CN110082463A - The detection method of 4-methylimidazole in flavouring - Google Patents
The detection method of 4-methylimidazole in flavouring Download PDFInfo
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- G—PHYSICS
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Abstract
The present invention provides a kind of detection methods of 4-methylimidazole in flavouring.The detection method includes being detected using LC/MS/MS internal standard method to the 4-methylimidazole in flavouring.The 4-methylimidazole in flavouring is detected by using LC/MS/MS internal standard method, so that testing result is more acurrate.
Description
Technical field
The present invention relates to non-staple food detection fields, in particular to a kind of detection side of 4-methylimidazole in flavouring
Method.
Background technique
The detection method of 4-methylimidazole generally uses HPLC external standard method in existing non-staple food, the inspection for burnt sugar coloring
It surveys, detects limit for height, qualitative and quantitative can not be carried out for the low sample of content.In addition, the rate of recovery is low, and tight by matrix interference
Weight, retention time is qualitative to be easy to appear false positive.
Therefore, it is still necessary to the detection method of the 4-methylimidazole in existing non-staple food be improved, to improve detection
Accuracy.
Summary of the invention
The main purpose of the present invention is to provide a kind of detection methods of 4-methylimidazole in flavouring, to solve existing skill
The low problem of detection method accuracy in art.
To achieve the goals above, the present invention provides a kind of detection method of 4-methylimidazole in flavouring, the detections
Method includes: to be detected using LC/MS/MS internal standard method to the 4-methylimidazole in flavouring.
Further, with 4-methylimidazole-d6As internal standard, using LC/MS/MS internal standard method to the 4- methyl in flavouring
The step of imidazoles is detected includes: with 4-methylimidazole-d6As internal standard, the standard solution work for establishing 4-methylimidazole is bent
Line；The 4-methylimidazole in flavouring is detected using standard solution working curve.
Further, with 4-methylimidazole-d6As internal standard, the step of the standard solution working curve of 4-methylimidazole is established
It suddenly include: with 4-methylimidazole concentration and 4-methylimidazole-d6The ratio of concentration is abscissa, with 4-methylimidazole quota ion
Pair peak area and 4-methylimidazole-d6The ratio of quota ion pair peak area is ordinate, establishes the standard of 4-methylimidazole
Solution working curve.
Further, before being detected using LC/MS/MS internal standard method to the 4-methylimidazole in flavouring, detection side
Method further include: the step of preparing the sample to be tested of flavouring, it is preferable that the step of preparing the sample to be tested of flavouring include: to
4-methylimidazole-d is added in flavouring6, obtain just mixed object；It is preferred that 4-methylimidazole-d6With the addition mass ratio of flavouring are as follows:
(0.1~0.5) × 10-7: 1；Removal just mixes the matrix interference in object, obtains liquid to be purified；It treats refined solution and carries out column chromatography, obtain
To refined solution；Constant volume is carried out to refined solution, obtains the sample to be tested of flavouring.
Further, the matrix interference in just mixed object is removed by way of precipitating proteins and/or enzymatic starch, is obtained
Liquid to be purified；Preferably, using methanol extraction protein, using amylase enzymolysis starch；Preferably, flavouring includes sauce, sauce
Oil, oyster sauce and oyster juice are any one or more of.
Further, treating the step of refined solution carries out column chromatography, obtains refined solution includes: using MCX solid-phase extraction column
It treats refined solution and carries out column chromatography, obtain refined solution；Preferably, refined solution is treated using MCX solid-phase extraction column and carries out column chromatography,
Before obtaining refined solution, detection method further includes the steps that activating MCX solid-phase extraction column；It is highly preferred that successively using
First alcohol and water activates MCX solid-phase extraction column.
Further, treating the step of refined solution carries out column chromatography, obtains refined solution includes: that MCX is added in liquid to be purified
Column was carried out in solid-phase extraction column, it is preferable that the column flow rate of crossing for controlling liquid to be purified is 1/3~4s of drop；Successively use formic acid water
Solution and methanol solution elute the liquid to be purified in MCX solid-phase extraction column, obtain elution column；Using ammonium hydroxide methanol solution
Elution column is eluted, gained eluent is refined solution.
Further, the mass concentration of formic acid is 1.5~3% in aqueous formic acid, it is preferable that aqueous formic acid and methanol
The volume of solution is each independently 3~10mL；Preferably, the mass concentration of ammonium hydroxide is 4.5~6% in ammonium hydroxide methanol solution,
Volume is 8~15mL.
Further, the step of carrying out constant volume to refined solution, obtaining the sample to be tested of flavouring includes: to carry out to refined solution
It is dry, obtain purified；It is filtered after being dissolved using -0.1% formic acid solution of acetonitrile to purified, gained supernatant is to adjust
The sample to be tested of taste product.
Further, it is filtered after being dissolved using filter membrane to purified, preferably the aperture of filter membrane is 0.22~0.4 μm.
It applies the technical scheme of the present invention, detection method includes: using LC/MS/MS internal standard method to the 4- first in flavouring
Base imidazoles is detected.The 4-methylimidazole in flavouring is detected by using LC/MS/MS internal standard method, so that detection
As a result more acurrate.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
As background technique is previously mentioned, it is accurate to exist in the prior art to the detection method of the 4-methylimidazole in flavouring
The low problem of property in a kind of typical embodiment of the application, provides 4- in a kind of flavouring to improve this situation
The detection method of methylimidazole, the detection method include: using LC/MS/MS internal standard method to the 4-methylimidazole in flavouring into
Row detection.The 4-methylimidazole in flavouring is detected by using LC/MS/MS internal standard method, so that testing result is more quasi-
Really.
In a kind of preferred embodiment, with 4-methylimidazole-d6As internal standard, using LC/MS/MS internal standard method to seasoning
The step of 4-methylimidazole in product is detected includes: with 4-methylimidazole-d6As internal standard, the mark of 4-methylimidazole is established
Quasi- solution working curve；The 4-methylimidazole in flavouring is detected using standard solution working curve.Pass through 4- methyl
The standard solution working curve of imidazoles is detected come the 4-methylimidazole detected in flavouring, compares HPLC external standard method, not only
Testing result is more accurate, and detection limit it is low (for 10 μ g/kg, minimum detection limit be according to the signal shown in equipment with make an uproar
The ratio of sound, which is more than or equal to, 10 can quantify, and less than 10 when is no standard measure to determine), as content is 10 μ g/ in soy sauce
When kg, it can detect.
In the case where the mode above by standard curve is to carry out internal standard method detection, any one establishes 4- methyl miaow
The mode of the standard solution working curve of azoles is suitable for the application.In a kind of preferred embodiment, with 4-methylimidazole-d6
As internal standard, the step of establishing the standard solution working curve of 4-methylimidazole includes: with 4-methylimidazole concentration and 4- methyl
Imidazoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6It is quantitative from
Son is ordinate to the ratio of peak area, establishes the standard solution working curve of 4-methylimidazole.Pass through 4-methylimidazole and 4-
Methylimidazole-d6Relative parameter establish standard solution working curve, help to ensure that the accuracy of result.
Internal standard selects the reason of 4-methylimidazole-d6: (1) property is similar with 4-methylimidazole；(2) retention time and 4- first
Base imidazoles is close；(3) peak energy of other all substances and in sample is enough completely separable.
In a kind of preferred embodiment, with 4-methylimidazole-d6As internal standard, the standard solution of 4-methylimidazole is established
The step of working curve includes: with 4-methylimidazole concentration and 4-methylimidazole-d6The ratio of concentration is abscissa, with 4- methyl
The peak area and 4-methylimidazole-d of imidazoles quota ion pair6The ratio of quota ion pair peak area is ordinate, establishes 4- first
The standard solution working curve of base imidazoles.
In above preferred embodiment, due to using 4-methylimidazole-d6Concentration and quota ion pair peak area two
Index, thus, result error caused by matrix interference can be corrected, guarantee the accuracy of result.
In the step of being detected to the 4-methylimidazole in flavouring, established according to above-mentioned standard solution working curve
Basis, suitable pre-treatment operation can be carried out to the sample to be tested of flavouring, with the working curve with above-mentioned standard solution
It is adapted.In a kind of preferred embodiment, the 4-methylimidazole in flavouring is detected using LC/MS/MS internal standard method
Before, detection method further include: the step of preparing the sample to be tested of flavouring, it is preferable that prepare the sample to be tested of flavouring
Step includes: the addition 4-methylimidazole-d into flavouring6, obtain just mixed object；The first matrix interference in mixed object of removal, obtain to
Refined solution；It treats refined solution and carries out column chromatography, obtain refined solution；Constant volume is carried out to refined solution, obtains the sample to be tested of flavouring.
It has been observed that being with 4-methylimidazole-d according in the standard solution working curve of 4-methylimidazole6As reference pair
As, thus in sample to be tested, also accordingly add 4-methylimidazole-d6As interior target substance.In addition, just by removal
The matrix interferences such as protein and/starch in mixed object, so that the testing result of the 4-methylimidazole in sample to be tested is more acurrate.Tool
The step of body purifies is also so that test substance purity is higher, to keep testing result more acurrate.
In above preferred embodiment, 4-methylimidazole-d6Addition mass ratio with flavouring can be according in standard curve
4-methylimidazole-d6Concentration carry out reasonable set.In a kind of preferred embodiment, 4-methylimidazole-d6With flavouring
Add mass ratio are as follows: (0.1~0.5) × 10-7: 1, more preferably 0.25 × 10-7: 1.The mass ratio of the two is controlled above-mentioned
In range, convenient for 4-methylimidazole-d added in the standard solution working curve and flavouring according to 4-methylimidazole6's
Amount, accurately calculates the content of the 4-methylimidazole in flavouring.
Above-mentioned matrix can be the substances such as protein, starch, and content and type can be according to specific flavouring types
It is different and different.In a kind of preferred embodiment, removed by way of precipitating proteins and/or enzymatic starch just mixed
Matrix interference in object obtains liquid to be purified；Preferably, using methanol extraction protein, using amylase enzymolysis starch；It is preferred that
Ground, flavouring include that sauce, soy sauce, oyster sauce and oyster juice are any one or more of.Wherein, the difference of sauce and soy sauce is: sauce: using
A kind of semisolid flavouring that beans, wheat after fermentation etc. are made into；Soy sauce: with soybean and (or) defatted soybean (dregs of beans or soya-bean cake),
Wheat and (or) wheat bran are raw material, through having the liquid flavoring of special color made of microbial fermentation；.Oyster sauce and oyster
The difference of juice is oyster sauce: referring to mainly using oyster juice as seasoning made of raw material tanning；Oyster juice: refer to and boiled with oyster (oyster)
Made of liquid.
To soy sauce class sample, in a kind of specific embodiment, base can be extracted and removed by handling as follows
Matter interference: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the water that 1mL is added in sticky soy sample such as braise in soy sauce, stir
It mixes uniformly, column to be crossed.
To oyster sauce class sample, in a kind of specific embodiment, base can be extracted and removed by handling as follows
Matter interference: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, suitable amylase is added and carrys out starch-splitting, adds 5ml's
Water dissolution, stirs evenly, and 3500r/min centrifugation, supernatant waited for column.
To oyster juice class sample, in a kind of specific embodiment, base can be extracted and removed by handling as follows
Matter interference: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the methanol of addition 4mL 75%, vortex mixed 1min,
3500r/min centrifugation, supernatant waited for column.
To sauce class sample, in a kind of specific embodiment, matrix can be extracted and removed by handling as follows
Interference: adding 200 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, add the methanol of 4mL75%, vortex mixed 1min, 3500r/
Min centrifugation, accurate sample liquid (half) column to be crossed for drawing 2100 μ L.
The step of the step of purifying is chromatographed using column, column chromatography carry out column operation using existing mode of operation.?
In a kind of preferred embodiment, treating the step of refined solution carries out column chromatography, obtains refined solution includes: using MCX Solid Phase Extraction
Column treats refined solution and carries out column chromatography, obtains refined solution；Preferably, refined solution is treated using MCX solid-phase extraction column and carries out column layer
Analysis, before obtaining refined solution, detection method further includes the steps that activating MCX solid-phase extraction column；It is highly preferred that successively adopting
MCX solid-phase extraction column is activated with first alcohol and water.MCX solid-phase extraction column has compared to other extraction columns to 4-methylimidazole
With 4-methylimidazole-d6There is stronger absorption advantage, specific activation step is carried out according to the requirement of MCX solid-phase extraction column.
In a kind of preferred embodiment, treats refined solution and carry out column chromatography, the step of obtaining refined solution includes: will be to pure
Change in liquid addition MCX solid-phase extraction column and carried out column, it is preferable that the column flow rate of crossing for controlling liquid to be purified is 1/3~4s of drop；Successively
The liquid to be purified in MCX solid-phase extraction column is eluted using aqueous formic acid and methanol solution, obtains elution column；Using ammonia
Water beetle alcoholic solution elutes elution column, and gained eluent is refined solution.According to above-mentioned speed cross column help to improve it is pure
Change efficiency and quality.Aqueous formic acid and methanol solution is successively used to help to wash off acid impurities when elution, the application institute
The eluent property of selection is similar with 4-methylimidazole, can utmostly be eluted to object using similar compatibility principle.
Need rationally to be arranged the concentration and volume of formic acid in above-mentioned aqueous formic acid according to the effect of elution, correspondingly,
The concentration of ammonium hydroxide in ammonium hydroxide methanol solution can be rationally set according to elution effect.In a kind of preferred embodiment, formic acid water
The concentration of formic acid is 1.5~3wt% in solution, it is preferable that the volume of aqueous formic acid and methanol solution is each independently 3~
10mL；Preferably, the concentration of ammonium hydroxide is 4.5~6wt% in ammonium hydroxide methanol solution, and volume is 8~15mL.First in aqueous formic acid
The concentration of acid controls in the range of 1.5~3wt%, preferably in 2wt%, has the better advantage of effect.And ammonium hydroxide methanol
The concentration of ammonium hydroxide controls in the range of 4.5~6wt% in solution, when preferably 5wt%, elutes more thorough.
Since the application is detected to the content of the 4-methylimidazole in flavouring, in order to further increase its detection
Accuracy, before being detected to its sample to be tested to sample to be tested carry out constant volume.Specific constant volume step is using routine
Constant volume operation.In a kind of preferred embodiment, the step of carrying out constant volume to refined solution, obtain the sample to be tested of flavouring
Include: that refined solution is dried, obtains purified；Using -0.1% formic acid solution of acetonitrile, (acetonitrile of chromatographically pure contains with volume
Amount for 0.1% formic acid solution be 5:95 according to volume ratio ratio mixing) purified is dissolved after filter, gained supernatant
Liquid is the sample to be tested of flavouring.
Above-mentioned drying can be carried out using existing various drying modes, for example naturally dry or be air-dried etc..In the application
It is preferred that being dried by the way of nitrogen purging.More preferably it is dried with nitrogen using 40 DEG C.Be dried with nitrogen for liquid phase, gas phase and
The surface that nitrogen is blown into heating sample is usually carried out sample concentration, has time saving, operation side by the sample preparation in mass spectral analysis
Just the characteristics of and being easy to control can obtain expected result quickly, be very suitable to apply and the industries such as farming residual analysis.
In the step of carrying out dissolution filter to above-mentioned purified, filtering can use any existing suitable filtering side
Formula.It in a kind of preferred embodiment, is filtered after being dissolved using filter membrane to purified, the aperture of preferably organic filter membrane is
0.22~0.4 μm.
Had using membrane filtration using simple, efficiently advantage, is widely applied and field of food detection.Filter membrane is often referred to
Miillpore filter is added proper amount of acetic acid cellulose, acetone, n-butanol, ethyl alcohol etc. and is made mainly by purification nitrocotton, hydrophilic, tool
There is the advantages of non-toxic sanitary.The aperture of preferably used filter membrane is 0.22 μm in the application.
In a kind of preferred embodiment of the application, a kind of detection method of 4-methylimidazole in flavouring is provided,
The detection method is specific as follows:
Testing process are as follows: claim sample-sample extraction-sample purification-sample constant volume-LC/MS/MS detection.
1. claiming sample: the samples such as soy sauce, oyster sauce and oyster juice claim 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube, sauce
Class product accurately weighs 2.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
(1) soy sauce class sample: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the sticky soy sauce sample such as braised in soy sauce
The water of 1mL is added in product, stirs evenly, column to be crossed.
(2) oyster sauce class sample: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, suitable amylase is added
Starch-splitting adds the water of 5ml to dissolve, stirs evenly, and 3500r/min centrifugation, supernatant waited for column.
(3) oyster juice class sample: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the methanol of 4mL 75% is added,
Vortex mixed 1min, 3500r/min centrifugation, supernatant waited for column.
(4) sauce class sample: adding 200 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, add the methanol of 4mL75%, be vortexed
Mix 1min, 3500r/min centrifugation, accurate sample liquid (half) column to be crossed for drawing 2100ul.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
Flow velocity 1 processed drips/3~4s, is successively eluted with 2% aqueous formic acid 5mL and methanol 5mL, then washed with 5% ammonium hydroxide methanol solution 9mL
It is de-, eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R20.999 should be not less than.
It will illustrate the beneficial effect of the application further combined with specific embodiment below.
Embodiment 1
1. claiming sample: soy sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the water of the sticky soy sample addition 1mL such as braised in soy sauce,
It stirs evenly, column to be crossed.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/3s processed is successively eluted with 2% aqueous formic acid 5mL and methanol 5mL, then is eluted with 5% ammonium hydroxide methanol solution 9mL,
Eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the soy sample is 11.3 μ g/kg according to internal standard method.
Embodiment 2
1. claiming sample: oyster sauce sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, suitable amylase is added and carrys out starch-splitting, adds 5ml
Water dissolution, stir evenly, 3500r/min centrifugation, supernatant waited for column.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/4s processed is successively eluted with 1.5% aqueous formic acid 3mL and methanol 3mL, then is washed with 4.5% ammonium hydroxide methanol solution 8mL
It is de-, eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: acetonitrile -0.1% formic acid solution (volume ratio 5:95) 1mL dissolved residue is used, is vortexed and mixes, filter membrane
Filtering, filtrate wait for machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the oyster sauce sample is that (ND indicates that content is small to ND according to internal standard standard curve
, can not accurate quantitative analysis in 10 μ g/kg) (10 μ g/kg of detection limit).
Embodiment 3
1. claiming sample: oyster juice sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Oyster juice class sample: adding 100 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, the methanol of 4mL 75%, whirlpool is added
Rotation mixing 1min, 3500r/min centrifugation, supernatant waited for column.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/4s processed is successively eluted with 2.5% aqueous formic acid 5mL and methanol 5mL, then is washed with 5% ammonium hydroxide methanol solution 9mL
It is de-, eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the oyster sauce sample is 20.3 μ g/kg (detection limits according to internal standard standard curve
10μg/kg)。
Embodiment 4
1. claiming sample: sauce class sample accurately weighs 2.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Sauce class sample: adding 200 μ L concentration is the 4-methylimidazole-d of 250 μ g/L6, add the methanol of 4mL75%, vortex mixed
1min, 3500r/min centrifugation, accurate sample liquid (half) column to be crossed for drawing 2100ul.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/3s processed is successively eluted with 3% aqueous formic acid 10mL and methanol 10mL, then is washed with 6% ammonium hydroxide methanol solution 15mL
It is de-, eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the oyster sauce sample is that (detection is limited to 10 μ g/ to ND according to internal standard standard curve
Kg shows the content of 4-methylimidazole in the sample lower than detection limit).
Embodiment 5
1. claiming sample: soy sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Adding 100 μ L concentration is the 4-methylimidazole-d of 100 μ g/L6, the water of the sticky soy sample addition 1mL such as braised in soy sauce,
It stirs evenly, column to be crossed.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/3s processed is successively eluted with 2% aqueous formic acid 5mL and methanol 5mL, then is eluted with 5% ammonium hydroxide methanol solution 9mL,
Eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the oyster sauce sample is 11.5 μ g/kg (detection limits according to internal standard standard curve
10μg/kg)。
Embodiment 6
1. claiming sample: soy sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Adding 100 μ L concentration is the 4-methylimidazole-d of 500 μ g/L6, the water of the sticky soy sample addition 1mL such as braised in soy sauce,
It stirs evenly, column to be crossed.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/3s processed is successively eluted with 2% aqueous formic acid 5mL and methanol 5mL, then is eluted with 5% ammonium hydroxide methanol solution 9mL,
Eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 4- methyl miaow
Azoles-d6The ratio of concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 4-methylimidazole-d6Quota ion
Ratio to peak area is ordinate, establishes the standard solution working curve of 4-methylimidazole；To standard solution detection data into
Row linear regression acquires the regression equation of 4-methylimidazole, R2It is 0.999.
6. the content for measuring 4-methylimidazole in the soy sample is 11.4 μ g/kg according to internal standard method.
Embodiment 7
Embodiment 7 is that interior target is different from unique difference of embodiment 1, remaining is all the same.
1. claiming sample: soy sample claims 1.00g sample (being accurate to 0.01g) in 50ml centrifuge tube.
2. sample extracts
Adding 100 μ L concentration is the 2-methylimidazole of 250 μ g/L, and the water of 1mL, stirring is added in the sticky soy sample such as braised in soy sauce
Uniformly, column to be crossed.
3. sample purifies: taking MCX solid-phase extraction column, successively activated with methanol 3mL and water 5mL, liquid to be clean is taken to cross column, control
1 drop of flow velocity/3s processed is successively eluted with 2% aqueous formic acid 5mL and methanol 5mL, then is eluted with 5% ammonium hydroxide methanol solution 9mL,
Eluent is collected, is dried with nitrogen in 40 DEG C.
4. sample constant volume: using -0.1% formic acid solution 1mL dissolved residue of acetonitrile, be vortexed and mix, membrane filtration, filtrate is waited for
Machine testing.
5.LC/MS/MS internal mark method determination: establishing standard solution working curve, with 4-methylimidazole concentration and 2- methyl miaow
The ratio of azoles concentration is abscissa, with the peak area of 4-methylimidazole quota ion pair and 2-methylimidazole quota ion pair peak face
Long-pending ratio is ordinate, establishes the standard solution working curve of 4-methylimidazole；Standard solution detection data is carried out linear
It returns, acquires the regression equation of 4-methylimidazole, R2It is 0.995.
6. the content for measuring 4-methylimidazole in the soy sample is ND (practical such as embodiment 1 according to internal standard standard curve
Described, content may be up to 11.3 μ g/kg, because internal standard detection inaccuracy, thus not up to detection is limited and cannot be detected).
Embodiment 8
Embodiment 8 and unique difference of embodiment 3 are that sample does not carry out methanol extraction when extracting.
R in the regression equation of the 4-methylimidazole of its LC/MS/MS internal mark method determination2It is 0.999.
It is 18.6 μ g/kg according to the content for according to internal standard standard curve, measuring 4-methylimidazole in the soy sample.
It can be seen from the above description that the above embodiments of the present invention realized the following chievements: by
Reference material (4-methylimidazole-d is added in test sample6As internal standard), and utilize the phase of 4-methylimidazole and reference material
The standard solution working curve of 4-methylimidazole is established to parameter, thus according to reference material added in sample to be tested
Amount, accurately measures the content of the 4-methylimidazole in sample to be tested.Quantitative detection is carried out using the internal standard method, it is as a result more acurrate,
And detection limit is low (10 μ g/kg).
In addition, using methanol extraction protein and/or Amylase Hydrolysis starch when sample pre-treatments, matrix interference is reduced,
It can be that testing result is more acurrate.The detection method of the application is suitable for the detection of condiment base complex sample, has filled up seasoning
The blank of this technical field of conduct industry.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. the detection method of 4-methylimidazole in a kind of flavouring, which is characterized in that the detection method includes:
The 4-methylimidazole in the flavouring is detected using LC/MS/MS internal standard method.
2. detection method according to claim 1, which is characterized in that with 4-methylimidazole-d6As internal standard, using LC/
The step of MS/MS internal standard method detects the 4-methylimidazole in the flavouring include:
With 4-methylimidazole-d6As internal standard, the standard solution working curve of 4-methylimidazole is established；
The 4-methylimidazole in the flavouring is detected using the standard solution working curve.
3. detection method according to claim 2, which is characterized in that with 4-methylimidazole-d6As internal standard, 4- first is established
The step of standard solution working curve of base imidazoles includes:
With 4-methylimidazole concentration and 4-methylimidazole-d6The ratio of concentration is abscissa, with 4-methylimidazole quota ion pair
Peak area and 4-methylimidazole-d6The ratio of quota ion pair peak area is ordinate, establishes the standard of the 4-methylimidazole
Solution working curve.
4. detection method according to claim 3, which is characterized in that using LC/MS/MS internal standard method in the flavouring
4-methylimidazole detected before, the detection method further include: the step of preparing the sample to be tested of the flavouring,
Preferably, the step of preparing the sample to be tested of the flavouring include:
4-methylimidazole-d is added into the flavouring6, obtain just mixed object；It is preferred that 4-methylimidazole-the d6With the seasoning
The addition mass ratio of product are as follows: (0.1~0.5) × 10-7: 1；
The matrix interference in the just mixed object is removed, liquid to be purified is obtained；
Column chromatography is carried out to the liquid to be purified, obtains refined solution；
Constant volume is carried out to the refined solution, obtains the sample to be tested of the flavouring.
5. detection method according to claim 4, which is characterized in that pass through the side of precipitating proteins and/or enzymatic starch
Formula removes the matrix interference in the just mixed object, obtains the liquid to be purified；
Preferably, using protein described in methanol extraction, using starch described in amylase enzymolysis；
Preferably, the flavouring includes that sauce, soy sauce, oyster sauce and oyster juice are any one or more of.
6. detection method according to claim 5, which is characterized in that carry out column chromatography to the liquid to be purified, obtain pure
Change liquid the step of include:
Column chromatography is carried out to the liquid to be purified using MCX solid-phase extraction column, obtains refined solution；
Preferably, column chromatography, before obtaining refined solution, the detection are carried out to the liquid to be purified using MCX solid-phase extraction column
Method further includes the steps that activating the MCX solid-phase extraction column；
It is highly preferred that successively being activated using first alcohol and water to the MCX solid-phase extraction column.
7. detection method according to claim 6, which is characterized in that carry out column chromatography to the liquid to be purified, obtain pure
Change liquid the step of include:
The liquid to be purified is added in the MCX solid-phase extraction column and carried out column, it is preferable that the mistake of the control liquid to be purified
Column flow rate is 1/3~4s of drop；
Successively the liquid to be purified in the MCX solid-phase extraction column is eluted using aqueous formic acid and methanol solution,
Obtain elution column；
The elution column is eluted using ammonium hydroxide methanol solution, gained eluent is the refined solution.
8. detection method according to claim 7, which is characterized in that the mass concentration of formic acid is in the aqueous formic acid
1.5~3%, it is preferable that the volume of the aqueous formic acid and methanol solution is each independently 3~10mL；
Preferably, the mass concentration of ammonium hydroxide is 4.5~6% in the ammonium hydroxide methanol solution, and volume is 8~15mL.
9. detection method according to claim 8, which is characterized in that carry out constant volume to the refined solution, obtain the tune
The step of sample to be tested of taste product includes:
The refined solution is dried, purified is obtained；
It is filtered after being dissolved using -0.1% formic acid solution of acetonitrile to the purified, gained supernatant is the flavouring
Sample to be tested.
10. detection method according to claim 9, which is characterized in that after being dissolved using filter membrane to the purified
Filtering, the aperture of the preferably described filter membrane are 0.22~0.4 μm.
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