CN109655555A - Method for measuring 6-benzyladenine in bean sprouts - Google Patents
Method for measuring 6-benzyladenine in bean sprouts Download PDFInfo
- Publication number
- CN109655555A CN109655555A CN201910118067.8A CN201910118067A CN109655555A CN 109655555 A CN109655555 A CN 109655555A CN 201910118067 A CN201910118067 A CN 201910118067A CN 109655555 A CN109655555 A CN 109655555A
- Authority
- CN
- China
- Prior art keywords
- methanol
- benzyladenine
- solution
- measuring method
- feature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
本发明公开了一种测定豆芽中6‑苄基腺嘌呤的方法，包括如下步骤：(1)制备基质提取液；(2)制备供试品溶液；(3)制备标准工作溶液；(4)分别将标准工作溶液和供试品溶液注入超高效液相色谱‑质谱联用仪进行分析。本发明检测6‑苄基腺嘌呤稳定性高，检测限低，萃取效率高，富集倍数大，有机溶剂消耗量小，易于操作，适用于豆芽中6‑苄基腺嘌呤的快速定性和定量分析，具有良好的应用前景。
The invention discloses a method for determining 6-benzyladenine in bean sprouts, comprising the following steps: (1) preparing a matrix extract; (2) preparing a test solution; (3) preparing a standard working solution; and (4) respectively injecting the standard working solution and the test solution into an ultra-high performance liquid chromatography-mass spectrometer for analysis. The invention has high stability in detecting 6-benzyladenine, low detection limit, high extraction efficiency, large enrichment multiple, low organic solvent consumption, and is easy to operate. The invention is suitable for rapid qualitative and quantitative analysis of 6-benzyladenine in bean sprouts, and has good application prospects.
Description
Technical field
The invention belongs to technical field of food safety detection, are related to a kind of method for measuring 6-benzyladenine in bean sprouts.
Background technique
Bean sprouts also known as dish as one wishes, it is deep in recent years to be liked by the majority of consumers, the illegal businessman in part be shorten the production cycle,
Increase yield, improve bean sprouts appearance in order to sell, " AB powder ", " rootless bean sprouts element " are illegally used during bean sprout cultivation
It is then wherein main component, platymiscium growth regulator class Deng, 6-benzyladenine (6-benzylaminopurine, 6-BA)
Hormone residues, weak poison is accumulative, and certain embryo's toxic actions can be shown by reaching doses.6-benzyladenine uses in food
The remaining limitation mark of 6-benzyladenine has been formulated by more and more extensive concern, many countries and international organization with residual
It is quasi-.The U.S. allows 6-benzyladenine to be used for agricultural production as plant growth regulator, and noresidue limits, but use scope is all
It does not include bean sprouts；European Union allows a variety of agricultural product including mung bean sprouts to use using 6-benzyladenine, and residue limits are
0.01mg/kg；It is Japanese then 6-benzyladenine is included in " positive list " catalogue, and residue limits have been formulated respectively.2015 4
Months 13 days, state food pharmaceuticals administration general bureau, China, the Ministry of Agriculture, national health and Family Planning Committee's publication " about
The bulletin (o.11 in 2015) of the substances such as 6-benzyladenine is forbidden to use in the production process of bean sprouts ", forbid adding in bean sprouts
Or use 6-benzyladenine.Therefore, 6-benzyladenine residue detection should cause enough attention in bean sprouts.
In the sample detection of bean sprouts, because actual sample matrix components are complex, interfering substance is more and object content compared with
It is low, it usually needs analysis measurement just can be carried out using pretreatment technology appropriate.Currently about 6-benzyladenine in bean sprouts
In detection method, pretreatment technology mainly has Solid Phase Extraction, dispersed solid phase micro-extraction, liquid-liquid extraction etc., however these are traditional
There are many disadvantages for technology: experimental implementation is tedious, time-consuming, and extraction efficiency is low, a large amount of organic reagents is consumed in the process, easily to environment
Generate pollution；Meanwhile the detection method both at home and abroad for 6-benzyladenine in bean sprouts mainly has the chromatography of ions, thin layer at present
Chromatography, gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, high performance liquid chromatography-flight time matter
Spectrometry and high performance liquid chromatography-tandem mass method etc. have introducing to interfere and false positive results occur.
It is had no at present by ultrasonic wave added-dispersive liquid-liquid microextraction pretreatment technology combination ultra performance liquid chromatography-series connection matter
Spectrometer is applied to the research detected in bean sprouts using 6-benzyladenine.
Summary of the invention
To solve the above problems, the present invention provides a kind of methods of 6-benzyladenine in measurement bean sprouts.
The method that the present invention measures 6-benzyladenine in bean sprouts, it is super using ultrasonic wave added-dispersive liquid-liquid microextraction-
The measuring method of high performance liquid chromatography-tandem mass, includes the following steps:
(1) it prepares matrix extracting solution: taking bean sprouts sample comminution to be measured, methanol is added to extract, then plus anhydrous sodium sulfate and chlorination
Sodium extracts, and centrifugation takes supernatant liquid filtering, obtains matrix extracting solution；
(2) it prepares test solution: taking matrix extracting solution, sequentially add water and sodium chloride dissolution, it is mixed to reinject extractant
It is even, it extracts, centrifugation takes layer deposition to be mutually used as test solution；
(3) it prepares standard working solution: weighing 6-benzyladenine standard items, methanol is added to dissolve, dilute, with step (1)
Matrix extracting solution constant volume, as standard working solution；
(4) standard working solution and test solution injection ultra performance liquid chromatography-mass spectrometer are divided respectively
Analysis:
Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column；
Mobile phase: mobile phase A is methanol, and Mobile phase B is 0.1% aqueous formic acid；
Condition of gradient elution:
0~1min:10% methanol, 1~10min:90% methanol, 0~11min:10% methanol, 11~13min:10% first
Alcohol；
Mass Spectrometry Conditions:
Ionization mode, electric spray ion source；
Detection mode: cation scanning；
Scan pattern: selection reaction monitoring；
(5) qualitative and quantitative analysis.
Further, the mass volume ratio of step (1) sample, methanol, anhydrous sodium sulfate and sodium chloride is 2g:
10ml:5g:3g.
Further, step (1) is described is extracted as extraction of ocean eddies, and extraction of ocean eddies speed is 11000rpm, and methanol is added to be vortexed
The time of extraction is 3min, and adding the time of anhydrous sodium sulfate and sodium chloride extraction of ocean eddies is 1min.
Further, the revolving speed of step (1) described centrifugation is 4500r/min, time 5min；And/or it described was filtered into
0.45 μm of filter membrane.
Further, the mass volume ratio of step (2) the matrix extracting solution, water, sodium chloride and extractant are as follows: 1ml:
5ml:1.5g:100 μ l；And/or the extractant is tetrachloroethanes；And/or the extraction is ultrasonic extraction, the time is
10min；And/or the revolving speed of the centrifugation is 12000r/min, time 5min.
Further, the concentration of step (3) described standard working solution is 0.2ng/ml, 0.5ng/ml, 1ng/ml, 5ng/
Ml, 10ng/ml, 50ng/ml and/or 100ng/ml；The additional amount of matrix extracting solution is the 1/2 of constant volume when the constant volume.
Further, step (4) the C18 chromatographic column be Waters Atlantis T3 chromatographic column, specification be 150mm ×
2.1mm×3μm；And/or in the chromatographic condition, sample volume: 10 μ L, column temperature are 40 DEG C, flow velocity 0.5mL/min.
Further, in step (4) described Mass Spectrometry Conditions, spray voltage: 4500V, metal capillary temperature: 350 DEG C, sheath
Atmospheric pressure: 30psi, assist gas pressure power: 5psi, Q1 half-peak breadth: 0.7Da, Q3 half-peak breadth: 0.7Da.
Further, in the Mass Spectrometry Conditions, feature quantifies fragment ion N1 collision energy: 21eV, and feature is qualitative broken
Piece ion N2 collision energy: 16eV.
Further, the method for step (5) described qualitative and quantitative analysis are as follows: parent ion M is quantified by feature and feature is fixed
Property fragment ion N2 carry out it is qualitative；Fragment ion N1 peak area external standard method is quantified by feature to be quantified.
Further, it is 225.3 that the feature, which quantifies parent ion M charge-mass ratio, and feature quantifies fragment ion N1 and feature
Qualitative fragment ion N2 charge-mass ratio is respectively 91.0 and 148.0.
The method stability that the present invention detects 6-benzyladenine is high, and detection limit is low, and extraction efficiency is high, and enrichment times are big,
Organic solvent consumption is small, easily operated, the fast qualitative of 6-benzyladenine and quantitative analysis suitable for bean sprouts, has good
Good application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 ultra performance liquid chromatography-tandem mass spectrometry detection 6-benzyladenine standard ionomer stream (TIC) chromatogram；
Specific embodiment
The 6-benzyladenine qualitative detection of the present invention of embodiment 1
(1) it prepares matrix extracting solution: accurately weighing the bean sprouts sample 2.00g sufficiently crushed through tissue mashing machine and (be accurate to
0.01g) have in plug centrifuge tube to 50mL, 10mL methanol is added and is homogenized (revolving speed 11000rpm) 3min, it is anhydrous that 5g is then added
Sodium sulphate and 3g sodium chloride, are centrifuged 5min with 4500r/min after vortex 1min, and supernatant is transferred to 10mL after crossing 0.45 μm of filter membrane
Volumetric flask obtains matrix extracting solution with methanol constant volume；
(2) prepare test solution: take 1.00mL matrix extracting solution to 10mL have plug centrifuge tube in, sequentially add 5mL water,
1.5gNaCl, be vortexed dissolution, then 100 μ L tetrachloroethanes is injected into above-mentioned solution rapidly with the glass syringe of 1mL, whirlpool
Rotation mixes, formation emulsion, after ultrasound on extracting 10min, is centrifuged 5min with 12000r/min, takes 30 μ L with microsyringe
Layer deposition phase sample liquid, is added in the sample bottle containing interpolation pipe, measures for ultra performance liquid chromatography-tandem mass spectrometer；
(3) it prepares standard working solution: accurately weighing 6-benzyladenine standard items, be configured to 10mg/mL's with methanol
Standard reserving solution, it is spare.It is accurate to measure 100 μ L of 6-benzyladenine standard reserving solution when detection, 1 μ g/mL is configured to methanol
Standard working solution, then it is accurate measure 6-benzyladenine standard working solution 0.1mL, with methanol dilution to 0.5ml, then use step
(1) matrix extracting solution is settled to 1ml, be configured to concentration be 100ng/mL standard working solution to get；
(4) standard working solution and test solution injection ultra performance liquid chromatography-mass spectrometer are divided respectively
Analysis:
Ultra performance liquid chromatography condition, chromatographic column: Waters Atlantis T3 (3 μ m 2.1mm × 150mm)；Flowing
Phase: methanol, 0.1% aqueous formic acid；Gradient elution program (0~1min, 10% methanol；1~10min, 90% methanol；10~
11min, 10% methanol；11~13min, 10% methanol)；Flow velocity: 0.5mL/min；Column temperature: 40 DEG C；Sample volume: 10 μ L；
Tandem mass spectrum condition, ionization mode: electric spray ion source (ESI), cation scanning；Spray voltage: 4500V；
Metal capillary temperature: 350 DEG C；Sheath atmospheric pressure: 30psi；Assist gas pressure power: 5psi；Scan pattern: selection reaction monitoring
(SRM)；Q1 half-peak breadth: 0.7Da；Q3 half-peak breadth: 0.7Da；Feature quantifies fragment ion N1 collision energy: 21eV, feature are qualitative
Fragment ion N2 collision energy: 16eV；
(5) parent ion (m/z) M (charge-mass ratio 225.3) and qualitative qualitative analysis: is quantified by the feature of 6-benzyladenine
Fragment ion N2 (charge-mass ratio 148.0) carries out qualitative.
The 6-benzyladenine quantitative detection of the present invention of embodiment 2
(1) it prepares matrix extracting solution: accurately weighing the bean sprouts sample 2.00g sufficiently crushed through tissue mashing machine and (be accurate to
0.01g) have in plug centrifuge tube to 50mL, 10mL methanol is added and is homogenized (revolving speed 11000rpm) 3min, it is anhydrous that 5g is then added
Sodium sulphate and 3g sodium chloride, are centrifuged 5min with 4500r/min after vortex 1min, and supernatant is transferred to 10mL after crossing 0.45 μm of filter membrane
Volumetric flask obtains matrix extracting solution with methanol constant volume；
(2) prepare test solution: take 1.00mL matrix extracting solution to 10mL have plug centrifuge tube in, sequentially add 5mL water,
1.5gNaCl, be vortexed dissolution, then 100 μ L tetrachloroethanes is injected into above-mentioned solution rapidly with the glass syringe of 1mL, whirlpool
Rotation mixes, formation emulsion, after ultrasound on extracting 10min, is centrifuged 5min with 12000r/min, takes 30 μ L with microsyringe
Layer deposition phase sample liquid, is added in the sample bottle containing interpolation pipe, measures for ultra performance liquid chromatography-tandem mass spectrometer；
(3) it prepares standard working solution: accurately weighing 6-benzyladenine standard items, be configured to 10mg/mL's with methanol
Standard reserving solution, it is spare.It is accurate to measure 100 μ L of 6-benzyladenine standard reserving solution when detection, 1 μ g/mL is configured to methanol
Standard working solution, then respectively it is accurate measure 6-benzyladenine standard working solution 0.0002,0.0005,0.001,0.005,
0.01,0.05,0.1mL are settled to 1ml with methanol dilution to 0.5ml, then with step (1) matrix extracting solution, are configured to concentration point
It Wei not 0.2, the standard series working solution of 0.5,1,5,10,50,100ng/mL；
(4) respectively by standard series working solution and test solution inject ultra performance liquid chromatography-mass spectrometer into
Row analysis:
Ultra performance liquid chromatography condition, chromatographic column: Waters Atlantis T3 (3 μ m 2.1mm × 150mm)；Flowing
Phase: methanol, 0.1% aqueous formic acid；Gradient elution program (0~1min, 10% methanol；1~10min, 90% methanol；10~
11min, 10% methanol；11~13min, 10% methanol)；Flow velocity: 0.5mL/min；Column temperature: 40 DEG C；Sample volume: 10 μ L；
Tandem mass spectrum condition, ionization mode: electric spray ion source (ESI), cation scanning；Spray voltage: 4500V；
Metal capillary temperature: 350 DEG C；Sheath atmospheric pressure: 30psi；Assist gas pressure power: 5psi；Scan pattern: selection reaction monitoring
(SRM)；Q1 half-peak breadth: 0.7Da；Q3 half-peak breadth: 0.7Da；Feature quantifies fragment ion N1 collision energy: 21eV, feature are qualitative
Fragment ion N2 collision energy:
16eV；
(5) it quantitative analysis: is quantified outside fragment ion N1 (charge-mass ratio 91.0) peak area by the feature of 6-benzyladenine
Mark method is quantified.
Beneficial effects of the present invention are further illustrated below by way of the mode of experimental example:
1 present invention of experimental example detects the research of the 6-benzyladenine in bean sprouts sample to be checked
1, experimental material
(1) instrument: 100 type Ultra Performance Liquid Chromatography instrument of Ekspert ultraLC, AB Sciex Triple Quad
5500 mass spectrographs [are furnished in electrospray ionization source (source ESI)] (American AB Sciex)；Ultrasonic washing instrument (the white extra large scientific instrument instrument in Nanjing
Table Co., Ltd)；Supercentrifuge (German Thermo)；Homogenizer (German IKA)
(2) reagent: methanol: chromatographically pure (U.S. Honeywell)；Acetonitrile: chromatographically pure (U.S. Honeywell)；Four chloroethenes
Alkane: chromatographically pure (U.S. Honeywell)；Formic acid: chromatographically pure (Shanghai Jingchun Industrial Co., Ltd.)；Remaining reagent: analysis it is pure (on
The general tech equipment Co., Ltd in Hai'an)；Experimental water: Milli-Q deionized water；6-benzyladenine standard items, (German Dr
Ehremstorfer, purity >=99.0%)；
(3) sample: bean sprouts comes from domestic market.
2, experimental method
(1) preparing standard solution and matrix extracting solution
Standard reserving solution: accurately weighing that 6-benzyladenine standard items are appropriate, is stored up with the standard that methanol is configured to 10mg/mL
Standby liquid.Standard working solution: it is accurate to measure 100 μ L of 6-benzyladenine standard reserving solution, the standard of 1 μ g/mL is configured to methanol
Working solution.Standard works serial solution: it is accurate measure 6-benzyladenine standard working solution 0.0002,0.0005,0.001,
0.005,0.01,0.05,0.1mL are configured to standard series working solution with methanol dilution.
Matrix extracting solution: negative bean sprouts sample 2.00g to the 50mL tool plug centrifugation sufficiently crushed through tissue mashing machine is weighed
Guan Zhong is added 5g anhydrous sodium sulfate and 3g sodium chloride after 10mL methanol homogenate (revolving speed 11000rpm) 3min is added, is vortexed
After 1min with 4500r/min be centrifuged 5min, cross 0.45 μm of filter membrane after it is spare.
(2) detection method selects embodiment 1 or 2 detection methods
(3) sensitivity verifying accurately pipettes 6-benzyladenine standard working solution, with embodiment 1 or 2 experimental method matrix
Extracting solution constant volume, being configured to concentration is the 0.2, standard working curve of 0.5,1,5,10,50,100ng/mL, according to embodiment 1 or
2 detection method steps (4), using the peak area Y of quota ion as ordinate, mass concentration X (ng/mL) draws standard curve, with 3
Times signal-to-noise ratio determines that the method detection limit of object, 10 times of signal-to-noise ratio determine the method quantitative limit of object.
(4) rate of recovery, enrichment factor and precision verifying take bean sprouts sample to carry out mark-on reclaims test.Weigh blank sample
2g adds the horizontal 6-benzyladenine standard solution of 0.5,1.5,10 μ g/kg tri-, according to 2 detection method of embodiment respectively
Measurement, peak area external standard method calculate the rate of recovery and relative standard deviation of 6-benzyladenine.It is being extracted with 6-benzyladenine
Concentration determines enrichment factor EF with the ratio between the initial concentration in sample solution after enrichment in agent.
(5) matrix effect verifying takes negative bean sprouts sample to prepare anima extracting solution by embodiment 1 or 2 experimental methods,
Accurately pipette 6-benzyladenine standard working solution, after methanol dilution, then with anima extracting solution constant volume (volume ratio 1:
1) it, is configured to the object standard solution that mass concentration is 100ng/kg, it is dense to prepare another equal quality using methanol as constant volume liquid
The standard solution of degree is injected separately into ultra performance liquid chromatography-mass spectrometer and is analyzed, and calculates the two mass spectrum response intensity
Ratio.
3, experimental result
(1) the characteristic ion parameter of 6-benzyladenine
The results are shown in Table 1:
The characteristic ion parameter of table 1:6- benzyladenine
The results are shown in Table 1: the selection ion chromatography peak of sample prepare liquid and standard items at identical retention time (±
0.5%) occur, and the mass-to-charge ratio of corresponding mass spectrometric fragment ion is consistent with standard items.Wherein object ion N1 and N2 is to be divided
The quantitative fragment ion of object and the accurate mass number of qualitative fragment ion are analysed, object ion M is determining for analyzed object
Measure the accurate mass number of parent ion (m/z).
Standard ionomer stream (TIC) chromatogram of 6-benzyladenine, as shown in Figure 1.
(2) sensitivity
The results are shown in Table 2:
2 range of linearity of table, linear equation, related coefficient, method detection limit (LOD), method quantitative limit (LOQ)
As a result such as table 2,6-benzyladenine is to have good linear within the scope of 0.2ng/mL~100ng/mL in concentration
Response, detection limit is low, high sensitivity, can be used for 6-benzyladenine residue detection in bean sprouts.
(3), the rate of recovery, precision and enrichment factor
The results are shown in Table 3:
3 rate of recovery of table, precision and enrichment factor test result (n=6)
As a result such as table 3, the average recovery rate of 6-benzyladenine is 68.4%~70.3%, relative standard deviation (RSD)
It is 3.32%~7.05%, for enrichment factor EF between 39.1~44.3, this three indexs show the accurate of institute's choosing method of the present invention
Degree and accuracy are high, reproducible, and enrichment times are high, can be used for the fast enriching of 6-benzyladenine and residual inspection in bean sprouts
It surveys.
The results showed that the present invention detect bean sprouts in 6-benzyladenine method have it is easy to operate, using organic
The advantages that reagent is few, high sensitivity, favorable reproducibility, enrichment times are high, qualitative, quantitative is accurate, 6- benzyl gland is fast suitable for bean sprouts
Purine qualitative and quantitative analysis.
(4), matrix effect
The experimental results showed that constant volume, the mass spectrum response intensity ratio of 6-benzyladenine are 0.74 in different ways, i.e.,
Bean sprouts sample belongs to complex matrices, and there are stronger mechanism inhibiting effect.Therefore this experiment uses the extracting solution conduct of bean sprouts matrix
Constant volume liquid prepares standard working solution and is corrected compensation, and NaCl is added in an experiment and further decreases substrate inhibition effect,
Reduce influence and interference of the substrate inhibition to analysis test result.
2 detection method condition optimizing screening test of experimental example
1 experimental method
Influence dispersive liquid-liquid microextraction efficiency principal element have extractant type and dosage, Dispersant types and dosage,
Ultrasound on extracting time, salinity, pH value etc..Enrichment factor (Enrichment factors, EF): target compound is extracting
Take the ratio between the initial concentration of concentration and target compound in sample solution in agent, i.e. EF=csed/c0.The present invention is using rich
Collect factor parameter EF value to investigate method extraction efficiency, optimize the principal element for influencing dispersive liquid-liquid microextraction efficiency, realizes
To 6-benzyladenine fast enriching and measurement in bean sprouts.
(1) screening of extractant type and dosage
In dispersive liquid-liquid microextraction, the optimization of extractant has a major impact extraction efficiency and EF value.General extraction
The selection of agent need to meet following condition: density ratio water is big；There is preferable effect of extracting to target compound；It dissolves in water
It spends small；Stable two-phase system can be formed.The present invention is carried out using the bean sprouts without 6-benzyladenine as blank sample matrix
1 times of lower limit of measurement concentration level addition experiment, selects 4 kinds of common extractants: methylene chloride, carbon tetrachloride, tetrachloroethanes and chlorine
Benzene is compared, and investigates influence of the different extractant types to enrichment factor.Determine choose 20 after extractant type again, 50,
100,150,200 μ L difference Solvent quantity, investigates its influence to enrichment factor.
(2) screening of Dispersant types and dosage
The type of dispersing agent is also to influence a key factor of extraction efficiency.Since 6-benzyladenine dissolves in water
The dispersive liquid-liquid microextraction method low, present invention use improves is spent, bean sprouts is carried out first to extract the pre-treatment extracted afterwards, selection
Dispersing agent is also used as extracting solution.Requirement of the dispersive liquid-liquid microextraction to dispersing agent mainly has: having good dissolution in extractant
Property and can be miscible with water, so that extractant is dispersed into microballon in system, increase connecing for extractant and target solution to the maximum extent
Contacting surface product, and then improve extraction efficiency；Interference is not generated to the measurement of target compound.The present invention is to be free of 6-benzyladenine
Bean sprouts as blank sample matrix, carry out 1 times of lower limit of measurement concentration level addition experiment, select 3 kinds of common extractants: first
Alcohol, acetonitrile.Acetone is compared, and investigates influence of the different Dispersant types to enrichment factor.Dispersing agent is to " dispersing agent/water/extraction
Taking agent " formation of emulsion system has a direct impact, and volume can change degree of scatter of the dispersing agent in system and then influence
Extraction efficiency.0.5,1,1.5,2,3mL difference dispersant dosage are chosen again after determining Dispersant types, investigate it to enrichment factor
Influence.
(3) influence of ultrasound on extracting time
Ultrasound on extracting can make ionic liquid be dispersed into microballon, by constantly movement in the solution, increase and target
The contact area of object solution so that ionic liquid is contacted with object more rapidly, fully, and then reaches extraction equilibrium.Dividing
In dispersion liquid liquid extraction experiments, the foundation of balance needs the regular hour.The present invention is using the bean sprouts without 6-BA as blank sample
Matrix carries out 1 times of lower limit of measurement concentration level addition experiment, has investigated 2min, 5min, 10min, 15min and 20min five not
Influence of the time of same ultrasound on extracting to enrichment factor.
(4) influence of salinity
In extraction experiments, certain density salt, which is added, will affect aqueous ion intensity, generate certain salting-out effect: suitable
When reducing object in the solubility of water phase, extraction efficiency of the object in system is improved.The present invention is to be free of 6- benzyl gland
The bean sprouts of purine carries out 1 times of lower limit of measurement concentration level addition experiment as blank sample matrix, select 0,0.1,0.5,1.0,
1.5, the NaCl concentration of 2g/mL is compared, and investigates influence of the different salinity to enrichment factor.
(5) influence of pH value
Solution is under different ph values to the influence of enrichment factor when the present invention has investigated extraction.
2 experimental results
(1) the selection result of extractant type and dosage is shown in Table 4, table 5.
Influence of the different extractants of table 4 to EF value
Influence of the different Solvent quantities of table 5 to EF value
The results showed that relatively stable with dispersing agent formation two-phase system when tetrachloroethanes is as extractant, EF value is most
Greatly, extraction efficiency highest, followed by carbon tetrachloride, and more through chlorobenzene sample liquid impurity extracted, mass spectrogram baseline interference compared with
By force, the analysis for influencing object is quantitative, and methylene chloride can not then form stable two-phase system with dispersing agent.Therefore, four are chosen
Extractant of the chloroethanes as sample.Therefore, the present invention uses extractant of the tetrachloroethanes as sample.In Solvent quantity
On, when solvent volume is lower than 100 μ L, EF value and the solvent volume of object are positively correlated；When higher than 100 μ L, EF value is reduced.
When extract agent content it is very little when, extractant may lose, or too small lead to extraction efficiency with object contact area product
It is low；After extractant reaches certain volume, object extraction efficiency reaches peak value, increases extractant again after reaching extraction equilibrium
Amount, target concentration reduce instead cause EF value decline, therefore the present invention choose 100 μ L as Solvent quantity.
(2) the selection result of Dispersant types and dosage is shown in Table 6, table 7.
Influence of the different dispersing agents of table 6 to EF value
Influence of the different dispersing agent volumes of table 7 to EF value
The result shows that: when using methanol as dispersing agent, object EF value is maximum, and extraction efficiency is high at this time, is secondly third
Ketone.When using acetonitrile as dispersing agent, stable two-phase system can only be formed with a kind of extractant of carbon tetrachloride, the present invention chooses first
Alcohol is as dispersing agent.In dosage, using 1mL methanol dispersing agent when, the EF value of object is maximum.Due to dispersing in small size
Under agent effect, extractant is not dispersed also completely, it is difficult to form emulsion, EF value increases with the increase of methanol volume at this time
Greatly.The solubility that conference makes target compound in sample liquid is crossed when methanol volume to increase, and is not easy to be extracted agent extraction, is caused to extract
Efficiency reduces.Therefore the present invention is using 1mL as dispersant dosage.
(3) the influence result of ultrasound on extracting time
It the results are shown in Table 8
Influence of the different ultrasonic times of table 8 to EF value
The result shows that: dispersing agent is added in system and extractant, EF value can reach maximum after ultrasound on extracting 10min
It is worth, for extraction efficiency with the increase of extraction time without substantially changeing, this, which shows that extraction time is very short, can reach two-phase after 10min
The extraction equilibrium of system.
(4) the influence result of salinity
It is shown in Table 9
Influence of the different salinity of table 9 to EF value
The result shows that: it can effectively improve extraction efficiency after adding 1.5gNaCl in sample liquid, and EF value is maximum, such as table 9.
Lead to not be completely dissolved in system if NaCl additional amount is excessive, influences the experiment behaviour of layer deposition phase after subsequent centrifugation
Make, therefore the present invention chooses NaCl concentration of the 1.5g/mL as addition.
(5) the influence result of pH value
The result shows that enrichment factor EF value influenced by pH value it is little.Therefore the present invention is using under the conditions of neutral solution
It carries out.
The results showed that optimizing screening by the condition to detection method, detection method has operation
Simply, organic solvent consumption is small, and extraction efficiency is high, can effectively exclude the advantages of interference of other compounds in extraction process,
Fast enriching extraction, the qualitative and quantitative analysis of 6-benzyladenine suitable for bean sprouts are carried out to target compound.
To sum up, the present invention detects 6-benzyladenine stability height, and detection limit is low, and extraction efficiency is high, and enrichment times are big, have
Solvent consumption is small, easily operated, the fast qualitative of 6-benzyladenine and quantitative analysis suitable for bean sprouts, has good
Application prospect.
Claims (10)
1. a kind of method of 6-benzyladenine in measurement bean sprouts, it is characterised in that: it is micro- using ultrasonic wave added-dispersion liquid
Extraction-ultra performance liquid chromatography-tandem mass spectrum measuring method, includes the following steps:
(1) prepare matrix extracting solution: taking bean sprouts sample comminution to be measured, methanol is added to extract, then plus anhydrous sodium sulfate and sodium chloride mention
It takes, is centrifuged, takes supernatant liquid filtering, obtain matrix extracting solution；
(2) it prepares test solution: taking step (1) matrix extracting solution, sequentially add water and sodium chloride dissolution, reinject extractant
It mixes, extracts, centrifugation takes layer deposition to be mutually used as test solution；
(3) it prepares standard working solution: weighing 6-benzyladenine standard items, methanol is added to dissolve, dilute, with step (1) matrix
Extracting solution constant volume, as standard working solution；
(4) standard working solution and test solution injection ultra performance liquid chromatography-mass spectrometer are analyzed respectively:
Chromatographic condition is as follows:
Chromatographic column: C18 chromatographic column；
Mobile phase: mobile phase A is methanol, and Mobile phase B is 0.1% aqueous formic acid；
Condition of gradient elution:
0~1min:10% methanol, 1~10min:90% methanol, 0~11min:10% methanol, 11~13min:10% methanol；
Mass Spectrometry Conditions:
Ionization mode, electric spray ion source；
Detection mode: cation scanning；
Scan pattern: selection reaction monitoring；
(5) qualitative and quantitative analysis.
2. measuring method according to claim 1, it is characterised in that: step (1) sample, methanol, anhydrous sodium sulfate
Mass volume ratio with sodium chloride is 2g:10ml:5g:3g.
3. measuring method according to claim 1, it is characterised in that: step (1) is described to be extracted as extraction of ocean eddies, and vortex mentions
Taking speed is 11000rpm, and adding the time of methanol extraction of ocean eddies is 3min, add anhydrous sodium sulfate and sodium chloride extraction of ocean eddies when
Between be 1min.
4. measuring method according to claim 1, it is characterised in that: the revolving speed of step (1) described centrifugation is 4500r/
Min, time 5min；And/or described it was filtered into 0.45 μm of filter membrane.
5. measuring method according to claim 1, it is characterised in that: step (2) the matrix extracting solution, water, sodium chloride
With the mass volume ratio of extractant are as follows: 1ml:5ml:1.5g:100 μ l；And/or the extractant is tetrachloroethanes；And/or institute
Stating extraction is ultrasonic extraction, time 10min；And/or the revolving speed of the centrifugation is 12000r/min, time 5min.
6. measuring method according to claim 1, it is characterised in that: the concentration of step (3) described standard working solution is
0.2ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 50ng/ml and/or 100ng/ml；Matrix mentions when the constant volume
The additional amount for taking liquid is the 1/2 of constant volume.
7. measuring method according to claim 1, it is characterised in that: step (4) the C18 chromatographic column is Waters
Atlantis T3 chromatographic column, specification are 150mm × 2.1mm × 3 μm；And/or in the chromatographic condition, sample volume: 10 μ L, column
Temperature is 40 DEG C, flow velocity 0.5mL/min.
8. measuring method according to claim 1, it is characterised in that: in step (4) described Mass Spectrometry Conditions, spray voltage:
4500V, metal capillary temperature: 350 DEG C, sheath atmospheric pressure: 30psi, assist gas pressure power: 5psi, Q1 half-peak breadth: 0.7Da, Q3 half
Peak width: 0.7Da, feature quantify fragment ion N1 collision energy: 21eV, the qualitative fragment ion N2 collision energy of feature: 16eV.
9. measuring method according to claim 1, it is characterised in that: the method for step (5) described qualitative and quantitative analysis are as follows:
Parent ion M is quantified by feature and the qualitative fragment ion N2 progress of feature is qualitative；Fragment ion N1 peak area is quantified by feature
External standard method is quantified.
10. measuring method according to claim 8 or claim 9, it is characterised in that: the feature quantifies parent ion M charge-mass ratio and is
225.3, it is respectively 91.0 and 148.0 that feature, which quantifies fragment ion N1 and the qualitative fragment ion N2 charge-mass ratio of feature,.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910118067.8A CN109655555A (en) | 2019-02-15 | 2019-02-15 | Method for measuring 6-benzyladenine in bean sprouts |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910118067.8A CN109655555A (en) | 2019-02-15 | 2019-02-15 | Method for measuring 6-benzyladenine in bean sprouts |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109655555A true CN109655555A (en) | 2019-04-19 |
Family
ID=66122795
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910118067.8A Pending CN109655555A (en) | 2019-02-15 | 2019-02-15 | Method for measuring 6-benzyladenine in bean sprouts |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109655555A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101738441A (en) * | 2010-01-22 | 2010-06-16 | 大连民族学院 | Method for testing plant endogenous hormones |
| CN104535703A (en) * | 2015-01-06 | 2015-04-22 | 山东省分析测试中心 | Method for detecting 6-benzyladenine and 4-sodium chlophenoxycetate in bean sprouts at the same time |
| CN105929091A (en) * | 2016-06-30 | 2016-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Efficient detection method for illegally added medicines in bean sprouts |
| CN106053703A (en) * | 2016-08-17 | 2016-10-26 | 刘思洁 | Method of detecting various plant growth regulators in foods at same time |
| CN106404958A (en) * | 2016-10-21 | 2017-02-15 | 重庆市食品药品检验检测研究院 | Method for quickly detecting contents of plant growth regulators in vegetables |
| CN107621498A (en) * | 2016-07-14 | 2018-01-23 | 罗牛山莱茵检测认证服务（海南）有限公司 | The detection method of one plant growth regulators residual |
| CN108956794A (en) * | 2018-04-20 | 2018-12-07 | 公安部物证鉴定中心 | Detection method of plant growth regulator 6-benzyl adenine and paclobutrazol in biological fluid |
| CN109212064A (en) * | 2018-09-19 | 2019-01-15 | 遵义市产品质量检验检测院 | The detection method of one plant growth regulators residual volume |
-
2019
- 2019-02-15 CN CN201910118067.8A patent/CN109655555A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101738441A (en) * | 2010-01-22 | 2010-06-16 | 大连民族学院 | Method for testing plant endogenous hormones |
| CN104535703A (en) * | 2015-01-06 | 2015-04-22 | 山东省分析测试中心 | Method for detecting 6-benzyladenine and 4-sodium chlophenoxycetate in bean sprouts at the same time |
| CN105929091A (en) * | 2016-06-30 | 2016-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Efficient detection method for illegally added medicines in bean sprouts |
| CN107621498A (en) * | 2016-07-14 | 2018-01-23 | 罗牛山莱茵检测认证服务（海南）有限公司 | The detection method of one plant growth regulators residual |
| CN106053703A (en) * | 2016-08-17 | 2016-10-26 | 刘思洁 | Method of detecting various plant growth regulators in foods at same time |
| CN106404958A (en) * | 2016-10-21 | 2017-02-15 | 重庆市食品药品检验检测研究院 | Method for quickly detecting contents of plant growth regulators in vegetables |
| CN108956794A (en) * | 2018-04-20 | 2018-12-07 | 公安部物证鉴定中心 | Detection method of plant growth regulator 6-benzyl adenine and paclobutrazol in biological fluid |
| CN109212064A (en) * | 2018-09-19 | 2019-01-15 | 遵义市产品质量检验检测院 | The detection method of one plant growth regulators residual volume |
Non-Patent Citations (7)
| Title |
|---|
| SEMIHOTLES: "《食物成分与食品添加剂分析方法》", 31 August 2008 * |
| 张婧雯 等: "固相萃取_超高效液相色谱串联质谱法同时测定豆芽中6-苄基腺嘌呤、赤霉素和4-氯苯氧乙酸的残留量", 《中国食品卫生杂志》 * |
| 张雪莲 等: "分散液液微萃取技术及其在食品和环境农药残留检测中的应用", 《食品科学》 * |
| 曹江平 等: "分散液液微萃取技术在食品分析中的应用进展", 《分析测试学报》 * |
| 王宇成 等: "《最新色谱分析检测方法及应用技术实用手册 第1卷》", 31 December 2004 * |
| 谢寒冰 等: "高效液相色谱-四极杆飞行时间质谱法同时检测豆芽中的3种外源植物激素残留", 《色谱》 * |
| 黄少文 等: "6-苄基腺嘌呤对黄豆芽生长的影响及其残留量测定", 《南方农业学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106124643B (en) | The method for detecting tetrabromobisphenol A, decabromodiphenyl oxide and hexabromocyclododecane three classes bromide fire retardant content in aquatic products | |
| Xia et al. | 8-Hydroxyquinoline–chloroform single drop microextraction and electrothermal vaporization ICP-MS for the fractionation of aluminium in natural waters and drinks | |
| Gangjian et al. | Measurement on high-precision boron isotope of silicate materials by a single column purification method and MC-ICP-MS | |
| CN106124653B (en) | 5 kinds of Nitrofuran metatolites and the how remaining detection method of chloramphenicol in shrimp | |
| CN107300594B (en) | The method that liquid chromatography mass combination directly detects amino acid in biological tissue's Uniform Sample | |
| CN104931635B (en) | Detect method and the liquid matter data base of left drug in animal-derived food | |
| CN107727772A (en) | The more reaction detection patterns of Pyrolysis Mass Spectrometry are protonated using electron spray, and qualitative and quantitative method is carried out to triterpenoid saponin in Chinese medicine | |
| Shen et al. | Rapid quantification of four major bioactive alkaloids in Corydalis decumbens (Thunb.) Pers. by pressurised liquid extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry | |
| CN101865886A (en) | Method for measuring residual quantity of chloramphenicol in propolis by using high performance liquid chromatography tandem mass spectrum | |
| CN104897842B (en) | Liquid-mass database for detecting residual chemicals in animal-derived food and application method thereof | |
| Bergmann et al. | Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography | |
| Li et al. | A sensitive LC–MS/MS method for simultaneous determination of amygdalin and paeoniflorin in human plasma and its application | |
| Su et al. | Rapid and sensitive analysis of multiple bioactive constituents in tripterygium glycosides tablets using liquid chromatography coupled with time‐of‐flight mass spectrometry | |
| CN111208252B (en) | Quantitative detection method of tretinoin in human plasma | |
| CN101865887B (en) | Method for detecting nitromidazole residue in royal jelly by using high performance liquid chromatography tandem mass spectrum | |
| CN106645496A (en) | Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography | |
| CN109655555A (en) | Method for measuring 6-benzyladenine in bean sprouts | |
| Zhuo et al. | Rapid simultaneous quantitative determination of terpenoids in Tripterygium wilfordii Hook F by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry | |
| CN108760920A (en) | A method of cyazofamid and its metabolite residue amount are measured based on HPLC-MSMS methods | |
| CN112526049A (en) | Method for simultaneously detecting 10 sweetening agents in white spirit based on ultra-high performance liquid chromatography tandem electrostatic field orbit trap mass spectrum | |
| Kumar et al. | Determination of chloride in nuclear-grade boron carbide by ion chromatography | |
| CN110398554A (en) | A method and application for evaluating the matrix effect of endogenous compounds based on isotope internal standard | |
| Shi et al. | Determination of seven phenolic acids in honey by counter flow salting-out homogeneous liquid–liquid extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry | |
| CN104849383A (en) | Method for determining nitroimidazole drug in bee pollen powder through combination of rapid solvent extraction-gel chromatography purification-LC/MS/MS | |
| CN107144648A (en) | Detect the application of the Liquid Chromatography-Tandem Mass Spectrometry method of Pitavastatin and clinical pharmacokinetics research in human plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190419 |
|
| RJ01 | Rejection of invention patent application after publication |