CN104849383A - Method for determining nitroimidazole drug in bee pollen powder through combination of rapid solvent extraction-gel chromatography purification-LC/MS/MS - Google Patents
Method for determining nitroimidazole drug in bee pollen powder through combination of rapid solvent extraction-gel chromatography purification-LC/MS/MS Download PDFInfo
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- YZEUHQHUFTYLPH-UHFFFAOYSA-N 2-nitroimidazole Chemical compound [O-][N+](=O)C1=NC=CN1 YZEUHQHUFTYLPH-UHFFFAOYSA-N 0.000 title abstract 3
- 229940038481 bee pollen Drugs 0.000 title abstract 3
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- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 claims description 12
- 229960000282 metronidazole Drugs 0.000 claims description 12
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- 239000012086 standard solution Substances 0.000 claims description 10
- IBXPYPUJPLLOIN-UHFFFAOYSA-N dimetridazole Chemical compound CC1=NC=C(N(=O)=O)N1C IBXPYPUJPLLOIN-UHFFFAOYSA-N 0.000 claims description 9
- 229960000946 dimetridazole Drugs 0.000 claims description 9
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
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- IBXPYPUJPLLOIN-BMSJAHLVSA-N 2-methyl-5-nitro-1-(trideuteriomethyl)imidazole Chemical compound [2H]C([2H])([2H])N1C(C)=NC=C1[N+]([O-])=O IBXPYPUJPLLOIN-BMSJAHLVSA-N 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- WACQKHWOTAEEFS-UHFFFAOYSA-N cyclohexane;ethyl acetate Chemical compound CCOC(C)=O.C1CCCCC1 WACQKHWOTAEEFS-UHFFFAOYSA-N 0.000 claims description 5
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 claims description 3
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- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 24
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- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- NTAFJUSDNOSFFY-UHFFFAOYSA-N Ipronidazole Chemical compound CC(C)C1=NC=C([N+]([O-])=O)N1C NTAFJUSDNOSFFY-UHFFFAOYSA-N 0.000 description 1
- HJLSLZFTEKNLFI-UHFFFAOYSA-N Tinidazole Chemical compound CCS(=O)(=O)CCN1C(C)=NC=C1[N+]([O-])=O HJLSLZFTEKNLFI-UHFFFAOYSA-N 0.000 description 1
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- PQFRTXSWDXZRRS-UHFFFAOYSA-N ronidazole Chemical compound CN1C(COC(N)=O)=NC=C1[N+]([O-])=O PQFRTXSWDXZRRS-UHFFFAOYSA-N 0.000 description 1
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a detection method for a nitroimidazole drug in bee pollen powder, particularly to a method for determining a nitroimidazole drug in bee pollen powder through combination of rapid solvent extraction-gel chromatography purification-LC/MS/MS. According to the method, ethyl acetate is adopted as an extraction solvent, extraction with an accelerated solvent extractor (ASE) and gel chromatography purification (GPC) are performed, a liquid chromatography-mass spectrometry multiple reaction positive ion monitoring mode is used to determine, a matrix-matched standard curve isotope internal standard method is used to quantitate, and the results comprise the nitroimidazole compound detection limit is 1.5 [mu]g.kg<-1>, and the quantification limit is 5.0 [mu]g.kg<-1>. According to the present invention, the method recovery rate is 93.5-114.2%, and the relative standard deviation is 0.3-5.2%; and the conclusion is that the method has advantages of high automation degree, high sensitivity, accurate qualitative and quantitative result, and the like.
Description
Technical field
The present invention relates to the detection method of nitroimidazoles medicine in a kind of Bee Pollen.
Background technology
Nitroimidazoles medicine is that a class has antibacterial and antiprotozoal medicine, is mainly used in livestock and poultry animal cultivation field.Such drug main will comprise metronidazole, Dimetridazole, Lip river nitre reach azoles, Tinidazole, ipronidazole, hydroxylation metronidazole etc.Research shows that such medicine has potential cell mutation, carcinogenicity, and therefore, the U.S., European Union, Japan and China all will list in class medicine and prohibit the use compound inventory.Such drug main of the mensuration of current bibliographical information will concentrate in the matrix such as honey, milk, aquatic products, cosmetics, feed, and pre-treatment mainly adopts the organic reagents such as ethyl acetate manually to extract, complex operation step, consuming time.
Accelerate solvent extraction method is a kind of pretreatment technology of new development in recent years, is widely used in solid sample in pollutant, animal derived food veterinary drug residue etc., has that solvent load is few, extraction time is short and the advantage such as sample extraction robotization.GPC GPC cleanup system can be good at removing the macromolecular substances such as fat, pigment.
Summary of the invention
In order to solve above-mentioned technical matters, the object of this invention is to provide a kind of Accelerate solvent extraction-GPC cleanup system-LC/MS/MS and combine the method measuring nitroimidazoles medicine in Bee Pollen, the method has the advantages such as automaticity is high, highly sensitive, quantitative and qualitative analysis result is accurate, can meet the requirement of wild animal resources method.
In order to realize above-mentioned object, present invention employs following technical scheme:
Accelerate solvent extraction-GPC cleanup system-LC/MS/MS combines the method measuring nitroimidazoles medicine in Bee Pollen, and the method comprises the following steps:
1) preparation of standard solution
Standard reserving solution: accurately respectively take appropriate metronidazole, Dimetridazole, Lip river nitre reach azoles, hydroxyl Dimetridazole-D3, Dimetridazole-D3, Lip river nitre reaches azoles-D3 standard items, dissolve with methyl alcohol and be settled to 25 mL, being configured to 0.1 mgmL
-1standard reserving solution; This solution is preserved in the refrigerator of 4 DEG C;
Intermediate standard storing solution: accurately draw appropriate standard reserving solution respectively, become 0.1 μ gmL with methyl alcohol stepwise dilution
-1intermediate standard storing solution;
Standard working solution: the intermediate standard storing solution drawing different volumes, is diluted to 1.0,2.0,5.0,10.0,15.0 ngmL with blank Bee Pollen matrix extract
-1standard working solution, wherein inner mark solution is 5.0 ngmL
-1, now with the current;
2) sample-pretreating method
Accurately take Bee Pollen sample 2 g, in 150 mL beakers, be accurate to 0.01 g, add inner mark solution, add 2 g zeyssatite, sample is mixed with zeyssatite, the sample mixed is transferred to bottom and is covered with in the abstraction pool of silica sand, top repaves one deck silica sand, add a cover and tighten, abstraction pool is put into Accelerate solvent extraction instrument and extract, extract is collected in 240 mL receiving flasks, be transferred to by extract in heart bottle, 45 DEG C of backspins steam to dry; Add 10 mL Ethyl acetate-cyclohexane dissolved residues, Ethyl acetate-cyclohexane volume ratio 1:1, solution is crossed the filter membrane of 0.45 μm in GPC sample bottle, to be clean;
GPC gel permeation chrommatograph is used to purify the solution through membrane filtration, purification condition is shown in " 2.6 ", collect solution 5 mL after purification, at 45 DEG C, nitrogen dries up, and adds 1 mL methanol aqueous solution, the volume ratio 1:9 of methanol-water in methanol aqueous solution, ultrasonic 1 min, vortex mixed 1 min, crosses 0.22 μm of filter membrane, for liquid chromatography-Tandem Mass Spectrometry Analysis;
3) liquid phase chromatogram condition
Chromatographic column: Zorbax Eclipse Plus C18,2.1*100 mm, 3.5 um; Mobile phase: A phase is 0.15% aqueous formic acid; B phase is methyl alcohol; Column temperature 40 DEG C; Flow velocity: 0.3 mLmin
-1; Sample size 5 μ L; Liquid chromatography flow velocity and gradient elution as follows:
4) mass spectrometry parameters
Ion gun: electro-spray ionization (ESI) positive ion mode; Dry gas temperature: 325 DEG C; Dry gas flow velocity: 5 Lmin
-1; Spray pressure power: 45 psi; Sheath temperature degree: 400 DEG C; Sheath gas velocity: 12 Lmin
-1; Capillary voltage: 4000 V; Multiple-reaction monitoring scanning collection parameter is as follows:
5) Accelerate solvent extraction condition
Abstraction pool: 20 mL; Extraction solvent: ethyl acetate; Extracting pressure: 90 bar; Extraction temperature: 90 DEG C; Retention time: 5 min; Solvent washing: 30 s; Cycle index: 2 times;
6) GPC cleanup system condition
GPC decontaminating column; Filler 50.0 g Bio-Beads S-X3; Mobile phase: cyclohexane-ethyl acetate, volume ratio 1:1; Flow velocity: 5.0 mLmin
-1; The prewashing time: 10 s; Impurity abandons the time: 1200 s; Acquisition time: 800 s; Rinse pillar: 300 s; Sample size: 5 mL.
The present invention is owing to have employed above-mentioned technical scheme, and nitro glyoxaline compound detects and is limited to 1.5 μ gkg
-1, be quantitatively limited to 5.0 μ gkg
-1.The method recovery is between 93.5% ~ 114.2%, and relative standard deviation is between 0.3 ~ 5.2%.Conclusion: the advantages such as it is high, highly sensitive that the method has automaticity, and quantitative and qualitative analysis result is accurate.
Accompanying drawing explanation
The calibration curve of Fig. 1 metronidazole in matrix matching liquid and solvent.
Fig. 2 extraction temperature is on the impact of nitro glyoxaline extraction efficiency.
Fig. 3 extracting pressure is on the impact of nitro glyoxaline extraction efficiency.
Embodiment
instrument and reagent
1.1 materials and reagent
Zeyssatite; Methyl alcohol, acetonitrile, ethyl acetate, cyclohexane (chromatographically pure, Thermo Fisher Scientific Inc.); Ultrapure water.Standard substance: metronidazole (MNZ), Dimetridazole (DMZ), Lip river nitre reach azoles (RNZ) (purity > 99.0%, German Dr company); Hydroxyl Dimetridazole-D3 (DMZOH-D3), Dimetridazole-D3 (DMZ-D3), Lip river nitre reach azoles-D3 (RNZ-D3) (purity > 99.0%, German Dr company).
1.2 instrument and equipment
1260-6460 liquid chromatography-tandem mass spectrometry instrument (Anjelen Sci. & Tech. Inc of the U.S.); E-916 Accelerate solvent extraction instrument (BUCHI company of Switzerland); Gel purification chromatograph (German LC-Tech company); R-210 Rotary Evaporators (BUCHI company of Switzerland); MS3 Digital vortex oscillator (German IKA company); N-EVAP
tM111 Nitrogen evaporators (organomation company of the U.S.); BARNSTEAD NANOPURE ultrapure water instrument (Sai Mofei company of the U.S.); Analytical balance (Mettler Toledo Inc.).
test condition and method
The preparation of 2.1 standard solution
Standard reserving solution: accurately respectively take appropriate metronidazole, Dimetridazole, Lip river nitre reach azoles, hydroxyl Dimetridazole-D3, Dimetridazole-D3, Lip river nitre reaches azoles-D3 standard items, dissolve with methyl alcohol and be settled to 25 mL, being configured to 0.1 mgmL
-1standard reserving solution.This solution is preserved in the refrigerator of 4 DEG C;
Intermediate standard storing solution: accurately draw appropriate standard reserving solution respectively, become 0.1 μ gmL with methyl alcohol stepwise dilution
-1intermediate standard storing solution;
Standard working solution: the intermediate standard storing solution drawing different volumes, is diluted to 1.0,2.0,5.0,10.0,15.0 ngmL with blank Bee Pollen matrix extract (processing by under " 2.2 " item)
-1standard working solution, wherein inner mark solution is 5.0 ngmL
-1, now with the current.
2.2 sample-pretreating method
Accurately take Bee Pollen sample 2 g(and be accurate to 0.01 g) in 150 mL beakers, add inner mark solution, add 2 g zeyssatite, sample is mixed with zeyssatite, the sample mixed is transferred to bottom and is covered with in the abstraction pool of silica sand, top repaves one deck silica sand, add a cover and tighten, abstraction pool is put into Accelerate solvent extraction instrument and extract, extract is collected in 240 mL receiving flasks, be transferred to by extract in heart bottle, 45 DEG C of backspins steam to dry.Add 10 mL Ethyl acetate-cyclohexane (volume ratio 1:1) dissolved residues, solution is crossed the filter membrane of 0.45 μm in GPC sample bottle, to be clean.
GPC gel permeation chrommatograph is used to purify the solution through membrane filtration, purification condition is shown in " 2.6 ", collect solution 5 mL after purification, at 45 DEG C, nitrogen dries up, add 1 mL methanol aqueous solution (volume ratio 1:9), ultrasonic 1 min, vortex mixed 1 min, cross 0.22 μm of filter membrane, for liquid chromatography-Tandem Mass Spectrometry Analysis.
2.3 liquid phase chromatogram condition
Chromatographic column: Zorbax Eclipse Plus C18 (2.1 x 100 mm, 3.5 um); Mobile phase: A phase is 0.15% aqueous formic acid; B phase is methyl alcohol; Column temperature 40 DEG C; Flow velocity: 0.3 mLmin
-1; Sample size 5 μ L; Liquid chromatography flow velocity and gradient elution are in table 1;
table 1 liquid chromatography flow velocity and gradient elution program
2.4 mass spectrometry parameters
Ion gun: electro-spray ionization (ESI) positive ion mode; Dry gas temperature: 325 DEG C; Dry gas flow velocity: 5 Lmin
-1; Spray pressure power: 45 psi; Sheath temperature degree: 400 DEG C; Sheath gas velocity: 12 Lmin
-1; Capillary voltage: 4000 V; Multiple-reaction monitoring scanning (MRM) acquisition parameter is in table 2;
table 2 nitro glyoxaline liquid matter location parameter
2.5 Accelerate solvent extraction conditions
Abstraction pool: 20 mL; Extraction solvent: ethyl acetate; Extracting pressure: 90 bar; Extraction temperature: 90 DEG C; Retention time: 5 min; Solvent washing: 30 s; Cycle index: 2 times.
2.6 GPC cleanup system conditions
GPC decontaminating column (25 mm × 400 mm); Filler 50.0 g Bio-Beads S-X3(Bio-Rad); Mobile phase: cyclohexane-ethyl acetate (volume ratio 1:1); Flow velocity: 5.0 mLmin
-1; The prewashing time: 10 s; Impurity abandons the time: 1200 s; Acquisition time: 800 s; Rinse pillar: 300 s.Sample size: 5 mL.
methodological study
3.1 typical curves and detection limit
Get the standard working solution by preparing under " 2.1 " item, measure with the condition under " 2.3 " and " 2.4 " item, record object analyzes thing and interior target peak area, with each target analytes and interior target concentration ratio X for horizontal ordinate, with target analytes and interior target peak area ratio Y for ordinate, carry out regretional analysis.Nitro glyoxaline compound is at 1.0 ~ 15.0 ngmL
-1in good linear relationship in scope.
The detection limit of the method and quantitative limit are added target analytes by blank sample and are carried out recovery experiment acquisition, process by the condition under " 2.2 " item, condition under " 2.3 " and " 2.4 " item measures, with 3 times of signal to noise ratio (S/N ratio)s (S/N > 3) be detection limit, 10 times of signal to noise ratio (S/N ratio)s (S/N > 10) are for quantitative limit.The results are shown in Table 3.
the linear result of table 3 nitro glyoxaline compound and detection limit
3.2 recovery and precision
For measuring precision and the recovery of this method, experiment selects blank Bee Pollen to be sample substrate, and add the nitro glyoxaline standard solution of different amount, mark-on level is respectively 5.0,10.0,20.0 μ gkg
-1, each Pitch-based sphere all does 6 Duplicate Samples, and it the results are shown in Table 4;
table 4 recovery and Precision Experiment result (n=6)
The matrix effect of 3.3 methods
Get blank Bee Pollen sample, obtain vehicle solution by legal system below " 2.2 " item, use vehicle solution standard solution to be diluted to 1.0,2.0,5.0,10.0,15.0 ngmL
-1.Separately get standard solution, use methanol aqueous solution (volume ratio 1:9) to be diluted to 1.0,2.0,5.0,10.0,15.0 ngmL
-1.By above-mentioned extraction standard solution and solvent standard solution sample introduction respectively, drawing standard curve.After relatively, find that Dimetridazole and Lip river nitre reach azoles matrix curve more consistent with solvent curve, it is consistent that two kinds of curves carry out quantitative result to sample.And metronidazole matrix curve and solvent curve difference are comparatively greatly, there is obvious matrix effect, use matrix curve to carry out quantitatively, result conforms to actual, and use solvent curve to carry out quantitatively, result is less than normal.Therefore, the present invention uses matrix curve to carry out quantitatively.Fig. 1 is the calibration curve of metronidazole in matrix matching liquid and solvent.
actual sample is analyzed
The analytical approach adopting the present invention to set up, detects Bee Pollen sample (8 parts) commercially, and result is not tested with nitroimidazoles medicine and remains.
discuss
The selection of 5.1 liquid phase chromatogram conditions
Although Bee Pollen sample, through GPC purification, also also exists some small-molecule substances, can have an impact to the ionization of target analytes when carrying out LC-MS mensuration.Interfering material, by optimize chromatography condition, is separated with target analytes by the present invention as far as possible, reduces matrix effect.Choice for use Agilent Eclipse Plus-C18 (2.1 mm × 100 mm, 3.5 μm) chromatographic column of the present invention, and use gradient elution program to analyze.By optimizing, chaff interference and target analytes can be separated substantially.Condition after optimization is shown in liquid phase chromatogram condition under " 2.3 " item.
The foundation of 5.2 mass spectrometry parameters
The mass spectrometric advantage of triple level Four bars is quantitatively accurately, and the present invention adopts multiple-reaction monitoring pattern to carry out qualitative and quantitative analysis.By standard solution by liquid chromatography sample introduction, under one-level positive ion mode, carry out precursor scans, determine quasi-molecular ion peak [M+1]
-, then carry out cracked to the parent ion of target analytes respectively, carry out the scanning of secondary daughter ion, select 2 feature daughter ions, selection signal to noise ratio (S/N ratio) is large, peak shape good, disturb little ion as quota ion, and another ion is qualitative ion.Condition after optimization is shown in mass spectrometry parameters under " 2.4 " item.
The selection of 5.3 Accelerate solvent extraction conditions
Accelerate solvent extraction is a kind of modern sample preparation technology, is one of prefered method effectively extracting organic contaminant in food
[12].The ultimate principle of Accelerate solvent extraction utilizes raised temperature and pressure, increases Solubility of Substances and solutes accumulation efficiency, improves extraction efficiency.The present invention chooses 80,90,100,110 DEG C of 4 extraction temperature and tests.Fig. 2 is the impact of extraction temperature on nitroimidazoles medicine extraction efficiency.As shown in Figure 2,90 DEG C time, the effect of extracting of nitro glyoxaline is best, so choice for use 90 DEG C is as extraction temperature.When carrying out extracting pressure and optimizing, extraction temperature is set as 90 DEG C, extracting pressure is set as that 80,90,100,110 bar test respectively.Fig. 3 is the impact of extracting pressure on nitroimidazoles medicine extraction efficiency.As shown in Figure 3, when 90 bar, the effect of extracting of nitro glyoxaline is best, so choice for use 90 bar is as extracting pressure.Condition after optimization is shown in Accelerate solvent extraction condition under " 2.5 " item.
The selection of 5.4 GPC cleanup system conditions
Because Bee Pollen matrix is complicated, containing a large amount of natural colouring matters, flavones and fibre composition, in the analysis of liquid matter, there is serious matrix effect, need after the extraction to purify.Gel chromatography utilizes size exclusion to be separated, and effectively can remove the chaff interference of the high molecule mass such as grease, natural colouring matter
[13], therefore, the present invention uses gel chromatography to purify.
10.0 mL concentration are 6.0 ngmL by the present invention
-1the mixed mark of nitro glyoxaline cross GPC gel column as sample solution, carry out Fractional Collections, flow velocity: 5 mLmin
-1, start after rinsing gel column 960 s to collect, collect 20 pipes altogether, often 120 s collected by pipe.Dried up by collection liquid nitrogen, redissolve with 1 mL mobile phase, upper liquid matter is analyzed.
Through data analysis, from the 3rd pipe, after namely rinsing gel column 1200 s, nitro glyoxaline compound is washed out, and by the end of the 9th pipe, nitro glyoxaline compound by complete wash-out out.Therefore, the GPC condition after optimization is: impurity abandons the time, 1200 s; Acquisition time, 800 s.Condition after optimization is shown in GPC cleanup system condition under " 2.6 " item.
The elimination of 5.5 matrix effects
Matrix effect refers in LC/MS measures, the substance change Ionization Efficiency of composition to be measured of co-elute during chromatographic resolution, the suppression of caused signal or raising.The present invention measures matrix effect by the method extracting rear interpolation composition to be measured, according to the model that the people such as Matuszewski propose, its computing formula is as follows: Matrix effect (ME)=A/B × 100% (A: the response adding concentration known composition to be measured in the blank sample matrix through pre-treatment; B: the response of isoconcentration pure solution to be measured).
When concentration of standard solution is 5 ngmL
-1time, metronidazole, Dimetridazole, MCMN's matrix effect are respectively: ME (metronidazole)=18%, ME (Dimetridazole)=32%, ME (Lip river nitre reaches azoles)=12%.The method eliminating matrix effect has the suitable chromatographic separation technology of selection, selects suitable interior mark, improves Pretreatment etc.The present invention is by be marked with in using and the method such as bare substrate extract preparation typical curve reduces the impact of matrix effect, and interior mark it would be desirable Isotopic Internal Standard.Therefore, the present invention uses Isotopic Internal Standard in conjunction with bare substrate extract preparation typical curve, adopts this curve to carry out quantitatively.
Claims (1)
1. Accelerate solvent extraction-GPC cleanup system-LC/MS/MS combines the method measuring nitroimidazoles medicine in Bee Pollen, it is characterized in that the method comprises the following steps:
1) preparation of standard solution
Standard reserving solution: accurately respectively take appropriate metronidazole, Dimetridazole, Lip river nitre reach azoles, hydroxyl Dimetridazole-D3, Dimetridazole-D3, Lip river nitre reaches azoles-D3 standard items, dissolve with methyl alcohol and be settled to 25 mL, being configured to 0.1 mgmL
-1standard reserving solution; This solution is preserved in the refrigerator of 4 DEG C;
Intermediate standard storing solution: accurately draw appropriate standard reserving solution respectively, become 0.1 μ gmL with methyl alcohol stepwise dilution
-1intermediate standard storing solution;
Standard working solution: the intermediate standard storing solution drawing different volumes, is diluted to 1.0,2.0,5.0,10.0,15.0 ngmL with blank Bee Pollen matrix extract
-1standard working solution, wherein inner mark solution is 5.0 ngmL
-1, now with the current;
2) sample-pretreating method
Accurately take Bee Pollen sample 2 g, in 150 mL beakers, be accurate to 0.01 g, add inner mark solution, add 2 g zeyssatite, sample is mixed with zeyssatite, the sample mixed is transferred to bottom and is covered with in the abstraction pool of silica sand, top repaves one deck silica sand, add a cover and tighten, abstraction pool is put into Accelerate solvent extraction instrument and extract, extract is collected in 240 mL receiving flasks, be transferred to by extract in heart bottle, 45 DEG C of backspins steam to dry; Add 10 mL Ethyl acetate-cyclohexane dissolved residues, Ethyl acetate-cyclohexane volume ratio 1:1, solution is crossed the filter membrane of 0.45 μm in GPC sample bottle, to be clean;
GPC gel permeation chrommatograph is used to purify the solution through membrane filtration, purification condition is shown in " 2.6 ", collect solution 5 mL after purification, at 45 DEG C, nitrogen dries up, and adds 1 mL methanol aqueous solution, the volume ratio 1:9 of methanol-water in methanol aqueous solution, ultrasonic 1 min, vortex mixed 1 min, crosses 0.22 μm of filter membrane, for liquid chromatography-Tandem Mass Spectrometry Analysis;
3) liquid phase chromatogram condition
Chromatographic column: Zorbax Eclipse Plus C18,2.1*100 mm, 3.5 um; Mobile phase: A phase is 0.15% aqueous formic acid; B phase is methyl alcohol; Column temperature 40 DEG C; Flow velocity: 0.3 mLmin
-1; Sample size 5 μ L; Liquid chromatography flow velocity and gradient elution as follows:
4) mass spectrometry parameters
Ion gun: electro-spray ionization (ESI) positive ion mode; Dry gas temperature: 325 DEG C; Dry gas flow velocity: 5 Lmin
-1; Spray pressure power: 45 psi; Sheath temperature degree: 400 DEG C; Sheath gas velocity: 12 Lmin
-1; Capillary voltage: 4000 V; Multiple-reaction monitoring scanning collection parameter is as follows:
5) Accelerate solvent extraction condition
Abstraction pool: 20 mL; Extraction solvent: ethyl acetate; Extracting pressure: 90 bar; Extraction temperature: 90 DEG C; Retention time: 5 min; Solvent washing: 30 s; Cycle index: 2 times;
GPC cleanup system condition
GPC decontaminating column; Filler 50.0 g Bio-Beads S-X3; Mobile phase: cyclohexane-ethyl acetate, volume ratio 1:1; Flow velocity: 5.0 mLmin
-1; The prewashing time: 10 s; Impurity abandons the time: 1200 s; Acquisition time: 800 s; Rinse pillar: 300 s; Sample size: 5 mL.
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