CN110320308A - The measuring method of 2- methoxyacetophenone in honey - Google Patents
The measuring method of 2- methoxyacetophenone in honey Download PDFInfo
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- CN110320308A CN110320308A CN201810285909.4A CN201810285909A CN110320308A CN 110320308 A CN110320308 A CN 110320308A CN 201810285909 A CN201810285909 A CN 201810285909A CN 110320308 A CN110320308 A CN 110320308A
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- methoxyacetophenone
- honey
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- measuring method
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a kind of measuring methods of 2- methoxyacetophenone in honey, belong to technical field of food detection.The present invention is dissolved honey by buffer salt solution, adds organic solution extraction, extracting solution is concentrated on vacuum rotary evaporator and is closely done, be settled to certain volume, cross 0.22 μm of organic filter membrane, liquid phase chromatographic tandem mass spectrograph is measured.The features such as this method is compensated for currently without 2- methoxyacetophenone detection method in honey, and simple to operate, high sensitivity, the rate of recovery are high, and qualitative, quantitative is accurate, is able to satisfy experiment demand.
Description
[technical field]
The invention belongs to technical field of food detection, and in particular to one kind is measured by high performance liquid chromatography tandem mass spectrum instrument
The method of 2- methoxyacetophenone in honey.
[background technique]
Manuka honey is referred to as New Zealand " national treasure " grade specialty, currently, it is that have been approved by the world two kinds are medical
One of grade honey, is primarily to facilitate wound healing, improves oral cavity and gastrointestinal health etc., and because function is significant, nutritive value is high,
It is pursued by various countries consumer, price is tens times also more expensive than common honey.Since it and common honey price differential are excessive,
Fake producer goes after like a flock of ducks.According to the statistical data of beekeeper association, New Zealand, New Zealand's Manuka honey annual output is only 1700-
2000 tons.And in global range, the year sales volume of " New Zealand's Manuka honey " is more than 10,000 tons, it means that in the market at least
80% is fake products.
In order to retrieve the reputation of Manuka honey, primary industry portion, New Zealand convenes industry, enterprise and expert, by for many years
Research determines the standard for formulating Manuka honey, has selected methoxy benzoic acid (2-methoxybenzoic acid), hydroxyl
Phenyllactic acid (4-hydroxyphenyllactic acid), phenyllactic acid (3-phenyllactic acid), 2- methoxyacetophenone
5 characteristic indexs such as (2-methoxyacetophenone) and Mai Luka pollen DNA, only this 5 indexs all meet
Manuka honey can be.
It is reported that 2- methoxyacetophenone is flavor component exclusive in Manuka honey, if not containing this in honey
Substance, then proving this not is real Manuka honey, but there is presently no 2- methoxyacetophenones in measurement honey
Method, therefore, the method for developing 2- methoxyacetophenone in a measurement honey palms off imported and famous brand honey, dimension for strike
Shield consumers' rights and interests are of great significance.
[summary of the invention]
The object of the present invention is to provide a kind of methods of 2- methoxyacetophenone in measurement honey, make up currently without 2- first
The blank of oxygroup acetophenone detection method.The present invention measures the 2- methoxyl group in honey by high performance liquid chromatography tandem mass spectrometry
The features such as acetophenone content, this method is simple to operate, high sensitivity, the rate of recovery are high, and qualitative, quantitative is accurate, is able to satisfy experiment
Demand.
To achieve the above object, the present invention takes the following detecting step to include:
1, sample extraction:
10g uniform sample (being accurate to 0.01g) is weighed, is placed in 50mL centrifuge tube, 15mL ammonium acetate solution is added, is mixed
It is even, add 15mL ethyl acetate, oscillation mixes 10min, 5min is centrifuged in 6 000r/min, with sharp mouth suction pipe by upper layer acetic acid
Ethyl ester solution moves in 100mL chicken heart bottle, and water phase adds 15mL ethyl acetate, repeats aforesaid operations, merges extracting solution twice,
Be concentrated under reduced pressure into rotary evaporator in 40 DEG C of water-baths it is close dry, it is accurate that 2mL constant volume liquid is added, mix, cross 0.22 μm of organic filter membrane,
It is measured for high performance liquid chromatography tandem mass spectrum.
2, Liquid Chromatography-Tandem Mass Spectrometry measures
Liquid-phase condition and Mass Spectrometry Conditions are established, according to corresponding conditions metric works solution and prepare liquid.Pass through reservation
Time and abundance of ions carry out quantitative analysis than carrying out qualitative analysis, using external standard calibration curve method.
Preferably, the organic solution is ethyl acetate in step 1.
Preferably, the ammonium acetate solution concentration is 0.05_mol/LpH=4.0-5.5 in step 1.
Preferably, the constant volume liquid is 60_% methanol in step 1.
Preferably, the liquid phase analysis condition in step 2 are as follows:
A) chromatographic column: BEH C18Column, 2.1 × 50_mm, 1.7_ μm；
B) mobile phase A: methanol；Mobile phase B: 0.1% aqueous formic acid of 5_mmol/L ammonium acetate, gradient elution are detailed in down
Table 1.
1 eluent gradient elution program of table
| Time (min) | A (%) | B (%) |
| 0.0 | 95 | 5.0 |
| 1.0 | 95 | 5.0 |
| 15 | 5.0 | 95 |
| 19 | 5.0 | 95 |
| 20 | 95 | 5.0 |
C) column temperature: 40 DEG C
D) flow velocity: 0.2mL/min
E) sample volume: 5 μ L
Preferably, Mass Spectrometry Conditions in step 2 are as follows:
A) ion source: electric spray ion source
B) scanning mode: cation scanning
C) detection mode: multiple-reaction monitoring (MRM)
D) capillary voltage: 2400_V
E) ion source temperature: 150 DEG C
F) dry gas stream speed: 800L/h
G) dry temperature degree: 500 DEG C
H) ion pair, orifice potential and collision energy are monitored and is shown in Table 2
Table 2 monitors ion pair, orifice potential and collision energy
Note: * is quota ion pair
According to above-mentioned liquid-phase condition and Mass Spectrometry Conditions measurement sample and standard working solution, if the mass chromatography peak of detection
Retention time is consistent with standard items, and the relative abundance ratio of two daughter ions of target compound and the comparable standard solution of concentration
It compares, tolerance should meet range as defined in table 3.
With respect to the maximum allowable offset of abundance of ions ratio when 3 qualitative confirmation of table
| Relative ion abundance | > 50% | > 20%~50% | > 10%~20% | < 10% |
| It allow relative deviation | ± 20% | ± 25% | ± 30% | ± 50% |
3, calculation formula
The content of 2- methoxyacetophenone is calculated according to the following formula
Wherein:
X: the content (μ g/kg) of sample to be tested；
C: the concentration (μ g/L) of solution is tested；
V: the constant volume (mL) of solution is tested；
M: sample weighting amount (g)；
The invention has the advantages that using the content of the 2- methoxyacetophenone in method detection honey of the invention, it is easy
Fast, qualitative, quantitative is accurate, has filled up the blank at present without 2- methoxyacetophenone detection method, the detection of verified this method
Limit can achieve 0.1 μ g/kg, and for the rate of recovery in 80%-100% range, linear dependence and repeatability are good.It can be seen that this
The method of invention is to detect the content of the 2- methoxyacetophenone in honey, provides reliable method convenient to carry out, can
The monitoring requirement for meeting 2- methoxyacetophenone in import honey provides technical support for safeguarding the equity of consumer.
Detailed description of the invention
Fig. 1 2- methoxyacetophenone standard items are in the quota ion pair figure of high performance liquid chromatography tandem mass spectrum instrument and qualitative
Ion pair figure；
Fig. 2 2- methoxyacetophenone standard working curve；
Specific embodiment:
Invention is further described in detail combined with specific embodiments below, but the invention is not limited to specific implementations
Example.
The detection method of 2- methoxyacetophenone in 1 pagodatree flower honey of embodiment
1, sample extraction:
10g uniform sample (being accurate to 0.01g) is weighed, is placed in 50mL centrifuge tube, 15mL ammonium acetate solution is added, is mixed
It is even, add 15mL ethyl acetate, oscillation mixes 10min, and 6 000r/min are centrifuged 5min, with sharp mouth suction pipe by upper layer acetic acid second
Ester solution moves in 100mL chicken heart bottle, and water phase adds 15mL ethyl acetate, repeats aforesaid operations, merges extracting solution twice, uses
Rotary evaporator be concentrated under reduced pressure into 40 DEG C of water-baths it is close dry, it is accurate that 2mL constant volume liquid is added, mix, cross 0.22 μm of organic filter membrane, supply
High performance liquid chromatography tandem mass spectrum measurement.
2, Liquid Chromatography-Tandem Mass Spectrometry measures
Liquid-phase condition and Mass Spectrometry Conditions are established, according to corresponding conditions metric works solution and prepare liquid.Pass through reservation
Time and abundance of ions carry out quantitative analysis than carrying out qualitative analysis, using external standard calibration curve method.
Liquid-phase condition are as follows:
A) chromatographic column: BEH C18Column, 2.1 × 50mm, 1.7 μm；
B) mobile phase A: methanol；Mobile phase B: 5mmol/L ammonium acetate (containing 0.1% formic acid) aqueous solution, gradient elution are detailed in
Table 1.
C) column temperature: 40 DEG C
D) flow velocity: 0.2mL/min
E) sample volume: 5 μ L
Mass Spectrometry Conditions are as follows:
A) ion source: electric spray ion source
B) scanning mode: cation scanning
C) detection mode: multiple-reaction monitoring (MRM)
D) capillary voltage: 2400V
E) ion source temperature: 150 DEG C
F) dry gas stream speed: 800L/h
G) dry temperature degree: 500 DEG C
H) ion pair, orifice potential and collision energy are monitored and is shown in Table 2
3, linear relationship: standard reserving solution is diluted step by step, prepares the series of working liquids of six various concentrations, respectively
0.1 μ g/L, 0.2 μ g/L, 0.5 μ g/L, 1.0 μ g/L, 5.0 μ g/L and 10.0 μ g/L.From low to high by concentration, according to above-mentioned
Chromatographic condition and Mass Spectrometry Conditions measurement.Make canonical plotting with corresponding peak area Y by its concentration of standard solution X, calculates and return
Equation and related coefficient, regression equation Y=8958.56X-300.989, related coefficient R2=0.994, illustrate linear good
It is good.
4, the rate of recovery and precision: accurately weighing negative honey sample, adds the 2- methoxybenzene of 3 different content levels
Ethyl ketone standard solution, each concentration carry out 6 parallel sample measurements, are analyzed according to above-mentioned chromatographic condition and Mass Spectrometry Conditions.
The rate of recovery and precision calculated result are shown in Table 4.
The 2- methoxyacetophenone rate of recovery and Precision test result are added in 4 honey of table
It can be seen from Table 4 that the accuracy of the method for the present invention is between 80%-100%, precision is below 10%.
Illustrate that this method accuracy is high, and favorable reproducibility.
5, detection limit and quantitative limit: test is added with the blank sample of honey, method detection limit is estimated with signal-to-noise ratio >=3
It calculates；The determination basis of quantitative limit is signal-to-noise ratio >=10 between the practical recovery of standard addition 70%~120% of sample, relative standard
Deviation≤15%, it is final to determine that detection is limited to 0.5 μ g/kg, quantitatively it is limited to 1.0 μ g/kg.
Above-described embodiment is used for illustrative purposes only, and is not the limitation to the invention patent；It should be pointed out that
For those of ordinary skill in the art, without departing from the scope of the inventive concept, can with various changes can be made and
Modification, these are all within the scope of protection of the present invention；Therefore, all equivalent changes done with scope of the invention as claimed with repair
Decorations, should fall within the scope of the present invention.
Claims (6)
1. the measuring method of 2- methoxyacetophenone in honey, which comprises the following steps:
(1) 10g uniform sample is weighed in 50mL centrifuge tube, 15mL lysate is added, and is added 15mL and is extracted solution, oscillation is mixed
Even 10min, 6 000r/min are centrifuged 5min, move in 100mL chicken heart bottle upper layer extracting solution with sharp mouth suction pipe, add 15mL
Extracting solution repeats aforesaid operations, merges extracting solution twice, be concentrated under reduced pressure into rotary evaporator in 40 DEG C of water-baths it is close dry, with fixed
Hold liquid and be settled to 2mL, mix, cross 0.22 μm of organic filter membrane, forms prepare liquid.
(2) Liquid Chromatography-Tandem Mass Spectrometry measures: liquid-phase condition and Mass Spectrometry Conditions are established, it is molten according to corresponding conditions metric works
Liquid and prepare liquid carry out qualitative and quantitative analysis using external standard-calibration curve method.
2. the measuring method of 2- methoxyacetophenone in honey according to claim 1, which is characterized in that the lysate
For 0.05mol/L ammonium acetate solution pH=4.0-5.5.
3. the measuring method of 2- methoxyacetophenone in honey according to claim 1, which is characterized in that the extraction is molten
Liquid is ethyl acetate.
4. the measuring method of 2- methoxyacetophenone in honey according to claim 1, which is characterized in that the constant volume liquid
For 60% methanol.
5. the measuring method of 2- methoxyacetophenone in honey according to claim 1, which is characterized in that the liquid phase item
Part are as follows:
A) chromatographic column: BEH C18Column, 2.1 × 50mm, 1.7 μm；
B) mobile phase A: methanol；Mobile phase B: 0.1% aqueous formic acid of 5mmol/L ammonium acetate, gradient elution are detailed in the following table 1；
1 eluent gradient elution program of table
C) column temperature: 40 DEG C；
D) flow velocity: 0.2mL/min；
E) sample volume: 5 μ L.
6. the measuring method of 2- methoxyacetophenone in honey according to claim 1, which is characterized in that the mass spectrum item
Part are as follows:
A) ion source: electric spray ion source；
B) scanning mode: cation scanning；
C) detection mode: multiple-reaction monitoring (MRM)；
D) capillary voltage: 2400V；
E) ion source temperature: 150 DEG C；
F) dry gas stream speed: 800L/h；
G) dry temperature degree: 500 DEG C；
H) ion pair, orifice potential and collision energy are monitored and is shown in Table 2；
Table 2 monitors ion pair, orifice potential and collision energy
Note: * is quota ion pair.
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| CN201810285909.4A CN110320308A (en) | 2018-03-30 | 2018-03-30 | The measuring method of 2- methoxyacetophenone in honey |
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| CN201810285909.4A CN110320308A (en) | 2018-03-30 | 2018-03-30 | The measuring method of 2- methoxyacetophenone in honey |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110658286A (en) * | 2019-11-19 | 2020-01-07 | 浙江树人学院（浙江树人大学） | Method for simultaneously and rapidly detecting contents of phenyllactic acid and 4-hydroxy phenyllactic acid by RP-HPLC (reverse phase-high performance liquid chromatography) |
| CN114002363A (en) * | 2021-10-29 | 2022-02-01 | 尼勒克县种蜂繁殖场管委会 | Application of phenyllactic acid as characteristic marker of Neliger Xinjiang black bee honey |
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| CN1752751A (en) * | 2004-09-23 | 2006-03-29 | 上海市徐汇区中心医院 | Liquid chromatography tandom mass spectrometry determination method of chloromycetin residue in honey |
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| CN1752751A (en) * | 2004-09-23 | 2006-03-29 | 上海市徐汇区中心医院 | Liquid chromatography tandom mass spectrometry determination method of chloromycetin residue in honey |
| US20110287059A1 (en) * | 2008-12-24 | 2011-11-24 | Comvita New Zealand Limited | Honey analysis |
| CN102183596A (en) * | 2011-03-02 | 2011-09-14 | 江南大学 | Method for detecting antibiotic residue in honey based on rapid classification extraction technology |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110658286A (en) * | 2019-11-19 | 2020-01-07 | 浙江树人学院（浙江树人大学） | Method for simultaneously and rapidly detecting contents of phenyllactic acid and 4-hydroxy phenyllactic acid by RP-HPLC (reverse phase-high performance liquid chromatography) |
| CN110658286B (en) * | 2019-11-19 | 2022-07-08 | 浙江树人学院（浙江树人大学） | Method for simultaneously and rapidly detecting contents of phenyllactic acid and 4-hydroxy phenyllactic acid by RP-HPLC (reverse phase-high performance liquid chromatography) |
| CN114002363A (en) * | 2021-10-29 | 2022-02-01 | 尼勒克县种蜂繁殖场管委会 | Application of phenyllactic acid as characteristic marker of Neliger Xinjiang black bee honey |
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