CN109557230A - A method of three classes dithiocarbamate residual quantity in measurement tobacco - Google Patents
A method of three classes dithiocarbamate residual quantity in measurement tobacco Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of methods of three classes dithiocarbamate residual quantity in measurement tobacco.It will be filtered after the alkaline extracting solution processing of tobacco powder to be measured, collect filtrate and analyzed with LC-MS-MS, then obtain result through Specification Curve of Increasing and after calculating.The present invention realizes the Accurate Determining to three classes DCs substance residual quantity in tobacco for the first time.The range of linearity of three kinds of DCs substances is 20~100mg/L, related coefficient is not less than 0.99, detection limit is 2.3,4.3,5.2 μ g/kg respectively, quantitative limit is 7.7,14.3,17.3 μ g/kg respectively, matrix effect range is respectively 0.19~0.50,0.78~1.70,0.35~0.87, in a few days RSD11.33~16.80% of each DTCs of flue-cured tobacco matrix, in the daytime RSD5.84~6.54%, recovery of standard addition 84.5%~103.5%.This method is easy to operate, analysis speed is fast, and accuracy and precision height, stability are strong, meets the requirement of quantitative analysis.
Description
Technical field
The invention belongs to tobacco physical and chemical inspection technical field, being specifically related to one kind being capable of three classes two in Accurate Determining tobacco
The method of thiocarbamate residual quantity.
Background technique
Dithiocarbamate (DTCs) is organic sulfur class protective fungicide.It can inhibit pyruvic acid in germ body
Oxidation is mainly used for preventing and treating anthrax, founding withered, root rot, rust or balck shank etc. on tobacco to play bactericidal effect.
DTCs class pesticide is more stable in tobacco leaf surface, therefore is easy to cause to remain in tobacco.According to compound structure, DTCs points are
Three classes, it may be assumed that dimethyl dithiocarbamate (DMDC), ethylene bisdithiocarbamic ester (EBDC) and propylidene are double
(dithiocarbamates methyl esters) (PBDC).
The prior art mainly uses carbon disulfide method (including spectrophotometry, gas chromatography) to come to junior tobacco leaf, volume
Dithiocarbamate is detected in cigarette, but this method can only measure dithiocarbamate total residue, Bu Nengzhun
Really identification and quantitative specific ingredient.
Liquid chromatography-tandem mass spectrometry is combined method (LC-MS/MS), collects the high separation capacity of LC and the high sensitivity of MS, pole
Strong qualitative exclusive specificity can distinguish the variety classes of dithiocarbamate, can carry out such pesticide in one
It is qualitative to verify and quantitatively, can overcome the disadvantages that the deficiency of existing professional standard, if this method to be applied to the detection of tobacco, it should can
Deficiency existing for existing detection method is overcome well, but there has been no successful reports in the prior art.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide three classes dithiocarbamates first in a kind of measurement tobacco
The method of acid esters residual quantity, with LC-MS-MS method, under the premise of guaranteeing accuracy in detection, precision and stability, respectively
Accurate quantitative analysis is carried out for three kinds of different dithiocarbamates.
The purpose of the present invention is achieved by the following technical programs.
Unless otherwise indicated, percentage of the present invention is weight percent.
A method of three classes dithiocarbamate residual quantity in measurement tobacco, comprising the following steps:
(1) 10 mL alkalinity extracting solutions, whirlpool is added in 50mL centrifuge tube in the tobacco sample 0.5g to be measured after weighing grinding
1min is revolved, revolving speed 13000rpm is centrifuged 5min, and supernatant carries out LC-MS/MS measurement after 0.23 μm of membrane filtration；Described
Alkaline extracting solution: weighing 0. 6058g L-cysteine and 1.8612g EDTA-Na2, is dissolved with 450mL water, with 2 mol/L
NaOH be adjusted to pH 12.0, then be settled to 500mL with water, be made into the 10mmol/L EDTA and 10mmol/L L- of pH 12.0
The mixed liquor of cysteine；
(2) LC-MS/MS determination condition
Chromatographic condition mobile phase: Waters UPLC BEH HILIC chromatographic column (50 mm × 2.1mm × 2.8 μm)；Flowing
Phase A is acetonitrile, and B is 10mmol/L ammonium hydroxide, flow velocity: 0.2mL/min；Column temperature: 25 DEG C；Sampling volume: 5 μ L；Gradient elution: 4 points
Acetonitrile is linearly down to 5% by 15% in clock, is kept for 0.1 minute, is switched to acetonitrile 15%, keeps to 7 minutes；
Mass Spectrometry Conditions: electrospray ionisation source (ESI)；Scanning mode: anion scanning；Detection mode: multiple-reaction monitoring
(MRM)；Electron spray voltage: 500V；Dry temperature degree: 300 DEG C；Dry gas stream amount: 6L/min；Sheath temperature degree: 350 DEG C；Sheath gas
Flow: 10L/min；Capillary voltage: 4000V；
Table 1: the multiple-reaction monitoring of three classes DTC is qualitative, quota ion pair and mass spectrum relevant parameter
(3) preparation of standard solution
Single standard stock solution: according to each standard substance purity calculate, weigh respectively pure material fervam, thiram,
Ziram, zineb, maneb, Propineb and each 5mg of Polycarbamate dissolve simultaneously constant volume with the alkaline extracting solution respectively
To 100mL, ultrasound accelerates dissolution 5 minutes, single standard stock solution that be made into 7 kinds of pure materials respectively, that concentration is 50mg/L；
Single debugging solution: respectively diluting prepared each single standard stock solution alkaline extracting solution, point
Single debugging solution that do not obtain 7 kinds of pure materials, that concentration is 1mg/L；
The single mother liquor of standard working curve: it is calculated according to the purity of each standard substance, weighs pure material thiram, generation respectively
Gloomy zinc and each 100mg of Propineb dissolve respectively and are settled to 100mL with the alkaline extracting solution, and ultrasound accelerates 5 points of dissolution
Clock, the single mother liquor of standard working curve that be made into 3 kinds of pure materials respectively, that concentration is 1000mg/L；
Standard working solution: respectively pipetted from the single mother liquor of standard working curve of 3 kinds of pure materials respectively 0.2mL,
0.4mL, 0.6mL, 0.8mL and 1.0mL are respectively placed in 10mL volumetric flask, distinguish constant volume with the alkaline extracting solution, respectively
Obtain the series standard working solution that 3 kinds of DTCs concentration are 20 μ of μ g/mL~100 g/mL；It is measured with Liquid Chromatography-Tandem Mass Spectrometry,
The integrated peak areas of DTCs is obtained as ordinate, actual concentrations are calculated according to the purity of each standard substance, as abscissa,
Make standard curve；
Matrix matching standard working solution: the flue-cured tobacco without dithiocarbamate after weighing 0.5g grinding respectively
Each five parts of (burley tobaccos or Turkish tobaccos) powder, is respectively placed in 50mL polytetrafluoroethylene (PTFE) centrifuge tube, is separately added into 3 kinds of standard work
Curve single mother liquor 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, be then respectively adding 9.8mL, 9.6mL, 9.4mL,
Alkaline extracting solution described in 9.2mL, 9.0mL is vortexed 1 minute, and revolving speed 13000rpm is centrifuged 5 minutes, and supernatant is filtered through 0.23 μm
After film filtering, with liquid chromatography-mass spectrography/mass spectroscopy；
(4) qualitative results:
Standard solution and sample solution are first measured in same experimental conditions, then qualitatively judges sample object as follows
Object:
1) retention time of certain component peaks in the retention time and standard solution of the chromatographic peak detected in sample solution
It is almost the same, and the mass-to-charge ratio of selected ion pair is consistent；
2) sample ions are compared the be near the mark relative abundance of ion pair of working solution of relative abundance and concentration
When, relative deviation is no more than range as defined in table 2；Meet the above two o'clock, then can determine that there are the components in sample；
With respect to the maximum allowable offset of abundance of ions when table 2 is qualitative determining
(5) quantitative result:
Standard solution and sample solution are measured under same experimental conditions, make standard curve, the content of DTCs is used in sample
Quantified by external standard method is calculated by formula (1):
In formula: the content of certain component in X- sample, unit μ g/g；
ciThe concentration of certain component in the sample obtained by standard curve, unit μ g/mL；
c0Blank solution concentration, unit μ g/mL；
The final constant volume of V- sample, Unit/mL；
M- sample quality, unit g；
(6) result calculates: according to the abundance of sample solution quota ion to be measured, is read from standard curve as a result, deducting
The three classes dithiocarbamate content that sample is obtained after blank, using the arithmetic mean of instantaneous value of parallel determinations twice as most
Whole measurement result, the relative average debiation of parallel determinations should be less than 10% twice.
Compared with the existing technology, the invention has the following advantages that LC- MS/MS method is applied to cigarette initiatively by the present invention
The measurement of three classes dithiocarbamate in grass.With optimal conditions, dimethyl dithiocarbamate (DMDCs), second
Support the range of linearity of bisdithiocarbamic ester (EBDCs) and propylidene bis- (dithiocarbamates methyl esters) (PBDCs) for 20~
100mg/L, related coefficient are not less than 0.99.The detection limit of DMDCs, EBDCs, PBDCs are 2.3,4.3,5.2 μ g/ respectively
Kg, quantitative limit are 7.7,14.3,17.3 μ g/kg respectively.Matrix effect of 3 kinds of DTCs in burley tobaccos, Turkish tobaccos, flue-cured tobacco solution
Range is 0.19~0.50,0.78~1.70,0.35~0.87.The in a few days RSD of each DTCs of flue-cured tobacco matrix is 11.33
~16.80%, RSD is 5.84~6.54% in the daytime, and recovery of standard addition is 84.5%~103.5%.This method is easy to operate, divides
It is fast to analyse speed, accuracy and precision height, stability are strong, can meet the requirement of quantitative analysis, have a good application prospect.
Detailed description of the invention
The molecular structural formula of Fig. 1 three classes dithiocarbamate；
Fig. 2 fervam fragment ion figure；
Fig. 3 ziram fragment ion figure；
Fig. 4 thiram fragment ion figure；
Fig. 5 zineb fragment ion figure；
Fig. 6 maneb fragment ion figure；
Fig. 7 Propineb fragment ion figure；
Fig. 8 Polycarbamate fragment ion figure；
The cracking derivation process of Fig. 9 Polycarbamate；
The MRM chromatogram of 120 > 76 of Figure 10 DMDC quota ion；
The MRM chromatogram of 211 > 58 of Figure 11 EBDC quota ion；
The MRM chromatogram of 225 > 58 of Figure 12 PBDC quota ion；
The MRM chromatogram of 225 > 58 of Figure 13 burley tobaccos matrix PBDC quota ion；
The MRM chromatogram of 211 > 58 of Figure 14 burley tobaccos matrix EBDC quota ion；
The MRM chromatogram of 120 > 76 of Figure 15 burley tobaccos matrix DMDC quota ion；
The MRM chromatogram of 225 > 58 of Figure 16 flue-cured tobacco matrix PBDC quota ion；
The MRM chromatogram of 211 > 58 of Figure 17 flue-cured tobacco matrix EBDC quota ion；
The MRM chromatogram of 120 > 76 of Figure 18 flue-cured tobacco matrix DMDC quota ion；
The MRM chromatogram of 225 > 58 of Figure 19 fragrance tobacco matrix PBDC quota ion；
The MRM chromatogram of 211 > 58 of Figure 20 fragrance tobacco matrix EBDC quota ion；
The MRM chromatogram of 120 > 76 of Figure 21 fragrance tobacco matrix DMDC quota ion.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, but drawings and examples are not pair
The restriction of technical solution of the present invention, it is all based on present invention teach that made variation, all should belong to protection scope of the present invention.
Embodiment 1
1. experimental principle, material and method
1.1 principle
Alkaline extracting solution is added into the sample of grinding, takes supernatant after membrane filtration after vortex centrifugal, with efficient liquid
Phase chromatography-tandem mass spectrometer detection.
1.2 main materials and reagent
Experimental water is ultrapure water；Ammonium acetate, methanol are chromatographically pure, Beijing enlightening equine skill Products；Ammonium hydroxide: analysis
It is pure, the western Gansu Province scientific company product in Guangdong；Fervam (Ferbam, purity 75.0%), ziram (Ziram, purity 75.0%),
Thiram (Thiram, purity 97%), zineb (Zineb, purity 80.5%), maneb (mancozeb, 80%), Propineb
(Propineb, 71.3%), Polycarbamate (Polycarbamate, 88.4%) standard items: German Ehrenstorfer
GmbH Products.
Chromatographic column: ZIC-pHILIC column (150 × 4.6mmi.d., 5 μm), Waters UPLC BEH C18 (150 ×
2.1mm i.d., 2.8 μm) and Waters UPLC BEH HILIC (50 × 2.1mm i.d., 2.8 μm)；Syringe and filter；It is high
Pure nitrogen gas (purity is greater than 99.999%)；Have screw lid polytetrafluoroethylene (PTFE) centrifuge tube (50mL).
1.3 key instrument
- 6460 tandem mass spectrometer of 1290 high performance liquid chromatography: U.S.'s Agilent Products；Assay balance: Switzerland Mei Te
Strangle Products；Vortex vortex mixer: its Lindberg Optic Design A/S's product of Jiangsu；Universal 320R supercentrifuge: Germany
Hettich Products；PH meter: U.S.'s ThermoFisher Products；Water purification machine: U.S.'s Millipore Products.
The preparation of 1.4 standard solution
Alkaline extracting solution: weighing 0.6058g L-cysteine and 1.8612g EDTA-Na2, is dissolved with about 450mL water,
Be adjusted to pH 12.0 with the NaOH of 2mol/L, then be settled to 500mL with water, be made into the 10mmol/L EDTA of pH 12.0 with
The mixed liquor of 10mmol/L L-cysteine.
Single standard stock solution: it is calculated according to the purity of each standard substance, weighs pure material about 5mg fervam, good fortune respectively
U.S. double, ziram, zineb, maneb, Propineb, Polycarbamate, dissolve with alkaline extracting solution and are settled to 100mL, surpass
Sound accelerates dissolution 5 minutes, is made into the single standard stock solution that concentration is about 50mg/L.
Single debugging solution: respectively will by 50mg/L fervam, thiram, ziram, zineb, maneb, Propineb,
The single standard stock solution of Polycarbamate dilutes to obtain the single debugging solution that concentration is 1mg/L with alkaline extracting solution.
The single mother liquor of standard working curve: it is calculated according to the purity of each standard substance, weighs pure material about 100mg good fortune respectively
U.S. double, zineb, Propineb, dissolve with alkaline extracting solution and are settled to 100mL, and ultrasound accelerates dissolution 5 minutes, is made into concentration
The about single mother liquor of the standard working curve of 1000mg/L.
Standard working solution: 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0 mL, 3 kinds of DTCs standard work are pipetted respectively
The single mother liquor of curve is in 10mL volumetric flask, and using alkaline extracting solution constant volume, obtaining 3 kinds of DTCs concentration is about 20 g/mL~100 μ
The series standard working solution of μ g/mL.It is measured with Liquid Chromatography-Tandem Mass Spectrometry, the integrated peak areas for obtaining DTCs is sat as vertical
Mark calculates actual concentrations according to the purity of each standard substance, as abscissa, makes standard curve.
Matrix matching standard working solution: flue-cured tobacco (the white rib for not conforming to dithiocarbamate after weighing 0.5g grinding
Cigarette, Turkish tobaccos) each five parts of powder in 50mL polytetrafluoroethylene (PTFE) centrifuge tube, be separately added into 3 kinds of single mother liquor 0.2mL, 0.4mL,
Then 9.8mL, 9.6m L, 9.4mL, 9.2mL, 9.0mL alkalinity extracting solution is added in 0.6mL, 0.8mL and 1.0mL, be vortexed 1 point
Clock, revolving speed 13000rpm are centrifuged 5 minutes, and supernatant is after 0.23 μm of membrane filtration, with liquid chromatography-mass spectrography/mass spectroscopy.
1.5 experiment condition
1.5.1 chromatographic condition mobile phase: Waters UPLC BEH HILIC chromatographic column (50 mm × 2.1mm × 2.8 μm)；
Mobile phase A is acetonitrile, and B is 10mmol/L ammonium hydroxide, flow velocity: 0.2mL/min；Column temperature: 25 DEG C；Sampling volume: 5 μ L；Gradient is washed
De-: acetonitrile is linearly down to 5% by 15% in 4 minutes, is kept for 0.1 minute, is switched to acetonitrile 15%, keeps to 7 minutes.
1.5.2 Mass Spectrometry Conditions electrospray ionisation source (ESI)；Scanning mode: anion scanning；Detection mode: more reaction prisons
It surveys (MRM)；Electron spray voltage: 500V；Dry temperature degree: 300 DEG C；Dry gas stream amount: 6L/min；Sheath temperature degree: 350 DEG C；Sheath
Throughput: 10L/min；Capillary voltage: 4000V.
More reactions prison of 1 three classes DTC of table rinses qualitative, quota ion pair and mass spectrum relevant parameter
2. results and discussion
The optimization of 2.1 Mass Spectrometry Conditions
2.1.1 mass ion source condition optimizing DTCs exists in the form of anion in alkaline solution, therefore selects negative
Ion mode is detected.Fervam, zineb, maneb, Propineb, the Polycarbamate, good fortune for being respectively 1mg/L by concentration
U.S. double, the single debugging solution of ziram, 50%10mmol ammonium hydroxide-acetonitrile solution do mobile phase, flow velocity 0.2mL/min, sample introduction
5 μ L are measured, but chromatographic column optimizes electron spray voltage, dry temperature degree, dry gas stream amount, sheath gas directly into first mass spectrometric analysis
Temperature, sheath throughput, capillary voltage make the molecular ion peak obtained tend towards stability.Finally obtain the mass spectrometry parameters of 1.4.2.
2.1.2 second order ms condition optimizing take the fervam of 1mg/L, zineb, maneb, Propineb, Polycarbamate,
The single debugging solution continuous sample introduction of thiram, ziram, optimizes suitable orifice potential and collision energy.Then it is fixed to comprehensively consider
The selectivity and its sensitivity of ion are measured, selecting the maximum ion of abundance as far as possible is quota ion, to improve the spirit of entire method
Sensitivity, second largest ion of reselection abundance are to assist qualitative ion.DTCs mass spectroscopy parameter shown in table 1 is finally obtained
With quantitative/qualitative ion pair of all kinds of DTCs, the secondary ion figure of DTCs is shown in Fig. 2-8.
It can be seen that DMDCs (such as Sodium Dimethyldithiocarbamate, fervam, ziram, thiram), EBDCs (such as Dithane A40, Dai Sen
Manganese, zineb, Mancozeb etc.) it is consistent with the fragment ion of PBDCs (the such as third gloomy sodium, Propineb).According to Dai Senfumei
The ion situation of zinc speculate be decomposed into after the Polycarbamate of a molecule can be complexed by EDTA a molecule for gloomy class (EBDC)
With the good fortune U.S. class (DMDC) of two molecules.
2.1.3 qualitative results first measure standard solution and sample solution in same experimental conditions, then fixed as follows
Sex determination sample object object: 1) certain component peaks in the retention time and standard solution of the chromatographic peak detected in sample solution
Retention time is almost the same, and the mass-to-charge ratio of selected ion pair is consistent；2) sample ions connect relative abundance and concentration
When the relative abundance of the ion pair of nearly standard working solution is compared, relative deviation is no more than range as defined in table 2；Meet with
Upper two o'clock then can determine that there are the components in sample.
With respect to the maximum allowable offset of abundance of ions when table 2 is qualitative determining
The optimization of 2.2 liquid phase chromatogram conditions
2.2.1 the selection dithiocarbamate of mobile phase exists in the form of anion in alkaline solution, because
This uses anion scan pattern；Compared the most common negative ion mode mobile phase of LC-MS/MS: acetonitrile-water, methanol-water,
The separating effect of DTCs when acetonitrile -10mmol/L ammonium acetate, acetonitrile -10mmol/L ammonium hydroxide are as mobile phase.With methanol-water, second
Nitrile -10mmol/L ammonium acetate is mobile phase, and anyway DTCs changes mobile phase ratio, and object is without response.Analyze reason
Are as follows: cause object without response since the atomizing effect of methanol is bad when methanol-water is mobile phase；And with acetonitrile-
10mmol/L ammonium acetate be mobile phase when due in the water phase of the two respectively contain a large amount of acetic acid acid ammonium plasma, produce from
Sub- depression effect, causes object without response.When using acetonitrile-water, acetonitrile -10mmol/L ammonium hydroxide as mobile phase, object is equal
Show that very strong response, the response of -10 mmol/L ammonium hydroxide object of acetonitrile are greater than acetonitrile-water mobile phase, and acetonitrile -
10mmol/L ammonium hydroxide can preferably remove the impurity in chromatographic column, therefore test and finally use acetonitrile -10mmol/L ammonium hydroxide
As optimal flow phase.
2.2.2 the selection of chromatographic column due to the polarity of DTCs it is stronger, have different reservations in different chromatographic columns, therefore,
It investigates in Waters UPLC BEH C18 (150 × 2.1mm i.d., 2.8 μm), ZIC- pHILIC column (150 × 4.6mm
I.d., 5 μm) and Waters UPLC BEH HILIC (50 mm × 2.1mm × 2.8 μm) chromatographic column on DTCs separating effect.
In Waters UPLC BEH C18 chromatographic column, gradient is adjusted in any case, and DTCs is rushed out quickly it can be seen that C18 chromatography
Column does not retain DTCs, could not reach separating effect, and appearance quickly, is very easy to receive solvent effect；In ZIC-p
In HILIC chromatographic column, 3 kinds of DTCs peak types are poor, and peak type broadens；In Waters UPLC BEH HILIC chromatographic column,
HILIC chromatographic column has cracking separating effect for DTCs, and peak shape is preferable.To sum up, Waters UPLC BEH HILIC is selected
Separation liquid phase column as DTCs.
2.2.3 when the optimization of gradient elution uses isocratic elution, if being carried out etc. with 97: 3 acetonitrile -10mmol/L ammonium hydroxide
When degree elution, mobile phase is bad to the elution effect of object, will form very wide peak；60: 40 acetonitrile -10mmol/L ammonia
When water carries out isocratic elution, then since water content is too high, atomizing effect is bad, causes the response of object very low.DTCs is applicable in
Ammonia spirit/acetonitrile mobile phase system, separating effect are better than other flow visualizings.Optimize eluent gradient program repeatedly.Most
The chromatographic separation condition of 1.4.1 has been determined eventually, when the HILIC post separation three, although retention time is closer to, can pass through
The qualitative ion of the two respectively identifies it, has no effect on qualitative and quantitative determination.As can be seen from Figure in 2.5min
Quick separating, and peak shape is good.The quota ion MRM that Figure 10~12 are different DTCs schemes.
2.3 methodology validation
2.3.1 linear relationship Liquid Chromatography-Tandem Mass Spectrometry metric works solution, DTCs peak area as ordinate,
Each DTCs concentration is that abscissa makes standard curve, and equation of linear regression, the range of linearity and related coefficient are shown in Table 3.It can be with from table 3
Find out, three classes DTCs concentration and response regression equation related coefficient are all larger than 0.99, show in 20~100mg/L concentration range
There is good linear relationship.
Calibration curve equation, related coefficient and the range of linearity of 3 DTCs of table
Note: x is peak area in linear equation；Y is the concentration of each component, unit mg/L.
2.3.2 detection limit and quantitative limit will test corresponding dense when the response signal of device generation is 3 times, 10 times of noise figure
Degree substitutes into formula (1) respectively, and it is 2.3,4.3,5.2 μ g/kg, quantitative limit respectively that conversion, which obtains the detection limit of DMDC, EBDC, PBDC,
It is 7.7,14.3,17.3 μ g/kg respectively.This method has preferable sensitivity, lower detection limit, can preferably meet DTCs
Testing requirements.2.3.3 the LC-MS/MS method of Accurate Determining DTCs is established in the influence of matrix effect, and matrix influence is must to examine
Consider, matrix effect can sensitivity, precision and accuracy to analysis method impact, when ratio is close to 1, explanation
There is no the influences of matrix effect；When ratio is more than or less than 1, illustrate that matrix has enhancing or suppression to the response of component to be measured
Effect [17] processed.But in actually detected, relative ratio then thinks that matrix effect is unobvious [18] between 0.85~1.15.Choosing
Having taken burley tobaccos, Turkish tobaccos and flue-cured tobacco is matrix effect research object, using preparation matrix matching standard solution and optimization color
Spectrum-Mass Spectrometry Conditions reduce matrix effect.The slope of standard curve is prepared by the slope and pure solvent of matrix matching standard curve
The ratio between evaluated.Concrete outcome is shown in Table 4.As can be seen from the above table, different tobacco leaf matrix effects are different, and wherein DMDC matrix is imitated
Most serious is answered, in order to quantitative determine DTCs compound, is preferably selected the matrix configuration matrix working curve of same sample,
To eliminate matrix influence.
The different tobacco sample matrix effect situations of table 4
2.3.4 repeatability and reproducibility weigh 6 parts of a certain flue-cured tobacco sample without dithiocarbamate of 0.5g, point
Other mark-on makes DTCs extract concentration be about 15.0 μ g/mL, and according to 1.5 the step of is handled, and under the conditions of equality operation, uses LC-
MS/MS measures DTCs content, calculates in a few days RSD value.Divide by the same way 3 days and detected, calculates RSD value in the daytime.As a result
It is shown in Table 5.As seen from Table 5, the in a few days RSD of each DTCs is 11.33~16.80%, and RSD is 5.84~6.54% in the daytime, shows weight
Existing property is preferable.
5 repeatability of table and reproducibility
Note: numerical value is the relative deviation of six measurement results in table
2.3.5 the rate of recovery weighs a certain flue-cured tobacco sample without dithiocarbamate of 0.5g, DTCs is added, so that mentioning
Taking liquid concentration is respectively 3.0,6.0,15.0 μ g/mL, carries out rate of recovery experiment, and each concentration carries out 3 times in parallel, obtains DTCs's
The rate of recovery is shown in Table 6.Table 6 is it is found that the rate of recovery of DTCs is 84.5%~103.5%, and be averaged/rate of recovery 87.6%~97.7%
Illustrate that the analysis method rate of recovery established is preferable, meets the requirement of the residual detection method of agriculture, accurately and reliably.
The recycling plug of table 6
3. results and discussion
Methodology validation research has been carried out for the LC-MS/MS method for establishing DTCs in measurement tobacco, as the result is shown:
(1) it is 2.62%~6.60% that RSD, which is respectively less than RSD, in day, is continuously respectively less than 6.60% to the RSD of DTCs measurement,
Show that method has preferable repeatability and reproducibility.
(2) 2.3~5.2 μ g/kg of detection limit of this method；Quantitatively it is limited to 5.2~17.3 μ g/kg.Detection limit and quantitative limit
It is lower, show this method sensitivity with higher.
(3) the DTCs rate of recovery illustrates that the analysis method rate of recovery established is preferable in 84.5%~103.5% range.
In conclusion this method can be used for measuring DTCs in tobacco leaf simultaneously, it is sensitive, accurate to have the characteristics that, meets tobacco leaf
The testing requirements of middle DTCs content.
4. conclusion
This method can be used for measuring DTCs in tobacco leaf simultaneously, and it is sensitive, accurate to have the characteristics that, meet DTCs content in tobacco leaf
Testing requirements.
Claims (1)
1. a kind of method of three classes dithiocarbamate residual quantity in measurement tobacco, which is characterized in that including following rapid:
(1) 10mL alkalinity extracting solution is added in 50mL centrifuge tube in the tobacco sample 0.5g to be measured after weighing grinding, is vortexed
1min, revolving speed 13000rpm are centrifuged 5min, and supernatant carries out LC-MS/MS measurement after 0.23 μm of membrane filtration；The alkali
Property extracting solution: weigh 0.6058g L-cysteine and 1.8612g EDTA-Na2, with 450mL water dissolve, with 2mol/L's
NaOH is adjusted to pH 12.0, then is settled to 500mL with water, is made into half Guang of 10mmol/L EDTA and 10mmol/L L- of pH 12.0
The mixed liquor of propylhomoserin；
(2) LC-MS/MS determination condition
Chromatographic condition mobile phase: Waters UPLC BEH HILIC chromatographic column (50mm × 2.1mm × 2.8 μm)；Mobile phase A is
Acetonitrile, B are 10mmol/L ammonium hydroxide, flow velocity: 0.2mL/min；Column temperature: 25 DEG C；Sampling volume: 5 μ L；Gradient elution: second in 4 minutes
Nitrile is linearly down to 5% by 15%, is kept for 0.1 minute, is switched to acetonitrile 15%, keeps to 7 minutes；
Mass Spectrometry Conditions: electrospray ionisation source；Scanning mode: anion scanning；Detection mode: multiple-reaction monitoring；Electron spray voltage:
500V；Dry temperature degree: 300 DEG C；Dry gas stream amount: 6L/min；Sheath temperature degree: 350 DEG C；Sheath throughput: 10L/min；Capillary
Tube voltage: 4000V；
Table 1: the multiple-reaction monitoring of three classes DTC is qualitative, quota ion pair and mass spectrum relevant parameter
(3) preparation of standard solution
Single standard stock solution: it is calculated according to the purity of each standard substance, weighs pure material fervam, thiram, Fu Mei respectively
Zinc, zineb, maneb, Propineb and each 5mg of Polycarbamate, are dissolved and are settled to respectively with the alkaline extracting solution
100mL, ultrasound accelerate dissolution 5 minutes, single standard stock solution that be made into 7 kinds of pure materials respectively, that concentration is 50mg/L；
Single debugging solution: respectively diluting prepared each single standard stock solution alkaline extracting solution, respectively
To 7 kinds of pure materials, single debugging solution that concentration is 1mg/L；
The single mother liquor of standard working curve: it is calculated according to the purity of each standard substance, weighs pure material thiram, zineb respectively
With each 100mg of Propineb, 100mL is dissolved and is settled to respectively with the alkaline extracting solution, ultrasound accelerates dissolution 5 minutes, point
The single mother liquor of standard working curve that be not made into 3 kinds of pure materials, that concentration is 1000mg/L；
Standard working solution: respectively pipetted from the single mother liquor of standard working curve of 3 kinds of pure materials respectively 0.2mL, 0.4mL,
0.6mL, 0.8mL and 1.0mL are respectively placed in 10mL volumetric flask, are distinguished constant volume with the alkaline extracting solution, are respectively obtained 3
The series standard working solution that kind DTCs concentration is 20 μ of μ g/mL~100 g/mL；It is measured, is obtained with Liquid Chromatography-Tandem Mass Spectrometry
The integrated peak areas of DTCs calculates actual concentrations according to the purity of each standard substance as ordinate, as abscissa, production
Standard curve；
Matrix matching standard working solution: the flue-cured tobacco or white rib without dithiocarbamate after weighing 0.5g grinding respectively
Cigarette or each five parts of Turkish tobaccos powder, are respectively placed in 50mL polytetrafluoroethylene (PTFE) centrifuge tube, are separately added into 3 kinds of standard working curve lists
One mother liquor 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL, be then respectively adding 9.8mL, 9.6mL, 9.4mL, 9.2mL,
Alkaline extracting solution described in 9.0mL is vortexed 1 minute, and revolving speed 13000rpm is centrifuged 5 minutes, and supernatant is through 0.23 μm of membrane filtration
Afterwards, with liquid chromatography-mass spectrography/mass spectroscopy；
(4) qualitative results:
Standard solution and sample solution are first measured in same experimental conditions, then qualitatively judges sample object object as follows:
1) retention time of certain component peaks in the retention time and standard solution of the chromatographic peak detected in sample solution is basic
Unanimously, and the mass-to-charge ratio of selected ion pair is consistent；
2) sample ions to relative abundance and concentration be near the mark the relative abundance of ion pair of working solution be compared when, phase
Range as defined in table 2 is no more than to deviation；Meet the above two o'clock, then can determine that there are the components in sample；
With respect to the maximum allowable offset of abundance of ions when table 2 is qualitative determining
(5) quantitative result:
Standard solution and sample solution are measured under same experimental conditions, make standard curve, the content external standard of DTCs in sample
Standard measure is calculated by formula (1):
In formula: the content of certain component in X- sample, unit μ g/g；
ciThe concentration of certain component in the sample obtained by standard curve, unit μ g/mL；
c0Blank solution concentration, unit μ g/mL；
The final constant volume of V- sample, Unit/mL；
M- sample quality, unit g；
(6) result calculates: according to the abundance of sample solution quota ion to be measured, is read from standard curve as a result, deducting blank
The three classes dithiocarbamate content for obtaining sample afterwards is surveyed using the arithmetic mean of instantaneous value of parallel determinations twice as final
Determine as a result, the relative average debiation of parallel determinations should be less than 10% twice.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111366656A (en) * | 2020-04-07 | 2020-07-03 | 江苏省理化测试中心 | High performance liquid chromatography-tandem mass spectrometry method for detecting asomate residue in fruits and vegetables |
| CN112014504A (en) * | 2020-08-31 | 2020-12-01 | 河北双吉化工有限公司 | Analysis method of Dyson series |
| CN112113935A (en) * | 2019-06-19 | 2020-12-22 | 中国科学院大连化学物理研究所 | Fermat pesticide detection method based on manganese-containing inorganic-organic hybrid material |
| CN113408945A (en) * | 2021-07-15 | 2021-09-17 | 广西中烟工业有限责任公司 | Method and device for detecting purity of flue-cured tobacco, electronic equipment and storage medium |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101765771A (en) * | 2007-07-27 | 2010-06-30 | 株式会社津村 | Method for analysis of dithiocarbamate agrichemical by microwave-assisted thermal decomposition/extraction method |
| CN103852545A (en) * | 2014-03-07 | 2014-06-11 | 云南省烟草质量监督检测站 | Method for determining content of dithiocarbamate in tobaccos and tobacco products |
| CN104535672A (en) * | 2014-12-04 | 2015-04-22 | 苏州国环环境检测有限公司 | Method for detecting residues of mancozeb in cucumber |
| CN104807922A (en) * | 2015-05-26 | 2015-07-29 | 新希望六和股份有限公司 | Detecting method for five pesticides like avemectin in crops |
| CN105021737A (en) * | 2015-08-10 | 2015-11-04 | 云南省食品药品检验所 | Method for simultaneously detecting content of dicyandiamide and melamine in dairy products |
| CN107638871A (en) * | 2017-09-30 | 2018-01-30 | 福建中烟工业有限责任公司 | Adsorbing composition and the method for determining dithio amino formate compounds |
| CN108489968A (en) * | 2018-03-31 | 2018-09-04 | 中国热带农业科学院分析测试中心 | A kind of method of easy detection dithiocarbamates pesticide |
| CN108760929A (en) * | 2018-06-13 | 2018-11-06 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A method of detection 8 kinds of mycotoxins of FRUCTUS CITRI SARCODACTYLIS |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
-
2018
- 2018-12-07 CN CN201811494190.1A patent/CN109557230A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101765771A (en) * | 2007-07-27 | 2010-06-30 | 株式会社津村 | Method for analysis of dithiocarbamate agrichemical by microwave-assisted thermal decomposition/extraction method |
| CN103852545A (en) * | 2014-03-07 | 2014-06-11 | 云南省烟草质量监督检测站 | Method for determining content of dithiocarbamate in tobaccos and tobacco products |
| CN104535672A (en) * | 2014-12-04 | 2015-04-22 | 苏州国环环境检测有限公司 | Method for detecting residues of mancozeb in cucumber |
| CN104807922A (en) * | 2015-05-26 | 2015-07-29 | 新希望六和股份有限公司 | Detecting method for five pesticides like avemectin in crops |
| CN105021737A (en) * | 2015-08-10 | 2015-11-04 | 云南省食品药品检验所 | Method for simultaneously detecting content of dicyandiamide and melamine in dairy products |
| CN107638871A (en) * | 2017-09-30 | 2018-01-30 | 福建中烟工业有限责任公司 | Adsorbing composition and the method for determining dithio amino formate compounds |
| CN108489968A (en) * | 2018-03-31 | 2018-09-04 | 中国热带农业科学院分析测试中心 | A kind of method of easy detection dithiocarbamates pesticide |
| CN108760929A (en) * | 2018-06-13 | 2018-11-06 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | A method of detection 8 kinds of mycotoxins of FRUCTUS CITRI SARCODACTYLIS |
| CN108896694A (en) * | 2018-07-05 | 2018-11-27 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of remaining LC-QToF-MS Screening analysis method of pesticide in animal food |
Non-Patent Citations (2)
| Title |
|---|
| 彭丽娟等: "烟草中二硫代氨基甲酸酯的液相色谱-串联质谱测定法", 《云南化工》 * |
| 陈鹭平等: "非衍生化/液相色谱-串联质谱法测定食品中的二硫代氨基甲酸酯类农药残留", 《分析测试学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112113935A (en) * | 2019-06-19 | 2020-12-22 | 中国科学院大连化学物理研究所 | Fermat pesticide detection method based on manganese-containing inorganic-organic hybrid material |
| CN112113935B (en) * | 2019-06-19 | 2021-11-16 | 中国科学院大连化学物理研究所 | Fermat pesticide detection method based on manganese-containing inorganic-organic hybrid material |
| CN111366656A (en) * | 2020-04-07 | 2020-07-03 | 江苏省理化测试中心 | High performance liquid chromatography-tandem mass spectrometry method for detecting asomate residue in fruits and vegetables |
| CN111366656B (en) * | 2020-04-07 | 2023-11-10 | 江苏省理化测试中心 | High performance liquid chromatography tandem mass spectrometry for detecting residual amount of thiram in fruits and vegetables |
| CN112014504A (en) * | 2020-08-31 | 2020-12-01 | 河北双吉化工有限公司 | Analysis method of Dyson series |
| CN113408945A (en) * | 2021-07-15 | 2021-09-17 | 广西中烟工业有限责任公司 | Method and device for detecting purity of flue-cured tobacco, electronic equipment and storage medium |
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