CN109239228A - The detection method of chloramphenicol a kind of while health food suitable for propolis and using propolis as raw material - Google Patents
The detection method of chloramphenicol a kind of while health food suitable for propolis and using propolis as raw material Download PDFInfo
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- CN109239228A CN109239228A CN201811265355.8A CN201811265355A CN109239228A CN 109239228 A CN109239228 A CN 109239228A CN 201811265355 A CN201811265355 A CN 201811265355A CN 109239228 A CN109239228 A CN 109239228A
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Abstract
The invention discloses a kind of detection methods of chloramphenicol health food suitable for propolis and using propolis as raw material simultaneously.This detection method includes the following steps: 1) extracting: dissolving by propolis or by the health food sample of raw material of propolis, obtain extracting solution；2) it purifies: removing the flavonoids chaff interferent and impurity in extracting solution, be purified liquid；3) extract: extracting and purifying liquid obtains solution to be measured；4) it detects: with the chloramphenicol in liquid chromatography-mass spectrography/mass spectrometric determination solution to be measured.Detection method of the invention has versatile, easy to operate, high sensitivity, strong antijamming capability, qualitative, quantitative is accurate, the good advantage of popularization and application foreground, to work out propolis and providing strong technical support by the detection of veterinary drugs in food standard of the health food of raw material of propolis.
Description
Technical field
The present invention relates to a kind of detection sides of chloramphenicol health food suitable for propolis and using propolis as raw material simultaneously
Method.
Background technique
Propolis is the glue mixed after the secretion such as worker honeybees foraging activity propolis with secretion such as itself mandibular gland, wax glands
Emplastic.Propolis is the traditional Chinese medicine in China, active with various biological, such as anti-oxidant, anti-inflammatory, antibacterial, immunological regulation,
Liver protecting, anticancer etc. are widely used in the industries such as health food, drug and cosmetics, are that bee product is over the past decade
One of the hot spot developed to Natural products research.From after the honey detection chloramphenicol of China's export European Union in 2002, China bee is produced
The export trade situation of product is severeer, the residue of veterinary drug problem in bee product have become influence its edible safety it is most important because
Element becomes the research hotspot of academia.
Chloramphenicol (Chloramphenicol, C11H12Cl2N2O5, relative molecular mass 323.14, CAS:56-75-7), be
White or micro- yellowish leukorrhea green acicular crystal is slightly soluble in water (2.5mg/ml, 25 DEG C), is slightly soluble in propylene glycol (150.8mg/ml),
It is soluble in methanol, ethyl alcohol, butanol, ethyl acetate, acetone, does not dissolve in ether, benzene, petroleum ether, vegetable oil.Chloramphenicol is in aqueous solution
In it is sufficiently stable, it is heat-resisting, be not easily decomposed, be once widely used in treatment various sensitive bacterias infection, it is rear because having seriously not to hemopoietic system
Good reaction, therefore strict control has been made to its clinical application.Chloramphenicol is a kind of broad-spectrum antibiotic, is to prevent and treat honeybee
One of the active drug of bacteriosis (American foul brood, European foul brood etc.).But since chloramphenicol can be in bee
It is remained in product, agranulocytosis disease, alpastic anemia and hemolytic anemia, many countries of people is caused to be included in
The list of substance being forbidden to use in food.The Ministry of Agriculture of China issued and implemented in 2002 " food animal disabling veterinary drug and its
The inventory of his compound ", " pollution-free food bee rearing management guideline " and " pollution-free food bee rearing veterinary drug uses quasi-
Then ", it emphasizes to be forbidden to use the drugs such as chloramphenicol, streptomysin, dapsone, promotes the sound development of apiculture.
Currently, the report about residual chloromycetin quantity measuring method in honey, royal jelly and its correlated product is more, mainly
There are high performance liquid chromatography-tandem mass method, mass spectrometer, enzyme-linked immunization]Deng.But about propolis and guarantor made from it
The report of health food is less, and since the ingredient and honey, royal jelly of propolis have very big difference, matrix is more complicated, with reference to bee
Sweet, royal jelly pre-treating method cannot remove a large amount of flavones ingredients in propolis, to generate to the measurement of chloramphenicol dry
It disturbs.Currently, there is no at the same the health food suitable for propolis raw material, using propolis as raw material chloramphenicol detection method, propolis
Product residual chloromycetin supervision problem lacks effective technical support means, it would be highly desirable to supplement.
Summary of the invention
The present invention provides a kind of suitable for bee according to propolis virgin rubber and using propolis as the matrix feature of the health food of raw material
Glue and using propolis as the detection method of the health food these two types sample chloramphenicol of raw material.
The technical solution used in the present invention is:
The detection method of chloramphenicol a kind of while health food suitable for propolis and using propolis as raw material, including it is following
Step:
1) it extracts: being dissolved by propolis or by the health food sample of raw material of propolis, obtain extracting solution；
2) it purifies: removing the flavonoids chaff interferent and impurity in extracting solution, be purified liquid；
3) extract: extracting and purifying liquid obtains solution to be measured；
4) it detects: with the chloramphenicol in liquid chromatography-mass spectrography/mass spectrometric determination solution to be measured.
In step 1), the sampling method of sample is as follows: after propolis is first placed in -20 DEG C~-15 DEG C freezings, then breaking into pieces as confession
Test agent；Directly take capsule 's content as test sample by the health food of raw material of propolis.
In step 1), Extraction solvent used in dissolved samples is ethyl alcohol.
In step 2), purification is flavonoids chaff interferent of the first removing containing adjacent two phenolic hydroxyl structures, then under alkaline condition
The flavonoids chaff interferent without containing adjacent two phenolic hydroxyl structures is removed, then removes wax, lipid and fat-soluble miscellaneous with alkane solvent
Matter.
In step 2), removing the flavonoids chaff interferent containing adjacent two phenolic hydroxyl structures is specifically that acetic acid lead solution precipitating is used to contain
There is the flavonoids chaff interferent of adjacent two phenolic hydroxyl structures.
In step 2), removing the flavonoids chaff interferent without containing adjacent two phenolic hydroxyl structures under alkaline condition is specifically to be added
It is 10~11 that alkaline reagent, which adjusts pH, then is centrifuged.
In step 3), extracting solvent used is methyl tertiary butyl ether(MTBE).
In step 4), the testing conditions of liquid chromatogram are as follows:
A) chromatographic column: Accucore C18；
B) flow velocity: 0.25mL/min~0.35mL/min；
C) column temperature: 38 DEG C~42 DEG C；
D) sample volume: 8 μ of μ L~12 L；
E) mobile phase: water-acetonitrile.
In step 4), mass spectrum/mass spectrographic testing conditions are as follows:
A) ion source: electric spray ion source；
B) electron spray voltage: -4000V~-5000V；
C) ion source temperature: 500 DEG C~600 DEG C；
D) atomization gas pressure: 45psi~55psi；
E) gas curtain atmospheric pressure: 35psi~45psi；
F) atmospheric pressure: 8psi~10psi is collided；
G) scanning mode: anion scanning；
H) detection mode: multiple reaction monitoring.
In step 4), chloramphenicol is quantitative determined using internal standard method.
The beneficial effects of the present invention are:
Detection method of the invention has versatile, easy to operate, high sensitivity, strong antijamming capability, qualitative, quantitative
Accurately, the good advantage of popularization and application foreground, to work out propolis and using propolis as the detection of veterinary drugs in food of the health food of raw material
Standard provides strong technical support.
Detailed description of the invention
Fig. 1 is liquid chromatography-mass spectrography/mass spectrum total ion current figure of chloramphenicol, chloramphenicol-D5 reference material；
Fig. 2 is the reconstructed ion chromatogram figure of chloramphenicol reference material；
Fig. 3 is chloramphenicol-D5The reconstructed ion chromatogram figure of reference material.
Fig. 4 is the reconstructed ion chromatogram figure using 1 sample of test No.；
Fig. 5 is the reconstructed ion chromatogram figure using 2 sample of test No.；
Fig. 6 is the reconstructed ion chromatogram figure using 5 sample of test No.；
Fig. 7 is the reconstructed ion chromatogram figure using 6 sample of test No.；
Fig. 8 is the reconstructed ion chromatogram figure using 7 sample of test No.；
Fig. 9 is the reconstructed ion chromatogram figure using 12 sample of test No.；
Figure 10 is the reconstructed ion chromatogram figure using 13 sample of test No.；
Figure 11 is the reconstructed ion chromatogram figure using 17 sample of test No..
Specific embodiment
The detection method of chloramphenicol a kind of while health food suitable for propolis and using propolis as raw material, including it is following
Step:
1) it extracts: being dissolved by propolis or by the health food sample of raw material of propolis, obtain extracting solution；
2) it purifies: removing the flavonoids chaff interferent and impurity in extracting solution, be purified liquid；
3) extract: extracting and purifying liquid obtains solution to be measured；
4) it detects: with the chloramphenicol in liquid chromatography-mass spectrography/mass spectrometric determination solution to be measured.
Preferably, in step 1), the sampling method of propolis sample is: after propolis is first placed in -20 DEG C~-15 DEG C freezings,
It is broken into pieces again as test sample；It is further preferred that the sampling method of propolis sample is: propolis being first placed in -18 in step 1)
After DEG C freezing 1h~3h, then break into pieces as test sample.
Preferably, in step 1), propolis is that the health food of raw material includes soft capsule, hard capsule dosage form, this kind of sample
Sampling method is: health food capsule 's content of directly going bail for is as test sample.
Preferably, in step 1), Extraction solvent used in dissolved samples is ethyl alcohol.
Preferably, in step 2), purification is flavonoids chaff interferent of the first removing containing adjacent two phenolic hydroxyl structures, then in alkalinity
Under the conditions of remove the flavonoids chaff interferent without containing adjacent two phenolic hydroxyl structures, then remove wax, lipid and rouge with alkane solvent
Solubility impurity.
Preferably, in step 2), removing the flavonoids chaff interferent containing adjacent two phenolic hydroxyl structures is specifically to use lead acetate molten
Liquid precipitate contains the flavonoids chaff interferent of adjacent two phenolic hydroxyl structures；Further, the mass concentration of acetic acid lead solution be 3%~
5%, the preferably mass concentration of acetic acid lead solution is 4%.
Preferably, in step 2), the flavonoids chaff interferent tool without containing adjacent two phenolic hydroxyl structures is removed under alkaline condition
Body is that addition alkaline reagent adjusting pH is 10~11, then is centrifuged, and takes supernatant；It is further preferred that alkaline reagent
For ammonium hydroxide.
Preferably, in step 2), removing alkane solvent used in impurity is n-hexane.
Preferably, in step 3), extracting solvent used is methyl tertiary butyl ether(MTBE).
Preferably, in step 4), the testing conditions of liquid chromatogram are as follows:
A) chromatographic column: Accucore C18；
B) flow velocity: 0.25mL/min~0.35mL/min；
C) column temperature: 38 DEG C~42 DEG C；
D) sample volume: 8 μ of μ L~12 L；
E) mobile phase: water-acetonitrile.
It is further preferred that in step 4), the testing conditions of liquid chromatogram are as follows:
A) chromatographic column: Accucore C18 (100mm × 2.1mm, 2.6 μm) or the suitable person of performance；
B) flow velocity: 0.3mL/min；
C) column temperature: 40 DEG C；
D) sample volume: 10 μ L；
E) mobile phase: water-acetonitrile.
Preferably, in step 4), mass spectrum/mass spectrographic testing conditions are as follows:
A) ion source: electric spray ion source；
B) electron spray voltage: -4000V~-5000V；
C) ion source temperature: 500 DEG C~600 DEG C；
D) atomization gas pressure: 45psi~55psi；
E) gas curtain atmospheric pressure: 35psi~45psi；
F) atmospheric pressure: 8psi~10psi is collided；
G) scanning mode: anion scanning；
H) detection mode: multiple reaction monitoring.
It is further preferred that in step 4), mass spectrum/mass spectrographic testing conditions are as follows:
A) ion source: electric spray ion source；
B) electron spray voltage: -4500V；
C) ion source temperature: 550 DEG C；
D) atomization gas pressure: 50psi；
E) gas curtain atmospheric pressure: 40psi；
F) atmospheric pressure: 9psi is collided；
G) scanning mode: anion scanning；
H) detection mode: multiple reaction monitoring.
Preferably, in step 4), chloramphenicol is quantitative determined using internal standard method.
Pre-treating method used in the present invention is described further below:
For propolis virgin rubber, sodium hydroxide solution, ethyl alcohol, t-butyl methyl ether dissolution are generallyd use, with acetic acid lead solution, hydrogen
Sodium hydroxide solution is that precipitating reagent removes flavones, finally carries out pre-treatment in the method for ethyl acetate extraction.For being original with propolis
The health food of material, main dosage form be soft capsule and hard capsule, due to pharmaceutical adjunct (such as polyethylene glycol 400, gelatin, glycerol,
Vegetable oil, starch, microcrystalline cellulose etc.) addition, sample substrate is increasingly complex.For soft capsule, partial supplementary material is (as gathered
Ethylene glycol 400) additive amount it is larger, dissolution characteristics are similar to chloramphenicol, use common chloramphenicol Extraction solvent (acetic acid second
Ester, acetonitrile etc.) more serious total extraction will be generated, cause sample to be difficult to be concentrated, influences the sensitivity and anti-interference of method；It is right
In hard capsule, partial supplementary material (such as starch, microcrystalline cellulose) meets the lower organic solvent of polarity and is easy to produce jelly, no
Conducive to the extraction of chloramphenicol.Therefore, suitable Extraction solvent, purification means and extractant are selected, is pre-treating method research
Emphasis.
(1) selection of Extraction solvent: propolis dissolves in 2% sodium hydroxide solution, ethyl alcohol, and chloramphenicol is soluble in methanol, second
Alcohol, butanol, ethyl acetate, acetone.Since hard capsule generally uses starch as content auxiliary material, 2% sodium hydroxide is used
Solution processing can be such that starch is gelatinized, and lead to chloramphenicol package wherein, seriously affect the extraction of chloramphenicol in sample.And it is anhydrous
Ethyl alcohol can effectively dissolve propolis, and the hard capsules such as insoluble starch, microcrystalline cellulose often use auxiliary material, by being centrifuged, filtering
Remove the total extraction of a large amount of adjunct ingredients.For the versatility for improving pre-treating method, the difficulty of subsequent purification processing, this hair are reduced
It is bright that dehydrated alcohol is used to extract to propolis and by the chloramphenicol in the health food of raw material of propolis.
(2) selection of purification style: honey, royal jelly sample purification process mainly use liquid-liquid extraction, Solid Phase Extraction
Or both combine mode purify.Since the ingredient and honey, royal jelly of propolis have very big difference, matrix is more complicated, makes
A large amount of flavones ingredients in propolis cannot be removed with the mode of liquid-liquid extraction；It is eaten by the soft capsule health care of raw material of propolis
Product make chloramphenicol in solid-phase extraction column, molecular blotting column due to a large amount of additions of pharmaceutical adjunct (such as polyethylene glycol 400, glycerol)
In retention behavior change.Therefore, the present invention has the flavones of adjacent two phenolic hydroxyl structures using 4% acetic acid lead solution precipitating
Class chaff interferent further removes the flavonoids chaff interferent for being free of this structure under alkaline condition, meanwhile, it is more conducive under alkaline condition
Extraction of the subsequent organic solvent to chloramphenicol.In addition, being protected since propolis is containing about 30% beeswax by the soft capsule of raw material of propolis
Health food is often using vegetable oil as auxiliary material, therefore this method uses the interference of n-hexane removal wax, lipid and oil-soluble impurities.
(3) selection of extractant: the common extractant of chloramphenicol mainly has ethyl acetate, acetonitrile, alkaline acetic acid second
Ester and alkaline acetonitrile.For soft capsule, polyethylene glycol 400 is common pharmaceutical adjunct, is a kind of non-ionic water solubility
Polymer has good dissolubility in the biggish solvent of polarity, and additive amount is larger, use ethyl acetate, acetonitrile etc. as
Extractant can generate serious total extraction, and sample is caused to be difficult to be concentrated, and influence the sensitivity of method, bring serious matrix dry
It disturbs.Therefore, select a kind of suitable extractant most important.The present invention uses methyl tertiary butyl ether(MTBE) as solvent, in alkalinity
Under the conditions of the chloramphenicol in scavenging solution is extracted, effectively reduce polarity chaff interferent and pharmaceutical adjunct (polyethylene glycol in sample
400, glycerol etc.) total extraction.
The contents of the present invention are described in further detail below by way of specific embodiment.Original used in embodiment
Material unless otherwise specified, can be obtained from routine business approach.
One, detection method
1 principle
Propolis in sample is dissolved through ethyl alcohol, has the flavonoids of adjacent two phenolic hydroxyl structures with 4% acetic acid lead solution precipitating
Chaff interferent further removes the flavonoids chaff interferent for being free of this structure under alkaline condition, and is to extract with methyl tertiary butyl ether(MTBE)
Solvent effectively reduces the total extraction of polarity chaff interferent and health food auxiliary material (such as polyethylene glycol, glycerol) in sample.Sample is molten
Liquid is measured through liquid chromatography-mass spectrography/mass spectrograph, inner mark method ration.
2 reagents and material
Unless otherwise stated, agents useful for same of the present invention is that analysis is pure, and water used should meet one specified in GB/T 6682
Grade water.
2.1 reagent
2.1.1 acetonitrile (CH3CN): chromatographically pure.
2.1.2 dehydrated alcohol (CH3CH2OH)。
2.1.3 three acetate hydrate lead (Pb (CH3COO)2·3H2O)。
2.1.4 glacial acetic acid (CH3COOH)。
2.1.5 ammonium hydroxide (NH3)。
2.1.6 n-hexane (CH3(CH2)4CH3)。
2.1.7 methyl tertiary butyl ether(MTBE) (CH3OC(CH3)3): chromatographically pure.
2.2 preparation of reagents
2.2.1 acetic acid lead solution (4%): weighing tri- acetate hydrate lead (2.1.3) of 40g, dissolved with water, and 5.0mL ice is added
Acetic acid (2.1.4), adds water to 1000mL, mixes.
2.2.2 acetonitrile-water (1+1): 1:1 is mixed by volume for acetonitrile (2.1.1), ultrapure water.
2.3 reference material
2.3.1 chloramphenicol standard substance: purity >=99.0%.
2.3.2 the deuterated internal standard of chloramphenicol (chloramphenicol-D5) substance: purity >=99.0%.
The preparation of 2.4 reference solutions
2.4.1 it chloramphenicol Standard Stock solutions: accurately weighs suitable chloramphenicol standard substance (2.3.1) and (is accurate to
0.1mg), the Standard Stock solutions (4 DEG C be kept in dark place can be used 6 months) of 500 μ g/mL are made into acetonitrile (2.1.1).
2.4.2 chloramphenicol standard intermediate solution: suitable chloramphenicol Standard Stock solutions (2.4.1) are accurately pipetted, second is used
Nitrile (2.1.1) is diluted to the standard intermediate solution (4 DEG C be kept in dark place can be used 3 months) of 50 μ g/mL.
2.4.3 chloramphenicol standard uses solution: accurately pipetting suitable chloramphenicol standard intermediate solution (2.4.2), uses second
Nitrile (2.1.1) is diluted to the standard of 0.1 μ g/mL and uses solution (face with now match).
2.4.4 the deuterated internal standard of chloramphenicol (chloramphenicol-D5) stock solution: accurately weigh suitable chloramphenicol-D5Standard substance
(2.3.2) (is accurate to 0.1mg), and being made into the Standard Stock solutions of 100 μ g/mL with acetonitrile (2.1.1), (4 DEG C are kept in dark place and can make
With 12 months).
2.4.5 the deuterated internal standard of chloramphenicol (chloramphenicol-D5) intermediate solution: accurately pipette suitable chloramphenicol-D5Stock solution
(2.4.4) is diluted to the internal standard intermediate solution (4 DEG C be kept in dark place can be used 6 months) of 1 μ g/mL with acetonitrile (2.1.1).
2.4.6 the deuterated internal standard of chloramphenicol (chloramphenicol-D5) working solution: accurately pipette suitable chloramphenicol-D5Intermediate solution
(2.4.5) is diluted to the internal standard intermediate solution (4 DEG C be kept in dark place can be used 2 weeks) of 0.1 μ g/mL with acetonitrile (2.1.1).
2.4.7 chloramphenicol standard working solution: suitable chloramphenicol standard is accurately pipetted using solution (2.4.3) in 10mL
It is accurate that the deuterated internal standard of 1.0mL chloramphenicol (chloramphenicol-D is added in volumetric flask5) working solution (2.4.6), with acetonitrile-water (1+1)
(2.2.2) is diluted to suitable standard working solution (face with now match).
3 instrument and equipments
3.1 liquid chromatography-tandem mass spectrometry instruments: it is furnished with electric spray ion source.
3.2 assay balance.
3.3 turbula shaker.
3.4 centrifuge.
3.5 Rotary Evaporators.
4 analytical procedures
The preparation of 4.1 samples
Propolis sample is placed in -18 DEG C and freezes 2 hours, breaks into pieces immediately after taking-up, the sample after breaking into pieces is as test sample.With
Propolis is that the health food of raw material directly takes capsule 's content.
The preparation of 4.2 sample solutions
Sample 2g (being accurate to 0.01g) is weighed, is placed in 50mL centrifuge tube, the 100 deuterated internal standards of μ L chloramphenicol are added, and (chlorine is mould
Element-D5) working solution (2.4.6) and dehydrated alcohol (2.1.2, propolis, hard capsule add 4mL, and soft capsule adds 2mL), vortex oscillation
10min (hard capsule needed after dehydrated alcohol extracts 8000r/min be centrifuged 10min, supernatant with filter paper filter to another 50mL from
In heart pipe, the residue on filter paper is washed with 1~2mL dehydrated alcohol, and filtrate is spare).10mL water is added, vortex 3min is added
10mL acetic acid lead solution (2.2.1), vortex 3min, 8000r/min are centrifuged 5min.Supernatant is poured into another 50mL centrifuge tube, residual
5mL acetic acid lead solution (2.2.1) is added in slag, vortex 3min, and 8000r/min is centrifuged 5min, merge supernatant in same 50mL from
Heart pipe.Above-mentioned clear liquid adjusts pH to 10~11 with ammonium hydroxide (2.1.5), and 5min is stood after mixing, and 8000r/min is centrifuged 5min.On
Clear liquid is poured into 50mL centrifuge tube, is added 10mL n-hexane (2.1.6), and vortex oscillation 5min, 8000r/min are centrifuged 5min, is abandoned
Go upper solution.It adds 10mL methyl tertiary butyl ether(MTBE) (2.1.7), vortex oscillation 5min, 8000r/min centrifugation 5min takes
Layer clear liquid is in chicken heart bottle.It repeats to extract 1 time, combined extract is in same chicken heart bottle.Rotary evaporated to dryness in 40 DEG C of water-baths is added
1mL acetonitrile-water (1+1) (2.2.2) redissolves.Redissolve liquid by 0.22 μm of filter membrane to sample bottle, it is to be measured.
4.3 instrument reference conditions
4.3.1 liquid phase chromatogram condition
4.3.1.1 chromatographic column: Accucore C18 (100mm × 2.1mm, 2.6 μm) or suitable person.
4.3.1.2 flow velocity: 0.3mL/min.
4.3.1.3 column temperature: 40 DEG C.
4.3.1.4 sample volume: 10 μ L.
4.3.1.5 mobile phase: water-acetonitrile, gradient elution program are shown in Table 1.
1 gradient elution program of table
4.3.2 mass spectrum/Mass Spectrometry Conditions
4.3.2.1 ion source: electric spray ion source.
4.3.2.2 electron spray voltage: -4500V.
4.3.2.3 ion source temperature: 550 DEG C.
4.3.2.4 atomization gas pressure: 50psi.
4.3.2.5 gas curtain atmospheric pressure: 40psi.
4.3.2.6 atmospheric pressure is collided: 9psi.
4.3.2.6 scanning mode: anion scanning.
4.3.2.7 detection mode: multiple reaction monitors (MRM).Monitoring condition is shown in Table 2.
2 multiple reaction of table monitors the condition of (MRM)
Note in table 2: * is quota ion.
4.4 qualitative analysis
Sample and reference material solution are measured according to above-mentioned condition, if the mass chromatography peak retention time of sample and reference
Standard of physical solution is consistent；The relative abundance of ion pair is consistent with the relative abundance of the comparable reference material solution of concentration, relatively
Abundance deviation is no more than the regulation of table 3, then can determine whether that there are corresponding measured objects in sample.
The liquid chromatography-tandem mass spectrometry chromatogram of reference material solution is referring to attached drawing 1~3.Wherein Fig. 1 is chloramphenicol, chlorine
The liquid chromatography-mass spectrography of mycin-D5 reference material/mass spectrum total ion current figure；Fig. 2 is the reconstruct ion color of chloramphenicol reference material
Spectrogram；Fig. 3 is the reconstructed ion chromatogram figure of chloramphenicol-D5 reference material.
With respect to the maximum allowable offset of abundance of ions when 3 qualitative confirmation of table
| Relative ion abundance/% | >50 | >20-50 | >10-20 | ≤10 |
| Relative deviation/% of permission | ±20 | ±25 | ±30 | ±50 |
4.5 quantitative analysis
Sample solution and chloramphenicol standard series working solution are injected separately into liquid chromatography-mass spectrography/mass spectrograph, measured
Corresponding peak area, inner mark method ration obtain the chloramphenicol concentration in sample solution according to standard curve.
The statement of 5 analysis results
The content X of chloramphenicol in sample is calculated by formula (1):
In formula (1):
X --- the content of chloramphenicol in sample, unit are ng/kg (μ g/kg)；
C --- the mass concentration of chloramphenicol in sample solution is checked in from standard curve, unit is nanograms per milliliter (ng/
mL)；
V --- sample dilutes total volume, and unit is milliliter (mL)；
M --- sample mass, unit are gram (g).
Note: calculated result should deduct blank value.
6 other
Method detection is limited to 0.03 μ g/kg, and method is quantitatively limited to 0.1 μ g/kg.
Two, practical application
Using measuring method of the invention, to 5 batches of propolis raw materials being collected into, 13 batches using propolis as the health food of raw material
It is measured.Wherein, chloramphenicol is not detected in propolis raw material；It is positive using propolis as 5 batches of detection chloramphenicol of health food of raw material
Rate 38%, respectively 1 batch of hard capsule, 4 batches of soft capsules, chloromycetin content range the results are shown in Table 4 in 0.23~21.66 μ g/kg.
4 propolis of table and using propolis as the measurement result of chloramphenicol in the health food of raw material
The acquisition of sample is from supermarket and market, the visible attached drawing 4~11 of the test spectrogram of associated sample, wherein attached in table 4
Fig. 4 is the reconstructed ion chromatogram figure of 1 sample of number；Attached drawing 5 is the reconstructed ion chromatogram figure of 2 sample of number；Attached drawing 6 is number 5
The reconstructed ion chromatogram figure of sample；Attached drawing 7 is the reconstructed ion chromatogram figure of 6 sample of number；Attached drawing 8 is the reconstruct of 7 sample of number
Chromatography of ions figure；Attached drawing 9 is the reconstructed ion chromatogram figure of 12 sample of number (sample 12 contains polyethylene glycol)；10 number of attached drawing, 13 sample
The reconstructed ion chromatogram figure of product (sample 13 is free of polyethylene glycol)；11 number of attached drawing, 17 sample (sample 17 is free of polyethylene glycol)
Reconstructed ion chromatogram figure.
Three, methodological study
1 method detection limit and quantitative limit
The chloramphenicol standard solution that various concentration is added in vehicle solution, as signal-to-noise ratio S/N >=3, chloramphenicol
Concentration of standard solution is 0.06ng/mL, is 1mL calculating by sampling amount 2.0g, constant volume, determines that method detection is limited to 0.03 μ
g/kg；As signal-to-noise ratio S/N >=10, chloramphenicol concentration of standard solution is 0.20ng/mL, is by sampling amount 2.0g, constant volume
1mL is calculated, and determines that quantifying for method is limited to 0.1 μ g/kg.
2 calibration curves
Chloramphenicol Standard Stock solutions are diluted to the standard system of 0.2ng/mL~50ng/mL step by step with acetonitrile-water (1+1)
Column working solution, is added appropriate inner mark solution, inner mark method ration, and regression equation is y=0.08385x -0.01191, phase relation
Number r=0.9998.
3 rate of recovery
It differs greatly due to propolis raw material, by the matrix of the health food of raw material of propolis, health food different dosage forms
The composition significant difference of pharmaceutical adjunct, therefore, respectively to propolis virgin rubber, using propolis as the health food (hard capsule) of raw material,
It is investigated by health food (soft capsule) three classes sample of raw material of propolis.Wherein, due to system common in soft capsule
The low polarity auxiliary material such as agent auxiliary material polyethylene glycol 400 and beeswax, vegetable oil is incompatible, and usually has two by the soft capsule of raw material of propolis
Kind supplementary product compatibility mode, one is be to contain containing polyethylene glycol 400 but without low polar substances, another kinds such as beeswax, vegetable oil
There are beeswax, vegetable oil but is free of polyethylene glycol 400.This method investigates above-mentioned four kinds of matrix, and each matrix is basic, normal, high
The method rate of recovery of three concentration levels is shown in Table 5, and average recovery rate range is 95.6~108.3%.
The rate of recovery of chloramphenicol in 5 different substrates of table
4 precision
Four kinds of matrix repeat back to acceptance test 6 times in high concentration level in table 5, calculate method precision range be 1.9~
3.8%.Determination data is shown in Table 6.
Method precision in 6 different substrates of table
This detection method uses high performance liquid chromatography-tandem mass method, carries out according to retention time and mass spectrogram consistency
Qualitive test can be carried out accurately to propolis and using propolis as chloramphenicol in the health food of raw material qualitative using inner mark method ration
ELISA in determination of chloramphenicol with quantitatively, developing one for the first time while suitable for these two types of samples, has passed through method validation, has been suitble to
Carry out risk investigation for batch samples, meets supervision needs.
Claims (10)
1. the detection method of chloramphenicol, feature exist a kind of health food suitable for propolis and using propolis as raw material simultaneously
In: the following steps are included:
1) it extracts: being dissolved by propolis or by the health food sample of raw material of propolis, obtain extracting solution；
2) it purifies: removing the flavonoids chaff interferent and impurity in extracting solution, be purified liquid；
3) extract: extracting and purifying liquid obtains solution to be measured；
4) it detects: with the chloramphenicol in liquid chromatography-mass spectrography/mass spectrometric determination solution to be measured.
2. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1
Detection method, it is characterised in that: in step 1), the sampling method of sample is as follows: after propolis is first placed in -20 DEG C~-15 DEG C freezings,
It is broken into pieces again as test sample；Directly take capsule 's content as test sample by the health food of raw material of propolis.
3. chlorine is mould a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1 or 2
The detection method of element, it is characterised in that: in step 1), Extraction solvent used in dissolved samples is ethyl alcohol.
4. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1
Detection method, it is characterised in that: in step 2), purification is flavonoids chaff interferent of the first removing containing adjacent two phenolic hydroxyl structures, then
The flavonoids chaff interferent without containing adjacent two phenolic hydroxyl structures is removed under alkaline condition, then removes wax, rouge with alkane solvent
Class and oil-soluble impurities.
5. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 4
Detection method, it is characterised in that: in step 2), removing the flavonoids chaff interferent containing adjacent two phenolic hydroxyl structures is specifically to use acetic acid
Flavonoids chaff interferent of the lead solution precipitating containing adjacent two phenolic hydroxyl structures.
6. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 4
Detection method, it is characterised in that: in step 2), remove the flavonoids interference without containing adjacent two phenolic hydroxyl structures under alkaline condition
Object is specifically that addition alkaline reagent adjusting pH is 10~11, then is centrifuged.
7. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1
Detection method, it is characterised in that: in step 3), extracting solvent used is methyl tertiary butyl ether(MTBE).
8. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1
Detection method, it is characterised in that: in step 4), the testing conditions of liquid chromatogram are as follows:
A) chromatographic column: Accucore C18；
B) flow velocity: 0.25mL/min~0.35mL/min；
C) column temperature: 38 DEG C~42 DEG C；
D) sample volume: 8 μ of μ L~12 L；
E) mobile phase: water-acetonitrile.
9. chloramphenicol a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 1
Detection method, it is characterised in that: in step 4), mass spectrum/mass spectrographic testing conditions are as follows:
A) ion source: electric spray ion source；
B) electron spray voltage: -4000V~-5000V；
C) ion source temperature: 500 DEG C~600 DEG C；
D) atomization gas pressure: 45psi~55psi；
E) gas curtain atmospheric pressure: 35psi~45psi；
F) atmospheric pressure: 8psi~10psi is collided；
G) scanning mode: anion scanning；
H) detection mode: multiple reaction monitoring.
10. chlorine is mould a kind of health food suitable for propolis and using propolis as raw material simultaneously according to claim 8 or claim 9
The detection method of element, it is characterised in that: in step 4), chloramphenicol is quantitative determined using internal standard method.
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