CN113295785A - Method for detecting antiviral drug residue in milk and milk powder - Google Patents
Method for detecting antiviral drug residue in milk and milk powder Download PDFInfo
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- 239000008267 milk Substances 0.000 title claims abstract description 129
- 210000004080 milk Anatomy 0.000 title claims abstract description 129
- 235000013336 milk Nutrition 0.000 title claims abstract description 129
- 239000003443 antiviral agent Substances 0.000 title claims abstract description 69
- 239000000843 powder Substances 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 51
- 238000001514 detection method Methods 0.000 claims abstract description 42
- 238000010828 elution Methods 0.000 claims abstract description 24
- 150000001875 compounds Chemical class 0.000 claims abstract description 19
- 238000011084 recovery Methods 0.000 claims abstract description 19
- 238000011002 quantification Methods 0.000 claims abstract description 14
- 238000012545 processing Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000012421 spiking Methods 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 42
- 239000012086 standard solution Substances 0.000 claims description 36
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 150000002500 ions Chemical class 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 239000011259 mixed solution Substances 0.000 claims description 18
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 claims description 13
- 229960003805 amantadine Drugs 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 12
- UBCHPRBFMUDMNC-UHFFFAOYSA-N 1-(1-adamantyl)ethanamine Chemical compound C1C(C2)CC3CC2CC1(C(N)C)C3 UBCHPRBFMUDMNC-UHFFFAOYSA-N 0.000 claims description 10
- 229960002751 imiquimod Drugs 0.000 claims description 10
- DOUYETYNHWVLEO-UHFFFAOYSA-N imiquimod Chemical compound C1=CC=CC2=C3N(CC(C)C)C=NC3=C(N)N=C21 DOUYETYNHWVLEO-UHFFFAOYSA-N 0.000 claims description 10
- BUGYDGFZZOZRHP-UHFFFAOYSA-N memantine Chemical compound C1C(C2)CC3(C)CC1(C)CC2(N)C3 BUGYDGFZZOZRHP-UHFFFAOYSA-N 0.000 claims description 10
- 229960004640 memantine Drugs 0.000 claims description 10
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 claims description 10
- 229960003752 oseltamivir Drugs 0.000 claims description 10
- 229960000888 rimantadine Drugs 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 239000012496 blank sample Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
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- 238000012544 monitoring process Methods 0.000 claims description 6
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- 238000006297 dehydration reaction Methods 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
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- 239000011148 porous material Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000005185 salting out Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 2
- 239000003640 drug residue Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 description 6
- 238000004451 qualitative analysis Methods 0.000 description 3
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- 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
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- 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
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- 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
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a method for detecting antiviral drug residues in milk and milk powder, which belongs to the technical field of drug residue detection and comprises the following steps: s1, providing a milk sample to be detected, a milk powder sample to be detected, a blank milk sample, a blank milk powder sample and acidified acetonitrile; s2, homogenizing and extracting the target compounds in the milk sample to be detected and the milk powder sample to be detected; s3, purifying the first extracting solution and the second extracting solution respectively; s4 gradient elution treatment; s5 ultra performance liquid chromatography-tandem mass spectrometer detection processing; s6, detecting and calculating the standard curve, the correlation coefficient, the detection limit and the quantification limit of the 5 antiviral drugs; s7 is calculated for the recovery of the 5 antiviral drugs with spiking and the relative standard deviation RSD. The method for detecting the antiviral drug residue provided by the invention is simple to operate, high in sensitivity and good in selectivity, and is suitable for rapid detection of the antiviral drug residue in milk and milk powder.
Description
Technical Field
The invention belongs to the technical field of drug residue detection, and particularly relates to a method for detecting antiviral drug residues in milk and milk powder.
Background
In order to ensure the edible safety of the milk products, the antiviral drug residues in the milk and the milk powder need to be detected and analyzed, and whether each index of the milk and the milk powder is combined with the food health standard or not is evaluated through detection. When the existing drug residue detection method is used for detecting antiviral drugs in milk and milk powder, the operation is inconvenient, the sensitivity is low, the selectivity and the applicability are poor, and the separation, the identification and the rapid detection of the antiviral drug residues in the milk and the milk powder cannot be well carried out.
Disclosure of Invention
The invention aims to provide a method for detecting antiviral drug residues in milk and milk powder, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting antiviral drug residues in milk and milk powder comprises the following steps:
s1, providing a milk sample to be detected, a milk powder sample to be detected, a milk blank sample, a milk powder blank sample, acidified acetonitrile, anhydrous magnesium sulfate and sodium chloride;
s2, homogenizing and extracting the target compounds in the milk sample to be detected and the milk powder sample to be detected;
s21, extracting a target compound in a milk sample to be detected to obtain a mixed solution I;
s22, extracting a target compound in a milk powder sample to be detected to obtain a mixed solution II;
s23 extracting the supernatant of the first mixed solution and the supernatant of the second mixed solution, respectively marking as a first extracting solution and a second extracting solution for standby;
s3, purifying the first extracting solution and the second extracting solution respectively to obtain a first standard solution and a second standard solution;
s31, transferring 2ml of the first extracting solution into a plastic centrifuge tube, uniformly mixing the first extracting solution with the plastic centrifuge tube for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a first standard solution;
s32, transferring 2ml of the second extracting solution into a plastic centrifuge tube, uniformly mixing the two extracting solutions for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a second standard solution;
s4, performing gradient elution treatment on the first standard solution and the second standard solution;
s5, respectively carrying out detection processing on the standard solution I and the standard solution II after elution processing by using an ultra-high performance liquid chromatography-tandem mass spectrometer, and detecting 5 antiviral drugs;
the operating conditions of the tandem mass spectrometer include:
(1) the tandem mass spectrometer adopts an electrospray ionization source ESI, and the temperature of the ionization source is 550 ℃; the electric spray voltage is 5500V;
(2) the tandem mass spectrometer adopts a detection mode of multi-reaction monitoring MRM; the air curtain pressure is 35psi, the collision air pressure is 9psi, the atomizer pressure is 55psi, the auxiliary air pressure is 60 psi;
(3) the tandem mass spectrometer scans in a positive ion mode; the inlet voltage is 10.0V, and the outlet voltage of the collision chamber is 6V;
the 5 antiviral drugs are amantadine, imiquimod, oseltamivir, memantine and rimantadine respectively;
s6, detecting and calculating the standard curve, the correlation coefficient, the detection limit and the quantification limit of the 5 antiviral drugs;
s61, adding a series of first extracting solutions with mass concentrations into a blank milk sample, and drawing a standard curve by taking the mass concentrations of the first extracting solutions as abscissa and taking mass spectrum quantitative peak areas as ordinate;
s62, adding a series of second extracting solutions with mass concentrations into the blank milk powder sample, and drawing a standard curve by taking the mass concentrations of the second extracting solutions as abscissa and taking mass spectrum quantitative peak areas as ordinate;
s63, determining detection limit by S/N being 3, determining quantification limit by S/N being 10, and calculating correlation coefficient, detection limit and quantification limit of 5 antiviral drugs according to standard curve;
s7, standard recovery and relative standard deviation RSD of the 5 antiviral drugs were calculated:
s71, adding the first standard solution into the milk sample according to three addition levels of low, medium and high, parallelly measuring each addition level for 6 times, and calculating the average standard addition recovery rate and the relative standard deviation of the 5 antiviral drugs;
s72 adding the standard solution II into the milk powder sample according to three addition levels of low, medium and high, each addition level is measured in parallel for 6 times, and the average spiking recovery rate and the relative standard deviation of the 5 antiviral drugs are calculated.
As a preferred embodiment, 10ml of acetic acid is added to acetonitrile, and the volume is increased to 1L to obtain the acidified acetonitrile.
Weighing 10g of milk sample, placing the milk sample into a 50mL centrifuge tube, sequentially adding 20mL acidified acetonitrile, 5g anhydrous magnesium sulfate and 2g sodium chloride, shaking for 1min, and centrifuging for 3min by using a centrifuge to obtain a first mixed solution;
and/or weighing 2g of milk powder sample, placing the milk powder sample in a 50mL centrifuge tube, adding 10mL of water, performing vortex mixing, standing for 30min, sequentially adding 20mL of acidified acetonitrile, 5g of anhydrous magnesium sulfate and 2g of sodium chloride, performing oscillation treatment for 1min, and performing centrifugal treatment for 3min by using a centrifugal machine to obtain a mixed solution II.
In a preferred embodiment, the operating parameter of the centrifuge is 5000 r/min; when anhydrous magnesium sulfate and sodium chloride were added, the protein precipitated as the salt concentration in the solution increased, and the salting-out treatment was carried out by dehydration.
As a preferred embodiment, the purification process employs QuEChERS technology; the plastic centrifuge tube contains 200mg of anhydrous magnesium sulfate, 50mg of PSA and 50mg of C18; impurities are absorbed by anhydrous magnesium sulfate, PSA and C18 to remove impurities and purify.
As a preferred embodiment, the gradient elution process conditions include:
(1) the mobile phase of the gradient elution adopts 0.1 percent formic acid water as phase A and acetonitrile as phase B;
(2) the flow rate of the gradient elution was 0.3 mL/min.
As a preferred embodiment, the operating conditions of the ultra-high performance liquid chromatography comprise:
(1) the ultra-high performance liquid chromatography adopts an ACQUITY UPLC BEH C18 chromatographic column; the specification of the C18 chromatographic column is 2.1mm multiplied by 150mm, the column temperature of the C18 chromatographic column is 40 ℃ and the diameter is 1.7 mu m;
(2) the sample injection amount of the ultra-high performance liquid chromatography is 2 mu L.
As a preferred embodiment, the C18 chromatographic column has a packing pore size of 1.7 μm; the C18 column had an internal diameter of 2.1mm and a column length of 150 mm.
As a preferred embodiment, scanning is performed in a positive ion mode, detection is performed in a multi-reaction monitoring MRM mode, the standard solution one and the standard solution two after elution are subjected to detection processing by a high performance liquid chromatography-tandem mass spectrometer, and 5 target compounds in the solutions are separated on the C18 chromatographic column, so that an ion chromatogram of 5 antiviral drugs and an amantadine isotope is obtained.
As a preferred embodiment, the three addition levels are 5. mu.g/kg, 10. mu.g/kg and 50. mu.g/kg, respectively, in the milk sample; in the milk powder sample, the three addition levels are 25 mug/kg, 50 mug/kg and 250 mug/kg respectively.
Compared with the prior art, the invention has the beneficial effects that:
1. the method for detecting the antiviral drug residue in the milk and the milk powder establishes a system for detecting five antiviral drugs including amantadine, imiquimod, oseltamivir, memantine and rimantadine in the milk and the milk powder, and realizes the separation, identification and detection of the five antiviral drugs;
2. according to the detection method for the antiviral drug residue in the milk and the milk powder, detection is carried out under the three conditions of meeting the ultra-high performance liquid chromatography condition, the gradient elution condition and the tandem mass spectrometry condition, and qualitative and quantitative analysis basis of a target compound is provided according to the difference of peak heights in an obtained chromatogram, so that 5 antiviral drugs are identified and obtained;
3. according to the detection method for the antiviral drug residues in the milk and the milk powder, the marked average recovery rate is close to 100% and the relative deviation is small by calculating the average recovery rate and the relative standard deviation, so that the detection method provided by the invention is good in selectivity and applicability, and can be used for rapidly detecting the residues of five compounds, namely amantadine, imiquimod, oseltamivir, memantine and rimantadine, in two matrixes, namely the milk and the milk powder;
in conclusion, the method for detecting the antiviral drug residue provided by the invention is simple to operate, high in sensitivity and good in selectivity, and is suitable for quickly detecting the antiviral drug residue in milk and milk powder.
Drawings
FIG. 1 is a flow chart of the method for detecting residual antiviral drugs in milk and milk powder according to the present invention;
FIG. 2 is an ion chromatogram of amantadine-d 15 of the present invention;
FIG. 3 is an ion chromatogram of amantadine of the present invention;
FIG. 4 is an ion chromatogram of imiquimod according to the invention;
FIG. 5 is an ion chromatogram of oseltamivir of the present invention;
FIG. 6 is an ion chromatogram of memantine of the present invention;
FIG. 7 is an ion chromatogram of rimantadine of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The conditions in the embodiments can be further adjusted according to specific conditions, and simple modifications of the method of the present invention based on the concept of the present invention are within the scope of the claimed invention.
Referring to fig. 1, a method for detecting residual antiviral drugs in milk and milk powder includes the following steps:
s1, providing a milk sample to be detected, a milk powder sample to be detected, a milk blank sample, a milk powder blank sample, acidified acetonitrile, anhydrous magnesium sulfate and sodium chloride;
and adding 10ml of acetic acid into acetonitrile, and fixing the volume to 1L to obtain the acidified acetonitrile.
S2, homogenizing and extracting the target compounds in the milk sample to be detected and the milk powder sample to be detected;
s21, extracting a target compound in a milk sample to be detected to obtain a mixed solution I;
weighing 10g of milk sample, placing the milk sample into a 50mL centrifuge tube, sequentially adding 20mL acidified acetonitrile, 5g anhydrous magnesium sulfate and 2g sodium chloride, shaking for 1min, and centrifuging for 3min by using a centrifuge to obtain a mixed solution I.
S22, extracting a target compound in a milk powder sample to be detected to obtain a mixed solution II;
weighing 2g of milk powder sample, placing the milk powder sample in a 50mL centrifuge tube, adding 10mL of water, carrying out vortex mixing, standing for 30min, sequentially adding 20mL of acidified acetonitrile, 5g of anhydrous magnesium sulfate and 2g of sodium chloride, carrying out oscillation treatment for 1min, and then carrying out centrifugal treatment for 3min by using a centrifugal machine to obtain a mixed solution II.
The working parameter of the centrifugal machine is 5000 r/min; when anhydrous magnesium sulfate and sodium chloride were added, the protein precipitated as the salt concentration in the solution increased, and the salting-out treatment was carried out by dehydration.
S23 extracting the supernatant of the first mixed solution and the supernatant of the second mixed solution, respectively marking as a first extracting solution and a second extracting solution for standby;
s3, purifying the first extracting solution and the second extracting solution respectively to obtain a first standard solution and a second standard solution;
s31, transferring 2ml of the first extracting solution into a plastic centrifuge tube, uniformly mixing the first extracting solution with the plastic centrifuge tube for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a first standard solution;
s32, transferring 2ml of the second extracting solution into a plastic centrifuge tube, uniformly mixing the two extracting solutions for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a second standard solution;
the purification treatment adopts a QuEChERS technology; the plastic centrifuge tube contains 200mg of anhydrous magnesium sulfate, 50mg of PSA and 50mg of C18; impurities are absorbed by anhydrous magnesium sulfate, PSA and C18 to remove impurities and purify.
S4, performing gradient elution treatment on the first standard solution and the second standard solution;
the gradient elution process conditions include:
(1) the mobile phase of the gradient elution adopts 0.1 percent formic acid water as phase A and acetonitrile as phase B;
(2) the flow rate of the gradient elution was 0.3 mL/min.
Mobile phase gradient elution conditions, as shown in table 1:
table 1:
gradient elution is used to improve the separation degree, shorten the separation time, reduce the minimum detection amount and improve the separation precision.
S5, respectively carrying out detection processing on the standard solution I and the standard solution II after elution processing by using an ultra-high performance liquid chromatography-tandem mass spectrometer, and detecting 5 antiviral drugs;
the operating conditions of the ultra-high performance liquid chromatography comprise:
(1) the ultra-high performance liquid chromatography adopts an ACQUITY UPLC BEH C18 chromatographic column; the specification of the C18 chromatographic column is 2.1mm multiplied by 150mm, the column temperature of the C18 chromatographic column is 40 ℃ and the diameter is 1.7 mu m;
the pore diameter of the C18 chromatographic column is 1.7 mu m; the C18 column had an internal diameter of 2.1mm and a column length of 150 mm.
(2) The sample injection amount of the ultra-high performance liquid chromatography is 2 mu L.
The operating conditions of the tandem mass spectrometer include:
(1) the tandem mass spectrometer adopts an electrospray ionization source ESI, and the temperature of the ionization source is 550 ℃; the electrospray voltage was 5500V.
(2) The tandem mass spectrometer adopts a detection mode of multi-reaction monitoring MRM; the air curtain pressure was 35psi, the collision air pressure was 9psi, the atomizer pressure was 55psi, and the auxiliary air pressure was 60 psi.
(3) The tandem mass spectrometer scans in a positive ion mode; the inlet voltage was 10.0V and the collision cell outlet voltage was 6V.
Scanning in a positive ion mode, detecting in a multi-reaction monitoring MRM mode, detecting the standard solution I and the standard solution II after elution in a high performance liquid chromatography-tandem mass spectrometer, and separating 5 target compounds in the solution on the C18 chromatographic column to obtain the ion chromatogram of the 5 antiviral drugs and the amantadine isotope. The 5 antiviral drugs are amantadine, imiquimod, oseltamivir, memantine and rimantadine, respectively.
Referring again to FIGS. 2-7, FIG. 2 shows an ion chromatogram of amantadine-d 15; FIG. 3 shows an ion chromatogram of amantadine; FIG. 4 shows an ion chromatogram of imiquimod; FIG. 5 shows an ion chromatogram of oseltamivir; FIG. 6 shows an ion chromatogram of memantine; FIG. 7 shows an ion chromatogram of rimantadine. The chromatogram is obtained by performing detection processing under the conditions of meeting the ultra-high performance liquid chromatography condition, the gradient elution condition and the tandem mass spectrometry condition, and qualitative analysis basis of the target compound is provided according to the difference of peak heights in the chromatogram, so that 5 antiviral drugs are identified and obtained.
The external standard method is used for quantification, and the detection result of the high performance liquid chromatography-tandem mass spectrometer is shown in table 2:
table 2:
referring to table 2, it can be seen that 5 antiviral drugs can be well separated, identified and detected under the condition of detecting instrument parameters in the table.
S6, detecting and calculating the standard curve, the correlation coefficient, the detection limit and the quantification limit of the 5 antiviral drugs;
s61, adding a series of extracting solutions I with mass concentration into a blank milk sample, and drawing a standard curve by taking the mass concentration of the extracting solutions I as an abscissa and taking a mass spectrum quantitative peak area as an ordinate.
S62, adding a series of second extracting solutions with mass concentrations into the blank milk powder sample, and drawing a standard curve by taking the mass concentrations of the second extracting solutions as abscissa and taking the mass spectrum quantitative peak area as ordinate.
S63, determining detection limit by S/N being 3, determining quantification limit by S/N being 10, calculating correlation coefficient, detection limit and quantification limit of 5 antiviral drugs according to standard curve.
The results of the standard curves, correlation coefficients, detection limits and quantitation limits of the 5 antiviral drugs are shown in table 3.
Table 3:
referring to Table 3, it can be found that 5 antiviral drugs show good linear relationship in the range of 1-100 μ g/L, and the correlation coefficient R is greater than 0.995. The detection limit is determined by the signal-to-noise ratio S/N being 3, the quantification limit is determined by the signal-to-noise ratio S/N being 10, and the detection limit and the quantification limit of 5 antiviral drugs in the milk are respectively 0.03 mu g/kg and 0.1 mu g/kg. The detection limit of 5 antiviral drugs in the milk powder is 0.15 mug/kg, and the quantification limit is 0.5 mug/kg.
S7, standard recovery and relative standard deviation RSD of the 5 antiviral drugs were calculated:
s71, adding the first standard solution into the milk sample according to three addition levels of low, medium and high, parallelly measuring each addition level for 6 times, and calculating the average standard addition recovery rate and the relative standard deviation of the 5 antiviral drugs;
in the milk samples, the three addition levels were 5. mu.g/kg, 10. mu.g/kg and 50. mu.g/kg, respectively.
S72, adding the standard solution II into the milk powder sample according to three addition levels of low, medium and high, parallelly measuring each addition level for 6 times, and calculating the average standard addition recovery rate and the relative standard deviation of the 5 antiviral drugs;
in the milk powder sample, the three addition levels are 25 mug/kg, 50 mug/kg and 250 mug/kg respectively.
The results of the calculation of the spiked recovery and relative standard deviation RSD for the 5 antiviral drugs are shown in table 4.
Table 4:
referring to table 4, it can be seen that the normalized recovery of the 5 antiviral drugs at three addition levels (5, 10 and 50 μ g/kg) in the milk base is 87.2% to 105.8% with a relative standard deviation (RSD, n ═ 6) of 2.1% to 6.2%. The recovery rate of the 5 antiviral drugs at three addition levels (25, 50 and 250 mug/kg) in the milk powder base is 86.4-104.9%, and the relative standard deviation (RSD, n ═ 6) is 2.4-6.4%. The evaluation recovery rate is close to 100% and the relative deviation is small by calculating the average standard addition recovery rate and the relative standard deviation RSD, which shows that the detection method provided by the invention has good selectivity and applicability, can be used for detecting the residues of five compounds, namely amantadine, imiquimod, oseltamivir, memantine and rimantadine, in two matrixes, namely milk and milk powder.
In summary, compared to the following drug residue detection method, the drug residue detection method provided in this embodiment has the following advantages:
in this embodiment, a system for determining residues of five antiviral drugs including amantadine, imiquimod, oseltamivir, memantine, and rimantadine in milk and milk powder is established, so as to realize separation, identification, and detection of the five antiviral drugs. The method provides a qualitative and quantitative analysis basis for the target compound according to the difference of peak heights in the chromatogram by detecting under the conditions of meeting the ultra-high performance liquid chromatography condition, the gradient elution condition and the tandem mass spectrometry condition, thereby identifying and obtaining 5 antiviral drugs. The average recovery rate and the relative standard deviation are calculated to obtain the result that the added standard average recovery rate is close to 100%, and the relative deviation is small, so that the detection method provided by the embodiment has good selectivity and applicability, and can be used for rapidly detecting the residues of five compounds, namely amantadine, imiquimod, oseltamivir, memantine and rimantadine, in two matrixes, namely milk and milk powder.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The method for detecting the antiviral drug residue in milk and milk powder is characterized by comprising the following steps of:
s1, providing a milk sample to be detected, a milk powder sample to be detected, a milk blank sample, a milk powder blank sample, acidified acetonitrile, anhydrous magnesium sulfate and sodium chloride;
s2, homogenizing and extracting the target compounds in the milk sample to be detected and the milk powder sample to be detected;
s21, extracting a target compound in a milk sample to be detected to obtain a mixed solution I;
s22, extracting a target compound in a milk powder sample to be detected to obtain a mixed solution II;
s23 extracting the supernatant of the first mixed solution and the supernatant of the second mixed solution, respectively marking as a first extracting solution and a second extracting solution for standby;
s3, purifying the first extracting solution and the second extracting solution respectively to obtain a first standard solution and a second standard solution;
s31, transferring 2ml of the first extracting solution into a plastic centrifuge tube, uniformly mixing the first extracting solution with the plastic centrifuge tube for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a first standard solution;
s32, transferring 2ml of the second extracting solution into a plastic centrifuge tube, uniformly mixing the two extracting solutions for 1min in a vortex mode, centrifuging the mixture for 3min by using a centrifuge, absorbing supernate and filtering the supernate through a microporous filter membrane to obtain a second standard solution;
s4, performing gradient elution treatment on the first standard solution and the second standard solution;
s5, respectively carrying out detection processing on the standard solution I and the standard solution II after elution processing by using an ultra-high performance liquid chromatography-tandem mass spectrometer, and detecting 5 antiviral drugs;
the operating conditions of the tandem mass spectrometer include:
(1) the tandem mass spectrometer adopts an electrospray ionization source ESI, and the temperature of the ionization source is 550 ℃; the electric spray voltage is 5500V;
(2) the tandem mass spectrometer adopts a detection mode of multi-reaction monitoring MRM; the air curtain pressure is 35psi, the collision air pressure is 9psi, the atomizer pressure is 55psi, the auxiliary air pressure is 60 psi;
(3) the tandem mass spectrometer scans in a positive ion mode; the inlet voltage is 10.0V, and the outlet voltage of the collision chamber is 6V;
the 5 antiviral drugs are amantadine, imiquimod, oseltamivir, memantine and rimantadine respectively;
s6, detecting and calculating the standard curve, the correlation coefficient, the detection limit and the quantification limit of the 5 antiviral drugs;
s61, adding a series of first extracting solutions with mass concentrations into a blank milk sample, and drawing a standard curve by taking the mass concentrations of the first extracting solutions as abscissa and taking mass spectrum quantitative peak areas as ordinate;
s62, adding a series of second extracting solutions with mass concentrations into the blank milk powder sample, and drawing a standard curve by taking the mass concentrations of the second extracting solutions as abscissa and taking mass spectrum quantitative peak areas as ordinate;
s63, determining detection limit by S/N being 3, determining quantification limit by S/N being 10, and calculating correlation coefficient, detection limit and quantification limit of 5 antiviral drugs according to standard curve;
s7, standard recovery and relative standard deviation RSD of the 5 antiviral drugs were calculated:
s71, adding the first standard solution into the milk sample according to three addition levels of low, medium and high, parallelly measuring each addition level for 6 times, and calculating the average standard addition recovery rate and the relative standard deviation of the 5 antiviral drugs;
s72 adding the standard solution II into the milk powder sample according to three addition levels of low, medium and high, each addition level is measured in parallel for 6 times, and the average spiking recovery rate and the relative standard deviation of the 5 antiviral drugs are calculated.
2. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: and adding 10ml of acetic acid into acetonitrile, and fixing the volume to 1L to obtain the acidified acetonitrile.
3. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: weighing 10g of milk sample, placing the milk sample into a 50mL centrifuge tube, sequentially adding 20mL acidified acetonitrile, 5g anhydrous magnesium sulfate and 2g sodium chloride, shaking for 1min, and centrifuging for 3min by using a centrifuge to obtain a mixed solution I;
and/or weighing 2g of milk powder sample, placing the milk powder sample in a 50mL centrifuge tube, adding 10mL of water, performing vortex mixing, standing for 30min, sequentially adding 20mL of acidified acetonitrile, 5g of anhydrous magnesium sulfate and 2g of sodium chloride, performing oscillation treatment for 1min, and performing centrifugal treatment for 3min by using a centrifugal machine to obtain a mixed solution II.
4. The method for detecting antiviral drug residues in milk and milk powder according to claim 3, wherein the method comprises the following steps: the working parameter of the centrifugal machine is 5000 r/min; when anhydrous magnesium sulfate and sodium chloride were added, the protein precipitated as the salt concentration in the solution increased, and the salting-out treatment was carried out by dehydration.
5. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: the purification treatment adopts a QuEChERS technology; the plastic centrifuge tube contains 200mg of anhydrous magnesium sulfate, 50mg of PSA and 50mg of C18; impurities are absorbed by anhydrous magnesium sulfate, PSA and C18 to remove impurities and purify.
6. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: the gradient elution process conditions include:
(1) the mobile phase of the gradient elution adopts 0.1 percent formic acid water as phase A and acetonitrile as phase B;
(2) the flow rate of the gradient elution was 0.3 mL/min.
7. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: the operating conditions of the ultra-high performance liquid chromatography comprise:
(1) the ultra-high performance liquid chromatography adopts an ACQUITY UPLC BEH C18 chromatographic column; the specification of the C18 chromatographic column is 2.1mm multiplied by 150mm, the column temperature of the C18 chromatographic column is 40 ℃ and the diameter is 1.7 mu m;
(2) the sample injection amount of the ultra-high performance liquid chromatography is 2 mu L.
8. The method for detecting residual antiviral drugs in milk and milk powder according to claim 7, wherein the method comprises the following steps: the pore diameter of the C18 chromatographic column is 1.7 mu m; the C18 column had an internal diameter of 2.1mm and a column length of 150 mm.
9. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: scanning in a positive ion mode, detecting in a multi-reaction monitoring MRM mode, detecting the standard solution I and the standard solution II after elution in a high performance liquid chromatography-tandem mass spectrometer, and separating 5 target compounds in the solution on the C18 chromatographic column to obtain the ion chromatogram of the 5 antiviral drugs and the amantadine isotope.
10. The method for detecting antiviral drug residues in milk and milk powder according to claim 1, wherein the method comprises the following steps: in the milk sample, the three addition levels are respectively 5 mug/kg, 10 mug/kg and 50 mug/kg; in the milk powder sample, the three addition levels are 25 mug/kg, 50 mug/kg and 250 mug/kg respectively.
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