CN104730186A - Precolumn derivatization-UPLC(ultra performance liquid chromatography)-ESI(electronic spray ion)+-MS/MS (mass spectrometry) detection method of glyphosate and glufosinate-ammonium pesticide residue in tea - Google Patents
Precolumn derivatization-UPLC(ultra performance liquid chromatography)-ESI(electronic spray ion)+-MS/MS (mass spectrometry) detection method of glyphosate and glufosinate-ammonium pesticide residue in tea Download PDFInfo
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Abstract
The invention discloses a precolumn derivatization-UPLC(ultra performance liquid chromatography)-ESI(electronic spray ion)+-MS/MS (mass spectrometry) detection method of glyphosate and glufosinate-ammonium pesticide residue in tea, sodium borate is used as a buffer salt, a relatively pure target derived product can be obtained by derivatization of glyphosate and glufosinate-ammonium and FMOC-Cl and then ultra performance liquid chromatography (UPLC) washing out and separation, and response signals can be detected by tandem mass spectrometry (MS-MS). According to the method, competition between the two pesticide residue derivatization can be eliminated, the detection effect caused by matrix effect can be effectively weakened, and the derivatization time is shortened. By experimental verification, the detection method of glyphosate and glufosinate-ammonium pesticide (herbicide) residue in tea has the characteristics of being efficient, fast and accurate, good in sensitivity and the like, is applicable in detection of the glyphosate and glufosinate-ammonium pesticide residue in tea samples of sundried green crude tea, fermented tea and substitutional tea (green food substitutional tea of agricultural industry standards of NY / T2140-2012 of the people's Republic of China) and finished tea (brick tea, Tuo tea, melon-shaped tea, cake tea, scattered tea and bagged tea and the like).
Description
Technical field
The invention belongs to field of food detection, be specifically related to the column front derivation-UPLC-ESI of tealeaves (tealeaves matrix or similar tealeaves matrix) glyphosate and careless ammonium phosphine agricultural chemicals (herbicide) residual quantity
+-MS/MS detection method.
Background technology
Glyphosate and careless ammonium phosphine are two kinds of broad-spectrum touch-out herbicides, are widely used in the cultivation of agricultural, forestry and gardening.Because of the increase day by day of glyphosate, careless ammonium phosphine herbicide use amount and unreasonable, make it often be found to be present in environmental water sample, soil and plant, such long term accumulation can cause environmental pollution, and can cause serious threat to human health.Along with GB2763-2012(national food safety standard Pesticide maximum residue limit) issue implement, glyphosate, careless ammonium phosphine are by the forcible inspection of item increased newly as Pesticide Residues in Tea.
Glyphosate and careless ammonium phosphine similar, and all containing phosphonate group, hydroxyl, amino, be extremely strong amphoteric compound, difficult volatilization.At present, the detection method of glyphosate and careless ammonium phosphine has 1. liquid phase chromatography, 2. vapor-phase chromatography, gas chromatography-mass spectrography and liquid chromatography-mass spectrography/mass spectroscopy after column front derivation.
Based on the complicacy of tealeaves matrix, above method is used for tealeaves glyphosate, the mensuration of careless ammonium phosphine agricultural chemicals (herbicide) residual quantity exists following defect:
1. liquid phase chromatography: do not have conjugated double bond in the molecular structure of glyphosate and careless ammonium phosphine, only there is n-pi-conjugated, ultraviolet region substantially without absorption, directly measures poor sensitivity, can not meet the analytical test requirement of persticide residue.After carrying out tealeaves glyphosate and the residual extraction of careless ammonium phosphine agricultural chemicals (herbicide), the impurity such as Tea Polyphenols, caffeine, pectin, carbohydrate often introduced by extract, but existing purification style is difficult to again these impurity to remove completely, the final impurity do not eliminated can cause interference to detection, causes measurement result inaccurate.
2. pre-column derivatization: column front derivation ratio juris is: under certain condition, reacts glyphosate, careless ammonium phosphine object and derivative reagent, the test substance that generating detector can detect, responsiveness is high, sensitivity is good.Adopt vapor-phase chromatography, gas chromatography-mass spectrography mainly because glyphosate, careless ammonium phosphine object are by after column front derivation, generate volatile, to be soluble in organic solvent etc. determinand, although this detection method has higher sensitivity, but glyphosate and careless ammonium phosphine object are converted in the process of easy gasification substance, the reagent introduced is more, operating process is relatively loaded down with trivial details, and detection efficiency is extremely low.Adopt liquid chromatography-mass spectrography/mass spectroscopy, in ion gun ESI, detection method liquid chromatography-mass spectrography/the mass spectroscopy of standard SN/T1923-2007(import and export food glyphosate residual quantity) adopt the positive ion mode in ESI source, and document (glyphosate in LC-MS/MS rice and AminomethylphosphoniAcid Acid remain, Cao Zhaoyun, Mu Renxiang, Chen Mingxue, chromatogram, 28th volume the 8th phase in 2010, 743-748) result of study is peak type under negative ion mode, response repeatability is better, because of the disunity of literature research result, to subsequent processes grope cause certain puzzlement, in the extraction and cleanups such as tealeaves glyphosate, careless ammonium phosphine, available data needs the solution such as the potassium hydroxide with variable concentrations, hydrochloric acid to carry out the minute adjustment of pH, operating process is very complicated, and the recovery of standard addition adopting the analysis of ion-exchange little column purification to draw is low, instability (the detection method liquid chromatography-mass spectrography/mass spectroscopy of SN/T1923-2007 import and export food glyphosate residual quantity).
In addition, said method all only relates to the analysis of single residues of pesticides material, so far also do not occur the method that simultaneously can detect tealeaves glyphosate and careless ammonium phosphine (particularly careless ammonium phosphine) content, associated mechanisms or state-owned unit did not issue examination criteria yet, did not retrieve relevant Research Literature.
Summary of the invention
For solving the problem in existing tealeaves glyphosate and the determination of residual amount of careless ammonium phosphine agricultural chemicals (herbicide), the object of this invention is to provide a kind of sample pre-treatments simple and quick, and can efficient, accurate, reproducible, the highly sensitive column front derivation-UPLC-ESI of Simultaneously test tealeaves (tealeaves matrix or similar tealeaves matrix) glyphosate and careless ammonium phosphine (herbicide) residual quantity
+-MS/MS detection method.
Column front derivation-the UPLC-ESI of tealeaves glyphosate of the present invention and careless ammonium phosphine persticide residue
+-MS/MS detection method, tealeaves obtains tea sample by GB/T8303 and comprises the following steps:
A) tea sample ultrapure water and the ultrasonic extraction of methylene chloride or cyclotron oscillation, get supernatant;
B) supernatant is through C
18reverse phase silica gel bonding solid-phase extraction column (C
18pillar) purification be purified liquid;
C) with FMOC-Cl acetone derive liquid, sodium borate buffer salt solusion derives the obtained derivative sample (liquid to be measured) of scavenging solution;
D) liquid to be measured is carried out UPLC-ESI
+-MS/MS scans, and scanning result glyphosate, careless ammonium phosphine separately standards calibration curve correct, and draw the mass body volume concentrations c (μ g/mL) of liquid glyphosate to be measured, careless ammonium phosphine residual quantity respectively;
E) tealeaves glyphosate, careless ammonium phosphine residual quantity is calculated respectively with following formula:
In formula:
X-be tealeaves glyphosate or careless ammonium phosphine residual quantity, mg/kg;
The mass body volume concentrations of c-derivative liquid glyphosate to be measured or careless ammonium phosphine, μ g/mL;
V-abovementioned steps 6) in the volume of ultrapure water, mL;
D-be constant 3, the extension rate of extraction and cleaning liquid time derivative;
The quality of m-take tea sample to be measured, g.
In described detection method: step a) in tea sample, ultrapure water, methylene chloride materials than for 1g:20mL:5mL.Step a) operating process is: tea sample is placed in 80mL tool lid centrifuge tube, adds ultrapure water, and vortex mixing leaves standstill about 2min, then adds methylene chloride, mixing, and ultrasonic extraction or cyclotron oscillation are about 30min, and low speed centrifuge is centrifugal, get supernatant;
Step b) in purification run be: get 5mL supernatant and cross C
18purification pillar, discards front 1mL efflux, 2-3mL efflux after collecting, obtained scavenging solution.
Step c) mesoboric acid sodium buffer salt solution concentration is the preferred 50g/L of 30g/L-60g/L(), FMOC-Cl acetone soln concentration is the preferred 20g/L of 10g/L-30g/L().
Step c) in the derivative time be 2h or more.
Step c) operating process: by volume 1:1:1 ratio gets scavenging solution successively, sodium borate buffer solution, FMOC-Cl acetone solution be mixed to evenly, room temperature leaves standstill 2h or more, the centrifugal 5min of 4500r/min, gets supernatant and crosses 0.2 μm of organic filter membrane, obtains derivative sample (liquid to be measured).
UPLC-ESI described in step d)
+the parameter determined during-MS/MS scanning is:
1. mobile phase: mobile phase A is 0.1% formic acid-ammonium acetate-water, Mobile phase B is a kind of in pure acetonitrile, pure methyl alcohol, pure acetonitrile, 0.1% formic acid acetonitrile and 50% methanol acetonitrile etc.;
2. Ultra Performance Liquid Chromatography (UPLC) parameter is determined: the liquid phase gradient elution program that glyphosate and careless ammonium phosphine detect is: 5% mobile phase A keeps 0.5min, in 1.5min, mobile phase A becomes 95% from 5%, 95% mobile phase A keeps mobile phase A in 1.5min, 0.5min to become 5% from 95%; Flow velocity 0.3mL/min,
c
18post (specification 1.7 μm, 2.1 × 50mm), sample chamber temperature 15 DEG C, chromatogram column temperature 40 DEG C, sample size 1-6 μ L;
3. the determination of mass spectrum tuner parameters: electric spray ion source (ESI), determines with positive ion mode (ESI
+), multiple-reaction monitoring (MRM), capillary voltage 2.5kv, atomization gas and desolventizing gas are nitrogen, and collision gas is argon gas, desolventizing temperature degree 400 DEG C, desolventizing gas velocity is 800L/Hr, and taper hole gas velocity is 100L/Hr, and ion source temperature is 150 DEG C;
4. the determination of mass spectrometry parameters: determine that the characteristic ion of glyphosate derivative is to the taper hole voltage 25v of 392/88, collides voltage 20v, the characteristic ion of glyphosate derivative, to the taper hole voltage 25v of 392/214, collides voltage 10v; The characteristic ion of grass ammonium phosphine-derivatives, to the taper hole voltage 25v of 404/136, collides voltage 25v, and the characteristic ion of careless ammonium phosphine-derivatives, to the taper hole voltage 25v of 404/208, collides voltage 10v.
The acquisition of step d) Plays calibration curve comprises the following steps:
I) the mixed mark of preparation: with glyphosate and careless ammonium phosphine standard stock solution by volume 1:1 obtain the mixed mark that glyphosate and careless ammonium phosphine concentration are respectively 0.003125 μ g/mL, 0.00625 μ g/mL, 0.0125 μ g/mL, 0.025 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL separately;
Ii) mix mark by the method identical with step c) to each concentration to derive, obtained derivative mark liquid;
Iii) under same with derivative sample condition, UPLC-ESI is carried out to derivative mark liquid
+-MS/MS scans, with derivative mixed mark liquid concentration for horizontal ordinate, with the peak area of glyphosate 392/88 ion pair for ordinate draws glyphosate standards calibration curve, with the peak area of careless ammonium phosphine 404/136 ion pair for ordinate draws careless ammonium phosphine standards calibration curve.
Technical scheme of the present invention relates to: the 1. extracting method of herbicide persticide residue glyphosate and careless ammonium phosphine object in tealeaves; 2. the purification method of tea extract Pesticide Residues glyphosate and careless ammonium phosphine object; 3. the deriving method of tealeaves extraction and cleaning liquid Pesticide Residues glyphosate and careless ammonium phosphine object; 4. the UPLC method (chromatographic parameter) of glyphosate and the detection of careless ammonium phosphine; 5. the mass spectrum tuning methods (mass spectrum tuner parameters) of glyphosate and the detection of careless ammonium phosphine; 6. the mass spectrometry method (mass spectrometry parameters, qualitative and quantitative ion, taper hole and collision voltage etc.) of glyphosate and the detection of careless ammonium phosphine.
Under the present invention makes the condition of buffer salt with sodium borate, glyphosate and careless ammonium phosphine and FMOC-Cl derive, and form derivative products.The potpourris such as derivative products, buffer salt, derivating agent and related impurities inject UPLC and carry out chromatography eluant separation, obtain comparatively pure derivative goal product, adopt tandem mass spectrum probe response signal.The method is applicable to the detection of solar dried green tea, fermented tea, alternative tea (NY/T2140-2012 People's Republic of China (PRC) agricultural industry criteria pollution-free food alternative tea), become to sample tea (brick tea, Tuo tea, melon tea, cake tea, loose tea, tea in bag etc.) tea sample glyphosate, careless ammonium phosphine persticide residue.
The present invention adopts column front derivation-UPLC-ESI
+-MS/MS method determines glyphosate in Tea Samples and careless ammonium phosphine content, has the following advantages:
1, highly sensitive, good linearity, detection limit are low (can reach ng/mL level);
2, quantitative result is accurate, stable, reproducible;
3, experimental implementation is simple, step is few, consuming time short, analysis speed is fast, detection efficiency is high;
4, pollution-free, nontoxic, the safety of experiment reagent;
5, the impact that matrix detects object is effectively weakened;
What 6, prevent glyphosate, careless ammonium phosphine from deriving is incomplete, overcomes derivative competition.
Above advantage makes the method can be able to good application, and principal element comprises:
1) pre-treatment of Tea Samples, Tea Samples, after ultrapure water soaks, adds methylene chloride, effectively can remove the liposoluble substance in tealeaves, as Tea Pigment, Tea Polyphenols etc., alleviate load to the subsequent purification work of extract;
2) in purification, to nonpolar, low pole and middle polarity compound, there is strong reservation by adopting, and to decontaminating column CAX, C that the aqueous solution of object ion does not retain
18, and the test of comparing such as activated charcoal pillar, determine with C
18little column purification extract, optimizes the recovery of standard addition scope of sample, improves the accuracy of detection;
3) by the condition test to mobile phase, optimize the composition of mobile phase, simplify the preparation process of mobile phase;
4) by integrating existing glyphosate, careless ammonium phosphine analytical approach, effectively two residues of pesticides projects being detected and being put in Same Way, the detection that the Multiple Pesticides of same sample is residual can be realized;
5) by derivating agent solution concentration, dobell's solution concentration, the condition test of derivative time, determine that the best of derivative test derives parameter, thus effectively eliminate glyphosate, the competition of careless ammonium phosphine when derivative, shorten the derivative time, improve detection efficiency;
6) meet on the basis of pesticide residue analysis in method sensitivity, optimize instrument condition, alleviate instrument load, reach the invisible protection to high-accuracy detector (tandem mass spectrum and ion gun etc.).
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is the molecular structure of glyphosate herbicidal (agricultural chemicals)
Fig. 2 is the molecular structure of careless ammonium phosphine herbicide (agricultural chemicals)
Fig. 3 is the molecular structure of derivating agent FMOC-Cl
Fig. 4 is the derivative mechanism of glyphosate under dobell's solution condition, careless ammonium phosphine and FMOC-Cl acetone soln
Fig. 5 is the splitting mechanism of glyphosate derivative in tandem mass spectrum
Fig. 6 is the splitting mechanism of careless ammonium phosphine-derivatives in tandem mass spectrum
Fig. 7 is the UPLC-ESI of glyphosate standard solution under MRM pattern
+-MS/MS spectrogram
Fig. 8 is the UPLC-ESI of careless ammonium phosphine standard solution under MRM pattern
+-MS/MS spectrogram
Fig. 9 is the linear relationship of glyphosate concentration and response peak area under MRM pattern
Figure 10 is the linear relationship of careless ammonium phosphine concentration and response peak area under MRM pattern
Embodiment
The present invention adopts the residual quantity of LC-MS technology to tealeaves glyphosate and careless ammonium phosphine agricultural chemicals (herbicide) to carry out Simultaneously test.
Mass spectrum principle is first by substance ion, is separated by the mass-to-charge ratio of ion, then measures the various intensity at ionic spectrum peak and a kind of analytical approach of Realization analysis object.The present invention adopts LC-MS to be combined by qualitative function powerful to the separating power of chromatogram and mass spectrum, realize complex mixture quantitative and qualitative analysis more accurately, simplify the pre-treatment flow process of sample, make sample analysis easier, the analysis mainly for compounds such as involatile, polarity, thermally labile, macromolecules measures.LC-MS detection technique is highly sensitive, and tandem mass spectrum (triple level Four bar) is accurately qualitative, effectively can stop false positive phenomenon during micro-even trace species analysis, be usually used in the trace analysis of target substance.
Concrete, the invention provides the column front derivation-UPLC-ESI for tealeaves (tealeaves matrix or similar tealeaves matrix) glyphosate and careless ammonium phosphine (herbicide) residual quantity
+-MS/MS detection method.
Inventor's research is found out, and make the condition of buffer salt with sodium borate under, glyphosate, careless ammonium phosphine and FMOC-Cl are derivative, form derivative products.The potpourris such as derivative products, buffer salt, derivating agent and related impurities inject UPLC(superelevation liquid chromatography) carry out chromatography eluant separation, namely obtain comparatively pure derivative goal product.And then adopt tandem mass spectrum effectively to detect the response signal of derivative goal product, the mass spectrophotometry parameter to glyphosate and careless ammonium phosphine can be obtained.
Derivative mechanism as shown in Figure 4, under sodium borate buffer salt solusion condition, in glyphosate (molecular structure is as shown in Figure 1) and careless ammonium phosphine (molecular structure is as shown in Figure 2) ,-H of R-NH-R ' is by FMOC-Cl(molecular structure as shown in Figure 3) in FMOC-replace, generate
obtain derivative goal product glyphosate derivative and careless ammonium phosphine-derivatives.
The splitting mechanism of glyphosate derivative in tandem mass spectrum as shown in Figure 5, can find out that glyphosate derivative has two kinds of cracking modes after ion gun ionization, first kind of way is cracked into ion m/z179, the second way is cracked into ion m/z214, and ion m/z214 automatically can lose a CO in the process flowing through collision cell from first mass spectrometric
2molecule and become ion m/z170, ion m/z170 collides with inert gas argon gas in collision cell, and second pyrolysis is ion m/z88.
The splitting mechanism of grass ammonium phosphine-derivatives in tandem mass spectrum as shown in Figure 6, can find out that careless ammonium phosphine-derivatives has three kinds of cracking modes after ion gun ionization, first kind of way is cracked into ion m/z179, the second way is cracked into ion m/z208, the third mode is cracked into ion m/z226, and ion m/z226 automatically can lose a CO in the process flowing through collision cell from first mass spectrometric
2molecule and become ion m/z182, ion m/z182 collides with inert gas argon gas in collision cell, and second pyrolysis is ion m/z136.
On the basis of understanding research above, the present invention passes through chromatogram and the mass spectrometry parameters of constantly optimization deriving technology parameter, instrument repeatedly, probes into out the column front derivation-UPLC-ESI of a kind of glyphosate and careless ammonium phosphine
+-MS/MS detection method.The method not only eliminate two kinds of agricultures residual derivative between competition, also effectively weakening matrix effect to detecting the impact brought, shortening the derivative time.The present invention passes through the condition test of the relative recovery of standard addition of Tea Samples, absolute recovery of standard addition, sample size, derivative time, mobile phase composition, standards calibration curve etc., the features such as the detection method of the tealeaves glyphosate of finally probing into and careless ammonium phosphine agricultural chemicals (herbicide) residual quantity has efficiently, quick, accurate, sensitivity is good.
Below, the column front derivation-UPLC-ESI of tealeaves (tealeaves matrix or similar tealeaves matrix) glyphosate that the present invention determines and careless ammonium phosphine agricultural chemicals (herbicide) residual quantity is illustrated
+-MS/MS detection method.
Specifically, the column front derivation-UPLC-ESI of tealeaves provided by the present invention (tealeaves matrix or similar tealeaves matrix) glyphosate and careless ammonium phosphine agricultural chemicals (herbicide) residual quantity
+-MS/MS detection method, can comprise the following steps:
1) reagent preparation
1. mobile phase A: accurately take 0.154g (being accurate to 0.001g) anhydrous acetic acid ammonium and be dissolved in appropriate ultrapure water, add 1mL formic acid, be settled to 1000mL with ultrapure water, obtain 0.1% formic acid-ammonium acetate-water, cross 0.2 μm of organic film, be mobile phase A;
Mobile phase B: pure acetonitrile (also can select pure methyl alcohol, pure acetonitrile, 0.1% formic acid acetonitrile, 50% methanol acetonitrile etc.), crosses 0.2 μm of organic film, is Mobile phase B;
2. derivative reagent: accurately take appropriate (being accurate to 0.001g) FMOC-Cl, be settled to 100mL with acetone, obtain FMOC-Cl acetone soln, concentration range 1g/L-30g/L;
3. sodium borate buffer solution: accurately take appropriate (being accurate to 0.001g) sodium borate (Na
2b
4o
710H
2o), be settled to 100mL with ultrapure water, obtain sodium borate buffer solution, concentration range 20g/L-60g/L;
4. mixed mark: with micropipettor from 1000 μ g/mL standard stock solution (standard stock solution is bought from State center for standard matter) glyphosates, respectively pipette 10 μ L respectively in grass ammonium phosphine and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 1 μ g/mL, the mixed mark of grass ammonium phosphine, then, from the glyphosate of 1 μ g/mL, pipette 1mL in the mixed mark of grass ammonium phosphine and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 0.1 μ g/mL, the mixed mark of grass ammonium phosphine, again from the glyphosate of 0.1 μ g/mL, pipette 5mL in the mixed mark of grass ammonium phosphine and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 0.05 μ g/mL, the mixed mark of grass ammonium phosphine, the rest may be inferred, stepwise dilution, concentration can be obtained and be respectively 0.003125 μ g/mL, 0.00625 μ g/mL, 0.0125 μ g/mL, 0.025 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL glyphosate, the mixed mark of grass ammonium phosphine,
2) mark liquid-derived
The determination of derivatization conditions: with the concentration 20g/L of sodium borate buffer salt solusion, 30g/L, 40g/L, 50g/L, 60g/L, the concentration 1g/L of FMOC-Cl acetone soln, 5g/L, 10g/L, 20g/L, 30g/L make condition Comparability test, show that sodium borate buffer concentration of salt solution is the preferred 50g/L of 30g/L-60g/L(), FMOC-Cl acetone soln concentration is the preferred 20g/L of 10g/L-30g/L() time, derivative effect is better; Derivative effect is changed all better, between the two without being closely connected in FMOC-Cl acetone soln concentration and each comfortable scope of sodium borate buffer concentration of salt solution; During the preferred 20g/L of FMOC-Cl acetone soln concentration and the preferred 50g/L of sodium borate buffer concentration of salt solution, there is optimum derivative effect.Carry out the derivative time be 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 7h, 9h, the condition test such as spend the night, and after to draw when the derivative time be 2h, derivatively reaches balance.
Derivation operation: label taking liquid (glyphosate, careless ammonium phosphine are each to be singly marked with and the mixed mark of glyphosate grass ammonium phosphine), sodium borate buffer solution, each 1mL(of FMOC-Cl acetone solution by volume 1:1:1 ratio mixing successively) be placed in tool lid centrifuge tube, whirlpool is mixed to evenly, 1min and more than, room temperature leaves standstill and derives to completely, 2h and more than, the centrifugal 5min of low speed centrifuge 4500r/min, gets supernatant and crosses 0.2 μm of organic filter membrane, obtains derivative mark liquid;
3) instrument parameter is determined
1. the selection of mobile phase: be 0.1% formic acid water by mobile phase A, 0.1% formic acid-ammonium acetate-water, Mobile phase B is pure acetonitrile, 0.1% formic acid acetonitrile, pure methyl alcohol, the condition test of 50% methanol acetonitrile, show that in mobile phase A, ammonium acetate has a significant impact object chromatogram peak-to-peak type, there is serious hangover in the chromatographic peak of 0.1% formic acid water, therefore mobile phase A selects 0.1% formic acid-ammonium acetate-water, the detection of Mobile phase B on object does not almost affect, if unique difference has methyl alcohol in Mobile phase B, the sensitivity of instrument response can increase, but appearance time can be delayed a little, therefore Mobile phase B can experimentally be selected by room own actual situation.The present invention determines with 0.1% formic acid-ammonium acetate-water (mobile phase A), and pure acetonitrile (Mobile phase B) is tested for example;
2. Ultra Performance Liquid Chromatography (UPLC) parameter is determined: the liquid phase gradient elution program that glyphosate, careless ammonium phosphine detect is: 5% mobile phase A keeps 0.5min, in 1.5min, mobile phase A becomes 95% from 5%, 95% mobile phase A keeps mobile phase A in 1.5min, 0.5min to become 5% from 95%; Flow velocity 0.3mL/min, Waters Acquity
c
18post (specification 1.7 μm, 2.1 × 50mm), sample chamber temperature 15 DEG C, chromatogram column temperature 40 DEG C, sample size 1-6 μ L;
3. the determination of mass spectrum tuner parameters: electric spray ion source (ESI), determines with positive ion mode (ESI
+), multiple-reaction monitoring (MRM), capillary voltage 2.5kv, atomization gas and desolventizing gas are nitrogen, and collision gas is argon gas, desolventizing temperature degree 400 DEG C, desolventizing gas velocity is 800L/Hr, and taper hole gas velocity is 100L/Hr, and ion source temperature is 150 DEG C;
4. the determination of mass spectrometry method: step 2) derivative mark liquid through Ultra Performance Liquid Chromatography (UPLC) be separated after, first adopt the scanning of Daughters Scan(daughter ion) Mode scans, find out the stable characteristic ion of glyphosate derivative respectively to 392/88,392/214, the stable characteristic ion of careless ammonium phosphine-derivatives is to 404/136,404/208; Under positive ion (ESI+), full scan (MRM) pattern, doing taper hole voltage is 10v, 15v, 20v, 25v, 30v, 35v, 40v, collision voltage is the condition test of 10v, 15v, 20v, 25v, 30v, 35v, 40v, determines the optimum taper hole voltage that glyphosate, careless ammonium phosphine-derivatives characteristic ion are right and collide voltage as shown in table 1:
The mass spectrometry parameters of table 1 glyphosate, careless ammonium phosphine-derivatives
4) Specification Curve of Increasing
By step 2) derivative mark liquid by step 3) instrument condition determined carries out UPLC-ESI+-MS/MS test scan, from the analytical test parameter of scanning, qualitative with abundance ratio of retention time, quota ion, qualitative ion, quota ion and qualitative ion etc., be horizontal ordinate with mixed mark derivatives concentration, be that ordinate draws respective standards calibration curve with the peak area of quota ion (glyphosate 392/88 ion pair, careless ammonium phosphine 404/136 ion pair).
5) Tea Samples preparation
Tealeaves by GB/T8303 through grinding, sieving obtained tea sample to be measured (fermented tea first low temperature should remove moisture, makes sample be easy to grind);
6) tealeaves detects sample extraction
Accurately taking by step 5) the tea sample (being accurate to 0.001g) that processes is placed in 80mL tool lid centrifuge tube, add ultrapure water, vortex mixing leaves standstill about 2min, add methylene chloride, wherein the materials of tea sample, ultrapure water, methylene chloride are than being 1g:20mL:5mL, mixing, ultrasonic extraction or cyclotron oscillation are about 30min, low speed centrifuge is centrifugal, gets supernatant, obtained extraction supernatant;
7) sample purification is detected
To nonpolar, low pole and middle polarity compound, there is strong reservation by adopting, and to decontaminating column CAX, C that the aqueous solution of object ion does not retain
18, and the test of comparing such as activated charcoal pillar, finally determine with C
18little column purification extract, optimizes the recovery of standard addition scope of sample, improves the accuracy of detection.
Purification run: get step 6) in obtained 5mL extract supernatant and cross C
18purification pillar (C
18reverse phase silica gel bonding solid-phase extraction column), discard front 1mL efflux, 2-3mL efflux after collecting, obtained scavenging solution;
8) analyte derivative
Get 1mL scavenging solution obtained in step 7) in 10mL tool lid centrifuge tube, the FMOC-Cl acetone adding 1mL successively derives liquid, sodium borate buffer salt solusion, carry out and step 2) (vortex mixed is to even for same treatment, 1min and more than, room temperature is placed and is derived to completely, 2h and more than, the centrifugal 5min of low speed centrifuge 4500r/min, get supernatant and cross 0.2 μm of organic phase filter membrane), obtained derivative sample (liquid to be measured);
9) upper machine scanning
By step 8) obtained derivative sample is by step 3) instrument parameter determined carries out UPLC-ESI
+-MS/MS scan, scanning result adopt step 4) in standards calibration curve correct, draw the mass body volume concentrations c (μ g/mL) of liquid glyphosate to be measured, careless ammonium phosphine residual quantity respectively;
10) result calculates: the calculating formula of tealeaves glyphosate, careless ammonium phosphine residual quantity is:
In formula:
X-be tealeaves glyphosate or careless ammonium phosphine residual quantity, mg/kg;
The mass body volume concentrations of c-derivative liquid glyphosate to be measured or careless ammonium phosphine, μ g/mL;
V-abovementioned steps 6) in the volume of ultrapure water, mL;
D-be constant 3, the extension rate of extraction and cleaning liquid time derivative;
The quality of m-take tea sample to be measured, g.
The present invention is further illustrated below with embodiment.Embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.
Percent concentration of the present invention is mass/mass (W/W if no special instructions, unit is wt%) percent concentration, mass/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, unit is v%) percent concentration.
Main field of employment of the present invention is the laboratory possessing required instrument and equipment and experimental situation condition.Agents useful for same is and is purchased.
The detection of embodiment 1, fermented tea glyphosate, careless ammonium phosphine persticide residue
The detection of the present embodiment is by abovementioned steps 1)-9) complete, identical content no longer repeats, change part and be:
1) in, Mobile phase B is pure acetonitrile.
2) use derivative optimal conditions 50g/L sodium borate buffer salt solusion, 20g/L FMOC-Cl acetone soln in, the derivative time gets the shortest 2h.Mixing 1min, centrifugal 5min, cross 0.2 μm of organic filter membrane.
4) the UPLC-ESI+-MS/MS spectrogram of glyphosate standard solution under MRM pattern as shown in Figure 7, ion m/z88 (A width), the m/z214(B width of glyphosate can be found out) and the chromatogram peak-to-peak type of total ion figure (C width) good, symmetrical, highly sensitive, meet pesticide residue analysis test request completely; The UPLC-ESI of grass ammonium phosphine standard solution under MRM pattern
+-MS/MS spectrogram as shown in Figure 8, can find out ion m/z136 (A width), the m/z208(B width of careless ammonium phosphine) and the chromatogram peak-to-peak type of total ion figure (C width) good, symmetrical, highly sensitive, meet pesticide residue analysis test request completely.
With mixed mark derivatives concentration be horizontal ordinate, with the peak area of quota ion (corresponding response peak area) for ordinate drawing standard calibration curve, as shown in Figure 9, the linear relationship of careless ammonium phosphine concentration and quota ion 404/136 response peak area as shown in Figure 10 for the linear relationship of glyphosate concentration and quota ion 392/88 response peak area.
5), in before sample preparation, at first fermented tea being put in 60 DEG C in constant temperature blast drying oven, making fermented tea moisture control about 10%, be then made as tea sample to be measured by GB/T8303.
6) in, tealeaves is fermented tea, the quality extracting tea sample when detecting sample is that 1g (is designated as m), adds 20mL(and be designated as v) ultrapure water, add 5mL methylene chloride, ultrasonic extraction or cyclotron oscillation 30min, low speed centrifuge 4500r/min is centrifugal, and 5min gets supernatant.
8) derivation operation condition in: 50g/L sodium borate buffer salt solusion, 20g/L FMOC-Cl acetone soln and derivative time 2h.Mixing 1min, centrifugal 5min, cross 0.2 μm of organic filter membrane.
9) in, actual measurement glyphosate mass body volume concentrations c is 0.00512 μ g/mL, and the mass body volume concentrations c of careless ammonium phosphine is 0.00376 μ g/mL.
10) calculating tealeaves glyphosate residual quantity x in is 0.3172mg/kg, and careless ammonium phosphine residual quantity x is 0.2256mg/kg.
The residual quantity of embodiment 2, mensuration solar dried green tea glyphosate, careless ammonium phosphine agricultural chemicals
The detection of the present embodiment is by abovementioned steps 1)-9) complete.Step 1)-4) identical with embodiment 1.Detecting portion operation is as follows:
5)-6) Tea Samples preparation with extract: for solar dried green tea material sample, tea sample grinds through GB/T8303, sieve after obtained tea sample to be measured; Accurately take and grind tea sample 1g (being accurate to 0.001g) in 80mL tool lid centrifuge tube (two parallel sample numberings are respectively I and II), because considering may can not to detect glyphosate, careless ammonium phosphine in tealeaves, therefore in two tool lid centrifuge tubes, first add the mixed mark 1mL of isoconcentration 5.5 μ g/mL, add 20mL ultrapure water again, vortex mixing leaves standstill 2min, add 5mL methylene chloride again, ultrasonic or cyclotron oscillation extracts 30min, the centrifugal 5min of low speed centrifuge 4500r/min, get supernatant, obtained extraction supernatant.
7) purify: get 5mL and extract supernatant and cross C18 pillar (purchased from enlightening equine skill, if can meet the analytical test requirement of persticide residue, the pillar of other producers also can use.), discard precontract 1mL efflux, 2-3mL efflux after collecting, obtained scavenging solution.
8) derivative: to get 1mL scavenging solution in 10mL tool lid centrifuge tube, add the 10g/L FMOC-Cl acetone soln of 1mL, 30g/L sodium borate buffer salt solusion successively, carry out and step 2 in embodiment 1) (vortex mixed is to even for same treatment, 1min and more than, room temperature is placed and is derived to completely, 2h and more than, the centrifugal 5min of low speed centrifuge 4500r/min, get supernatant and cross 0.2 μm of organic phase filter membrane), obtained liquid to be measured.
9) upper machine scanning
By liquid to be measured obtained for step 8) by step 3 in embodiment 1) instrument parameter carry out UPLC-ESI
+-MS/MS scans, scanning result adopts embodiment 1 step 4) in standards calibration curve (Fig. 9, Figure 10) correct, draw the mass body volume concentrations c (μ g/mL) of liquid glyphosate to be measured, careless ammonium phosphine, the results are shown in Table 2, the mass body volume concentrations of its glyphosate is 0.08913 μ g/mL, 0.09018 μ g/mL, and the mass body volume concentrations of careless ammonium phosphine is 0.08376 μ g/mL, 0.08299 μ g/mL.
10) result calculates: calculate tealeaves glyphosate, careless ammonium phosphine residual quantity (mg/kg) by aforementioned formula.
The residual quantity of result solar dried green tea sample glyphosate, careless ammonium phosphine is as shown in table 2, data display tealeaves detects sample glyphosate, careless ammonium phosphine persticide residue and mixed target addition (5.5mg/kg) and coincide, the horizontal checkout data stabilization of same sample, shows that detection method is stablized.
The addition of table 2 solar dried green tea sample glyphosate, careless ammonium phosphine
The matrix effect test of test example 1, glyphosate, careless ammonium phosphine pesticide residue determination
Matrix effect is mainly derived from interfering material (comprising endogenous components and the exogenous components) impact on determinand Ionization Efficiency of flowing out altogether with determinand in chromatographic separation process.Endogenous components refers to the organic or inorganic molecule (as inorganic salts, phenols, pigment and lipid) be extracted in Tea Samples leaching process, exogenous components is mainly introduced by pretreatment process, the buffer salt introduced when comprising derivatization reaction and derivating agent and lysate etc. thereof.
For the matrix effect that analysis tealeaves Endogenous ground substance and derivatization reaction produce glyphosate and careless ammonium phosphine, spy carries out bare substrate Comparability test: glyphosate and careless ammonium phosphine pure water are diluted to the mixed mark of following concentration 0.009375 μ g/mL, 0.01875 μ g/mL, 0.075 μ g/mL, 0.15 μ g/mL and 0.3 μ g/mL by A, and glyphosate and careless ammonium phosphine bare substrate liquid are diluted to the mixed mark of following concentration 0.009375 μ g/mL, 0.01875 μ g/mL, 0.075 μ g/mL, 0.15 μ g/mL and 0.3 μ g/mL by B.Get the above-mentioned mixed mark of 1mL, 1mL40g/L sodium borate buffer solution, 1mL30g/L FMOC-Cl acetone soln successively in 10mL tool lid centrifuge tube, vortex mixing 1min, room temperature derives 2h, the centrifugal 5min of low speed centrifuge 4500r/min, organic filter membrane of 0.2 μm crossed by supernatant, carries out the analysis of UPLC-ESI+-MS/MS sample introduction by the method in embodiment 1.
Response criteria calibration curve is as shown in table 3, and matrix effect (matrix effect, ME) can calculate with ME=B/A × 100%, and in formula, B is extraction standard rate of curve, and A is slope of standard curve.Generally, there is not obvious matrix effect in the ratio of extraction standard curve response and its typical curve response between 85% ~ 115%.Judging accordingly, there is not obvious matrix effect in the detection of method of the present invention to glyphosate, careless ammonium phosphine.
The typical curve of table 3 glyphosate, careless ammonium phosphine and extraction standard curve
Test example 2, glyphosate, careless ammonium phosphine pesticide residue determination limit
By the detection method of embodiment 1, estimate LOD (detection limit) and LOQ (detected level) by the standard deviation of the linear equation set up between series standard sample and chromatographic peak response (peak area).
Standard substance concentration and chromatographic peak response peak area, data are as shown in table 4, table 5, table 6.Standards calibration curve is see Fig. 9, Figure 10.
Table 4 standard substance concentration and peak area detailed data
Table 5 glyphosate detection limit calculates
By y=314548x-198.68, Sy/x=236.78, can response be drawn:
YLOD=3×Sy/x-198.68=511.66
YLOQ=10×Sy/x-198.68=2169.12
Carry it into typical curve equation and calculate glyphosate concentration value, detection limit LOD=0.002258 μ g/mL, detected level LOQ=0.007528 μ g/mL.
Table 6 careless ammonium phosphine detection limit calculates
By y=316029x-356.95, Sy/x=222.71, can response be drawn:
YLOD=3×Sy/x-356.95=311.18
YLOQ=10×Sy/x-356.95=1870.15
Carry it into typical curve equation and calculate careless ammonium phosphine concentration value, detection limit LOD=0.002114 μ g/mL, detected level LOQ=0.007047 μ g/mL.
Test example 3, glyphosate, the test of careless ammonium phosphine pesticide residue determination Tea Samples recovery of standard addition
For Menhai Ba Da high mountain organic tea, accurately take the same tea sample 1g (being accurate to 0.001g) that grinds in the tool lid centrifuge tube of 4 80mL, numbering is followed successively by 1#-4#.Respectively to adding glyphosate in 2# sample, careless ammonium phosphine mix in mark 0.375 μ g, 3# sample add glyphosate, careless ammonium phosphine mix in mark 1.5 μ g, 4# sample add glyphosate, careless ammonium phosphine mixes mark 4.5 μ g; Separately in 3 empty tool lid centrifuge tube 5#, add glyphosate, careless ammonium phosphine mix in mark 0.375 μ g, 6# add glyphosate, careless ammonium phosphine mix in mark 1.5 μ g, 7# add glyphosate, careless ammonium phosphine mixes mark 4.5 μ g.7 centrifuge tubes all add 20mL ultrapure water, and vortex mixing leaves standstill about 2min, adds 5mL methylene chloride, ultrasonic or cyclotron oscillation extracts 30min, in the centrifugal 5min of low speed centrifuge 4500r/min, gets 5mL supernatant and crosses C18 purification pillar, discard precontract 1mL efflux, about 2-3mL efflux after collection.Accurately get 1mL efflux in 10mL tool lid centrifuge tube, add 1mL sodium borate buffer solution (50g/L), 1mL FMOC-Cl acetone soln (10g/L) successively, vortex mixing 1min, room temperature derives 2h, the centrifugal 5min of low speed centrifuge 4500r/min, organic filter membrane of 0.2 μm crossed by supernatant, carries out the analysis of UPLC-ESI+-MS/MS sample introduction by the method in embodiment 1.
Scanning result corrects (Fig. 9, Figure 10) through standards calibration curve and draws the content of glyphosate, careless ammonium phosphine, thus calculate glyphosate, careless ammonium phosphine sample relative to recovery of standard addition and absolute recovery of standard addition, detailed data is as shown in table 7.
Table 7 sample recovery of standard addition test findings
As can be seen from Table 7, the recovery of standard addition of glyphosate, careless ammonium phosphine is reasonable, and data show that this pre-treating method Detection accuracy is high; And comparing analysis from relative recovery of standard addition and absolute recovery of standard addition, result shows that this pre-treating method reduces tealeaves matrix effect to the impact detected, and matrix effect is not remarkable.
The precision test of test example 4, glyphosate, careless ammonium phosphine pesticide residue determination
Cake tea in sampling tea for one-tenth, 7542 raw cakes (Tea Samples is provided by Menghai Tea Co., Ltd.) are divided into four pieces, get diagonal angle tea sample, after breaking into pieces, mixing, grind through GB/T8303, sieve after obtained tea sample to be measured, accurately take and grind tea sample 1g (being accurate to 0.001g) 6 parts in 80mL tool lid centrifuge tube, sample number into spectrum is 1-6.In each centrifuge tube, first add 2.0 μ g glyphosate and careless ammonium phosphine mix mark (because not detecting glyphosate and careless ammonium phosphine in Tea Samples, therefore when doing precision test, the mode of adding standard substance is adopted to implement), add 20mL ultrapure water, vortex mixing leaves standstill 2min, add 5mL methylene chloride again, ultrasonic or cyclotron oscillation extracts the centrifugal 5min of 30min, low speed centrifuge 4500r/min.Get 5mL extraction supernatant and cross C
18pillar, discards precontract 1mL efflux, 2-3mL efflux after collecting.Get 1mL scavenging solution, 1mL sodium borate buffer salt solusion (60g/L), 1mL FMOC-Cl acetone soln (20g/L) successively in 10mL centrifuge tube, vortex 1min, room temperature leaves standstill derivative 2h, in the centrifugal 5min of low speed centrifuge 4500r/min, get supernatant and cross 0.2 μm of organic phase filter membrane, upper machine carries out UPLC-ESI by the method in embodiment 1
+-MS/MS scans.Scanning result standards calibration curve (Fig. 9, Figure 10) corrects, and obtains the mass body volume concentrations of derivative liquid glyphosate to be measured, careless ammonium phosphine, brings formula into
can calculate the content of cake tea sample glyphosate, careless ammonium phosphine, correlation computations data are as shown in table 8.
Table 8 Precision test result
As can be seen from Table 8, the RSD (n=6)=1.55% that glyphosate detects, the RSD (n=6)=1.88% that grass ammonium phosphine detects, testing result shows favorable reproducibility, the data stabilization of glyphosate of the present invention, careless ammonium phosphine pesticide residue determination method, and precision is good.
More than test shows, the present invention adopts column front derivation-UPLC-ESI
+-MS/MS method determines glyphosate in Tea Samples and careless ammonium phosphine residual quantity, effectively can weaken the impact that matrix detects object, highly sensitive, good linearity, detection limit are low (can reach ng/mL level), quantitative result accurately, stable, reproducible.On the other hand, avoid the derivative competition of glyphosate and careless ammonium phosphine, can Simultaneously test, simple to operate, step is few, consuming time short, analysis speed is fast, detection efficiency is high, and detects that reagent is pollution-free, nontoxic, safety.The present invention is applicable to the detection of solar dried green tea, fermented tea, alternative tea (see NY/T2140-2012 agricultural industry criteria pollution-free food alternative tea), become to sample tea (brick tea, Tuo tea, melon tea, cake tea, loose tea, tea in bag etc.) tea sample glyphosate, careless ammonium phosphine persticide residue.
Claims (10)
1. column front derivation-the UPLC-ESI of tealeaves glyphosate and careless ammonium phosphine persticide residue
+-MS/MS detection method, tealeaves obtains tea sample by GB/T8303, it is characterized in that, comprises the following steps:
A) tea sample ultrapure water and the ultrasonic extraction of methylene chloride or cyclotron oscillation, get supernatant;
B) supernatant is through C
18reverse phase silica gel bonding solid-phase extraction column (C
18pillar) purification be purified liquid;
C) with FMOC-Cl acetone derive liquid, sodium borate buffer salt solusion derives the obtained derivative sample (liquid to be measured) of scavenging solution;
D) liquid to be measured is carried out UPLC-ESI+-MS/MS scanning, scanning result glyphosate, careless ammonium phosphine separately standards calibration curve correct, and draw the mass body volume concentrations c (μ g/mL) of liquid glyphosate to be measured, careless ammonium phosphine residual quantity respectively;
E) tealeaves glyphosate, careless ammonium phosphine residual quantity is calculated respectively with following formula:
In formula:
X-be tealeaves glyphosate or careless ammonium phosphine residual quantity, mg/kg;
The mass body volume concentrations of c-derivative liquid glyphosate to be measured or careless ammonium phosphine, μ g/mL;
V-abovementioned steps 6) in the volume of ultrapure water, mL;
D-be constant 3, the extension rate of extraction and cleaning liquid time derivative;
The quality of m-take tea sample to be measured, g.
2. detection method according to claim 1, is characterized in that, step a) in tea sample, ultrapure water, methylene chloride materials than being 1g:20mL:5mL.
3. detection method according to claim 1 or 2, it is characterized in that, step a) operating process is: tea sample is placed in 80mL tool lid centrifuge tube, add ultrapure water, vortex mixing leaves standstill about 2min, then adds methylene chloride, mixing, ultrasonic extraction or cyclotron oscillation are about 30min, and low speed centrifuge is centrifugal, get supernatant.
4. detection method according to claim 1 or 2 or 3, is characterized in that, step b) in purification run be: get 5mL supernatant and cross C
18purification pillar, discards front 1mL efflux, 2-3mL efflux after collecting, obtained scavenging solution.
5. detection method according to claim 1 or 2 or 3 or 4, is characterized in that, step c) mesoboric acid sodium buffer salt solution concentration is the preferred 50g/L of 30g/L-60g/L(), FMOC-Cl acetone soln concentration is the preferred 20g/L of 10g/L-30g/L().
6., according to the arbitrary described detection method of claim 1 to 5, it is characterized in that, step c) in the derivative time be 2h or more.
7. according to the arbitrary described detection method of claim 1 to 6, it is characterized in that, step c) operating process: by volume 1:1:1 ratio gets scavenging solution successively, sodium borate buffer solution, FMOC-Cl acetone solution be mixed to evenly, room temperature leaves standstill 2h or more, the centrifugal 5min of 4500r/min, get supernatant and cross 0.2 μm of organic filter membrane, obtain derivative sample (liquid to be measured).
8., according to the arbitrary described detection method of claim 1 to 7, it is characterized in that, UPLC-ESI described in step d)
+the parameter determined during-MS/MS scanning is:
1. mobile phase: mobile phase A is 0.1% formic acid-ammonium acetate-water, Mobile phase B is a kind of in pure acetonitrile, pure methyl alcohol, pure acetonitrile, 0.1% formic acid acetonitrile and 50% methanol acetonitrile etc.;
2. Ultra Performance Liquid Chromatography (UPLC) parameter is determined: the liquid phase gradient elution program that glyphosate and careless ammonium phosphine detect is: 5% mobile phase A keeps 0.5min, in 1.5min, mobile phase A becomes 95% from 5%, 95% mobile phase A keeps mobile phase A in 1.5min, 0.5min to become 5% from 95%; Flow velocity 0.3mL/min,
c
18post (specification 1.7 μm, 2.1 × 50mm), sample chamber temperature 15 DEG C, chromatogram column temperature 40 DEG C, sample size 1-6 μ L;
3. the determination of mass spectrum tuner parameters: electric spray ion source (ESI), determines with positive ion mode (ESI
+), multiple-reaction monitoring (MRM), capillary voltage 2.5kv, atomization gas and desolventizing gas are nitrogen, and collision gas is argon gas, desolventizing temperature degree 400 DEG C, desolventizing gas velocity is 800L/Hr, and taper hole gas velocity is 100L/Hr, and ion source temperature is 150 DEG C;
4. the determination of mass spectrometry parameters: determine that the characteristic ion of glyphosate derivative is to the taper hole voltage 25v of 392/88, collides voltage 20v, the characteristic ion of glyphosate derivative, to the taper hole voltage 25v of 392/214, collides voltage 10v; The characteristic ion of grass ammonium phosphine-derivatives, to the taper hole voltage 25v of 404/136, collides voltage 25v, and the characteristic ion of careless ammonium phosphine-derivatives, to the taper hole voltage 25v of 404/208, collides voltage 10v.
9. according to the arbitrary described detection method of claim 1 to 8, it is characterized in that, the acquisition of step d) Plays calibration curve comprises the following steps:
I) the mixed mark of preparation: with glyphosate and careless ammonium phosphine standard stock solution by volume 1:1 obtain the mixed mark that glyphosate and careless ammonium phosphine concentration are respectively 0.003125 μ g/mL, 0.00625 μ g/mL, 0.0125 μ g/mL, 0.025 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL separately;
Ii) mix mark by the method identical with step c) to each concentration to derive, obtained derivative mark liquid;
Iii) under same with derivative sample condition, UPLC-ESI+-MS/MS scanning is carried out to derivative mark liquid, with derivative mixed mark liquid concentration for horizontal ordinate, with the peak area of glyphosate 392/88 ion pair for ordinate draws glyphosate standards calibration curve, with the peak area of careless ammonium phosphine 404/136 ion pair for ordinate draws careless ammonium phosphine standards calibration curve.
10., according to the arbitrary described detection method of claim 1 to 9, it is characterized in that, experimental implementation is as follows:
1) preparation of reagents
1. mobile phase A: accurately take 0.154g (being accurate to 0.001g) anhydrous acetic acid ammonium and be dissolved in appropriate ultrapure water, add 1mL formic acid, be settled to 1000mL with ultrapure water, obtain 0.1% formic acid-ammonium acetate-water, cross 0.2 μm of organic film, be mobile phase A;
Mobile phase B: pure acetonitrile (or with pure methyl alcohol, pure acetonitrile, 0.1% formic acid acetonitrile, 50% methanol acetonitrile etc.) crosses 0.2 μm of organic film, is Mobile phase B;
2. derivative reagent: accurately take 2g (being accurate to 0.001g) FMOC-Cl, be settled to 100mL with acetone, obtains 20g/L FMOC-Cl acetone soln;
3. sodium borate buffer solution: accurately take 5g (being accurate to 0.001g) sodium borate (Na
2b
4o
710H
2o), be settled to 100mL with deionized water, obtain 50g/L sodium borate buffer solution;
4. mixed mark: with micropipettor from 1000 μ g/mL glyphosates, respectively pipette 10 μ L respectively in grass ammonium phosphine standard stock solution and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 1 μ g/mL, the mixed mark of grass ammonium phosphine, then, from the glyphosate of 1 μ g/mL, pipette 1mL in the mixed mark of grass ammonium phosphine and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 0.1 μ g/mL, the mixed mark of grass ammonium phosphine, again from the glyphosate of 0.1 μ g/mL, pipette 5mL in the mixed mark of grass ammonium phosphine and be placed in the brown volumetric flask of 10mL, be settled to scale with ultrapure water and shake up, the i.e. glyphosate of obtained 0.05 μ g/mL, the mixed mark of grass ammonium phosphine, the rest may be inferred, stepwise dilution, concentration can be obtained and be respectively 0.003125 μ g/mL, 0.00625 μ g/mL, 0.0125 μ g/mL, 0.025 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL glyphosate, the mixed mark of grass ammonium phosphine,
2) mark liquid-derived
Get mixed mark, 50g/L sodium borate buffer solution, each 1mL(of 20g/L FMOC-Cl acetone solution by volume 1:1:1 ratio mixing successively) be placed in tool lid centrifuge tube, whirlpool is mixed to evenly, 1min and more than, room temperature leaves standstill and derives to completely, 2h and more than, the centrifugal 5min of low speed centrifuge 4500r/min, gets supernatant and crosses 0.2 μm of organic filter membrane, obtains derivative mark liquid;
3) instrument parameter is determined
1. the selection of mobile phase: with 0.1% formic acid-ammonium acetate-water for mobile phase A, pure acetonitrile is Mobile phase B;
2. Ultra Performance Liquid Chromatography (UPLC) parameter is determined: the liquid phase gradient elution program that glyphosate, careless ammonium phosphine detect is: 5% mobile phase A keeps 0.5min, in 1.5min, mobile phase A becomes 95% from 5%, 95% mobile phase A keeps mobile phase A in 1.5min, 0.5min to become 5% from 95%; Flow velocity 0.3mL/min, Waters Acquity
c18 post (specification 1.7 μm, 2.1 × 50mm), sample chamber temperature 15 DEG C, chromatogram column temperature 40 DEG C, sample size 1-6 μ L;
3. the determination of mass spectrum tuner parameters: electric spray ion source (ESI), determines with positive ion mode (ESI
+), multiple-reaction monitoring (MRM), capillary voltage 2.5kv, atomization gas and desolventizing gas are nitrogen, and collision gas is argon gas, desolventizing temperature degree 400 DEG C, desolventizing gas velocity is 800L/Hr, and taper hole gas velocity is 100L/Hr, and ion source temperature is 150 DEG C;
4. the determination of mass spectrometry method: step 2) derivative mark liquid through Ultra Performance Liquid Chromatography (UPLC) be separated after, first adopt the scanning of Daughters Scan(daughter ion) Mode scans, find out the stable characteristic ion of glyphosate derivative respectively to 392/88,392/214, the stable characteristic ion of careless ammonium phosphine-derivatives is to 404/136,404/208; Under positive ion (ESI+), full scan (MRM) pattern, doing taper hole voltage is 10v, 15v, 20v, 25v, 30v, 35v, 40v, collision voltage is the condition test of 10v, 15v, 20v, 25v, 30v, 35v, 40v, determines the optimum taper hole voltage that glyphosate, careless ammonium phosphine-derivatives characteristic ion are right and collide voltage as shown in the table:
The mass spectrometry parameters of glyphosate, careless ammonium phosphine-derivatives
4) Specification Curve of Increasing
By step 2) derivative mark liquid by step 3) instrument condition determined carries out UPLC-ESI
+-MS/MS test scan, from the analytical test parameter of scanning, qualitative with abundance ratio of retention time, quota ion, qualitative ion, quota ion and qualitative ion etc., be horizontal ordinate with mixed mark derivatives concentration, be that ordinate draws respective standards calibration curve with the peak area of quota ion (glyphosate 392/88 ion pair, careless ammonium phosphine 404/136 ion pair).
5) Tea Samples preparation
Tealeaves by GB/T8303 through grinding, sieving obtained tea sample to be measured; Fermented tea should remove moisture by first low temperature, makes sample be easy to grind;
6) tealeaves detects sample extraction
Accurately taking by step 5) the tea sample (being accurate to 0.001g) that processes is placed in 80mL tool lid centrifuge tube, add ultrapure water, vortex mixing leaves standstill about 2min, add methylene chloride, wherein the materials of tea sample, ultrapure water, methylene chloride are than being 1g:20mL:5mL, mixing, ultrasonic extraction or cyclotron oscillation are about 30min, low speed centrifuge is centrifugal, gets supernatant, obtained extraction supernatant;
7) sample purification is detected
Get step 6) in obtained 5mL extract supernatant and cross C
18purification pillar, discards front 1mL efflux, 2-3mL efflux after collecting, obtained scavenging solution;
8) analyte derivative
Get 1mL scavenging solution obtained in step 7) in 10mL tool lid centrifuge tube, the 20g/L FMOC-Cl acetone adding 1mL successively derives liquid, 50g/L sodium borate buffer salt solusion, carry out and step 2) (vortex mixed is to even for same treatment, 1min and more than, room temperature is placed and is derived to completely, 2h and more than, the centrifugal 5min of low speed centrifuge 4500r/min, get supernatant and cross 0.2 μm of organic phase filter membrane), obtained derivative sample (liquid to be measured);
9) upper machine scanning
By step 8) obtained derivative sample is by step 3) instrument parameter determined carries out UPLC-ESI+-MS/MS scanning, scanning result adopt step 4) in standards calibration curve correct, draw the mass body volume concentrations c (μ g/mL) of liquid glyphosate to be measured, careless ammonium phosphine residual quantity respectively;
10) result calculates: the calculating formula of tealeaves glyphosate, careless ammonium phosphine residual quantity is:
In formula:
X-be tealeaves glyphosate or careless ammonium phosphine residual quantity, mg/kg;
The mass body volume concentrations of c-derivative liquid glyphosate to be measured or careless ammonium phosphine, μ g/mL;
V-abovementioned steps 6) in the volume of ultrapure water, mL;
D-be constant 3, the extension rate of extraction and cleaning liquid time derivative;
The quality of m-take tea sample to be measured, g.
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