monitoring (SRM), with parent, daughter and collision energies, shown in table 2;
5. sample detection and result calculation
(1) Qualitative determination
Under the same test conditions, the retention time of the target compound in the sample solution and the retention time of the target compound in the standard working solution are within +/-5%, and the relative abundance of the detected qualitative ions is consistent with the relative abundance of the qualitative ions in the standard working solution with similar concentration, and the deviation is in accordance with the requirement of table 3.
(2) Quantitative determination
Taking the sample solution and the corresponding standard working solution, performing multi-point calibration, and quantifying by peak area according to an internal standard method. The response values of the semicarbazide in the standard solution and the sample solution should be within the linear range detected by the instrument.
(3) Blank experiment
The measurement was carried out under the above-mentioned conditions and procedures except that no sample was added.
(4) Result calculation and presentation
Taking the concentration as a horizontal coordinate, taking the ratio of peak areas of an external standard and a corresponding internal standard as a vertical coordinate, drawing a standard curve, then analyzing and processing sample peaks to obtain the concentration of a target compound in a sample liquid to be detected, and calculating the residual quantity of the target compound in the sample according to a formula (a):
in the formula:
x is the residual amount of the target compound in the sample, μ g/kg;
c-concentration of target compound in sample solution, ng/mL;
v, the volume of the final sample liquid is determined to be mL;
f, dilution multiple;
m-sample weight, g.
Note: blank values need to be deducted from the calculation result.
The total amount of semicarbazide and the bound semicarbazide content were directly calculated according to the formula (a), and the free semicarbazide content was the total amount of semicarbazide-bound semicarbazide content.
6. Results
The method has semicarbazide detection limit of 0.5 mug/kg and quantitative limit of 1.0 mug/kg. The semicarbazide has a recovery rate of 70-120% in the added concentration range of 1.0-10 mug/kg, and the relative standard deviation in batches and the relative standard deviation between batches of the method are both less than or equal to 15%.
The results of measuring the recovery and precision of semicarbazide in scallop tissue are shown in Table 4 and FIGS. 1-5.
TABLE 4 measurement of recovery and precision of scallop tissue standard
7. Applications of
The established method is used for measuring the existence form and the content of the semicarbazide in the scallop tissues exposed for 20 days in the environment, and the results are shown in the table 5.
TABLE 5 semicarbazide content in scallop tissue after 20 days semicarbazide exposure
Claims (4)
1. A method for measuring the existence form and content of semicarbazide in shellfish tissues is characterized in that the method comprises the steps of measuring the total amount of semicarbazide, drawing a binding state semicarbazide, drawing a standard curve, and using instrument conditions, qualitative determination and quantitative determination; the method comprises the following specific steps:
1) measurement of Total amount of semicarbazide
(1) Sample hydrolysis and derivatization
Adding hydrochloric acid, derivatization reagent and SEM-13C,15N2Carrying out hydrolysis and derivatization for 16h at 37 ℃ on an isotope internal standard, taking out and placing to room temperature;
(2) extraction and purification
Adding a dipotassium phosphate solution into the sample treated in the step (1) to ensure that the pH value of the sample solution is 7.0-7.5, adding ethyl acetate liquid-liquid extraction, carrying out vortex mixing, centrifuging, taking supernate, transferring the supernate into nitrogen for drying at 45 ℃, adding a methanol aqueous solution with the volume ratio of 5% to carry out vortex oscillation to dissolve residues, transferring the supernate into an ultrafiltration centrifugal tube, centrifuging at 14000r/min, and determining by using an efficient liquid chromatography-tandem mass spectrometer;
2) determination of bound semicarbazide
(1) Washing the sample with methanol/water, methanol and water respectively to obtain free semicarbazide;
(2) then, measuring according to the step 1);
3) drawing of standard curve
Respectively transferring semicarbazide standard solutions with certain concentrations, carrying out sample hydrolysis and derivatization, extraction and purification according to the operation steps of (1) and (2) in the step 1) except that no sample is added, then carrying out LC-MS/MS analysis, and drawing a standard curve;
4) conditions of the apparatus used
The chromatographic conditions were as follows:
a) a chromatographic column: c18A reversed phase chromatographic column with the diameter of 2.1mm multiplied by 100mm and the diameter of 2.6-5 mu m;
b) column temperature: room temperature;
c) flow rate: 0.3 mL/min;
d) sample introduction amount: 10 mu L of the solution;
e) mobile phase: a: methanol, B: 2mmol/L ammonium acetate in water, gradient elution procedure is shown in Table 1;
TABLE 1 mobile phase gradient elution conditions
The mass spectrometry conditions were as follows:
an electrospray ion source; spraying voltage: 5500V, positive ion scan; ion source temperature: 550 ℃, collision gas: medium; air curtain air: 30 psi; atomizing: 35 psi; auxiliary heating gas: 35 psi; ion mode: multiple reaction selective monitoring, wherein parameters of selected reaction monitoring parent ions, ionic ions, collision energy and cone hole voltage mass spectra are shown in a table 2;
table 2 selective reaction monitoring of parent ions, daughter ions and collision energy
Note: represents quantitative fragment ions
5) Qualitative and quantitative
(1) Qualitative determination
Under the same test conditions, the deviation of the retention time of the target compound in the sample solution and the retention time of the target compound in the standard working solution is within +/-5 percent, and the relative abundance of the detected qualitative ions is consistent with the relative abundance of the qualitative ions in the standard working solution with similar concentration, and the deviation is in accordance with the requirement of table 3;
TABLE 3 base peak to sub-intense fragment ion abundance ratio requirements
(2) Quantitative determination
Taking a sample solution and a corresponding standard working solution, performing multi-point calibration, and quantifying according to an internal standard method; the response values of the target compounds in the standard solution and the sample solution are within the linear range detected by the instrument; the semicarbazide is quantified by taking an isotope thereof as an internal standard;
(3) blank experiment
The measurement is carried out according to the measurement conditions and steps except that no sample is added;
(4) result calculation and presentation
The residual amount of the target compound in the sample was calculated according to the formula (a):
in the formula:
x is the residual amount of the target compound in the sample, μ g/kg;
c-concentration of target compound in sample solution, ng/mL;
v, the volume of the final sample liquid is determined to be mL;
f, dilution multiple;
m-sample size, g;
note: blank values need to be deducted from the calculation result;
the total amount of semicarbazide and the bound semicarbazide content were calculated according to the formula (a), and the free semicarbazide content was the total amount of semicarbazides-bound semicarbazides content.
2. The method according to claim 1, wherein the derivatizing reagent in step (1) is 2-nitrobenzaldehyde at a concentration of 100 mmol/L2-nitrobenzaldehyde, and the volume to sample mass ratio is 3:40(mL: g).
3. The method according to claim 1, wherein the concentration of hydrochloric acid in the step (1) is 0.5mol/L, and the mass ratio of the volume of the added hydrochloric acid to the sample is 5:2(mL: g).
4. The method according to claim 1, wherein the dipotassium hydrogen phosphate solution added in the step (2) has a concentration of 1.0mol/L and a ratio of volume added to mass of the sample of 3:1(mL: g).
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|---|---|---|---|---|
| CN103698435A (en) * | 2013-12-30 | 2014-04-02 | 江苏省环境监测中心 | Method for detecting ultrahigh performance liquid chromatography-triple quadrupole mass spectrum of nitrofuran metabolic product |
| CN104297384A (en) * | 2014-09-05 | 2015-01-21 | 北京华都肉鸡公司 | Detection method of nitrofuran drug metabolites in meat product |
| CN106124653A (en) * | 2016-06-16 | 2016-11-16 | 中国水产科学研究院黄海水产研究所 | 5 kinds of Nitrofuran metatolites and the detection method of the many residuals of chloromycetin in shrimp |
-
2020
- 2020-09-29 CN CN202011053485.2A patent/CN112198254A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698435A (en) * | 2013-12-30 | 2014-04-02 | 江苏省环境监测中心 | Method for detecting ultrahigh performance liquid chromatography-triple quadrupole mass spectrum of nitrofuran metabolic product |
| CN104297384A (en) * | 2014-09-05 | 2015-01-21 | 北京华都肉鸡公司 | Detection method of nitrofuran drug metabolites in meat product |
| CN106124653A (en) * | 2016-06-16 | 2016-11-16 | 中国水产科学研究院黄海水产研究所 | 5 kinds of Nitrofuran metatolites and the detection method of the many residuals of chloromycetin in shrimp |
Non-Patent Citations (5)
| Title |
|---|
| CHRISTOF VAN POUCKE 等: "Investigation into the possible natural occurence of semicarbazide in Macro brachium rosenbergii prawns", 《J. AGRIC. FOOD CHEM.》 * |
| 中华人民共和国农业部: "《中华人民共和国国家标准 农业部783号公告-1-2006》", 19 December 2006 * |
| 于召强 等: "四十里湾海洋贝类对氨基脲的生物富集特性", 《海洋环境科学》 * |
| 宋蓓 等: "日本沼虾中氨基脲含量检测方法的优化", 《食品安全质量检测学报》 * |
| 李虞明: "超高效液相色谱_质谱联用仪检测贝类产品中硝基呋喃代谢物", 《食品安全导刊》 * |
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