CN115290764B - Method for simultaneously detecting 10 kinds of hormone residues in edible tissues of cattle and sheep by UPLC-MS/MS - Google Patents
Method for simultaneously detecting 10 kinds of hormone residues in edible tissues of cattle and sheep by UPLC-MS/MS Download PDFInfo
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Abstract
The invention discloses a method for simultaneously detecting 10 kinds of assimilation hormone residues in edible tissues of cattle and sheep by UPLC-MS/MS, which relates to the technical field of assimilation hormone detection and comprises the following steps: taking edible tissue of cattle and sheep, homogenizing to obtain tissue homogenate; adding an ammonium acetate buffer solution into the edible muscle tissue homogenate, adding the ammonium acetate buffer solution and beta-glucuronidase into the edible non-muscle tissue homogenate, and carrying out water bath and cooling; adding ethyl acetate, vortexing, ultrasonic processing, vortexing, centrifuging, collecting supernatant, repeating the above vortexing, ultrasonic processing, vortexing, centrifuging steps, mixing the supernatants, and freezing; freezing and centrifuging the frozen supernatant, evaporating to dryness in a water bath, adding acetonitrile dissolution residues, performing ultrasonic dissolution, diluting with water, freezing and centrifuging, and taking the supernatant as an extracting solution; purifying the extract by a C18 solid phase extraction column; UPLC-MS/MS detection is adopted. The method is simple, convenient, quick and sensitive, has low detection limit, and can meet the requirements of quick detection and trace analysis in veterinary drug residue analysis.
Description
Technical Field
The invention relates to the technical field of anabolic hormone detection, in particular to a method for simultaneously detecting 10 kinds of anabolic hormone residues in edible tissues of cattle and sheep by UPLC-MS/MS.
Background
Anabolic hormone is a steroid substance with strong protein assimilation, can inhibit dissimilation or oxidative metabolism and can raise protein deposition, and has important residual toxicological significance. The most common assimilating hormones in livestock and poultry raising industry mainly comprise testosterone, methyltestosterone, progesterone, trenbolone, boehmeria, nandrolone, emasculone, kang Lilong, nandrolone propionate, testosterone propionate, nandrolone phenylpropionate and the like. Currently, the most widely used methods for detecting animal-derived isohormone residues include High Performance Liquid Chromatography (HPLC), gas chromatography tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), and High Performance Capillary Electrophoresis (HPCE), time-resolved fluoroimmunoassay (TR-FIA), chemiluminescent immunoassay (CLIA) and the like. Only HPLC, GC-MS, LC-MS/MS and ELISA are reviewed here.
(1) High Performance Liquid Chromatography (HPLC)
HPLC is popular in analysis and detection, and has the characteristics of high speed, high pressure, high efficiency and the like, and some macromolecular compounds with poor thermal stability and strong polarity can be analyzed by HPLC. In a laboratory, HPLC is commonly used for detecting and analyzing single drug residues in samples, and has the advantages of simple operation and wide application range. However, the HPLC method requires a long time to detect various hormone residues due to low sensitivity and poor specificity, and cannot meet the requirements of rapid detection and trace analysis in veterinary drug residue analysis. Therefore, the substances to be detected are subjected to gradient elution by a liquid chromatograph, and then are scanned by a mass spectrometer for analysis and detection of the medicines.
Wang Jingzhi and the like, the residual quantity of estradiol, estrone, estriol and testosterone in eggs are simultaneously measured by adopting an ultraviolet absorption-high performance liquid chromatography, the samples are extracted by methanol, the recovery rate of the samples is 79.23-102.26% by a method of 4 hormones, the detection limit is 0.06-0.17 mg/kg, the relative standard deviation is 1.05-4.21%, and the method has high accuracy and simple operation. Wang Lian and the like establish a method for detecting 11 steroid hormones in meat products and milk by using a diode array-high performance liquid chromatography, the samples are extracted by acetonitrile ultrasonic, and are enriched by adopting SPE small columns after enzymolysis and dilution, the detection limit of the 11 hormones is 0.009-0.020 mg/kg, and the average recovery rate is 51.0% -107.0%. Zhang Lan and the like establish a high performance liquid chromatography method for simultaneously separating and detecting 10 protein assimilation hormones, adopts a C18 reversed phase chromatographic column, adopts acetonitrile and water as mobile phases, separates 10 medicines in a gradient elution mode, analyzes the medicines by an ultraviolet absorption detector, realizes rapid separation of components to be detected within 10min, has the detection limit of 0.01-0.10 mug/mL, and has the average recovery rate of 70.3-120 percent.
(2) Gas chromatography tandem mass spectrometry (GC-MS)
The principle of gas chromatography tandem mass spectrometry is that a mixed substance is separated into individual components by a gas chromatograph and then enters a mass spectrometer for detection. The method is only suitable for detecting the medicines with good thermal stability, volatilizable and higher boiling point, but most hormone medicines have poor thermal stability and are difficult to volatilize, so that the GC-MS is not suitable for residual analysis and detection of the hormone medicines. And (3) detecting 9 steroid anabolic hormones remained in animal muscle tissues by adopting a gas chromatography tandem mass spectrometry method, wherein after solid phase extraction purification and derivatization, a sample is subjected to gas chromatography tandem mass spectrometry analysis detection, the quantitative limit of the 9 steroid hormones such as methyltestosterone, testosterone propionate and the like is 1.0-2.0 mug/kg, the average recovery rate is 62.5% -80.5%, the relative standard deviation is 12.5% -26.8%, and the requirements of residual detection can be met.
(3) Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
LC-MS/MS is based on liquid chromatography coupled to one or two mass spectrometers, combining the advantages of high separation capacity of HPLC and strong structural discrimination of MS. After the liquid chromatography is used for effectively separating and eluting a plurality of compounds, the compounds enter a mass spectrometer for electrospray or atmospheric pressure chemical ionization, so that trace chemical substances in complex matrixes such as animal tissues, blood plasma, urine and the like can be accurately determined and quantified. Compared with liquid chromatography, the hormone medicine can obtain lower detection limit and lower quantitative limit in an LC-MS/MS method, and the sensitivity of the instrument is higher. Compared with GC-MS, the medicine does not need to carry out complicated derivatization treatment before entering LC-MS/MS, and the problem that the anabolic hormone medicine is easy to limit the volatility and the thermal stability of a sample in the GC-MS is solved. For several years, with the development of pretreatment technology, LC-MS/MS is widely used in residual detection analysis of protein assimilation hormone to monitor illicit addition use of assimilation hormone in animal-derived foods.
He Limin and the like establish an ultra-high performance liquid chromatography-tandem mass spectrometry method for measuring steroid hormone medicines remained in animal muscle tissues and eggs, extract a sample matrix by tert-butyl methyl ether under alkaline conditions, freeze, centrifuge, degreasing and purifying, and perform gradient elution by taking acetonitrile and formic acid water solution as mobile phases, wherein the detection limit of the method is 0.3-0.4 mug/Kg, the recovery rate is more than 50%, and the relative standard deviation is less than 16%. YouY et al established a rapid assay for two androgens in horse plasma using ultra high performance liquid chromatography-tandem mass spectrometry, liquid-liquid extraction of horse urine using a mixed solution of methyl tert-butyl ether and ethyl acetate (50:50, v/v), separation elution on a C18 chromatographic column, and detection on a triple quadrupole mass spectrometer using a positive electrospray ionization mode and a selected reaction monitoring scan, the detection limit for both analytes being 50pg/0.5mL. Kaabiaz et al established a rapid assay for detecting more than 20 steroid hormones in serum and plasma in trace amounts (less than 50 pg/mL) and was useful for the detection of agonists in cattle and horses. After the pretreatment step, the samples were submitted to Solid Phase Extraction (SPE) and then analyzed using UPLC-MS/MS. Chen Junhui A super high performance liquid chromatography-tandem mass spectrometry method for 12 steroid hormones in beef and chicken is established, the sensitivity, recovery rate and reproducibility of the method under two different matrix purification modes of a solid phase extraction method and a matrix solid phase extraction method are compared, the traditional solid phase extraction method is optimized, the optimal condition of the matrix solid phase extraction method is researched, the detection limit of the solid phase extraction method is between 0.3 and 1.0 mug/L, the average recovery rate is between 64.75 and 112.53 percent, the detection limit of the matrix solid phase extraction method is between 0.2 and 0.8 mug/L, and the average recovery rate is between 72.7 and 123.3 percent. Gao Xudong A high performance liquid chromatography-tandem mass spectrometry of androgens and anabolic hormones in livestock and poultry meat is established, acetonitrile-0.1% of formic acid water is used as a mobile phase for gradient elution, ZORBAX Eclipse Plus C chromatographic column pairs are used for separation, positive ion mode scanning is adopted, a multi-reaction monitoring mode (MRM) is adopted for collecting signals, the detection limit of 10 target hormones is 1ng/kg, the standard deviation is between 51.06% and 91.34%, and the relative standard deviation is between 0.64% and 3.62%, so that the method is rapid and simple compared with the traditional pretreatment technology. Wang Fei and the like rapidly measure 3 steroid assimilating hormones in beef and beef kidney by adopting an ultrahigh pressure liquid chromatography-tandem mass spectrometry method, the method adopts tert-butyl methyl ether as an extraction solvent, adopts a method of freezing a sample at a low temperature after rotary evaporation and then centrifuging to remove fat, does not need solid phase extraction and purification, and has the average recovery rate of 73.6% -82.8% and the relative standard deviation of less than 10% at 3 additive concentration levels of 1.0, 2.0 and 10.0 mug/kg.
(4) Enzyme-linked immunosorbent assay (ELISA)
ELISA is a qualitative and quantitative analysis method by utilizing the specific combination of antibodies and antigens, and the method has the advantages of no need of using complex instruments, low cost and high efficiency, is suitable for rapid detection and analysis of a large number of samples, and has little influence on the purification degree of a matrix. For residual detection of multiple targets, it is often necessary to use multiple different kits, which have limitations in multi-residual analysis of drugs.
10 androgenic protein assimilating hormones in this study: testosterone, methyltestosterone, trenbolone, boehmeria, nandrolone, emasculone, kang Lilong, nandrolone propionate, testosterone propionate, nandrolone propionate. In the laboratory sampling inspection of the inventor, the current situation that assimilation hormone exceeds standard exists in the edible organization of cattle and sheep in the market, and the establishment of the national detection standard is imperative.
However, the detection method is complicated and difficult to implement in the multi-residue detection process of the edible tissues of the cattle and the sheep, and no method for simultaneously detecting the 10 androgen protein assimilation hormones in the edible tissues of the cattle and the sheep is available at present, so that the method for determining the residual quantity of the 10 assimilation hormones in the edible tissues (including muscles, kidneys, livers and fats) of the cattle and the sheep by using the ultra-high performance liquid chromatography-tandem mass spectrometry is established, and technical reserve is provided for detecting the residual of the assimilation hormones in animal-derived foods in China.
Disclosure of Invention
Based on the technical problems in the background technology, the invention provides a method for simultaneously detecting 10 kinds of isohormone residues in edible tissues of cattle and sheep by UPLC-MS/MS.
The invention provides a method for simultaneously detecting 10 kinds of homologous hormone residues in edible tissues of cattle and sheep by UPLC-MS/MS, which comprises the following steps:
sampling: taking edible tissues of cattle and sheep, cutting the edible tissues, and homogenizing the edible tissues to obtain tissue homogenate;
extracting: adding an ammonium acetate buffer solution with the pH of=5.2 to the edible muscle tissue homogenate, and adding an ammonium acetate buffer solution with the pH of=5.2 and beta-glucuronidase to the edible non-muscle tissue homogenate; then water bath, cooling, adding ethyl acetate into tissue homogenate, vortex, ultrasonic extraction, vortex, centrifuging, taking supernatant, adding acetonitrile solution of methanol into precipitate, repeating the steps of vortex, ultrasonic extraction, vortex and centrifuging, combining the supernatant, and freezing at-80 ℃ for 2 hours for standby; then freezing and centrifuging the frozen supernatant, evaporating to dryness in a water bath, finally adding acetonitrile dissolution residues, performing ultrasonic dissolution, diluting with water, freezing and centrifuging, and taking the supernatant as an extracting solution;
purifying: purifying the extract on a C18 solid phase extraction column, activating with methanol and water, passing the extract through the column, eluting with 30vt% methanol water solution, drying under negative pressure, eluting with acetonitrile, and collecting the eluate; blowing the eluent with nitrogen, adopting 30vt percent acetonitrile water solution to carry out ultrasonic redissolution, and passing through a 0.22 mu m filter membrane for UPLC analysis;
and (3) measuring: adopting UPLC-MS/MS detection;
the UPLC chromatographic conditions are: the chromatographic column is Agilent ZORBAX SB-Aq,2.1mm×150mm,3.5 μm; column temperature 37 ℃; mobile phase a was an acetonitrile solution of 0.1vt% formic acid and mobile phase B was an aqueous solution of 0.1vt% formic acid; the gradient elution procedure was: 0.0 to 1.0min,60vt percent of A;1.0 to 3.0min,45vt percent of A; 3-4 min,25vt%A; 4-8 min,0vt% A; 8-10.5 min,60vt%A;10.5 to 12.5min,60% vt A; sample injection volume 5. Mu.L; the flow rate is 0.25mL/min;
MS/MS conditions: adopting an electrospray ion source positive ion detection mode, a mass spectrum scanning mode and a multi-reaction monitoring mode; atomization air pressure 65psi; electrospray voltage 5500V; air curtain pressure 30psi; the flow rate of the auxiliary device is 60L/rain; collision cell pressure 8psi; the ion source temperature was 550 ℃.
Preferably, the edible tissue comprises muscle tissue, adipose tissue, liver tissue, kidney tissue.
Preferably, the 10 assimilating hormones are testosterone, methyltestosterone, trenbolone, boehmeria, nandrolone, emasculone, kang Lilong, nandrolone propionate, testosterone propionate, nandrolone phenylpropionate.
Preferably, in the extraction step, the mass-volume ratio g/mL of the edible tissue of the cattle and the sheep to the ammonium acetate buffer solution is 1:2-3; the mass volume ratio of the edible tissue of the cattle and the sheep to the beta-glucosaccharase is 1:0.04-0.06.
Preferably, in the method, the centrifugal speed is 11000-13000r/min, and the centrifugal speed is 8-12min; wherein the temperature of the refrigerated centrifugation is-5 ℃.
The beneficial effects are that: the invention has established a UPLC-MS/MS and examined 10 kinds of anabolic hormone residual quantity in the edible tissue of cattle and sheep (including muscle, kidney, liver, fat) at the same time, homogenate sample, add ammonium acetate buffer and beta-glucuronidase (muscle tissue does not need to add), add ethyl acetate to draw, and adopt the ultrasonic treatment to make the anabolic hormone that remains on glass redissolve in blow-dry or spin-steaming process, and freeze the supernatant to remove fat first and transfer to the centrifuge to freeze and centrifuge rapidly, improve the recovery rate of anabolic hormone through the above sample pretreatment step, and then reduce the quantitative limit; especially in the extraction process of sample pretreatment, acetonitrile is firstly adopted to dissolve residues, after sufficient ultrasonic dissolution, water is added for dilution, and compared with the direct dilution by adopting acetonitrile aqueous solution, the recovery rate can be improved by about 10 percent; the detection limit of the assimilation hormone in the tissue is 0.1-0.5 mug/kg, the quantitative limit is 0.5-1 mug/kg, which is far higher than the quantitative limit of 10 mug/kg in the prior method, and the invention also obtains the recovery rate data with the quantitative limit of 0.5 mug/kg and below 1 mug/kg, which can meet the requirements of national regulation and can not be detected, and the invention can not only detect the residual amount of the assimilation hormone in the muscle tissue of cattle and sheep, but also be used for detecting kidney, liver and fat tissue, and is also suitable for poultry such as chickens, pigs and the like; the method is simple, convenient, quick and sensitive, can meet the requirements of quick detection and trace analysis in veterinary drug residue analysis, and provides an effective detection method for detecting the residues of anabolic hormone in animal-derived foods in China.
Drawings
FIG. 1 is a UPLC spectrum of 6 of 10 assimilation hormone standards of the invention detected simultaneously;
FIG. 2 is a UPLC spectrum of 4 of the 10 assimilation hormone standards of the present invention detected simultaneously.
Detailed Description
The technical scheme of the invention is described in detail through specific embodiments.
Example 1
1. UPLC-MS/MS conditions
(1) Ultra-high performance liquid chromatography conditions:
flow rate: 0.25mL/min
Chromatographic column: agilent ZORBAX SB-Aq (2.1 mm. Times.150 mm,3.5 μm)
Sample injection volume: 5 mu L
Column temperature: 37 DEG C
Mobile phase: the phase A is 0.1% formic acid acetonitrile, the phase B is 0.1% formic acid aqueous solution, a gradient elution mode is adopted, and the gradient elution program is shown in table 1.
TABLE 1 gradient elution program table
| Time (min) | Flow rate | A％ | B％ |
| 0 | 0.25 | 60 | 40 |
| 1.0 | 0.25 | 45 | 55 |
| 3.0 | 0.25 | 45 | 55 |
| 4.0 | 0.25 | 25 | 75 |
| 8.0 | 0.25 | 0 | 100 |
| 10.5 | 0.25 | 60 | 40 |
| 12.5 | 0.25 | 60 | 40 |
(2) Mass spectrometry conditions:
adopting an electrospray ion source positive ion detection mode; a primitive scanning mode; multiple reaction monitoring mode. The main mass spectrum conditions are:
atomization gas pressure (GSl, nebulizer gas 1): 65psi;
electrospray voltage (IS, ionospay): 5500V:
curtain air pressure (CUR): 30psi;
helper flow rate (GS 2, ion source gas 2): 60L/rain;
collision cell pressure (CAD): 8psi;
ion source temperature (TEM, the source temperature): 550 ℃;
mass spectrometry of 10 kinds of assimilating hormones was performed to determine the declustering voltage, collision energy, and mass of parent ion Q1 and child ion Q3, and retention time of each medicine are shown in Table 2.
TABLE 2 Multi-reaction monitoring Mass Spectrometry parameters for target Compounds
Note that: "a" is labeled as a quantitative ion pair.
2. Sample pretreatment
The pretreatment method comprises the following steps: accurately weighing 2.00g of homogenized tissue in a 50mL polypropylene centrifuge tube, adding 5.0mL (pH=5.2) of ammonium acetate buffer solution and 100 mu L (muscle tissue is not used) of beta-glucosidase, and cooling to room temperature after water bath at 37 ℃ for 12 hours; and adding 10mL of ethyl acetate into the tissue homogenate, carrying out vortex for 30s, carrying out ultrasonic extraction for 10min, continuing to vortex for 3min, and centrifuging at 12000r/min for 10min. The supernatant was transferred to a 50mL centrifuge tube and the above extraction steps were repeated. Mixing the extractive solutions, standing at-80deg.C, and freezing for 2 hr. The centrifugation was continued at 12000r/min at-5℃for 10min. The supernatant is distilled to near dryness in a water bath at 37 ℃, 1mL of acetonitrile dissolution residue is added, sufficient ultrasonic dissolution (the sufficient ultrasonic is key, the recovery rate of ultrasonic dissolution and non-ultrasonic dissolution is 20% -30%) is added, 3mL of water is added for dilution (part of the method is directly diluted by 25% acetonitrile aqueous solution, the recovery rate is 10% different), and the supernatant is taken for standby after refrigerated centrifugation at a rotation speed of 12000r/min for 10min. The solution is added into a C18 solid phase extraction column for purification (activated by 3mL of methanol and 3mL of water in sequence, and eluted by 6mL of 30% methanol water), and after being pumped to dryness under negative pressure for 3min, 4mL of acetonitrile is added for elution, and the eluent is slowly dried by nitrogen in a water bath at 37 ℃. 1.0mL of 30% acetonitrile-water (30:70, v/v) was added and sonicated well (with well sonication being critical, recovery rate of sonicated and non-sonicated was 20% -30%), and the supernatant was filtered through a 0.22 μm filter membrane, awaiting detection.
3. Standard solution preparation
Respectively dissolving 10 assimilation hormone reference substances in 30vt% acetonitrile water solution, ultrasonically dissolving, and filtering with 0.22 μm filter membrane to obtain standard solution.
4. Sample detection
The edible organizations of cattle and sheep purchase from the large supermarket in Xingxiang county. After the sample is selected and primarily treated, the sample is homogenized by a homogenizer, and then is packaged by a sample bag and stored in a refrigerator at the temperature of minus 20 ℃. Each tissue is selected from several batches and initially screened by liquid chromatography using existing literature methods, and as the individual samples contain the target anabolic hormone, it is necessary to reject the sample with the target compound, leaving a sample without the target compound.
5. Methodology investigation
5.1 quantitative limit and detection limit
By adopting the detection method, liquid chromatogram of 10 assimilation hormone (testosterone, methyltestosterone, trenbolone, bombricus, nandrolone, metaandrosterone, kang Lilong, nandrolone propionate, testosterone propionate, and nandrolone phenylpropionate) standard substances are obtained, and are shown in figures 1-2, which are detection results of the same needle insertion sample. The test covers 8 samples in total, and the result shows that the detection limit of the anabolic hormone in tissues is generally 0.1-0.5 mug/kg and the quantitative limit is 0.5-1 mug/kg; the specific results are shown in tables 3 and 4.
TABLE 3 detection limit LOD and quantitative limit LOQ (Unit: μg/kg) of assimilating hormone in bovine edible tissues
TABLE 4 detection limit LOD and quantitative limit LOQ (Unit: μg/kg) of assimilating hormone in edible sheep tissue
5.2 recovery and precision
The average recovery rate of edible tissues (muscle, fat, liver and kidney) of chickens, cattle and sheep at Low (LOQ), medium (20 mug/kg) and high (100 mug/kg) three adding levels is 61.9% -101.6%, the precision in batch is 1.6% -21.2%, and the precision between batches is less than 13.9%. The result shows that the method has better repeatability and meets the veterinary drug residue detection requirement. Specific results are shown in tables 5-10, and recovery data of less than 0.5 and 1 μg/kg were obtained for the present invention. According to the national requirements, the quantitative limit recovery rate data are required to be approved, but most of the prior art only has 10 mug/kg recovery rate data, which does not meet the requirements of veterinary drug residue guidelines.
TABLE 5 recovery rate and coefficient of variation of 10 assimilation hormones in bovine muscle
TABLE 6 recovery rate, coefficient of variation and matrix effect of 10 assimilating hormones in bovine fat
TABLE 7 recovery rate, coefficient of variation and matrix effect of 10 assimilating hormones in bovine liver
TABLE 8 recovery rate, coefficient of variation and matrix effect of 10 assimilating hormones in sheep muscle
TABLE 9 recovery rate and coefficient of variation of 10 assimilation hormones in sheep liver
TABLE 10 recovery rate, coefficient of variation and matrix effect of 10 assimilation hormones in sheep fat
5.3 Standard Curve and quantitative Range
Assimilation hormone in edible tissues (muscle, fat, liver and kidney) of pigs, cattle and sheep is in the range of LOQ (0.5-1.0 mug/kg) to 100 mug/kg, the method shows good linear relation, the correlation coefficient is more than 0.99, and the residual detection requirement is met; the linear regression equations of assimilating hormones in each edible tissue are shown in tables 11 to 18.
TABLE 11 Linear regression equation data for bovine liver
Table 12 linear regression equation data for beef
| Assimilation hormone | Concentration range (ug/kg) | Linear regression equation | Correlation coefficient |
| Testosterone (Testosterone) | 0.5～100 | y＝1.72×10 5 x+2.68×10 5 | R 2 ＝0.9978 |
| Testosterone propionate | 1～100 | y＝1.34×10 4 x-1.79×10 4 | R 2 ＝0.9934 |
| Methyltestosterone | 0.5～100 | y＝1.28×10 5 x+2.22×10 5 | R 2 ＝0.9946 |
| Kang Lilong | 0.5～100 | y＝1.95×10 5 x-5.66×10 4 | R 2 ＝0.9988 |
| Trenbolone (Bo-Bo) | 0.5～100 | y＝1.14×10 5 x+1.87×10 5 | R 2 ＝0.9946 |
| Bo Lumbricus | 0.5～100 | y＝3.05×10 5 x+7.00×10 5 | R 2 ＝0.9928 |
| Mexiong ketone | 0.5～100 | y＝5.54×10 5 x-6.85×10 4 | R 2 ＝0.9999 |
| Norong | 0.5～100 | y＝5.10×10 4 x+5.28×10 4 | R 2 ＝0.9989 |
| Phenylpropanoid nandrolone | 0.5～100 | y＝1.30×10 5 x+1.56×10 5 | R 2 ＝0.9996 |
| Norilon propionate | 1～100 | y＝2.60×10 4 x-1.69×10 4 | R 2 ＝0.9966 |
TABLE 13 Linear regression equation data for bovine kidneys
| Assimilation hormone | Concentration range (ug/kg) | Linear regression equation | Correlation coefficient |
| Testosterone (Testosterone) | 1～100 | y＝1.46×10 5 x+3.34×10 5 | R 2 ＝0.9951 |
| Testosterone propionate | 1～100 | y＝1.41×10 4 x+1.45×10 4 | R 2 ＝0.9993 |
| Methyltestosterone | 1～100 | y＝6.51×10 4 x+9.06×10 4 | R 2 ＝0.9956 |
| Kang Lilong | 0.5～100 | y＝1.78×10 5 x+4.27×10 5 | R 2 ＝0.9939 |
| Trenbolone (Bo-Bo) | 0.5～100 | y＝1.04×10 5 x+2.08×10 5 | R 2 ＝0.9947 |
| Bo Lumbricus | 0.5～100 | y＝2.50×10 5 x+5.18×10 5 | R 2 ＝0.9936 |
| Mexiong ketone | 1～100 | y＝2.01×10 5 x+3.85×10 5 | R 2 ＝0.9925 |
| Norong | 0.5～100 | y＝4.53×10 4 x+9.01×10 4 | R 2 ＝0.9950 |
| Phenylpropanoid nandrolone | 0.5～100 | y＝1.09×10 5 x+1.44×10 5 | R 2 ＝0.9979 |
| Norilon propionate | 1～100 | y＝2.45×10 4 x+4.98×10 4 | R 2 ＝0.9953 |
TABLE 14 Linear regression equation data for bovine fat
| Assimilation hormone | Concentration range (ug/kg) | Linear regression equation | Correlation coefficient |
| Testosterone (Testosterone) | 0.5～100 | y＝1.82×10 5 x+2.74×10 5 | R 2 ＝0.9979 |
| Testosterone propionate | 1～100 | y＝1.52×10 4 x-1.86×10 3 | R 2 ＝1 |
| Methyltestosterone | 0.5～100 | y＝1.55×10 5 x+1.00×10 5 | R 2 ＝0.9990 |
| Kang Lilong | 0.5～100 | y＝2.15×10 5 x+2.85×10 5 | R 2 ＝0.9979 |
| Trenbolone (Bo-Bo) | 0.5～100 | y＝1.14×10 5 x+1.51×10 5 | R 2 ＝0.9973 |
| Bo Lumbricus | 0.5～100 | y＝2.94×10 5 x+7.19×10 5 | R 2 ＝0.9947 |
| Mexiong ketone | 0.5～100 | y＝4.89×10 5 x+9.49×10 4 | R 2 ＝0.9999 |
| Norong | 0.5～100 | y＝5.77×10 4 x+7.21×10 4 | R 2 ＝0.9987 |
| Phenylpropanoid nandrolone | 0.5～100 | y＝1.64×10 5 x-2.80×10 4 | R 2 ＝0.9999 |
| Norilon propionate | 1～100 | y＝3.06×10 4 x+2.77×10 4 | R 2 ＝0.9996 |
TABLE 15 Linear regression equation data for sheep liver
| Assimilation hormone | Concentration range (ug/kg) | Linear regression equation | Correlation coefficient |
| Testosterone (Testosterone) | 0.5～100 | y＝1.84×10 5 x-1.71×10 5 | R 2 ＝0.9986 |
| Testosterone propionate | 1～100 | y＝1.53×10 4 x-2.18×10 4 | R 2 ＝0.9980 |
| Methyltestosterone | 0.5～100 | y＝1.34×10 5 x-1.25×10 5 | R 2 ＝0.9984 |
| Kang Lilong | 0.5～100 | y＝2.05×10 5 x-3.92×10 4 | R 2 ＝0.9936 |
| Trenbolone (Bo-Bo) | 0.5～100 | y＝8.55×10 4 x+1.32×10 4 | R 2 ＝0.9939 |
| Bo Lumbricus | 0.5～100 | y＝3.03×10 5 x-3.39×10 5 | R 2 ＝0.9991 |
| Mexiong ketone | 0.5～100 | y＝2.91×10 5 x-4.97×10 4 | R 2 ＝0.9975 |
| Norong | 0.5～100 | y＝5.70×10 4 x-6.01×10 4 | R 2 ＝0.9991 |
| Phenylpropanoid nandrolone | 0.5～100 | y＝1.54×10 5 x-2.25×10 5 | R 2 ＝0.9998 |
| Norilon propionate | 1～100 | y＝2.33×10 4 x-2.18×10 4 | R 2 ＝0.9987 |
Table 16 Linear regression equation data for mutton
Table 17 Linear regression equation data for sheep kidney
| Assimilation hormone | Concentration range (ug/kg) | Linear regression equation | Correlation coefficient |
| Testosterone (Testosterone) | 0.5～100 | y＝1.91×10 5 x+1.12×10 5 | R 2 ＝0.9999 |
| Testosterone propionate | 1～100 | y＝1.02×10 4 x+3.87×10 2 | R 2 ＝0.9999 |
| Methyltestosterone | 0.5～100 | y＝1.59×10 5 x+1.07×10 5 | R 2 ＝0.9994 |
| Kang Lilong | 0.5～100 | y＝1.63×10 5 x+4.01×10 5 | R 2 ＝0.9963 |
| Trenbo (trenbo)Dragon | 0.5～100 | y＝1.22×10 5 x+2.54×10 5 | R 2 ＝0.9960 |
| Bo Lumbricus | 0.5～100 | y＝3.45×10 5 x+2.04×10 5 | R2＝0.9999 |
| Mexiong ketone | 0.5～100 | y＝4.46×10 5 x+5.66×10 5 | R2＝0.9981 |
| Norong | 0.5～100 | y＝6.02×10 4 x+4.32×10 4 | R 2 ＝0.9996 |
| Phenylpropanoid nandrolone | 0.5～100 | y＝1.26×10 5 x+3.47×10 4 | R 2 ＝0.9998 |
| Norilon propionate | 1～100 | y＝2.60×10 4 x+1.51×10 4 | R2＝0.9995 |
Table 18 Linear regression equation data for sheep fat
5.4 the specificity and specificity meet the veterinary drug residue detection requirements
5.5 stability
The 10 standard stock solutions of assimilation hormones are stored in a refrigerator at the temperature of minus 20 ℃ for 1 month, and all compounds are not degraded basically through the analysis of LC-MS/MS and the comparison of the peak areas of two sample injections before and after. Whereas short term stability studies have found that only a weak degradation of the labeled sample at a concentration (10. Mu.g/kg) occurs when the labeled sample is left to stand at 4℃for 24 hours. Considering the influence of temperature and time on the stability of medicaments with different physicochemical properties, in order to ensure the accuracy of test results, the processed samples should be stored in a refrigerator at 4 ℃ and detected as soon as possible.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (5)
1. A method for simultaneously detecting 10 kinds of isohormone residues in edible tissues of cattle and sheep by UPLC-MS/MS is characterized by comprising the following steps:
sampling: taking edible tissues of cattle and sheep, cutting the edible tissues, and homogenizing the edible tissues to obtain tissue homogenate;
extracting: adding an ammonium acetate buffer solution with the pH of=5.2 to the edible muscle tissue homogenate, and adding an ammonium acetate buffer solution with the pH of=5.2 and beta-glucuronidase to the edible non-muscle tissue homogenate; then water bath, cooling, adding ethyl acetate into tissue homogenate, vortex, ultrasonic extraction, vortex, centrifuging, taking supernatant, adding acetonitrile solution of methanol into precipitate, repeating the steps of vortex, ultrasonic extraction, vortex and centrifuging, combining the supernatant, and freezing at-80 ℃ for 2 hours for standby; then freezing and centrifuging the frozen supernatant, evaporating to dryness in a water bath, finally adding acetonitrile dissolution residues, performing ultrasonic dissolution, diluting with water, freezing and centrifuging, and taking the supernatant as an extracting solution;
purifying: purifying the extract on a C18 solid phase extraction column, activating with methanol and water, passing the extract through the column, eluting with 30vt% methanol water solution, drying under negative pressure, eluting with acetonitrile, and collecting the eluate; blowing the eluent with nitrogen, adopting 30vt percent acetonitrile water solution to carry out ultrasonic redissolution, and passing through a 0.22 mu m filter membrane for UPLC analysis;
and (3) measuring: adopting UPLC-MS/MS detection;
the UPLC chromatographic conditions are: the chromatographic column is Agilent ZORBAX SB-Aq,2.1mm×150mm,3.5 μm; column temperature 37 ℃; mobile phase a was an acetonitrile solution of 0.1vt% formic acid and mobile phase B was an aqueous solution of 0.1vt% formic acid; the gradient elution procedure was: 0.0 to 1.0min,60vt percent of A;1.0 to 3.0min,45vt percent of A; 3-4 min,25vt%A; 4-8 min,0vt% A; 8-10.5 min,60vt%A;10.5 to 12.5min,60% vt A; sample injection volume 5. Mu.L; the flow rate is 0.25mL/min;
MS/MS conditions: adopting an electrospray ion source positive ion detection mode, a mass spectrum scanning mode and a multi-reaction monitoring mode; atomization air pressure 65psi; electrospray voltage 5500V; air curtain pressure 30psi; the flow rate of the auxiliary device is 60L/rain; collision cell pressure 8psi; the ion source temperature was 550 ℃.
2. The method for simultaneously detecting 10 kinds of isohormone residues in edible tissues of cattle and sheep by UPLC-MS/MS according to claim 1, wherein the edible tissues comprise muscle tissues, fat tissues, liver tissues and kidney tissues.
3. The method for simultaneously detecting 10 kinds of assimilating hormone residues in edible tissues of cattle and sheep according to claim 1, wherein the 10 kinds of assimilating hormone are testosterone, methyltestosterone, trenbolone, boehmite, nandrolone, mesterone, kang Lilong, nandrolone propionate, testosterone propionate and nandrolone propionate.
4. The method for simultaneously detecting 10 kinds of isohormone residues in edible tissues of cattle and sheep by UPLC-MS/MS according to claim 1, wherein in the extraction step, the mass-volume ratio g/mL of the edible tissues of cattle and sheep to the ammonium acetate buffer solution is 1:2-3; the mass volume ratio of the edible tissue of the cattle and the sheep to the beta-glucosaccharase is 1:0.04-0.06.
5. The method for simultaneously detecting 10 kinds of isohormone residues in edible tissues of cattle and sheep by UPLC-MS/MS according to claim 1, wherein the centrifugal speed is 11000-13000r/min and the centrifugal speed is 8-12min; wherein the temperature of the refrigerated centrifugation is-5 ℃.
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