CN111650310A - Method for determining residual quantity of ribavirin and amantadine compounds in poultry food - Google Patents
Method for determining residual quantity of ribavirin and amantadine compounds in poultry food Download PDFInfo
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- CN111650310A CN111650310A CN202010311449.5A CN202010311449A CN111650310A CN 111650310 A CN111650310 A CN 111650310A CN 202010311449 A CN202010311449 A CN 202010311449A CN 111650310 A CN111650310 A CN 111650310A
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- 229960000329 ribavirin Drugs 0.000 title claims abstract description 94
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 title claims abstract description 91
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical class C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 title claims abstract description 82
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- SDWIOXKHTFOULX-PDNLFSCWSA-N ribavirin monophosphate Chemical class N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](COP(O)(O)=O)O1 SDWIOXKHTFOULX-PDNLFSCWSA-N 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for determining residual quantity of ribavirin and amantadine compounds in poultry food, which comprises the steps of establishing an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to detect the residual quantity of ribavirin and amantadine compounds in poultry food, carrying out enzymolysis on a sample, precipitating protein with trichloroacetic acid, carrying out low-temperature high-speed centrifugation, regulating the pH value of supernatant, purifying the supernatant by a PBA/PCX composite solid phase extraction column, and analyzing ribavirin and Waters BEH C by an Agilent ZORBAX SB-Aq column (3.0mm × 100mm, 1.8 mu m)18The method is characterized in that a chromatographic column (2.1mm × 100mm,1.7 mu m) is used for analyzing amantadine, the tandem mass spectrometry is adopted, and the isotope internal standard method is used for quantification.
Description
Technical Field
The invention belongs to the technical field of detection, and relates to a method for determining residual amounts of ribavirin and amantadine compounds in poultry food.
Background
Ribavirin (also called ribavirin), is a broad-spectrum strong synthetic nucleoside antiviral drug, has an inhibiting effect on DNA and RNA viruses, and is mainly used for treating diseases such as human viral influenza, pneumonia, hepatitis A, viral encephalitis and the like. The amantadine compounds mainly comprise amantadine (amantadine), adamantanemethylamine (amantanemethylamine), rimantadine (rimantadine) and 3, 5-dimethyladamantadine (memantine), wherein the amantadine is mainly used for inhibiting Asian influenza A virus; the effect of the adamantanamine on resisting A2 type viruses and parainfluenza viruses is better than that of the amantadine; the effect of the amantadine on inhibiting influenza A virus is also stronger than that of amantadine, but the central nervous side effect is weaker than that of amantadine; 3, 5-dimethyl amantadine is also called memantine and is clinically used for treating Alzheimer disease in moderate and severe degree, but the memantine has similar structure of amantadine and also has the function of inhibiting influenza A virus.
Because ribavirin and amantadine compounds are abused in the livestock breeding link, drug residues can cause virus drug resistance, and harm is caused to human health through a food chain, so that a large safety risk exists, antiviral drugs such as ribavirin and amantadine compounds are prohibited from being used in the livestock breeding link in the 560 th bulletin issued by the ministry of agriculture in 2005 in the original Ministry of China, and human antiviral drugs are prohibited from being used in the livestock breeding link in 2006 by the United states Food and Drug Administration (FDA). China explodes a fast-growing chicken event in 2012, and media expose part of farms to illegally feed antiviral drugs such as ribavirin and amantadine, so that great social reverberation is caused. Because the antiviral drugs such as ribavirin and amantadine compounds are low in price and good in effect, the drugs are still applied to the prevention and treatment of animal diseases in a breeding link.
Because ribavirin is metabolized into phosphorylated ribavirin in an organism, phosphorylated ribavirin needs to be converted into free ribavirin when analyzing the total residual quantity of ribavirin, and amantadine compounds exist in the organism almost in a prototype form. At present, related research reports of detecting the total residual quantity of ribavirin and amantadine compounds in animal-derived food in China are few, and liquid chromatography-tandem mass spectrometry is mostly adopted to detect the ribavirin or amantadine compounds.
Disclosure of Invention
The invention aims to provide a method for determining the residual quantity of ribavirin and amantadine compounds in poultry food.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for determining the residual quantity of ribavirin and amantadine compounds in poultry food comprises the following steps:
step 1: placing a sample in a centrifuge tube, adding a mixed isotope internal standard working solution, adding an ammonium acetate buffer solution and an acid phosphatase solution, covering a cover tightly, carrying out vortex mixing for 30s, carrying out dark enzymolysis for 2h in a constant temperature shaking table at 37 ℃ to obtain an enzymolysis solution, then adding 4ml of a trichloroacetic acid solution with the mass fraction of 5% in the enzymolysis solution, carrying out vortex mixing for 30s, centrifuging for 10min at 10000r/min at the temperature of 2 ℃, then transferring a supernatant into another centrifuge tube, regulating the pH value of the solution to 8.4-8.6 by using an ammonia water solution with the mass fraction of 5%, fixing the volume to 10ml by using water, carrying out centrifugation for 5min at 10000r/min after mixing, and filtering the supernatant through a regenerated cellulose microporous filter membrane with the diameter of 0.45 mu m to obtain a sample extracting solution;
step 2: activating the PBA/PCX solid-phase extraction column by using acetonitrile, 0.5% by mass of formic acid aqueous solution and ammonium acetate buffer solution in sequence to obtain an activated PBA/PCX solid-phase extraction column; enabling the sample extracting solution to pass through the activated PBA/PCX solid phase extraction column, sequentially leaching with an ammonium acetate buffer solution and water, and pumping to dry the PBA/PCX solid phase extraction column; eluting the PBA/PCX solid phase extraction column in a 5ml volumetric flask by using a formic acid aqueous solution with the mass fraction of 0.5%, draining the PBA/PCX solid phase extraction column, fixing the volume to a scale by using the formic acid aqueous solution with the mass fraction of 0.5%, uniformly mixing, and then passing through a 0.22 mu m regenerated cellulose filter membrane for UPLC-MS/MS analysis of ribavirin; and leaching the PBA/PCX solid phase extraction column by using methanol, draining the column, eluting by using an ammoniated methanol solution containing 10% of isopropanol and having the volume fraction of 5%, drying by blowing nitrogen in a water bath at 50 ℃, redissolving by using a formic acid aqueous solution with the mass fraction of 0.5%, fixing the volume to 5ml, uniformly mixing, and passing through a 0.22 mu m hydrophilic polytetrafluoroethylene filter membrane for UPLC-MS/MS analysis of the amantadine compounds.
The preferable technical scheme is as follows: the filler of the PBA/PCX solid phase extraction column is phenylboronic acid and polymeric cation exchange resin, the mass ratio of the phenylboronic acid to the polymeric cation exchange resin is 200mg/100mg, and the volume of the PBA/PCX solid phase extraction column is 3 ml.
The preferable technical scheme is as follows: chromatographic column for detecting ribavirin: agilent ZORBAX SB-Aq column, mobile phase: a is formic acid water solution with the mass fraction of 0.2%, B is acetonitrile, the column temperature is 40 ℃, the sample injection amount is 20 mu L, and the mobile phase gradient elution procedure is as follows:
| time/min | Flow rate/(ml/min) | A/％ | B/％ |
| 0.0 | 0.40 | 100 | 0 |
| 2.5 | 0.40 | 100 | 0 |
| 3.0 | 0.40 | 95 | 5 |
| 4.0 | 0.40 | 10 | 90 |
| 5.0 | 0.40 | 10 | 90 |
| 5.5 | 0.40 | 100 | 0 |
| 6.5 | 0.40 | 100 | 0 |
。
The preferable technical scheme is as follows: a chromatographic column for detecting the amantadine compounds: waters BEH C18 column, mobile phase: a is 0.2% formic acid aqueous solution, B is methanol, the column temperature is 40 ℃, the sample amount is 10 mul, and the mobile phase gradient elution procedure is as follows:
the preferable technical scheme is as follows: mass spectrum: an ionization source: electrospray ion source, ionization mode: in a positive ion mode (ESI +), the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolvation gas temperature is 500 ℃, the desolvation gas flow is 800L/h, the cone hole back blowing gas flow is 50L/h, and the collision gas flow is 0.15ml/min, wherein the detection mode is as follows: monitoring the multi-ion reaction; the retention time, the qualitative and quantitative ion pairs, the taper hole voltage and the collision energy of the ribavirin and the amantadine compounds are as follows:
due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the invention establishes an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection technology of ribavirin, amantadine, adamantanemethylamine, rimantadine and 3, 5-dimethyladamantadine, and effectively solves the technical problem of detection of ribavirin and amantadine compounds in animal-derived foods.
2. The ribavirin of the invention is in a linear relationship of 1.0-100 ng/ml, the amantadine compound is in a linear relationship of 0.2-20 ng/ml, and the correlation coefficients (r) are all larger than 0.999. The detection limit and the quantification limit of ribavirin are respectively 0.5 and 1.5 mug/kg; the detection limit and the quantification limit of the amantadine compounds are respectively 0.1 and 0.3 mug/kg. The detection results of adding 3 concentrations of ribavirin (1.5-15 mug/kg) and amantadine compounds (0.3-3.0 mug/kg) can obtain that the recovery rate of ribavirin is 91.4-103.7%, the recovery rate of amantadine compounds is 94.3-108.2%, and the Relative Standard Deviation (RSD) is less than 10%.
3. The method has the characteristics of simplicity, convenience, rapidness, high sensitivity, accurate qualitative determination and the like.
Drawings
FIG. 1 is a chromatogram of a blank chicken sample with ribavirin as a labeling agent. A is an Agilent ZORBAX SB-Aq column (3.0 mm. times.100 mm, 1.8 μm); b is an Acquity UPLC HSS T3 column (3.0 mm. times.150 mm, 1.8 μm).
FIG. 2 shows an adamantanamine compound and its isotope internal standard chromatogram (WatersBEH C)18A chromatography column).
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: method for determining residual quantity of ribavirin and amantadine compounds in poultry food
1. The main instruments and reagents.
Acquity in ultra high performance liquid chromatography-tandem mass spectrometerTMUPLC ultra-high performance liquid chromatograph, XEVOTMTQMS was purchased from Waters, Milli-Q ultra pure water system (Millipore, USA), vortex mixer, electronic balance, GX-271 solid phase extractor (Gilson, USA), high speed refrigerated centrifuge, MTN-2800D nitrogen blow concentration device, Phenyl Boronic Acid (PBA)/polymeric cation exchange resin (PCX) composite solid phase extraction column (homemade, 200mg/100mg, 3ml, PBA and PCX packing: Agilent, USA). Pressing a polyethylene sieve plate (aperture: 20 μm) into the bottom of a polypropylene solid phase extraction hollow column tube (3mL type), filling 100mg of polymeric cation exchange resin filler (PCX) into the column tube in a fixed amount, lightly tapping the column tube and tamping, pressing a polyethylene sieve plate (aperture: 20 μm) into the column tube and sealing the PCX; quantitatively filling 200mg of silica gel bonded benzene boric acid filler (PBA) into the column tube again, slightly beating the column tube for tamping, and then pressing a polyethylene sieve plate (aperture: 20 mu m) to seal the PBA, thus obtaining the benzene boric acid (PBA)/polymeric cation exchange resin (PCX) composite solid phase extraction column.
And (3) standard substance: ribavirin (C16813570, Dr. Ehretroffer, Germany), amantadine (PHR1711), amantadine (180378), rimantadine (390593), and 3, 5-dimethyladamantadine (PHR1886) are all available from Sigma-Adrich, USA,13C5-ribavirin (R414477),D15Amantadine (A575822), D4Rimantadine (R517002) and D 63, 5-Dimethylamantadine (M218002) was purchased from Toronto Research Chemicals, Canada; acetonitrile, methanol and isopropanol were chromatographically pure, formic acid, ammonia and glacial acetic acid were of equal grade of merit, trichloroacetic acid was analytically pure, acid phosphatase (3.0U/mg, Sigma-Adrich in usa), 0.2mol/L ammonium acetate buffer (pH 4.8), 0.2mol/L ammonium acetate buffer (pH 8.5).
2. Preparation of Standard solutions
Accurately transferring a proper amount of single standard stock solution (100 mu g/mL) of amantadine, rimantadine and 3, 5-memantine and standard stock solution (100 mu g/mL) of ribavirin, and preparing mixed standard working solution (0.2 mu g/mL of amantadine compounds and 1.0 mu g/mL of ribavirin) by using methanol; accurately moving and taking proper amount D15Amantadine, D4Rimantadine and D6Isotope single standard stock solution (100. mu.g/mL) of 3, 5-memantine and13C5ribavirin standard stock solution (100. mu.g/mL), mixed isotope internal standard working solution (amantadine-like isotope compound: 0.2. mu.g/mL,13C5-ribavirin: 1.0. mu.g/mL).
Accurately transferring a proper amount of mixed standard working solution and mixed isotope internal standard working solution, and preparing a mixed standard series by using 0.5% formic acid aqueous solution, wherein the amantadine, the adamantanemethanamine, the adamantanethylamine and the 3, 5-dimethyladamantadine are as follows: 0.2, 0.4, 1.0, 2.0, 4.0, 10 and 20 μ g/L; the ribavirin concentration was: 1.0, 2.0, 5.0, 10, 20, 50 and 100. mu.g/L. Each standard series solution contained 2.0. mu.g/L of D15Amantadine, D4Rimantadine and D6-3, 5-memantine, 10. mu.g/L13C5-ribavirin mixed isotope internal standard.
3. Sample pretreatment
Mincing chicken, chicken liver, etc. and its product, and homogenizing. 500g of the sample is separated and stored at a temperature below-20 ℃ in the dark. Taking a plurality of eggs, shelling, and fully and uniformly beating the yolk and the egg white. 500g of the sample is separated and stored at a temperature below-20 ℃ in the dark.
Enzymolysis and extraction: weighing 2g of sample into a 50ml graduated centrifuge tube, adding the mixed isotope internal standard working solution, adding 5ml of ammonium acetate buffer solution (pH 4.8) and 40 mul of acid phosphatase solution, covering the lid tightly, mixing uniformly by vortex for 30s, and performing enzymolysis for 2h in a constant temperature shaking table at 37 ℃ in a dark place. Adding 4mL of 5% trichloroacetic acid solution into the enzymolysis solution, mixing uniformly by vortex for 30s, centrifuging at 2 ℃ at 10000r/min for 10min (the centrifugal radius is 10cm), transferring the supernatant into another 50mL graduated centrifuge tube, adjusting the pH value of the solution to 8.5 +/-0.1 by using 5% ammonia water solution, fixing the volume to 10mL by using water, mixing uniformly, centrifuging at 10000r/min for 5min (the centrifugal radius is 10cm), and passing the supernatant through a 0.45 mu m Regenerated Cellulose (RC) microporous filter membrane to obtain a clarified sample extracting solution.
Purifying: the PBA/PCX solid phase extraction column was activated with 3ml acetonitrile, 3ml 0.5% aqueous formic acid and 3ml ammonium acetate buffer (pH 8.5) in that order. 5.0ml of the sample extract was passed through the column, washed with 3ml of an ammonium acetate buffer (pH 8.5) and 3ml of water in this order, and the column was drained. The PBA/PCX solid phase extraction column is firstly eluted in a 5ml volumetric flask by using 3ml of 0.5 percent formic acid aqueous solution, the column is drained, the volume is fixed to a scale by using 0.5 percent formic acid aqueous solution, the mixture is uniformly mixed, and the mixture passes through a 0.22 mu m Regenerated Cellulose (RC) filter membrane for UPLC-MS/MS analysis of ribavirin. And eluting the PBA/PCX solid phase extraction column with 3ml of methanol, draining the column, eluting with 3ml of 5% ammoniated methanol solution (containing 10% of isopropanol), blowing the column to be nearly dry by nitrogen in a water bath at 50 ℃, redissolving the column with 0.5% formic acid aqueous solution, fixing the volume to 5ml, uniformly mixing, and filtering the mixture through a 0.22 mu m hydrophilic Polytetrafluoroethylene (PTFE) filter membrane for UPLC-MS/MS analysis of the amantadine compounds. Preparing an ammoniated methanol solution containing 10% of isopropanol and having a volume fraction of 5%, namely a 5% ammoniated methanol solution (containing 10% of isopropanol): 5mL of ammonia water and 10mL of isopropanol are taken, methanol is used for fixing the volume to 100mL, and the mixture is uniformly mixed.
4. Conditions of the apparatus
Chromatography (ribavirin): a chromatographic column: agilent ZORBAX SB-Aq column (3.0 mm. times.100 mm, 1.8 μm), mobile phase: a is 0.2% formic acid water solution, B is acetonitrile, column temperature 40 deg.C, sample size 20 μ L, mobile phase gradient elution procedure is shown in Table 1.
TABLE 1 ribavirin ultra performance liquid chromatography gradient elution procedure
Chromatography (amantadine compounds): a chromatographic column: waters BEH C18Column (2.1mm × 100mm,1.7 μm), mobile phase A0.2% aqueous formic acid, B methanol, column temperature 40 deg.C, sample size 10 μ l, mobile phase gradient elution procedure as shown in Table 2.
TABLE 2 ultra performance liquid chromatography gradient elution procedure for amantadine compounds
Mass spectrum: an ionization source: electrospray ion source, ionization mode: positive ion mode (ESI)+) Capillary voltage 3.0kV, ion source temperature 150 ℃, desolventizing gas temperature 500 ℃, desolventizing gas flow 800L/h, cone hole back blowing gas flow 50L/h, collision gas flow 0.15ml/min, detection mode: multiple ion reaction monitoring (MRM). The retention time, the qualitative and quantitative ion pairs, the taper hole voltage and the collision energy of the ribavirin and amantadine compounds are shown in Table 3.
TABLE 3 Mass Spectrometry parameters for ribavirin and amantadine Compounds
Note: are quantitative ions.
And (3) sample enzymolysis: the pharmacokinetic research of ribavirin shows that ribavirin is absorbed rapidly through digestive tract and enters host cells to be phosphorylated rapidly to generate active metabolites of Ribavirin Monophosphate (RMP), Ribavirin Diphosphate (RDP) and Ribavirin Triphosphate (RTP), so that when ribavirin in poultry food is measured, the phosphorylated ribavirin in a sample needs to be subjected to enzymolysis to obtain free ribavirin. The experiment adopts acid phosphatase to carry out enzyme digestion reaction to obtain a ribavirin prototype, the biological activity of the phosphatase is greatly influenced by the environment and should be prepared at present, and the activity value of the enzyme is determined by an acid phosphatase activity detection kit (a fluorescence method) to ensure the enzyme digestion reaction efficiency.
Sample extraction: in order to prevent the substances from blocking a sieve plate of a solid phase extraction column and purifying the substances through the solid phase extraction column after the proteins are precipitated, the test tries to add 1-5 ml of 5% trichloroacetic acid solution into various poultry food enzymatic hydrolysates to precipitate the proteins, and the result shows that when the addition amount of the 5% trichloroacetic acid solution is 4ml and 5ml, the bioactive substances such as the proteins and the polypeptides in the various poultry food enzymatic hydrolysates can be completely precipitated, and 4ml of 5% trichloroacetic acid solution is selected in consideration of the influence of the addition amount of the 5% trichloroacetic acid solution on the final constant volume of a sample extraction solution.
Because the sample enzymolysis liquid also contains certain fat, particularly the enzymolysis liquid of the egg sample contains a large amount of fat, after adding trichloroacetic acid solution to precipitate protein, centrifuging at 10000r/min for 10min at low temperature (2-4 ℃) to obtain a satisfactory clear solution, adjusting the pH value of the supernatant to 8.5 +/-0.1 by using 5% ammonia water solution, slightly turbidity of the sample solution at the moment, and filtering by using a 0.45 mu m RC microporous filter membrane to obtain a clear sample extracting solution.
Sample purification: the PBA/PCX composite solid phase extraction column is used for purification, so that the ionization efficiency in liquid chromatography of ribavirin is improved as much as possible, and the matrix inhibition effect of co-effluent (mainly endogenous free nucleotide) is effectively reduced. When the pH value of the solution of the upper PBA filler of the composite solid phase extraction column is 8.5 +/-0.1, ribavirin, amantadine, adamantanemethylamine, adamantanethylamine and 3, 5-dimethyladamantadine can be retained in the PBA filler, when the ribavirin is eluted, the lower PCX filler can adsorb the amantadine, the adamantanemethylamine, the 3, 5-dimethyladamantadine and endogenous free nucleotides in the enzymatic hydrolysate under the acidic condition, and finally the amantadine, the adamantanemethylamine and the 3, 5-dimethyladamantadine adsorbed by the PCX filler are eluted by ammoniated methanol, so that the solid phase extraction column and the solid phase extraction column are realizedThe sample solution was purified of ribavirin, amantadine, adamantanemethylamine, adamantanethylamine and 3, 5-dimethyladamantadine. Tests show that after the PBA/PCX composite solid phase extraction column is used for purification,13C5ribavirin, D15Amantadine, D4Rimantadine and D6The absolute recovery rate of the-3, 5-dimethyladamantane can reach more than 60 percent.
The PBA/PCX composite solid phase extraction column has the function of an ion exchange column, so that the flow rate of all other solutions passing through the solid phase extraction column is strictly controlled to be about 1.0ml/min (namely about 3 seconds and 1 drop) except for the activation of the solid phase extraction column. Experiments show that the absolute recovery rate of the internal standard in the sample is low due to the excessively high flow rate, so that the accuracy of the detection result is influenced.
Optimization of chromatographic conditions:
1. ribavirin chromatographic condition optimization
Because ribavirin contains hydroxyl and amido and is a compound with strong polarity, a polar compound separation chromatographic column, namely Agilent ZORBAX SB-Aq and Acquity, is adopted during chromatographic analysis
HSST 3 columns were all able to achieve retention of polar compounds under conditions of 100% aqueous phase through experimental optimization, Agilent ZORBAX SB-Aq columns (3.0mm × 100mm, 1.8 μm) and Acquity were determined
The HSS T3 chromatographic column (3.0mm × 150mm, 1.8 mu m) can realize complete baseline separation of an interference peak and a component to be detected, so that qualitative and quantitative analysis of trace ribavirin is met, as shown in figure 1, the column pressure of the SB-Aq chromatographic column is lower than that of the HSS T3 chromatographic column, and the separation degree of a ribavirin chromatographic peak and an impurity peak is better, so that the Agilent ZORBAX SB-Aq chromatographic column is finally adopted for analyzing ribavirin in the test.
Because Ribavirin has a stronger affinity for sodium ions than for hydrogen ions, [ Ribavirin + H ] is present in mass spectrometry]+In the case where the peak (excimer peak) is relatively small, [ Ribavirin + Na ] may also be used]+Detecting the peak as parent ion to ensure [ Ribavirin + Na]+The stability of the peak is changed, the water phase of the mobile phase is changed into sodium acetate solution (0.1mmol/L, containing 0.2% formic acid), and the mass spectrum [ Ribavirin + Na ] can be satisfied]+Stability of the peak. [ Ribavirin + H]+When the peak is the parent ion, the relative abundance of the two fragment daughter ions m/z is 113.1 and 96.1 is relatively small (about 4:1), while [ Ribavirin + Na ]]+When the peak is a parent ion, the relative abundance ratio of the two fragment daughter ions m/z is 135.1 and 155.1 is relatively large (about 20:1), which is not beneficial to the qualitative and quantitative analysis of low-content samples; therefore, the laboratory should use Ribavirin + H as much as possible]+Peaks (excimer peaks) were subjected to mass spectrometry.
2. Amantadine compound chromatographic condition optimization
When the amantadine compounds are analyzed, because the amantadine and the 3, 5-memantine are isomers of each other, the parent ions and the main daughter ions are completely the same on mass spectrometry, and the complete baseline separation on UPLC is needed to carry out the detection. Waters BEH C was used for this experiment18And the column is subjected to complete baseline separation of the rimantadine and the 3, 5-memantine on the UPLC by optimizing a gradient elution program, so that the method for simultaneously detecting the rimantadine and the 3, 5-memantine is realized. The adamantanamines chromatogram is shown in FIG. 2.
Linear range, detection limit, quantitation limit:
and injecting the standard curve series solution and the sample solution into a UPLC-MS/MS instrument to obtain chromatograms and peak areas of ribavirin, amantadine, adamantanemethylamine, adamantanethylamine and 3, 5-dimethyladamantadine. Taking the concentrations of ribavirin, amantadine, rimantadine and 3, 5-memantine as horizontal coordinates, and taking the peak areas of ribavirin, amantadine, rimantadine and 3, 5-memantine and the peak area ratio of each internal standard as vertical coordinates, drawing standard curves of ribavirin, amantadine, rimantadine and 3, 5-memantine. Because the isotope internal standard of the adamantanamine is lacked, the standard addition recovery test shows that the recovery rate of the adamantanamine in the matrix sample is very close to that of the adamantanamine, and therefore, the concentration of the adamantanamine is taken as the abscissa, and the adamantanamine is taken as the abscissaPeak area of (2) and D4-the peak area ratio of the adamantanamine is the ordinate, and an adamantanamine standard curve is drawn. The result shows that the ribavirin is 1.0-100 ng/ml, the amantadine, the rimantadine and the 3, 5-memantine are in good linear relation in the range of 0.2-20 ng/ml, and the correlation coefficients (r) are all larger than 0.999. The linear equation, correlation coefficient, detection Limit (LOD) and quantification Limit (LOQ) are shown in Table 4.
TABLE 4 Linear equations, correlation coefficients, detection limits and quantitation limits for ribavirin and amantadine compounds
Recovery and precision:
9 parts of chicken and egg samples without the substances to be detected are accurately weighed respectively to carry out addition recovery rate and precision tests, the concentrations of ribavirin added are respectively 1.5, 3.0 and 15 mu g/kg, the concentrations of amantadine compounds added are respectively 0.3, 0.6 and 3.0 mu g/kg, 3 parallel tests are carried out on each concentration level, and the results are shown in table 5. As can be seen from the table, the recovery rate of ribavirin is 91.4-103.7%, the recovery rate of amantadine compounds is 94.3-108.2%, and the Relative Standard Deviation (RSD) of the measurement results of 3 times of parallel tests is 2.7-5.8% and less than 10%, so that the method has good recovery rate, precision and accuracy and is suitable for measuring the residue of ribavirin and amantadine in poultry food.
Table 5 ribavirin and amantadine compound recovery and precision in chicken and eggs (n ═ 3)
Determination of actual samples
80 parts of chicken and 80 parts of eggs collected in the market are determined by the detection method established by the test. The result shows that the detection rate of ribavirin in chicken is 1.3 percent (1/80), the detection rate of amantadine is 17.5 percent (14/80), the detection rate of rimantadine is 5.0 percent (4/80), and other compounds are not detected; the detection rate of the amantadine in the eggs is 8.8 percent (7/80), the detection rate of the amantadine is 2.5 percent (2/80), and other compounds are not detected; therefore, the illegal addition of ribavirin and amantadine compounds also exists in the poultry breeding link.
In the embodiment, an acid phosphatase enzymolysis sample, trichloroacetic acid precipitated protein, PBA/PCX composite solid phase extraction column purification and isotope internal standard method quantification are adopted, so that a detection method for rapidly determining ribavirin, amantadine, adamantanemethylamine, adamantanethylamine and 3, 5-dimethyladamantadine in poultry food is established. The method can extract ribavirin and amantadine compounds at one time, is simple and rapid to operate, solves the problem of interference in ribavirin analysis, and simultaneously uses an isotope internal standard method for quantification, and has accurate and reliable results. The method is suitable for rapid detection of ribavirin, amantadine, adamantanemethylamine, rimantadine and 3, 5-dimethyladamantadine in large-batch poultry food.
Reference documents:
[1] zhuyonglin, Shaodejia, Jiang Tianmei, and the like, high performance liquid chromatography-tandem mass spectrometry is used for determining the total amount of ribavirin and metabolite residues thereof in chicken liver [ J ]. Chinese veterinary medicine, 2008,42(7): 22-25.
[2] Adamantanamide derivatives were studied and developed [ J ] scientific consultation (science and technology management), 2016(40) 59-60.
[3]ANTOINE S,FRANCOISE P.Gas chromatographic determination ofamantadine hydrochloride(symmetrel)in human plasma and ruine[J].J ChromatogrB:Biomed Sci and Appl,1980,183(1):33-39。
[4] Lissamine, Wangwei, liquid chromatography-mass spectrometry combined method for determining amantadine hydrochloride concentration in rats and in vivo pharmacokinetics research [ J ] J.Med.C.2007, 37(1): 66-68.
[5]WILLS R J,FAROLINO D A,CHOMA N,et al.Rimantadine pharmacokineticsafter single and multiple doses[J].Antimicrob Agents Chemother,1987,31(5):826-828。
[6] Ministry of agriculture of the people's republic of China bulletin No. 560 [ EB/OL ] (2005-11-01) [2019-10-20]. http:// www.moa.gov.cn/zwlmg/tzgg/gg/2005/t 20051117_496523. htm.
[7]U.S.FDA prohibits use of antiviral drugs in poultry to help keepdrugs effective for humans[EB/OL]。
[8]PAGE T,CONNOR J D.The Metabolism of Ribavirin in Erythrocytes andNucleated Cells[J].Int J Biochem,1990,22:379-383。
[9]Yeh LT,Nguyen M,Lourenco D,et al.A sensitive and specific methodfor the determination of total ribavirin in monkey liver by high-performanceliquid chromatography with tandem mass spectrometry.J Pharm BiomedAnal.2005Jun 1；38(1):34-40。
[10] The ultra-high performance hydrophilic chromatography-tandem mass spectrometry method is used for rapidly detecting the total residue of ribavirin and metabolites thereof in chicken and products thereof [ J ] chromatography, 2013,31(10): 934-938.
[11] Zhuyonglin, Shaodejia, Jiang Tianmei, and the like, high performance liquid chromatography-tandem mass spectrometry is used for determining the total amount of ribavirin and metabolite residues thereof in chicken liver [ J ]. Chinese veterinary medicine, 2008,42(7): 22-25.
[12] Shaanjinzhi, Yao Suzhou gao, Xianminling, etc. hydrophilic interaction chromatography-tandem mass spectrometry method simultaneously determines amantadine and ribavirin [ J ] in animal tissues, the academic paper of analytical tests, 2013,32(12):1448 and 1452.
[13] Wuyinliang, Zhaojian, Yeyufei, etc. isotope dilution liquid chromatography-tandem mass spectrometry is used to determine the residual quantity of adamantanamine drugs in eggs [ J ]. analysis and test report 2014,33(8):905 and 910.
[14] The method is characterized in that the liquid chromatography-tandem mass spectrometry is used for determining the residual quantity [ J ] of amantadine and rimantadine in animal tissues, and the mass spectrometry is reported in 2013,34(4): 226-.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (5)
1. A method for determining the residual quantity of ribavirin and amantadine compounds in poultry food is characterized in that: comprises the following steps:
step 1: placing a sample in a centrifuge tube, adding a mixed isotope internal standard working solution, adding an ammonium acetate buffer solution and an acid phosphatase solution, covering a cover tightly, carrying out vortex mixing for 30s, carrying out dark enzymolysis for 2h in a constant temperature shaking table at 37 ℃ to obtain an enzymolysis solution, then adding 4ml of a trichloroacetic acid solution with the mass fraction of 5% in the enzymolysis solution, carrying out vortex mixing for 30s, centrifuging for 10min at 10000r/min at the temperature of 2 ℃, then transferring a supernatant into another centrifuge tube, regulating the pH value of the solution to 8.4-8.6 by using an ammonia water solution with the mass fraction of 5%, fixing the volume to 10ml by using water, carrying out centrifugation for 5min at 10000r/min after mixing, and filtering the supernatant through a regenerated cellulose microporous filter membrane with the diameter of 0.45 mu m to obtain a sample extracting solution;
step 2: activating the PBA/PCX solid-phase extraction column by using acetonitrile, 0.5% by mass of formic acid aqueous solution and ammonium acetate buffer solution in sequence to obtain an activated PBA/PCX solid-phase extraction column; enabling the sample extracting solution to pass through the activated PBA/PCX solid phase extraction column, sequentially leaching with an ammonium acetate buffer solution and water, and pumping to dry the PBA/PCX solid phase extraction column; eluting the PBA/PCX solid phase extraction column in a 5ml volumetric flask by using a formic acid aqueous solution with the mass fraction of 0.5%, draining the PBA/PCX solid phase extraction column, fixing the volume to a scale by using the formic acid aqueous solution with the mass fraction of 0.5%, uniformly mixing, and then passing through a 0.22 mu m regenerated cellulose filter membrane for UPLC-MS/MS analysis of ribavirin; and leaching the PBA/PCX solid phase extraction column by using methanol, draining the column, eluting by using an ammoniated methanol solution containing 10% of isopropanol and having the volume fraction of 5%, drying by blowing nitrogen in a water bath at 50 ℃, redissolving by using a formic acid aqueous solution with the mass fraction of 0.5%, fixing the volume to 5ml, uniformly mixing, and passing through a 0.22 mu m hydrophilic polytetrafluoroethylene filter membrane for UPLC-MS/MS analysis of the amantadine compounds.
2. The method for determining the residual amount of ribavirin and amantadine compounds in poultry food according to claim 1, wherein: the filler of the PBA/PCX solid phase extraction column is phenylboronic acid and polymeric cation exchange resin, the mass ratio of the phenylboronic acid to the polymeric cation exchange resin is 200mg/100mg, and the volume of the PBA/PCX solid phase extraction column is 3 ml.
3. The method for determining the residual amount of ribavirin and amantadine compounds in poultry food according to claim 1, wherein: chromatographic column for detecting ribavirin: agilent ZORBAX SB-Aq column, mobile phase: a is formic acid water solution with the mass fraction of 0.2%, B is acetonitrile, the column temperature is 40 ℃, the sample injection amount is 20 mu L, and the mobile phase gradient elution procedure is as follows:
。
4. The method for determining the residual amount of ribavirin and amantadine compounds in poultry food according to claim 1, wherein: a chromatographic column for detecting the amantadine compounds: waters BEH C18 column, mobile phase: a is 0.2% formic acid aqueous solution, B is methanol, the column temperature is 40 ℃, the sample amount is 10 mul, and the mobile phase gradient elution procedure is as follows:
。
5. The method for determining the residual amount of ribavirin and amantadine compounds in poultry food according to claim 1, wherein: mass spectrum: an ionization source: electrospray ion source, ionization mode: in a positive ion mode (ESI +), the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolvation gas temperature is 500 ℃, the desolvation gas flow is 800L/h, the cone hole back blowing gas flow is 50L/h, and the collision gas flow is 0.15ml/min, wherein the detection mode is as follows: monitoring the multi-ion reaction; the retention time, the qualitative and quantitative ion pairs, the taper hole voltage and the collision energy of the ribavirin and the amantadine compounds are as follows:
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| CN113655153B (en) * | 2021-09-09 | 2023-02-03 | 浙江公正检验中心有限公司 | Method for detecting adamantanamine drug residues in animal-derived products |
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