Patent Application
20210063375
This application claims the benefit of priority from Chinese Patent Application No. 201910891387.7, filed on Sep. 20, 2019. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein by reference in its entirety.
This application relates to detection of animal-derived food, and more particularly to a screening and confirmation method for veterinary drugs and additives in animal-derived food.
Veterinary drugs refer to substances used to prevent, treat, diagnose animal diseases or purposely regulate animal physiological functions, which generally include antibiotics, anthelmintics, growth promoters, antiprotozoals, trypanosome-killing substances, sedatives and β-adrenoceptor blockers.
Recently, the animal husbandry has achieved rapid development in China, which is attributed to the development and rational use of veterinary drugs. Veterinary drugs have played an important role in reducing the morbidity and mortality of livestock, promoting animal growth, improving the quality of animal-derived products and improving feed utilization. However, at present, the veterinary drugs are often abused with the dosage far exceeding the therapeutic dose for animal diseases, resulting in drug residues in the animal-derived food. Through the long-term accumulation, most of the veterinary drugs show significant toxicity. For example, residual lincosamines can cause renal dysfunction and increased drug resistance of Gram-positive bacteria; residual macrolide drugs can cause allergic reactions and also lead to the spread of strains carrying drug-resistant factors; and chloramphenicol can cause anemia, gray baby syndrome and leukemia, etc. Therefore, the European Union and China have established markers for related drug residues and maximum residue limits in different animal-derived foods, and also stipulated the types of veterinary drugs banned from animal husbandry, but the abuse of veterinary drugs still cannot be precluded.
Public notice No. 235 of the Ministry of Agriculture of China has clearly stipulated the maximum residue limit of each drug in animal tissues. However, the irrational and illegal use of veterinary drugs still can be found in the veterinary drug inspection or in the farms. In addition to veterinary drugs, the pesticide residues in animal foods also raised widespread concern in 2017 due to the “fipronil” poison egg incident. Since the residues of pesticides and veterinary drugs in animal-derived foods directly or indirectly endanger the human health, and destroy the ecological environment, it is necessary to comprehensively regulate and monitor the use of veterinary drugs and additives in animal-derived foods.
The development of the detection technology enables the simultaneous detection of multiple categories of pesticides and veterinary drugs, and also diversifies the pre-processing technologies and instrument analysis tools. Developed countries such as Europe and the United States have made a great progress in the detection of residual pesticides and veterinary drugs. Specifically, the Food and Drug Administration (FDA) have established a method for detecting more than 150 kinds of veterinary drugs in milk using LC-TOF-MS, which has a detection limit of 10 ng/mL or lower for 50% of the veterinary drugs. The U.S. Department of Agriculture have established a detection method for 120 kinds of veterinary drugs in 11 categories in bovine kidney using HPLC-Quadrupole Mass Spectrometry, which has a detection limit of 1-100 ng/g and a recovery rate of 29%-192%.
At present, the multi-residue detection in China is mainly performed by HPLC-Quadrupole Mass Spectrometry and liquid chromatography-high resolution mass spectrometry (LC-HRMS). Among them, the traditional HPLC-Quadrupole Mass Spectrometry has the characteristics of high sensitivity and accurate quantification. However, it also has the shortcomings of low resolution, high occurrence rate of false positives, limited detectable drugs and large complexity in the establishment of methods.
An object of this application is to provide a screening and confirmation method for 207 veterinary drugs and additives in animal-derived food to solve the above problems.
The technical solutions of the application are specifically described as follows.
This application provides a screening and confirmation method for veterinary drugs and additives in animal-derived food, comprising:
(1) dissolving 10 mg of each reference standard of the veterinary drugs and additives followed by diluting to 10 mL in a volumetric flask to prepare a 1 mg/mL stock solution of each reference standard;
wherein the veterinary drugs and additives are selected from:
(a) sulfonamides: sulfadimidine, sulformethoxine, sulfisoxazole, sulfaguanidine, sulfamerazine, sulfamethylthiadizaole, sulfamethoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfamethazole, sulfapyridine, sulfaquinoxaline, sulfathiazole, sulfisomidine, trimethoprim, diaveridine, phthalylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachloropyrazine, sulfadiazine and sulfadimethoxine;
(b) fluoroquinolones: lomefloxacin, marbofloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pipemidic acid, pefloxacin, cinoxacin, sarafloxacin, sparfloxacin, ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine and gatifloxacin;
(c) benzimidazoles: mebendazole, mebendazole-amine, 2-aminofluorobenzimidazole, oxfendazole, oxibendazole, parbendazole, 5-hydroxythiabendazole, thiabendazole, albendazole, albendazole sulfone, albendazole sulfoxide, albendazole aminosulfone, benzimidazole, febantel and lobendazole;
(d) stimulants: cyproheptadine, fenoterol, labetalol, penbutolol, phenylethanolamine A, ractopamine, salbutamol, salmeterol, terbutaline, mabuterol, tulobuterol, bambuterol, brombuterol, cimaterol, cimbuterol, clenbuterol, clenproperol and clonidine;
(e) hormones: chlormadinone acetate, 17a-methyltestosterone, clobetasol propionate, clobetasone butyrate, corticosterone, hydrocortisone, cortisone acetate, cortisone, deflazacort, dexamethasone, diflorasone diacetate, epitestosterone, estradiol benzoate, fludrocortisone acetate, flumetasone pivalate, flumethasone, fluocinolone acetonide, fluorometholone, flurandrenolide, fluticasone propionate, nandrolone propionate, halcinonide, dihydrodiethylstilbestrol, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, methandienone, methylprednisolone, norethindrone, nandrolone propionate, levonorgestrel, prednicarbate, prednisolone, prednisone, progesterone, dehydrotestosterone, testosterone, trenbolone, triamcinolone acetonide, beclomethasone, betamethasone dipropionate, betamethasone valerate and betamethasone;
(f) nitroimidazoles: dimetridazole, ipronidazole, metronidazole, ornidazole, hydroxydimetridazole, ronidazole, secnidazole and tinidazole;
(g) antiviral substances: amantadine and rimantadine;
(h) lincosamides: clindamycin and lincomycin;
(i) amphenicols: chloramphenicol, florfenicol and thiamphenicol;
(j) penicillins: ceftiofur, flucloxacillin, cloxacillin, nafcillin, piperacillin and penicillin G;
(k) quinoxalines: quinocetone;
(l) antipyretics and analgesics: phenacetin, salicylic acid, amidopyrine, antipyrine, dapsone, flunixin meglumine and acetaminophen;
(m) psychoactive drugs: haloperidol, imipramine, nitrazepam, oxazepam, pemoline, perphenazine, promethazine, propionylpromazine hydrochloride, sulpiride, xylazine, acepromazine, anisopirol, azaperone, carbamazepine, chloropromazine, diazepam, diphenhydramine, droperidol, estazolam, clarithromycin, tiamulin, erythromycin, kitasamycin, tilmicosin and tylosin;
(n) anthelmintics: toltrazuril sulfoxide, clopidol, dinitolmide, halofuginone and levamisole;
(o) pesticides: buprofezin, carboxin, 3-hydroxycarbofuran, clothianidin, cyromazine, diuron, ethopabate, fipronil, fluridone, acephate, hexazinone, imazalil, imidacloprid, linuron, metribuzin, myclobutanil, acetamiprid, norflurazon, propyzamide, atrazine, simazine, desethyl atrazine, azoxystrobin and benoxacor;
(p) other substances: scopolamine, valnemulin and atropine;
the norfloxacin is dissolved with water and diluted with methanol to 10 mL; each of the pesticides is dissolved with acetone and then diluted with methanol to 10 mL; each of the diaveridine, albendazole, albendazole sulfone and febantel is dissolved with formic acid and diluted with methanol to 10 mL; and the rest substances are dissolved with a mixed solvent of methanol and ammonia water or a mixed solvent of methanol and dimethyl sulfoxide, and then diluted with methanol to 10 mL;
(2) pipetting 100 μL of the stock solution of each reference standard in the same category in Table 1 to a volumetric flask followed by diluting with methanol to 10 mL to accordingly prepare 16 first mixed standard solutions, wherein a concentration of each reference standard in the corresponding first mixed standard solution thereof is g/mL; pipetting 100 μL of each first mixed standard solution to another volumetric flask followed by diluting with methanol to 10 mL to prepare a second mixed standard solution, wherein each reference standard in the second mixed standard solution has a concentration of 100 ng/mL; and pipetting 100 μL, 500 μL, 1 mL and 5 mL, respectively, to a volumetric flask followed by diluting with a 20% aqueous methanol solution to 10 mL to prepare a series of mixed standard working solutions, wherein each reference standard in the series of mixed standard working solutions has a concentration of 1 ng/mL, 5 ng/mL, 10 ng/mL and 50 ng/mL, respectively;
(3) adding 5 g of a sample to a 50 mL centrifuge tube; adding an extracting solution followed by vortex mixing; shaking and centrifuging the centrifuge tube; transferring a first supernatant to another 50 mL centrifuge tube;
if the sample is muscle or poultry egg, treating a precipitate with the extracting solution again to collect an extract; combining the first supernatant with the extract to produce a mixture; adding 6 g of anhydrous sodium sulfate and 1.5 g of sodium chloride to the mixture followed by vortex mixing for 30 s and standing; and centrifuging the another 50 mL centrifuge tube to produce a second supernatant;
if the sample is cow's milk or goat's milk, adding 4 g of anhydrous sodium sulfate and 1 g of sodium chloride to the first supernatant followed by vortex mixing for 30 s and standing; and centrifuging the another 50 mL centrifuge tube to produce a second supernatant;
(4) activating and equilibrating a solid phase extraction column sequentially with 4 mL of methanol and 4 mL of water; rinsing the solid phase extraction column with 2 mL of the second supernatant and discarding a first effluent; loading 5 mL of the second supernatant to the solid phase extraction column and collecting a second effluent with a centrifuge tube; drying 4 mL of the second effluent in nitrogen flow at 40° C.; adding 200 μL of methanol to the dried product followed by vortex mixing for 10 s; adding 800 μL of pure water followed by vortex mixing for 30 s to produce a sample solution; and filtering the sample solution with 0.22 μm nylon filter membrane and collecting a filtrate for use;
(5) diluting each of the first mixed standard solutions such that each reference standard in the corresponding diluted first mixed standard solution thereof has a concentration of 1 μg/mL; subjecting each of the diluted first mixed standard solutions to full scan and secondary scan in positive and negative modes using a ultra high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometer; analyzing the scan results to obtain information of each reference standard using Trace Finder software of Q-Exactive plus; and establishing a mass spectrometry database based on the information of each reference standard; wherein the information of each reference standard comprises retention time, precise molecular weight of parent ion, addition mode, fragment ions, isotope abundance ratio and a fragment ion mass spectrometry generated through the superposition of fragment ion mass spectrometries obtained at collision energies of 20 eV, 40 eV and 60 eV; the isomerides are further identified through the analysis of single standard reference; and the mass spectrometry database is shown in Table 1;
(6) analyzing the filtrate by the ultra high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometer to obtain original data of the sample;
(7) subjecting the original data of the sample to automatic extraction using Trace Finder software within a mass window of 5 ppm; wherein when a compound meets the following conditions at the same time, it indicates that the compound is a suspected positive compound in the sample:
(i) a signal-noise ratio (S/N) of the compound is greater than 3;
(ii) a difference between the measured retention time of the compound and the retention time of the compound in the mass spectrometry database is equal to or less than 0.2 min or within ±2.5% (not to exceed 0.5 min);
(iii) the measured parent ion and one fragment ion of the compound simultaneously match the information in the mass spectrometry database, and a mass accuracy of the parent ion is less than 5 ppm and a mass accuracy of the fragment ion is less than 10 ppm; and
(8) comparing a fragment ion mass spectrometry of the suspected positive compound in the sample with a fragment ion mass spectrometry of standard for further identification; wherein if the mass accuracy between main fragment ions in the two fragment ion mass spectrometry is less than 10 ppm and the main fragment ions in the two fragment ion mass spectrometries have identical relative abundance, it confirms that the suspected positive compound is present in the sample.
In an embodiment, in step (3), when the sample is muscle or poultry egg, the extracting solution is a mixed solution of 3.0 mL of Mcllvaine-Na2EDTA buffered solution and 10 mL of acetonitrile, where a concentration of EDTA in the Mcllvaine-Na2EDTA buffered solution is 0.1 mol/L; when the sample is cow's milk or goat's milk, the extracting solution is 20 mL of acetonitrile.
In an embodiment, in step (4), the solid phase extraction column is a HyperSep Retain PEP column.
In an embodiment, in steps (5) and (6), parameters of the ultra high performance liquid chromatography are set as follows: chromatographic column: C18 (150 mm×3.0 mm, particle size 1.8 m); column temperature: 30° C.; injection volume: 10 μL; flow rate: 0.3 mL/min; mobile phase A: 0.1% aqueous formic acid solution for positive ion mode and 0.03% aqueous ammonia solution for negative ion mode; mobile phase B: a solution of 0.1% formic acid in acetonitrile for positive ion mode and a solution of 0.03% ammonia in acetonitrile for negative ion mode; a gradient elution program in positive ion mode is shown in Table 2, and a gradient elution program in negative ion mode is shown in Table 3.
In an embodiment, in steps (5) and (6), parameters of the mass spectrometry are set as follows:
ion source: electron spray ion source (ESI+ and ESI−);
scan mode: full scan and automatically-triggered secondary scan mode;
capillary voltage: 3.2 kv (ESI+) and 2.8 kv (ESI−);
ion transmission capillary temperature: 325° C.;
atomizing gas temperature: 350° C.;
sheath gas pressure: 40 arb;
auxiliary gas pressure: 40 arb;
dwell time of the primary mass spectrometry: 100 ms;
dwell time of the secondary mass spectrometry: 60 ms;
scanning range (m/z): 50-1000; and
collision energy: 20, 40 and 60 eV.
In an embodiment, in step (3), the shaking is performed at 6,000 rpm for 10 min; and the centrifugation is performed at 4,000-7,000 rpm for 5 min.
Compared with the prior art, the invention has the following beneficial effects.
1. The invention facilitates collecting high-quality and high-accuracy data, reducing the false positive rate.
2. The sample is directly analyzed in full scan mode, and there is no limit to the number of compounds scanned per unit time. Moreover, it is convenient to establish a method for the instrumental analysis.
3. The invention enables the rapid screening of compounds in the sample by comparing the measured data with the mass spectrometry database, and is particularly suitable for the screening and confirmation of multiple types of compounds.
4. The invention is suitable for the detection of veterinary drugs and additives in animal-derived food, which enriches the detection and screening technical means for multiple residues in animal-derived food, facilitating ensuring the safety of animal husbandry and food industry.
Provided herein was a screening and confirmation method for veterinary drugs and additives in animal-derived food, where the veterinary drugs and additives were selected from:
(a) sulfonamides: sulfamethazine, sulformethoxine, sulfisoxazole, sulfaguanidine, sulfamerazine, sulfamethylthiadizaole, sulfamethoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfamethazole, sulfapyridinee, sulfaquinoxaline, sulfathiazole, sulfisomidine, trimethoprim, diaveridine, phthalylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachloropyrazine, sulfadiazine and sulfadimethoxine;
(b) fluoroquinolones: lomefloxacin, marbofloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pipemidic acid, pefloxacin, cinoxacin, sarafloxacin, sparfloxacin, ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, flumequine and gatifloxacin;
(c) benzimidazoles: mebendazole, mebendazole-amine, 2-aminofluorobenzimidazole, oxfendazole, oxibendazole, parbendazole, 5-hydroxythiabendazole, tiabendazole, albendazole, albendazole sulfone, albendazole sulfoxide, amino albendazole sulfone, benzimidazole, febantel and lobendazole;
(d) stimulants: cyproheptadine, fenoterol, labetalol, penbutolol, phenylethanolamine A, ractopamine, salbutamol, salmeterol, terbutaline, mabuterol, tulobuterol, bambuterol, brombuterol, cimaterol, cimbuterol, clenbuterol, clenproperol and clonidine;
(e) hormones: chlormadinone acetate, 17a-methyltestosterone, clobetasol propionate, clobetasone butyrate, corticosterone, hydrocortisone, cortisone acetate, cortisone, deflazacort, dexamethasone, diflorasone diacetate, epitestosterone, estradiol benzoate, fludrocortisone acetate, flumetasone pivalate, flumethasone, fluocinolone acetonide, fluorometholone, flurandrenolide, fluticasone propionate, nandrolone propionate, halcinonide, dihydrodiethylstilbestrol, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, methandienone, methylprednisolone, norethindrone, nandrolone propionate, levonorgestrel, prednicarbate, prednisolone, prednisone, progesterone, dehydrotestosterone, testosterone, trenbolone, triamcinolone acetonide, beclomethasone, betamethasone dipropionate, betamethasone valerate and betamethasone;
(f) nitroimidazoles: dimetridazole, ipronidazole, metronidazole, ornidazole, hydroxydimetridazole, ronidazole, secnidazole and tinidazole;
(g) antiviral agents: amantadine and rimantadine;
(h) lincosamide: clindamycin and lincomycin;
(i) amphenicols: chloramphenicol, florfenicol and thiamphenicol;
(j) penicillins: ceftiofur, flucloxacillin, cloxacillin, nafcillin, piperacillin and penicillin G;
(k) quinoxalines: quinocetone;
(l) antipyretic and analgesics: phenacetin, salicylic acid, amidopyrine, antipyrine, dapsone, flunixin meglumine and acetaminophen;
(m) psychoactive agents: haloperidol, imipramine, nitrazepam, oxazepam, pemoline, perphenazine, promethazine, propionylpromazine hydrochloride, sulpiride, xylazine hydrochloride, acepromazin, anisopirol, azaperone, carbamazepine, chloropromazine, diazepam, diphenhydramine hydrochloride, droperidol, estazolam, clarithromycin, tiamulin, erythromycin, kitasamycin, tilmicosin and tylosin;
(n) anthelmintics: toltrazuril sulfocide, clopidol, dinitolmide, halofuginone and levamisole;
(o) pesticides: buprofezin, carboxin, 3-hydroxycarbofuran, clothianidin, cyromazine, diuron, ethopabate, fipronil, fluridone, acephate, hexazinone, imazalil, imidacloprid, linuron, metribuzin, myclobutanil, acetamiprid, norflurazon, propyzamide, atrazine, simazine, desethyl atrazine, azoxystrobin and benoxacor; and
(p) other substances: scopolamine, valnemulin and atropine.
The method was specifically described as follows.
1.1 Preparation of Stock Solutions
10 mg of each reference standard was weighed accurately, dissolved and diluted with methanol to 10 mL in a volumetric flask to prepare a 1 mg/mL stock solution of each reference standard; where norfloxacin was dissolved with water; the pesticides were dissolved with acetone; diaveridine, albendazole, albendazole sulfone and febantel were all dissolved with formic acid and then diluted with methanol to 10 mL; and the rest substances with lower solubility were dissolved with a mixed solvent of methanol and ammonia water or a mixed solvent of methanol (main solvent) and dimethyl sulfoxide (auxiliary solvent), and then diluted to 10 mL.
1.2 Preparation of Standard Working Solutions
(1) 100 μL of the stock solution of each reference standard in the same category in Table 1 was accurately pipetted to a volumetric flask, and then diluted to 10 mL with methanol to accordingly prepare 16 first mixed standard solutions, where each reference standard in the corresponding first mixed standard solution thereof had a concentration of 10 μg/mL. 100 μL of each first mixed standard solution was accurately pipetted to another volumetric flask and then diluted to 10 mL with methanol to prepare a second mixed standard solution, where each reference standard in the second mixed standard solution had a concentration of 100 ng/mL. (2) accurately 100 μL, 500 μL, 1 mL and 5 mL of the second mixed standard solution were pipetted, respectively, and, then diluted to 10 mL with a 20% aqueous methanol solution to prepare a series of mixed standard working solutions, where each reference standard in the series of mixed standard working solutions had a concentration of 1 ng/mL, 5 ng/mL, 10 ng/mL and 50 ng/mL, respectively.
2.1 Sample Preparation
2.1.1 Animal Tissues
An appropriate amount of fresh/thawed blank or test tissue was collected, minced and homogenized.
2.1.2 Cow's Milk and Goat's Milk
An appropriate amount of fresh/thawed blank or test cow's milk was collected and mixed uniformly.
2.1.3 Poultry Eggs
An appropriate number of fresh test eggs were collected, shelled and homogenized.
The above samples were all stored below −20° C.
2.2 Extraction
2.2.1 Muscle or Poultry Egg
5 g of the sample was added into a 50 mL centrifuge tube, to which a mixed solution of 3.0 mL Mcllvaine-Na2EDTA buffered solution and 10 mL acetonitrile was added for extraction, where the Mcllvaine-Na2EDTA buffered solution was prepared through steps of: dissolving 44.08 g of disodium hydrogen phosphate, 37.2 g of Na2EDTA and 8.08 g of citric acid followed by diluting to 1000 mL to prepare 0.1 mol/L Mcllvaine-Na2EDTA buffered solution. The centrifuge tube was subjected to vortex mixing, shook at 6,000 rpm for 10 min and centrifuged at 4,000 rpm for 5 min. The supernatant was transferred to another 50 mL centrifuge tube, and the precipitate was repeatedly treated with the extraction solution. The two supernatants were combined, added with 6 g of anhydrous sodium sulfate and 1.5 g of sodium chloride, mixed under vortex for 30 s, subjected to standing for 10 min and centrifuged at 7,000 rpm for 5 min, and the supernatant was collected for further use.
2.2.2 Cow's Milk or Goat's Milk
5 g of cow's milk or goat's milk was added into a 50 mL centrifuge tube, to which 20 mL of acetonitrile was added for extraction. The centrifuge tube was subjected to vortex mixing, shook at 6,000 rpm for 10 min and centrifuged at 7,000 rpm for 5 min, and the supernatant was transferred to another 50 mL centrifuge tube, added with 4 g of anhydrous sodium sulfate and 1 g of sodium chloride, mixed under vortex for 30 s, subjected to standing for 10 min and centrifuged at 7,000 rpm for 5 min. The supernatant was collected for further use.
2.3 Purification
The solid phase extraction column was activated and equilibraed sequentially with 4 mL of methanol and 4 mL of water and then rinsed with 2 mL of the supernatant prepared in step (2.2.1) or (2.2.2). The effluent was discarded, and then 5 mL of the supernatant was loaded to the solid phase extraction column. The effluent was collected by a centrifuge tube, and 4 mL of the effluent was accurately pipetted, dried at 40° C. in nitrogen flow, added with 200 μL of methanol, mixed under vortex for 10 s, added with 800 μL of pure water, mixed under vortex for 30 s and filtered through a 0.22 m nylon filter membrane. The filtrate was collected and stored for further use.
3.1 Liquid Chromatography
The gradient elution programs in positive and negative ion modes were respectively shown in Table 4 and Table 5.
3.2 Mass Spectrometry
Parameters of the mass spectrometry were set as follows: ion source: electron spray ion source (ESI+ and ESI−); capillary voltage: 3.2 kv (ESI+), 2.8 kv (ESI−); ion transmission capillary temperature: 325° C.; atomizing gas temperature: 350° C.; sheath gas pressure: 40 arb; auxiliary gas pressure: 40 arb; dwell time of the primary mass spectrometry: 100 ms; dwell time of the secondary mass spectrometry: 60 ms; scanning range (m/z): 50-1000; collision energy: 20, 40, 60 eV; scan mode: full scan and automatically-triggered secondary scan mode.
The 16 first mixed standard solutions were diluted such that each reference standard in the corresponding diluted first mixed standard solution thereof had a concentration of 1 μg/mL. Each of the diluted first mixed standard solutions was subjected to full scan and secondary scan in the positive and negative modes using ultra high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometer. The obtained scan results were analyzed using Trace Finder software of Q-Exactive plus to obtain information of each reference standard, where the information of each reference standard included retention time, precise molecular weight of parent ion, addition mode, fragment ions, isotope abundance ratio and a fragment ion mass spectrometry generated through the superposition of fragment ion mass spectrometries obtained at collision energies of 20 eV, 40 eV and 60 eV. A mass spectrometry database was established based on the information of each reference standard, which was shown in Table 1. Those isomerides were further identified through the analysis of single reference standard. Enrofloxacin (1 μg/mL) was used as an example, and its parent ion extraction chromatogram, parent ion and fragment ion mass spectrometry were shown in
Table 1 Mass spectrometry library information
The filtrate obtained in step (2.3) was analyzed by ultra high performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometer to obtain original data of the sample.
The original data of the sample was automatically extracted within a mass window of 5 ppm using the Trace Finder software, where when a compound met the following conditions at the same time, it indicated that the compound was a suspected positive compound in the sample:
(i) a signal-noise ratio (S/N) is greater than 3;
(ii) a difference between the measured retention time and the corresponding retention time in the mass spectrometry database is <0.2 min or within ±2.5% (no more than 0.5 min);
(iii) the measured parent ion and one fragment ion simultaneously matched the corresponding information in the mass spectrometry database, and a mass accuracy of the parent ion was less than 5 ppm and a mass accuracy of the fragment ion was less than 10 ppm.
A fragment ion mass spectrometry of the suspected positive compound in the sample was compared with a fragment ion mass spectrometry of standard for further identification, where if the mass accuracy between main fragment ions in the two fragment ion mass spectrometries is less than 10 ppm and the main fragment ions in the two fragment ion mass spectrometries have identical relative abundance, it confirmed that the suspected positive compound was present in the sample.
The detection limit of each compound of the veterinary drugs and additives in the invention was the lowest concentration that the compound can be detected in the sample in the screening in step (6) and in the confirmation in step (7). Further, the detection limits of the veterinary drugs and additives were shown in Table 6.
The parent ion extraction chromatograms of reference standards of the veterinary drugs and additives were shown in
It can be seen from above that the method of the invention mainly achieved the screening and confirmation of compounds, that was, this method was suitable for the qualification of the residues in the animal-derived food.
Therefore, the validity of the method of the invention was assessed mainly in terms of detection limit, recovery rate, precision and matrix effect.
9.1 Detection Limit
The blank sample was processed in the same manner and then analyzed. The measurement results showed that the blank sample has no interference with the drug to be tested at the corresponding retention time. If the blank sample had a peak at the retention time of the drug to be tested, the value of the blank sample should be deducted before the calculation.
An appropriate amount of the mixed standard solution was added into 5 g of the blank matrix, which was then processed by the above pretreatment method and analyzed. The detection limit of each compound in chicken, pork, mutton, beef, chicken eggs, duck eggs, cow's milk and goat's milk was determined according to the conditions of the compound confirmation. The results were shown in Table 6.
9.2 Recovery Rate and Precision
A standard solution having a concentration near the detection limit was added to the blank sample according to the spiking method. 5 parallel samples of each concentration were tested 3 times for the calculation of intra- and inter-batch relative standard deviations (RSD). The results were shown in Table 6, in which the recovery rates of the 207 veterinary drugs and additives were all in the range of 40%-130%, and intra- and inter-batch relative standard deviations were less than 30%.
9.3 Matrix Effect
The matrix effect of the sample was evaluated by comparing the standard solution and the spiked sample liquid before the nitrogen blowing in the chromatographic peak area. The results showed that the substances in the matrix may show matrix inhibition or matrix enhancement, where a matrix effect value between 0.85 and 1.15 indicated that there was no significant matrix effect; a matrix effect value greater than 1.15 indicated matrix enhancement; and a matrix effect value less than 0.85 indicated matrix inhibition. The matrix effect values measured herein ranged from 0.11 to 8.03, therefore, it was required to plot a matrix-matched standard curve for the quantification.
Described above are merely preferred embodiments of the invention. It should be noted that any modification and change made by those skilled in the art without departing from the spirit of the invention should fall within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910822246.X | Aug 2019 | CN | national |
| 201910891387.7 | Sep 2019 | CN | national |
