CN118348137A - Method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma - Google Patents
Method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma Download PDFInfo
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- CN118348137A CN118348137A CN202410286910.4A CN202410286910A CN118348137A CN 118348137 A CN118348137 A CN 118348137A CN 202410286910 A CN202410286910 A CN 202410286910A CN 118348137 A CN118348137 A CN 118348137A
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- glufosinate
- glyphosate
- diquat
- paraquat
- blood plasma
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- 210000002381 plasma Anatomy 0.000 title claims abstract description 58
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 239000005630 Diquat Substances 0.000 title claims abstract description 54
- 239000005562 Glyphosate Substances 0.000 title claims abstract description 54
- SYJFEGQWDCRVNX-UHFFFAOYSA-N diquat Chemical compound C1=CC=[N+]2CC[N+]3=CC=CC=C3C2=C1 SYJFEGQWDCRVNX-UHFFFAOYSA-N 0.000 title claims abstract description 54
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 229940097068 glyphosate Drugs 0.000 title claims abstract description 54
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 title claims abstract description 54
- 239000005561 Glufosinate Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 150000002500 ions Chemical class 0.000 claims abstract description 29
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 4
- 230000006920 protein precipitation Effects 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 14
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000007921 spray Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- ADCGAPKUMAQOLJ-UHFFFAOYSA-N azane;formic acid Chemical compound N.OC=O.OC=O ADCGAPKUMAQOLJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- 238000000889 atomisation Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 230000005540 biological transmission Effects 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims 1
- 239000004009 herbicide Substances 0.000 abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 9
- 230000002363 herbicidal effect Effects 0.000 abstract description 6
- 231100000572 poisoning Toxicity 0.000 abstract description 5
- 230000000607 poisoning effect Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000002574 poison Substances 0.000 abstract description 3
- 231100000614 poison Toxicity 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 19
- 238000012544 monitoring process Methods 0.000 description 19
- 230000000694 effects Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 230000000155 isotopic effect Effects 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- XDDAORKBJWWYJS-QZJDPFNNSA-N 2-(phosphonomethylamino)acetic acid Chemical compound O[13C](=O)[13CH2][15NH]CP(O)(O)=O XDDAORKBJWWYJS-QZJDPFNNSA-N 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 235000020354 squash Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 229940054967 vanquish Drugs 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
- G01N2030/3007—Control of physical parameters of the fluid carrier of temperature same temperature for whole column
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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- General Health & Medical Sciences (AREA)
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Abstract
The invention provides a method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma, and relates to the technical field of clinical medicine detection. The method comprises the following specific experimental steps: plasma samples were pre-treated by protein precipitation and liquid-liquid back extraction and analyzed by liquid chromatography tandem mass spectrometry using Thermo AcclaimTrinityQ chromatographic columns. The detection method provided by the invention can effectively detect paraquat, diquat, glufosinate and glyphosate in blood plasma at the same time, does not need to use an ion pair reagent as a mobile phase, is simple and convenient to operate, accurate in result, high in sensitivity and strong in specificity, is suitable for poison screening and quantitative detection of patients with herbicide poisoning in clinic, and has higher clinical application value.
Description
Technical Field
The invention relates to the technical field of clinical medicine detection, in particular to a method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma.
Background
Paraquat (PQ), diquat (DQ), glufosinate (glufosinate), and glyphosate (glyphosate) are water-soluble herbicides widely used in the agricultural field for controlling weeds and increasing crop yield. However, improper use of these herbicides poses serious hazards to human health. The type of herbicide poisoning can be accurately judged in time, and is important for clinical accurate diagnosis and effective treatment. In addition, the determination of the concentration of the herbicide is of great significance to the evaluation of curative effect and prognosis discrimination.
The liquid chromatography tandem mass spectrometry (high performance liquid chromatography-TANDEM MASS spectrometry) has the advantages of high specificity, high sensitivity and the like, and is widely applied to detection of water-soluble herbicides. However, the physical and chemical properties of paraquat, diquat, glufosinate and glyphosate are greatly different, and the paraquat, diquat, glufosinate and glyphosate have the characteristics of strong polarity, low volatility, different ionic forms and the like, and are difficult to analyze simultaneously under the same detection method. The use of ion pair reagents as mobile phases can realize simultaneous detection of four water-soluble herbicides, however, the use of ion pair reagents can cause mass spectrum pollution, generate ion inhibition to remarkably reduce the sensitivity of mass spectrum detection, thereby affecting detection of other subsequent projects and being difficult to popularize and apply in clinical detection. Therefore, it is necessary to establish a simple, convenient and rapid method without using ion pair reagents to detect paraquat, diquat, glufosinate and glyphosate in blood plasma simultaneously, which has important significance for practical clinical application and related toxicology studies.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a detection method of paraquat, diquat, glufosinate and glyphosate in blood plasma, solves the dilemma that the simultaneous detection of four herbicides needs to use ion pair reagents as mobile phases, is suitable for poison screening and quantitative detection of patients with herbicide poisoning in clinic, and has important significance for clinical accurate diagnosis and treatment, curative effect evaluation and prognosis judgment.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma, the method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma comprising:
Plasma samples were pre-treated by protein precipitation and liquid-liquid back extraction and analyzed by liquid chromatography tandem mass spectrometry using Thermo AcclaimTrinityQ chromatographic columns.
Further, the method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma comprises the following specific experimental steps:
Step 1, quantitatively taking a plasma sample, adding an internal standard solution, adding acetonitrile, uniformly mixing by vortex, and centrifuging to obtain a supernatant;
step 2, adding dichloromethane into the supernatant in the step 1, oscillating, centrifuging, and taking the supernatant to obtain a solution to be detected;
And 3, detecting the solution to be detected in the step 2 by using a liquid chromatography tandem mass spectrometry to obtain mass spectrometry detection data of paraquat, diquat, glufosinate-ammonium, glyphosate and corresponding internal standards in blood plasma, and calculating the content of the paraquat, diquat, glufosinate-ammonium and glyphosate.
Preferably, the volume ratio of the plasma sample, the internal standard solution, the acetonitrile solution and the dichloromethane solution is 180-220:15-25:550-650:900-1100.
Preferably, the chromatographic column is Thermo AcclaimTrinityQ1 mixed chromatographic column, and the specification is 3X 100mm and 3 μm.
Preferably, the mobile phase A is an ammonium formate-formic acid aqueous solution, and the mobile phase B is an acetonitrile solution; in the ammonium formate-formic acid aqueous solution, the concentration of ammonium formate is 45-55mmol/L, and the concentration of formic acid is 0.45% -0.55%, and the gradient elution is carried out.
Preferably, the mass spectrometry conditions include: the ion source is an electrospray ion source, the detection modes of paraquat, diquat and glufosinate are positive ion modes, the detection mode of glyphosate is negative ion modes, the sheath gas is 60arb, the auxiliary gas is 16arb, the CID gas is 1.5mTorr, the atomization temperature is 450 ℃, the temperature of an ion transmission tube is 350 ℃, the positive ion spray voltage is 2500V, and the negative ion spray voltage is-2000V.
The invention provides a method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma.
The beneficial effects are as follows:
1. according to the invention, thermo AcclaimTrinityQ mixed chromatographic columns are used, the simultaneous detection of paraquat, diquat, glufosinate-ammonium and glyphosate can be realized without using ion pair reagents as mobile phases, the detection of other mass spectrum items is not influenced, the popularization and application in clinical detection can be realized, and the method has higher clinical application value.
2. The invention establishes the detection method of paraquat, diquat, glufosinate and glyphosate in blood plasma, and the method has the advantages of simple pretreatment process, high detection speed and high sensitivity, is suitable for poison screening and quantitative detection of patients with herbicide poisoning in clinic, and has important significance for clinical accurate diagnosis and treatment, curative effect evaluation and prognosis judgment.
Drawings
FIG. 1 is a typical multi-reaction monitoring chromatogram of paraquat, diquat, glufosinate, glyphosate and their isotopic internal standard in plasma according to an embodiment of the present invention;
wherein A is a multi-reaction monitoring chromatogram of paraquat in blood plasma;
B is a multi-reaction monitoring chromatogram of diquat in blood plasma;
c is a multi-reaction monitoring chromatogram of glufosinate-ammonium in plasma;
d is a multi-reaction monitoring chromatogram of glyphosate in plasma;
e is a multi-reaction monitoring chromatogram of paraquat-d 8 in blood plasma;
f is a multi-reaction monitoring chromatogram of diquat-d 4 in blood plasma;
G is a multi-reaction monitoring chromatogram of glufosinate-d 3 in plasma;
h is a multi-reaction monitoring chromatogram of glyphosate- 13C2,15 N in blood plasma;
FIG. 2 is a typical multi-reaction monitoring chromatogram of paraquat, diquat, glufosinate, glyphosate and their isotopic internal standard in blank plasma according to an embodiment of the invention;
wherein A is a multi-reaction monitoring chromatogram of paraquat in blank plasma;
b is a multi-reaction monitoring chromatogram of diquat in blank plasma;
c is a multi-reaction monitoring chromatogram of glufosinate-ammonium in blank plasma;
d is a multi-reaction monitoring chromatogram of glyphosate in blank plasma;
e is a multi-reaction monitoring chromatogram of paraquat-d 8 in blank plasma;
f is a multi-reaction monitoring chromatogram of diquat-d 4 in blank plasma;
g is a multi-reaction monitoring chromatogram of glufosinate-d 3 in blank plasma;
h is a multi-reaction monitoring chromatogram of glyphosate- 13C2,15 N in blank plasma;
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment one:
The embodiment of the invention provides a method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma, which comprises the following specific experimental steps:
1. Standard substance and reagent
Standard substance: paraquat (not less than 98%), diquat (not less than 95%), glufosinate (not less than 98%) and glyphosate (not less than 98%) are all purchased from Sigma-Aldrich, USA; paraquat-d 8, diquat-d 4, glufosinate-d 3, and glyphosate- 13C2,15 N are all available from Toronto RESEARCH CHEMICALS, canada. Reagent: acetonitrile (Thermo FISHER SCIENTIFIC, chromatographically pure), dichloromethane (Thermo FISHER SCIENTIFIC, chromatographically pure), ammonium formate (Sigma-Aldrich, chromatographically pure), formic acid (Sigma-Aldrich, chromatographically pure).
2. Sample pretreatment
200. Mu.L of plasma was taken, 20. Mu.L of internal standard working solution was added, 600. Mu.L of acetonitrile was added, and the mixture was vortexed and homogenized for 1min, and 12000g was centrifuged for 5min. Taking 720 mu L of supernatant, adding 1mL of dichloromethane, mixing uniformly by vortex for 5min, centrifuging 12000g for 5min, and taking 100 mu L of supernatant to be detected.
3. Preparation of a Standard Curve
A certain amount of standard was added to the blank plasma to obtain 8 standard additive plasma samples (paraquat and diquat concentrations of 5, 10, 20, 50, 100, 200, 500 and 1000ng/mL, and glufosinate and glyphosate concentrations of 25, 50, 100, 250, 500, 1000, 2500 and 5000 ng/mL) and the samples were pre-treated as described in 2 and then tested.
4. Liquid chromatography tandem mass spectrometry detection
4.1 Chromatographic conditions
Liquid chromatograph tandem mass spectrometer: thermo Scientific Vanquish UHPLC and Thermo SCIENTIFIC TSQ ALTIS triple quadrupole mass spectrometers (company Thermo Scientific usa); chromatographic column: thermo ACCLAIM TRINITY Q column (3X 100mm,3 μm); column temperature: 35 ℃; mobile phase a: 50mmol/L ammonium formate aqueous solution containing 0.5% formic acid; mobile phase B: acetonitrile; flow rate: 0.5mL/min; gradient elution was used, gradient set as follows: 0min (80% B), 0-4min (80% B to 60% B), 4-4.5min (60% B to 5% B), 4.5-6.5min (5% B), 6.5-7min (5% B to 80% B), 7-8min (80% B); sample injection amount: 10 mu L.
4.2 Mass Spectrometry conditions
Ion source: an electrospray ion source; detection mode of paraquat, diquat and glufosinate: a positive ion mode; mode of glyphosate detection: a negative ion mode; sheath gas: 60arb; auxiliary gas: 16arb; CID gas: 1.5mTorr; atomization temperature: 450 ℃; ion transport tube temperature: 350 ℃; positive ion spray voltage: 2500V; negative ion spray voltage: -2000V. The detailed mass spectrum parameters are shown in table 1.
Table 1 mass spectral parameters of paraquat, diquat, glufosinate, glyphosate and their isotopic internal standard
* Indicating quantitative ion
5. Methodological validation and results
5.1 Selectivity
Typical multi-reaction monitoring chromatograms of paraquat, diquat, glufosinate, glyphosate and their isotopic internal standards in plasma are shown in figure 1. The results show that all the objects to be detected achieve better separation effect under the condition of not using ion pair reagents.
5.2 Linear Range, detection Limit and quantitative Limit
The results of the linear range, detection limit and quantification limit are shown in table 2. The results show that paraquat, diquat, glufosinate and glyphosate all show good correlation (R 2 > 0.999) in the linear range; the detection limit of paraquat and diquat is 1ng/mL, and the quantitative limit is 5ng/mL; the detection limit and the quantitative limit of the glufosinate are 5ng/mL and 25ng/mL respectively; the limit of detection and the limit of quantification of glyphosate were 10ng/mL and 25ng/mL, respectively.
TABLE 2 Linear ranges, detection limits and quantification limits for paraquat, diquat, glufosinate and glyphosate
5.3 Accuracy and precision
Quality control products with low, medium and high concentration levels (paraquat and diquat concentrations of 15, 80 and 800ng/mL and glufosinate and glyphosate concentrations of 75, 400 and 4000 ng/mL) are prepared in a standard and blank plasma mixing mode for precision and accuracy verification. Measuring each concentration level for 5 times, and calculating the daily precision; each concentration level was measured 5 times daily for 3 consecutive days to calculate the daytime precision. The daily precision and accuracy of paraquat, diquat, glufosinate and glyphosate are all less than 10%, and the precision and accuracy of the method are all within acceptable ranges. The results are shown in Table 3.
TABLE 3 daily, daytime precision and accuracy of paraquat, diquat, glufosinate and glyphosate
5.4 Extraction recovery and matrix Effect
Quality control materials with low, medium and high concentration levels (paraquat and diquat concentrations of 15, 80 and 800ng/mL and glufosinate and glyphosate concentrations of 75, 400 and 4000 ng/mL) are prepared in a standard and blank plasma mixing mode, and extraction recovery rate and matrix effect verification are carried out. The following 3 sets of samples were made and tested: (A) Adding a standard substance solution and an internal standard working solution before extracting a blank plasma sample; (B) After the blank plasma sample is extracted, adding a standard substance solution and an internal standard working solution; (C) Adding a standard substance solution and an internal standard working solution into ultrapure water; the extraction recovery and matrix effect were calculated as A/Bx100% and B/Cx100%, respectively. The extraction recovery rate and the matrix effect of paraquat, diquat, glufosinate and glyphosate are all between 85% and 115%, and the paraquat, diquat, glufosinate and glyphosate have good extraction recovery rate and acceptable matrix effect. The results are shown in Table 4.
Table 4 extraction recovery and matrix effect of paraquat, diquat, glufosinate and glyphosate (n=5)
6. Dilution consistency
For clinical samples with concentrations above the linear range, quantitative detection requires verification of dilution consistency. Plasma samples containing paraquat, diquat, glufosinate and glyphosate (paraquat and diquat concentrations of 5000ng/mL, glufosinate and glyphosate concentrations of 25000 ng/mL) were diluted with ultrapure water at different dilution factors (1:5, 1:10, 1:20, 1:50, 1:100). The results show that the precision and accuracy of all dilution factors of paraquat, diquat, glufosinate and glyphosate are less than 15%, and the highest dilution dilutable factor is 1:100. the results are shown in Table 5.
Table 5 dilution consistency verification of paraquat, diquat, glufosinate and glyphosate in plasma (n=5)
7. Sample detection
166 Plasma samples were tested using the methods for testing paraquat, diquat, glufosinate and glyphosate in plasma. Wherein, the diquat is detected in 99 samples, and the concentration range is 12.65-415261.90ng/mL; paraquat is detected in 99 samples, and the concentration range is 21.94-44929.80ng/mL; glufosinate is detected in 16 samples, and the concentration range is 47.05-65354.30ng/mL; glyphosate was detected in 5 samples at a concentration ranging from 292.06 to 473050.10ng/mL. The method can realize popularization and application in clinical detection, and has higher clinical application value. The results are shown in Table 6.
Table 6166 plasma sample test results for patients with herbicide poisoning
* The concentration is expressed as mean.+ -. Standard deviation, the minimum and maximum concentrations are in brackets
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. The method for detecting the paraquat, the diquat, the glufosinate and the glyphosate in the blood plasma is characterized by comprising the following steps of:
Pretreatment of the plasma sample by protein precipitation and liquid-liquid back extraction, analysis by liquid chromatography tandem mass spectrometry using Thermo AcclaimTrinityQ chromatographic column;
The specific experimental steps are as follows:
Step 1, quantitatively taking a plasma sample, adding an internal standard solution, adding acetonitrile, uniformly mixing by vortex, and centrifuging to obtain a supernatant;
step 2, adding dichloromethane into the supernatant in the step 1, oscillating, centrifuging, and taking the supernatant to obtain a solution to be detected;
And 3, detecting the solution to be detected in the step 2 by using a liquid chromatography tandem mass spectrometry to obtain mass spectrometry detection data of paraquat, diquat, glufosinate-ammonium, glyphosate and corresponding internal standards in blood plasma, and calculating the content of the paraquat, diquat, glufosinate-ammonium and glyphosate.
2. The method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma according to claim 1, wherein the volume ratio of the blood plasma sample, the internal standard solution, the acetonitrile solution and the dichloromethane solution is 180-220:15-25:550-650:900-1100.
3. The method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma according to claim 1, wherein said chromatographic column is Thermo AcclaimTrinityQ.times.100 mm,3 μm.
4. The method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma according to claim 1 wherein mobile phase a is an aqueous ammonium formate-formic acid solution and mobile phase B is an acetonitrile solution; in the ammonium formate-formic acid aqueous solution, the concentration of ammonium formate is 45-55mmol/L, and the concentration of formic acid is 0.45% -0.55%, and the gradient elution is carried out.
5. The method for detecting paraquat, diquat, glufosinate and glyphosate in blood plasma according to claim 1, wherein said mass spectrometric conditions comprise: the ion source is an electrospray ion source, the detection modes of paraquat, diquat and glufosinate are positive ion modes, the detection mode of glyphosate is negative ion modes, the sheath gas is 60arb, the auxiliary gas is 16arb, the CID gas is 1.5mTorr, the atomization temperature is 450 ℃, the temperature of an ion transmission tube is 350 ℃, the positive ion spray voltage is 2500V, and the negative ion spray voltage is-2000V.
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