CN104483425A - Method for detecting maleic hydrazide residue in tobacco and tobacco product - Google Patents
Method for detecting maleic hydrazide residue in tobacco and tobacco product Download PDFInfo
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Abstract
The invention belongs to the field of quality safety of tobacco and particularly relates to a method for detecting maleic hydrazide residue in tobacco and a tobacco product. According to the method for detecting maleic hydrazide residue in tobacco and the tobacco product provided by the invention, microwave-assisted extraction as well as high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) are adopted to determine the maleic hydrazide residue in tobacco and the tobacco product, and the residue is quantified by virtue of an internal standard curve method. Compared with the prior art, the method for detecting maleic hydrazide residue in tobacco and the tobacco product is high in extraction efficiency, simple and convenient to operate and accurate to quantify, has relatively high sensitivity, accuracy and precision, can be used for effectively improving the detection efficiency of maleic hydrazide residue in tobacco and the tobacco product and is very worth popularizing and applying.
Description
Technical field
The invention belongs to tobacco quality security fields, be specifically related to one and grow tobacco and the detection method of residual maleic hydrazide in tobacco product.
Background technology
Tobacco is as sucking product, its persticide residue problem has become the important component part in smoking safety issue, tobacco and tobacco product residues of pesticides figureofmerit are the important contents of various countries in tobacco product quality control, the key factor having become international market tobacco leaf evaluation and chosen also is the important content carrying out commodity inspection in tobacco international trade.Agricultural chemicals maleic acid hydrazide was the important indicator of the residual detection of tobacco agriculture, and international tobacco scientific research Cooperation Centre (CORESTA) the agricultural chemicals council (ACAC) just to propose in tobacco and tobacco product maleic acid hydrazide in the residual limitation of recommendatory agriculture of interior 136 kinds of agricultural chemicals in 2008.
Maleic acid hydrazide (maleic hydrazide) has another name called maleic hydrazide, is a Plants Suckering agents.Because maleic acid hydrazide is comparatively easily formed in conjunction with state maleic acid hydrazide with the material such as the carbohydrate in tobacco, and in tobacco and tobacco product, chemical substance is more, therefore is often difficult to realization for the extraction of maleic acid hydrazide and analysis.
At present, the On Analysis of Maleic Hydrazide Residues method in tobacco, mainly extracts in the mode heating boiling reflux under acid (or alkalescence) condition, or with acid or the ultrasonic extraction of alkali organic solvent.Use aqueous hydrochloric acid solution to add hot reflux through boiling water bath by the Zhengzhou Tobacco Research Institute of CNTC industry standard C/T 405.5-2011 " mensuration the 5th part of tobacco and tobacco product Multi-pesticide residues: the mensuration high performance liquid chromatography of maleic acid hydrazide persticide residue " drafted that takes the lead, measured by HPLC after using SPE-C18 column chromatography purification; The patent delivered by the Chinese Academy of Agricultural Sciences " one grows tobacco or the detection method of residual maleic hydrazide in tobacco product " adopts concussion extraction, the little column purification of C18, HPLC method mensuration.There is following several shortcoming in these methods: the more difficult control solution ph of (1) circumfluence method, needs high-temperature heating, have certain potential safety hazard; (2) adopt quantified by external standard method, each step of experiment is needed accurately to control or constant volume, complicated operation; (3) extraction efficiency of ultrasonic extraction is lower, can not extract the maleic acid hydrazide in conjunction with state completely.Therefore, be necessary to do further research and exploitation.
Summary of the invention
The shortcoming of prior art in view of the above, one is the object of the present invention is to provide to grow tobacco and the detection method of residual maleic hydrazide in tobacco product, accurate control ph is lacked for solving in prior art, easy and simple to handle, efficiency is high, the problem of the detection method of residual maleic hydrazide in the tobacco that quantivative approach is accurate, easy and tobacco product.
For achieving the above object and other relevant objects, the invention provides one to grow tobacco and the detection method of residual maleic hydrazide in tobacco product, adopt microwave auxiliary extraction, adopt tablets by HPLC-MS (HPLC-MS/MS) to measure the residual content of maleic acid hydrazide in tobacco and tobacco product again, and use Internal standard curve method quantitative.
Described one grows tobacco and the detection method of residual maleic hydrazide in tobacco product, comprises the following steps:
1) preparation of standard solution: preparation inner mark solution and standard solution;
A, take the standard items of maleic acid hydrazide, add solvent constant volume, be made into Standard Stock solutions;
Preferably, the content of described maleic acid hydrazide standard items is 250mg.
Preferably, in described Standard Stock solutions, the concentration of maleic acid hydrazide is 10 μ g/ml.Described Standard Stock solutions keeps in Dark Place in 4 DEG C of refrigerators, the term of validity 6 months.
B, take the standard items of maleic acid hydrazide-d2, add solvent constant volume, be made into inner mark solution;
Preferably, the content of described maleic acid hydrazide-d2 standard items is 50mg.
Preferably, in described inner mark solution, the concentration of maleic acid hydrazide-d2 is 1g/L.Described inner mark solution keeps in Dark Place in 4 DEG C of refrigerators, the term of validity 6 months.
Preferably, described maleic acid hydrazide-d2 is deuterated reagent.Concrete, described maleic acid hydrazide-d2 is deuterated maleic acid hydrazide.Described maleic acid hydrazide-d2 is commercial reagents, and its stable chemical nature is suitable for as interior mark.
C, pipette the steps A Plays stock solution of certain volume respectively, then inner mark solution in the step B adding certain volume, the standard solution of a series of variable concentrations is formulated as with solvent constant volume.
Preferably, in described standard solution, the concentration of maleic acid hydrazide is 0.01-1 μ g/ml.
Preferably, the addition of described inner mark solution is 20 μ l.
Preferably, in steps A, B or C, described solvent is acetic acid-aqueous solution.Further, described acetic acid-aqueous solution to be percent by volume be 1% acetic acid-aqueous solution.
2) sample pre-treatments: take tobacco sample, adds hydrochloric acid and step 1) described in inner mark solution, carry out microwave auxiliary extraction, cooled and filtered, to be measured;
Preferably, the sampling amount of described tobacco sample is 0.4-0.6g.Preferably, the sampling amount of described tobacco sample is 0.5g.
Preferably, the concentration of described hydrochloric acid is 1.5-2.5mol/L.Further, the concentration of described hydrochloric acid is 2mol/L.Described hydrochloric acid acid decomposable tobacco leaf fiber, is extracted into the maleic acid hydrazide in conjunction with state wherein in solution.
Preferably, described tobacco sample, hydrochloric acid, inner mark solution are positioned in micro-wave diminishing pot.
Preferably, the condition of described microwave auxiliary extraction is: the power of Microwave Extraction Apparatus: 1100-1300W; Microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 160-200 DEG C in 10min with the speed of 14-18 DEG C/min, keeps 30min.
Preferably, the condition of described microwave auxiliary extraction is: the power of Microwave Extraction Apparatus: 1200W; Microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 180 DEG C in 10min with the speed of 16 DEG C/min, keeps 30min.
Preferably, room temperature is cooled to described in.Described room temperature is 20-25 DEG C.
Preferably, described filtration adopts membrane filtration.Preferably, described filter membrane is 0.22 μm of filter membrane.
3) sample qualitative detection: respectively by step 1) standard solution prepared and step 2) in liquid to be measured carry out HPLC-MS/MS detection, compare retention time and carry out qualitative, determine maleic acid hydrazide composition in liquid to be measured;
4) sample amounts detects: respectively by step 1) standard solution prepared and step 2) in liquid to be measured carry out HPLC-MS/MS detection, adopt Internal standard curve method to carry out quantitatively, obtaining the levels of maleic acid hydrazide in liquid to be measured.
Preferably, described Internal standard curve method comprises the following steps:
Described Internal standard curve method refers to and add internal standard compound matter in standard solution, carries out Instrument measuring, obtains standard working curve, and then measures the method for the actual sample concentration containing internal standard compound matter.
I, by step 1) C in the standard solution of a series of variable concentrations carry out HPLC-MS/MS detection respectively, obtain the linear relationship of the quota ion peak area ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2 and the mass concentration ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2 respectively, draw corresponding standard working curve, calculate the regression equation of maleic acid hydrazide ingredient standard working curve respectively.
Further, in described typical curve, with the quota ion peak area ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for ordinate (Y-axis), with the mass concentration ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for horizontal ordinate (X-axis).
II, by step 2) in liquid to be measured, carry out HPLC-MS/MS detection, by the quota ion peak area ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2 in the liquid to be measured of acquisition, substitute into the regression equation of corresponding maleic acid hydrazide ingredient standard working curve in step I, and according to adding the known quality concentration of internal standard compound maleic acid hydrazide-d2, calculate the mass concentration of maleic acid hydrazide composition in liquid to be measured.
Preferably, described step 3) or 4) in high-efficient liquid phase chromatogram condition be: chromatographic column: Thermo Fisher Hypercarb C18 (4.6mm × 100mm, 5 μm) chromatographic column; Column temperature: 25-35 DEG C, preferably 30 DEG C; Flow velocity: 250-350 μ l/min, preferably 300 μ l/min; Sample size: 10 μ l; Mobile phase: A phase is acetic acid-aqueous solution, B phase is acetic acidacetonitrile solution; Analysis time: 30min; Linear gradient elution, the volume ratio of mobile phase A and B gradually changes in time.
Preferably, described A phase is 1% acetic acid-aqueous solution (v/v), and described B phase is 1% acetic acidacetonitrile solution (v/v).
Preferably, described linear gradient elution at 0-30min, described A phase: the graded wash-out of B phase meets following condition:
0-8min A phase: B phase volume ratio: 0:100-15:85;
8-15min A phase: B phase volume ratio: 15:85-15:85;
15-16min A phase: B phase volume ratio: 15:85-90:10;
16-20min A phase: B phase volume ratio: 90:10-90:10;
20-20.1min A phase: B phase volume ratio: 90:10-0:100;
20.1-30min A phase: B phase volume ratio: 0:100-0:100.
Preferably, described step 3) or 4) in Mass Spectrometry Conditions be:
Mass ions source dates: ionization mode: ESI; Ionization energy: 42eV; Scan mode: MRM; Ion source temperature (TEM): 550 DEG C; Ion spray voltage (IS): 5500V; Atomization gas (gas curtain gas): 30psi; Assisted gas (GS1): 70psi; Assisted gas (GS2): 55psi; Collision gas (CAD): 5psi; Filament current: nothing; Solvent delay: nothing;
Amalyzing substances spectrum parameter: quota ion (MH): 113.1/85; Internal standard compound quota ion (MH-d2): 115.1/87; Remove a bunch voltage (DP): 73V; Compound impact energy (CE): 42eV; Quadrupole rod exit potential (CXP): 10V.
As mentioned above, of the present invention one grows tobacco and the detection method of residual maleic hydrazide in tobacco product, utilizes microwave auxiliary extraction and HPLC-MS/MS to detect, and establishes one and grows tobacco and the method for quantitatively determining of residual maleic hydrazide in tobacco product.The present invention compared with prior art, extracts, simultaneously compared to hydrochloric acid heating reflux method apparently higher than ultrasonic and concussion by adopting the extraction efficiency of microwave auxiliary extraction, easy and simple to handle, efficiency is high, the highlyest can process 12 samples simultaneously, saves a large amount of manpower and materials.In addition, the present invention adopts Internal standard curve method quantitative, more easy compared to external standard method operation, walks all constant volumes without each, and it uses deuterated interior mark can eliminate the impact of matrix effect, does not affect quantitative accuracy.The coefficient R of standard working curve of the present invention
2>0.999, method detection limit (LOD) is 3ng/ml, and quantitative detection limit (LOQ) is 10ng/ml, has higher sensitivity.The recovery of method is 98.41-106.4%, and average relative standard's deviation (RSD%) is less than 5%, has good accuracy and precision, effectively improves the detection efficiency of residual maleic hydrazide in tobacco and tobacco product.
Accompanying drawing explanation
Fig. 1 is shown as the total ions chromatogram of maleic acid hydrazide standard items
Fig. 2 is shown as the total ionic chromatographic schematic diagram of maleic acid hydrazide in actual sample
Embodiment
Set forth the present invention further below in conjunction with specific embodiment, should be understood that these embodiments are only not used in for illustration of the present invention and limit the scope of the invention.
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this instructions can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this instructions also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Embodiment 1
1 reagent and instrument
1.1 reagent
Tobacco sample (Shanghai Tobacco Group Co., Ltd technique center provides); Object: maleic acid hydrazide (250mg, Toronto specializes in chemistry product company); Interior mark: maleic acid hydrazide-d2 (50mg, Toronto specializes in chemistry product company); Hydrochloric acid, acetic acid (analyzing pure, Merck company); Acetonitrile (chromatographically pure, Merck company); Pure water (analyzing pure, Merck company)
1.2 instrument
1200 type high performance liquid chromatographs (Agilent Science and Technology Ltd. of the U.S.); API 4000 type mass spectrometer (Agilent Science and Technology Ltd. of the U.S.); MARS 12 Microwave Extraction Apparatus (U.S. CE M company)
2 assay methods
The preparation of 2.1 standard solution
Take the standard items of maleic acid hydrazide, add 1% acetic acid-aqueous solution constant volume, be made into Standard Stock solutions.Wherein, first take the standard items of the maleic acid hydrazide of 10mg, be placed in 10ml volumetric flask, add 1% acetic acid-aqueous solution constant volume, be made into the intermediate of Standard Stock solutions.In Standard Stock solutions intermediate, the concentration of maleic acid hydrazide is 1mg/ml.Accurately pipette 100 μ l Standard Stock solutions intermediates again, be placed in 10ml volumetric flask, with 1% acetic acid-aqueous solution constant volume, be finally mixed with Standard Stock solutions.The Standard Stock solutions concentration of preparation is: 10 μ g/ml.
Meanwhile, pipette the maleic acid hydrazide-d2 internal standard compound of certain volume, add 1% acetic acid-aqueous solution constant volume, be made into inner mark solution, in inner mark solution, the concentration of maleic acid hydrazide-D2 is 1mg/ml.
The hybrid standard working solution of preparation suitable concn is stand-by.Accurately pipette 10 μ l, 25 μ l, 50 μ l, 100 μ l, 300 μ l, 500 μ l, 1000 μ l Standard Stock solutions respectively, be placed in 10ml volumetric flask, accurately add 20 μ l inner mark solutions more respectively, mix with after 1% acetic acid-aqueous solution constant volume, be formulated as series of standards solution.The series standard solution concentration of preparation is respectively: 0.01 μ g/ml, 0.025 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.3 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, internal standard compound concentration is 20 μ g/ml.
2.2 sample pre-treatments
Accurately take 0.5g tobacco sample, be placed in micro-wave diminishing pot, then to add 20ml concentration be 2mol/L hydrochloric acid and 20 μ l concentration be 1mg/ml maleic acid hydrazide-d2 in mark, carry out microwave auxiliary extraction.Wherein, the condition of described microwave auxiliary extraction is: the power of Microwave Extraction Apparatus: 1100-1300W; Microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 160-200 DEG C in 10min with the speed of 14-18 DEG C/min, keeps 30min.The optimum condition of described microwave auxiliary extraction is: the power of Microwave Extraction Apparatus: 1200W; Microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 180 DEG C in 10min with the speed of 16 DEG C/min, keeps 30min.
Then, take out after extract being cooled to room temperature, use 0.22 μm of membrane filtration, to be measured.
The mensuration of 2.3 sample sizes
Respectively liquid to be measured in 2.1 standard solution and 2.2 prepared is carried out HPLC-MS/MS detection, compare retention time and carry out qualitative, determine maleic acid hydrazide composition in liquid to be measured; Adopt Internal standard curve method to carry out quantitatively, obtain the levels of maleic acid hydrazide composition in liquid to be measured, concrete data are shown in Fig. 1-2 simultaneously.
Specifically, Internal standard curve method first the standard solution of variable concentrations a series of in above-mentioned 2.1 is carried out HPLC-MS/MS detection respectively, obtain the linear relationship of the quota ion peak area ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2 and the mass concentration ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2, draw corresponding standard working curve, with the quota ion peak area ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for ordinate (Y-axis), with the mass concentration ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for horizontal ordinate (X-axis), calculate the regression equation of maleic acid hydrazide ingredient standard working curve respectively.Again liquid to be measured in above-mentioned 2.2 is carried out HPLC-MS/MS detection, by the quota ion peak area ratio of maleic acid hydrazide composition/internal standard compound maleic acid hydrazide-d2 in the liquid to be measured of acquisition, substitute into the regression equation of corresponding maleic acid hydrazide ingredient standard working curve, and according to adding the known quality concentration of internal standard compound maleic acid hydrazide-d2, calculate the mass concentration of maleic acid hydrazide composition in liquid to be measured.
Wherein, high-efficient liquid phase chromatogram condition is: chromatographic column: Thermo Fisher Hypercarb C18 (4.6mm × 100mm, 5 μm) chromatographic column; Column temperature: 25-35 DEG C, preferably 30 DEG C; Flow velocity: 250-350 μ l/min, preferably 300 μ l/min; Sample size: 10 μ l; Mobile phase: A phase is 1% acetic acid-aqueous solution (v/v), B phase is 1% acetic acidacetonitrile solution (v/v); Analysis time: 30min; Linear gradient elution.
Described A phase: the graded wash-out of B phase meets following condition:
0-8min A phase: B phase volume ratio: 0:100-15:85;
8-15min A phase: B phase volume ratio: 15:85-15:85;
15-16min A phase: B phase volume ratio: 15:85-90:10;
16-20min A phase: B phase volume ratio: 90:10-90:10;
20-20.1min A phase: B phase volume ratio: 90:10-0:100;
20.1-30min A phase: B phase volume ratio: 0:100-0:100.
Wherein, Mass Spectrometry Conditions is:
Mass ions source dates: ionization mode: ESI; Ionization energy: 42eV; Scan mode: MRM; Ion source temperature (TEM): 550 DEG C; Ion spray voltage (IS): 5500V; Atomization gas (gas curtain gas): 30psi; Assisted gas (GS1): 70psi; Assisted gas (GS2): 55psi; Collision gas (CAD): 5psi; Filament current: nothing; Solvent delay: nothing;
Amalyzing substances spectrum parameter: quota ion (MH): 113.1/85; Internal standard compound quota ion (MH-d2): 115.1/87; Remove a bunch voltage (DP): 73V; Compound impact energy (CE): 42eV; Quadrupole rod exit potential (CXP): 10V.
In addition, the mass spectrometry parameters of object and internal standard compound is in table 1.
The mass spectrometry parameters of table 1 object and internal standard compound
3 results and discussion
The comparison of 3.1 different extracting process
Get tobacco blank sample, add the maleic acid hydrazide standard solution of concentration known, and add the interior mark of maleic acid hydrazide-d2 that 20 μ l concentration are 1mg/ml respectively, ultrasonic extraction is adopted (to take 0.5g sample respectively, add the hydrochloric acid that 20mL concentration is 2mol/L, ultrasonic extraction 40min), concussion extraction (takes 0.5g sample, add 40mL acidified methanol, concussion 1h), hydrochloric acid heating reflux method (takes 0.5g sample, add the hydrochloric acid that 20mL concentration is 2mol/L, boiling water bath adds hot reflux 1h) and microwave auxiliary extraction (take 0.5g sample, add the hydrochloric acid that 20mL concentration is 2mol/L, the power of Microwave Extraction Apparatus: 1200W, microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 180 DEG C in 10min with the speed of 16 DEG C/min, keeps 30min) carry out pre-treatment, and adopt the condition determination of the HPLC-MS/MS of regulation in 2.3, adopt inner mark method ration.Testing result is in table 2.In table 2, extraction ratio=sample determination concentration/sample actual concentrations × 100%.
From data in table 2, adopt ultrasonic extraction and concussion extraction to carry out pre-treatment, its extraction efficiency is starkly lower than microwave auxiliary extraction.And the extraction efficiency of hydrochloric acid heating reflux method is close with microwave auxiliary extraction, but the hydrochloric acid heating reflux method time is long, consumes quantity of solvent greatly, complex operation.As can be seen here, the extraction efficiency of microwave auxiliary extraction is best.
The measurement result of the different extracting process of table 2 compares
| Extracting process | Sample actual concentrations (μ g/g) | Sample determination concentration (μ g/g) | Extraction ratio (%) |
| Ultrasonic extraction | 80 | 20.3 | 25.4 |
| Concussion extraction | 80 | 18.5 | 23.1 |
| Hydrochloric acid heating reflux method | 80 | 79.4 | 99.3 |
| Microwave auxiliary extraction | 80 | 81.2 | 101.5 |
The comparison of 3.2 different extraction conditionss
As everyone knows, the extraction conditions of microwave auxiliary extraction is different, and the efficiency of its extraction is also different.Particularly, the microwave heating program in microwave auxiliary extraction, different temperature and times is very large on extraction efficiency impact.Get tobacco blank sample, add the maleic acid hydrazide standard solution of concentration known, and add the interior mark of maleic acid hydrazide-d2 that 20 μ l concentration are 1mg/ml respectively, adopt different extraction conditionss respectively, and adopt the condition determination of the HPLC-MS/MS of regulation in 2.3, adopt Internal standard curve method quantitative.Concrete data are in table 3.The microwave heating program of scheme 1 is: initial temperature 20 DEG C keeps 1min, in 10min, rise to 180 DEG C with the speed of 16 DEG C/min, keeps 30min.The microwave heating program of scheme 2 is: initial temperature 20 DEG C keeps 1min, in 10min, rise to 160 DEG C with the speed of 14 DEG C/min, keeps 30min.The microwave heating program of scheme 3 is: initial temperature 20 DEG C keeps 1min, in 10min, rise to 200 DEG C with the speed of 18 DEG C/min, keeps 30min.In table 3, extraction ratio=sample determination concentration/sample actual concentrations × 100%.From data in table 3, only have when selection scheme 1, in the present invention, the extraction efficiency of microwave auxiliary extraction is best.
The measurement result of the different extraction conditions of table 3 compares
| Extraction scheme | Sample actual concentrations (μ g/g) | Sample determination concentration (μ g/g) | Extraction ratio (%) |
| Scheme 1 | 80 | 81.2 | 101.5 |
| Scheme 2 | 80 | 69.5 | 86.9 |
| Scheme 3 | 80 | 74.8 | 93.5 |
3.3 standard working curves, detection limit and quantitative limit
As shown in above-mentioned 2.1, accurately pipette 10 μ l, 25 μ l, 50 μ l, 100 μ l, 300 μ l, 500 μ l, 1000 μ l Standard Stock solutions respectively, be placed in 10ml volumetric flask, accurately add 20 μ l inner mark solutions more respectively, mix with after 1% acetic acid-aqueous solution constant volume, be formulated as series of standards solution.The series standard solution concentration of preparation is respectively: 0.01 μ g/ml, 0.025 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.3 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, internal standard compound concentration is 20 μ g/ml.
By above-mentioned prepare a series of variable concentrations standard solution carry out HPLC-MS/MS detection, with the quota ion peak area ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for ordinate (Y-axis), with the mass concentration ratio of maleic acid hydrazide composition and internal standard compound maleic acid hydrazide-d2 for horizontal ordinate (X-axis), carry out regretional analysis, obtain standard working curve and the coefficient R thereof of maleic acid hydrazide
2>0.99, concrete outcome is as shown in table 4.
As shown in Table 4, to the object response signal in standard solution, least concentration standard specimen is adopted to repeat sample introduction 10 times, carry out HPLC-MS/MS analysis, the detection limit being method with 3 times of signal to noise ratio (S/N ratio)s (LOD), 10 times of signal to noise ratio (S/N ratio)s are quantitative limit (LOQ), show that the method for object detects and be limited to 3ng/ml after being scaled sample size, quantitatively be limited to 10ng/ml, there is higher sensitivity.
Table 4 working curve and detection limit
Y: ion peak areas ratio; X: concentration ratio
3.4 recovery and precision
Get tobacco blank sample, add the maleic acid hydrazide standard solution of 3 variable concentrations, and add the interior mark maleic acid hydrazide-d2 of the concentration known 20 μ g/ml of same volume, then the pre-treatment of above-mentioned 2.2 is carried out, and carry out HPLC-MS/MS analysis according to above-mentioned 2.3, according to adding scalar (low concentration 0.5mg/L, middle concentration 2.5mg/L, high concentration 5.0mg/L) and measured value calculates its recovery.Simultaneously to same sample solution replicate determination 5 times (n=5), obtain the precision determination data of maleic acid hydrazide composition, the results are shown in Table 5.As can be seen from Table 5, the average recovery rate of object is between 98.41 ~ 106.4%, and relative standard deviation (RSD) is all less than 5%, illustrates that the recovery of the inventive method is high, accuracy and reproducible, can meet quantitative needs.
The recovery of table 5 maleic acid hydrazide composition and precision
3.5 the mensuration of actual sample
Adopt this method to measure the content that in actual tobacco sample, maleic acid hydrazide is residual to measure, specifically in table 6.As shown in Table 6, the method effectively can measure the content that in tobacco sample, maleic acid hydrazide is residual, and method is easy and simple to handle, applicability good, and result accurately, reliably.
The residual quantity of maleic acid hydrazide in the actual tobacco sample of table 6
| Sequence number | Components Name | The content (μ g/g) of sample 1 | The content (μ g/g) of sample 2 | The content (μ g/g) of sample 3 |
| 1 | Maleic acid hydrazide | 40.6 | 81.2 | 140.3 |
In sum, of the present invention one grows tobacco and the detection method of residual maleic hydrazide in tobacco product, compared with prior art, extraction efficiency is high, easy and simple to handle, quantitatively accurately, there is higher sensitivity, accuracy and precision, effectively improve the detection efficiency of residual maleic hydrazide in tobacco and tobacco product, highly apply.So the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (10)
1. one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, adopt microwave auxiliary extraction, then adopt tablets by HPLC-MS to measure the residual content of maleic acid hydrazide in tobacco and tobacco product, and use Internal standard curve method quantitative.
2. according to claim 1 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, comprise the following steps:
1) preparation of standard solution: preparation inner mark solution and standard solution;
2) sample pre-treatments: take tobacco sample, adds hydrochloric acid and step 1) described in inner mark solution, carry out microwave auxiliary extraction, cooled and filtered, to be measured;
3) sample qualitative detection: respectively by step 1) standard solution prepared and step 2) in liquid to be measured carry out HPLC-MS/MS detection, compare retention time and carry out qualitative, determine maleic acid hydrazide composition in liquid to be measured;
4) sample amounts detects: respectively by step 1) standard solution prepared and step 2) in liquid to be measured carry out HPLC-MS/MS detection, adopt Internal standard curve method to carry out quantitatively, obtaining the levels of maleic acid hydrazide in liquid to be measured.
3. according to claim 2 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described step 1) be:
A) take the standard items of maleic acid hydrazide, add solvent constant volume, be made into Standard Stock solutions;
B) take the standard items of maleic acid hydrazide-d2, add solvent constant volume, be made into inner mark solution;
C) the steps A Plays stock solution of certain volume is pipetted respectively, then inner mark solution in the step B adding certain volume, the standard solution of a series of variable concentrations is formulated as with solvent constant volume.
4. according to claim 3 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, steps A), B) or C) in, described solvent is acetic acid-aqueous solution.
5. according to claim 2 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described step 2) in, the concentration of described hydrochloric acid is 1.5-2.5mol/L.
6. according to claim 2 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described step 2) in, the condition of described microwave auxiliary extraction is: the power of Microwave Extraction Apparatus: 1100-1300W; Microwave heating program: initial temperature 20 DEG C keeps 1min, rises to 160-200 DEG C in 10min with the speed of 14-18 DEG C/min, keeps 30min.
7. according to claim 2 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described step 3) or 4) in, high-efficient liquid phase chromatogram condition is: chromatographic column: Thermo Fisher Hypercarb C18 chromatographic column; Column temperature: 25-35 DEG C; Flow velocity: 250-350 μ l/min; Sample size: 10 μ l; Mobile phase: A phase is acetic acid-aqueous solution, B phase is acetic acidacetonitrile solution; Analysis time: 30min; Linear gradient elution, the volume ratio of mobile phase A and B gradually changes in time.
8. according to claim 7 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described A phase is 1% acetic acid-aqueous solution, and described B phase is 1% acetic acidacetonitrile solution.
9. according to claim 7 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, it is characterized in that, described linear gradient elution at 0-30min, described A phase: the graded wash-out of B phase meets following condition:
0-8min A phase: B phase volume ratio: 0:100-15:85;
8-15min A phase: B phase volume ratio: 15:85-15:85;
15-16min A phase: B phase volume ratio: 15:85-90:10;
16-20min A phase: B phase volume ratio: 90:10-90:10;
20-20.1min A phase: B phase volume ratio: 90:10-0:100;
20.1-30min A phase: B phase volume ratio: 0:100-0:100.
10. according to claim 2 one grow tobacco and the detection method of residual maleic hydrazide in tobacco product, its feature exists
In, described step 3) or 4) in, Mass Spectrometry Conditions is:
Mass ions source dates: ionization mode: ESI; Ionization energy: 42eV; Scan mode: MRM; Ion source temperature TEM:550 DEG C; Ion spray voltage IS:5500V; Gas curtain gas: 30psi; Assisted gas GS1:70psi; Assisted gas GS2:55psi; Collision gas: 5psi; Filament current: nothing; Solvent delay: nothing;
Amalyzing substances spectrum parameter: quota ion MH:113.1/85; Internal standard compound quota ion MH-d2:115.1/87; Remove a bunch voltage DP:73V; Compound impact energy CE:42eV; Quadrupole rod exit potential CXP:10V.
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