CN113155986B - Method for detecting perchlorate and chlorate in panax notoginseng - Google Patents
Method for detecting perchlorate and chlorate in panax notoginseng Download PDFInfo
- Publication number
- CN113155986B CN113155986B CN202011567975.4A CN202011567975A CN113155986B CN 113155986 B CN113155986 B CN 113155986B CN 202011567975 A CN202011567975 A CN 202011567975A CN 113155986 B CN113155986 B CN 113155986B
- Authority
- CN
- China
- Prior art keywords
- perchlorate
- solution
- mobile phase
- chlorate
- ion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a method for detecting perchlorate and chlorate in panax notoginseng, belonging to the technical field of traditional Chinese medicine detection. The invention takes precious Chinese medicinal material pseudo-ginseng as a research object, establishes an HPLC-MS/MS detection method for detecting perchlorate and chlorate in Chinese medicinal materials such as pseudo-ginseng and the like, fills the research blank of perchlorate which is an environmental pollutant in the field of Chinese medicinal materials, provides technical support for the research of perchlorate in Chinese medicinal materials, protects the medication safety of consumers, and promotes and develops great economic and social benefits of Chinese medicinal materials. The detection method of the invention has strong practicability, simple and convenient operation, accurate result and good reproducibility.
Description
Technical Field
The invention relates to a method for detecting perchlorate and chlorate in panax notoginseng, belonging to the technical field of traditional Chinese medicine detection.
Background
The record of the compendium of materia Medica is Shi Yi: ginseng, radix Ginseng and Notoginseng are the most valuable Chinese herbs because they tonify qi the first and tonify blood the first, and both the flavor and action of Notoginseng are the same. Since ancient times, china has a statement that ginseng exists in the north and pseudo-ginseng exists in the south, and pseudo-ginseng is a rare Chinese medicinal material and is a large variety of Chinese medicinal materials next to ginseng. At present, more than 400 Chinese patent medicine varieties using pseudo-ginseng as raw materials exist in China, and 1300 manufacturers involved need 800 kilo of pseudo-ginseng every year, and the sales of pseudo-ginseng-containing Chinese patent medicine preparations reaches 320 billion yuan. The pseudo-ginseng has good hemostatic effect and remarkable hematopoietic function; can strengthen and improve coronary microcirculation, and dilate blood vessel; analgesic, antifatigue, and learning and memory improving effects; anti-inflammatory effects; anti-tumor effect; anti-aging and anti-oxidation effects; reducing blood lipid and cholesterol; has liver protecting effect. Notoginseng radix is dry root and rhizome of Panax notoginseng belonging to Araliaceae, and is prepared by cleaning Notoginseng radix in soil with water, sun drying, and pulverizing into powder for administration. At present, most middle-aged and elderly people paying attention to health preservation take the pseudo-ginseng powder for a long time, and take the pseudo-ginseng powder with warm boiled water once in the morning and at night, but the pollution of perchlorate in tap water, irrigation water, soil, ore, surface water, sludge and other environments is serious. Perchlorate is a persistent environmental pollutant, is extremely difficult to degrade under natural conditions, can interfere the synthesis and secretion of thyroxine, influences normal metabolism, and has serious harm to nerves, reproduction, heredity and the like.
At present, the research on perchlorate pollution in the fields of environment, food, water quality and the like is widely regarded, but the research reports in the field of traditional Chinese medicinal materials are few. The method aims to establish an HPLC-MS/MS detection method for detecting perchlorate and chlorate in traditional Chinese medicinal materials such as pseudo-ginseng by taking a precious traditional Chinese medicinal material pseudo-ginseng as a research object, fills the research blank of perchlorate which is an environmental pollutant in the field of traditional Chinese medicinal materials, provides technical support for the research of perchlorate in traditional Chinese medicinal materials, protects the medication safety of consumers, and promotes and develops great economic and social benefits of traditional Chinese medicinal materials.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the method for detecting the perchlorate and the chlorate in the panax notoginseng, which has the advantages of strong practicability, simple and convenient operation, accurate result and good reproducibility.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for detecting perchlorate and chlorate in pseudo-ginseng, the method comprising the steps of:
(1) Preparation of control solutions:
preparing external standard solution from perchlorate solution and chlorate solution 18 Preparation of O-labeled perchlorate intermediate solutionPreparing standard series working solution by using the external standard solution and the internal standard solution;
(2) Preparing a test solution:
drying Notoginseng radix, pulverizing, and sieving to obtain Notoginseng radix powder; mixing Notoginseng radix powder, alkaline aluminum oxide, graphite carbon black powder, 18 Adding the O-labeled perchlorate intermediate solution and diatomite into an extraction tank, taking water as an extraction solvent, carrying out rapid extraction, transferring an extraction solution after extraction and purification are finished, carrying out constant volume with water, shaking up, filtering, and measuring by a liquid chromatography-tandem mass spectrometer;
(3) And (3) detection: precisely measuring the test solution and the reference solution, and injecting into a liquid chromatogram-tandem mass spectrometer for qualitative detection or quantitative detection.
As a preferred embodiment of the method of the present invention, in the step (1), the preparation of the reference solution is specifically as follows:
preparation of external standard solution: preparing a mixed solution, wherein the concentrations of a perchlorate solution and a chlorate solution in the mixed solution are both 10 mu g/mL; then preparing the mixed solution into a mixed standard intermediate solution with the concentration of 1 mu g/mL;
preparation of internal standard solution: prepared at 10. Mu.g/mL 18 Marking the perchlorate intermediate solution with O, and then adding 18 The O-marked perchlorate intermediate solution is prepared into 50ng/mL 18 Marking perchlorate working solution with O;
standard series working solutions: mixing standard intermediate solution and 18 marking perchlorate working solution with O, preparing standard series working solution with the concentration of 0.4, 1.0, 2.0, 5.0, 10.0, 15.0, 30.0 and 60.0ng/mL by pure water, wherein, 18 the concentration of the O-labeled perchlorate solution is 1-4 ng/mL.
Preferably, the control solution is prepared as follows:
preparing an external standard solution: accurately sucking 100 mu L of perchlorate standard solution and chlorate standard solution into a 10mL volumetric flask respectively, fixing the volume to the scale by using water, preparing 10 mu g/mL mixed solution, and storing at 4 ℃; precisely sucking 1.0mL of the mixed solution into a 10mL volumetric flask, adding water to a constant volume to a scale, preparing a mixed standard intermediate solution with the volume of 1 mu g/mL, and storing at 4 ℃;
preparing an internal standard solution: precise suction 18 Marking 100 mu L of perchlorate solution in a 10mL volumetric flask, adding water to constant volume to scale, and preparing into 1 mu g/mL 18 Marking the perchlorate intermediate solution with O, and storing at 4 ℃; then precisely absorb 18 Marking 500 mu L of perchlorate intermediate solution in a 10mL volumetric flask, adding water to constant volume to scale, and preparing into 50ng/mL 18 Marking perchlorate working solution with O, and storing at 4 ℃;
standard series working solutions: sucking a certain amount of mixed standard intermediate liquid 18 Marking perchlorate working solution with O, preparing standard series working solution with concentration of 0.4, 1.0, 2.0, 5.0, 10.0, 15.0, 30.0 and 60.0ng/mL by pure water, wherein 18 The concentration of the O-marked perchlorate solution is 1-4 ng/mL, and the solution is prepared and used, preferably, 18 the concentration of the O-labeled perchlorate solution is 2ng/mL.
As a preferred embodiment of the method of the present invention, in the step (2), the fast extraction conditions are: the extraction temperature is 80-200 ℃, the volume of the flushing solvent is 30-60%, the heat balance time is 6min, the static extraction time is 3-10 min, the extraction pressure is 1500psi, the cyclic extraction is carried out for 1-2 times, and the nitrogen purging time is 100-200 s; the filter membrane used for filtration was a 0.22 μm polyethersulfone filter membrane.
Preferably, in the step (2), the fast extraction conditions are as follows: the extraction temperature is 120 ℃, the volume of the flushing solvent is 30%, the heat balance time is 6min, the static extraction time is 5min, the extraction pressure is 1500psi, the cyclic extraction is carried out for 2 times, and the nitrogen purging time is 150s.
As a preferred embodiment of the method of the present invention, in the step (2), 0.5 to 5.0g of notoginseng powder is placed in a 34mL extraction cell, and 0.5 to 2.0g of alkaline alumina, 0.2 to 1.0g of graphite carbon black powder and 50 to 200 μ L of 1 μ g/mL-concentration notoginseng powder are added 18 Marking the perchlorate intermediate solution by O, and using diatomite as the rest in the extraction tank.
Preferably, in the step (2), dried pseudo-ginseng is taken and crushed, and is screened by a No. 4 screen for standby; precisely weighing 2g (to 0.01 g) of Notoginseng radix powder sampleThe mixture was placed in a 34mL stainless steel extraction cell (a glass fiber filter membrane was placed in the extraction cell in advance), and then 1g of basic alumina and 0.4g of graphite carbon black powder were sequentially placed therein, followed by addition of 100. Mu.L 18 Marking the perchlorate intermediate solution (1 mu g/mL) by O, and filling the rest part with diatomite; and (3) placing the extraction pool on an extraction disc of a rapid solvent extraction instrument after the extraction pool is assembled, performing rapid extraction and online purification, transferring all liquid into a 50mL volumetric flask after extraction and purification are finished, performing constant volume with water, shaking up, filtering through a 0.22 mu m polyether sulfone filter membrane, and determining by a liquid chromatography-tandem mass spectrometer.
Preferably, the blank matrix solution: taking a negative pseudo-ginseng sample, and obtaining a solution which is a matrix solution according to the operation; blank test: the preparation steps are carried out according to the preparation steps of the test sample except that no sample is added.
As a preferred embodiment of the method of the present invention, in the step (3), the liquid chromatography-tandem mass spectrometer has the following liquid chromatography conditions:
a chromatographic column: a C18 chromatographic column;
mobile phase: taking 0.1-1.0% formic acid water solution as mobile phase A and 0.1-0.5% acetic acid methanol solution as mobile phase B to carry out gradient elution; the conditions of the mobile phase gradient elution are as follows: 0-1 min, A (100-50%) in the mobile phase and B (0-50%) in the mobile phase; 1-5.5 min, mobile phase A (100-50% → 60% -0), mobile phase B (0-50% → 40% -100%); 5.5-9.5 min, A (60-0) in the mobile phase and B (40-100) in the mobile phase; 9.5-10.0 min, mobile phase A (60% -0 → 20% -0), mobile phase B (40% -100% → 80% -100%); 10.0-11.0 min, A (20-0) in the mobile phase and B (80-100) in the mobile phase; 11.0-11.1 min, A (20% -0 → 100% -50%) in the mobile phase and B (80% -100% → 0-50%) in the mobile phase; 11.1-14.0 min, A (100-50%) in the mobile phase and B (0-50%) in the mobile phase;
flow rate: 0.4-0.6 mL/min;
sample injection amount: 10 mu L of the solution;
column temperature: 35 ℃ is carried out.
As a preferred embodiment of the method of the present invention, in the step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometer are as follows:
ionization mode: electrospray ion source, scanning mode: negative ion mode, detection mode: multiple reactive ion monitoring, ionization voltage: -4.0 to-5.0 kV, air curtain gas: 69 to 276kPa, collision gas: 34 to 62kPa, ionization temperature: 250-450 ℃, auxiliary heating gas 1:275 to 482kPa, auxiliary heating gas 2:275 to 482kPa.
As a preferred embodiment of the method of the present invention, in the liquid chromatography conditions, the flow rate is 0.5mL/min, and the chromatographic column is Phenomenex Synergi MAX-RP 80A (4.6 mm. Times.250mm, 4 μm); in the mobile phase of the liquid chromatogram, 0.5% formic acid aqueous solution is taken as a mobile phase A, and 0.2% acetic acid methanol solution is taken as a mobile phase B for gradient elution; the conditions of the mobile phase gradient elution are as follows: 0-1 min, A (85%) in mobile phase and B (15%) in mobile phase; 1-5.5 min, A in mobile phase (85% → 40%), B in mobile phase (15% → 60%); 5.5-9.5 min, A (40%) in the mobile phase and B (60%) in the mobile phase; 9.5-10.0 min, A in mobile phase (40% → 10%), B in mobile phase (60% → 90%); 10.0-11.0 min, A (10%) in the mobile phase and B (90%) in the mobile phase; 11.0-11.1 min, A in mobile phase (10% → 85%), B in mobile phase (90% → 15%); 11.1-14.0 min, A (85%) in mobile phase and B (15%) in mobile phase.
As a preferred embodiment of the method of the present invention, in the mass spectrometry conditions, the ionization voltage: 4.5kV, gas curtain gas: 137kPa, collision gas: 48kPa, ionization temperature: 450 ℃, auxiliary heating gas 1:413kPa, auxiliary heating gas 2:482kPa.
As a preferred embodiment of the method of the present invention, the mass spectrometric conditions wherein the internal standard quantitation of ion pairs comprises 18 O-labeled perchlorate quantitative ion pair, and internal standard qualitative ion pair comprising 18 Marking a perchlorate qualitative ion pair by O, wherein the target quantitative ion pair comprises the perchlorate quantitative ion pair and the chlorate quantitative ion pair, and the target qualitative ion pair comprises the perchlorate qualitative ion pair and the chlorate qualitative ion pair;
multiple reactions of internal standard quantitative ion pairs and internal standard qualitative ion pairsThe mass-to-charge ratio conditions for monitoring the ion scan MRM are: 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate quantitative ion pair is 106.9, and the mass-to-charge ratio of the corresponding daughter ion is 88.9; 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate qualitative ion pair is 108.9, and the mass-to-charge ratio of the corresponding daughter ion is 90.9;
the mass-to-charge ratio conditions of the multi-reaction monitoring ion scanning MRM of the target quantitative ions and the target qualitative ions are as follows: the mass-to-charge ratio of parent ions of the perchlorate quantitative ion pairs is 98.8, and the mass-to-charge ratio of corresponding daughter ions is 83.0; the mass-to-charge ratio of parent ions of the chlorate quantitative ion pairs is 82.9, and the mass-to-charge ratio of corresponding daughter ions is 66.9; the mass-to-charge ratio of parent ions of the perchlorate qualitative ion pairs is 100.8, and the mass-to-charge ratio of corresponding daughter ions is 84.9; the mass-to-charge ratio of the parent ion of the chlorate qualitative ion is 84.9, and the mass-to-charge ratio of the corresponding daughter ion is 68.9.
The mass spectrum parameters of the compounds are shown in table 1.
TABLE 1
* Quantitative ion
As a preferred embodiment of the method of the present invention, in the step (3), the qualitative detection method comprises: measuring a test solution and a reference solution, and judging the existence of perchlorate and chlorate by taking the retention time and the relative abundance of perchlorate and chlorate as qualitative basis; the quantitative detection method comprises the following steps: according to perchlorates and chlorates and 18 and marking the peak area ratio of the perchlorate on the internal standard curve by O, and calculating the content of the perchlorate and the chlorate in the sample.
The qualitative determination of the invention is as follows: and (3) measuring the test sample and the standard series working solution according to the chromatographic mass spectrum conditions, and if the retention time of the detected mass spectrum peak is consistent with that of the standard sample, the relative abundance of each qualitative ion is consistent with that of the standard working solution with equivalent concentration, and the allowable deviation of the relative abundance does not exceed the range specified in the table 2, judging that the corresponding measured object exists in the sample.
TABLE 2
| Relative ion abundance | >50 | 20-50 | 10-20 | <10 |
| Allowable relative deviation% | ±20 | ±25 | ±30 | ±50 |
The quantitative determination of the invention is: the response values of the tested object in the standard series working solution and the test sample solution are both in the linear range of the instrument, the equal volume of the standard series working solution and the test sample solution is added into the sample for measurement, the internal standard method is used for quantification, 18 and marking the perchlorate as an internal standard substance of the perchlorate and chlorate by using O, and if the content of the measured substance in the sample exceeds the linearity of the working curve, diluting the sample as required and then measuring the sample. Blank values need to be deducted from the calculation result. The liquid chromatogram tandem mass spectrogram of the standard solution is shown in figures 1-6, 18 the MRM map of the O-labeled perchlorate quantitative ion pair is shown in figure 1, 18 o-labelled perchloric acidThe MRM map of the root qualitative ion pair is shown in figure 2, the MRM map of the perchlorate quantitative ion pair is shown in figure 3, the MRM map of the perchlorate qualitative ion pair is shown in figure 4, the MRM map of the chlorate quantitative ion pair is shown in figure 5, and the MRM map of the chlorate qualitative ion pair is shown in figure 6.
Compared with the prior art, the invention has the beneficial effects that: the invention takes precious Chinese medicinal material pseudo-ginseng as a research object, establishes an HPLC-MS/MS detection method for detecting perchlorate and chlorate in Chinese medicinal materials such as pseudo-ginseng and the like, fills the research blank of perchlorate which is an environmental pollutant in the field of Chinese medicinal materials, provides technical support for the research of perchlorate in Chinese medicinal materials, protects the medication safety of consumers, and promotes and develops great economic and social benefits of Chinese medicinal materials. The detection method of the invention has strong practicability, simple and convenient operation, accurate result and good reproducibility.
Drawings
FIG. 1 is a drawing of 18 And (3) marking the MRM map of the perchlorate quantitative ion pair by O.
FIG. 2 is 18 And (3) marking the MRM map of the perchlorate qualitative ion pair.
FIG. 3 is an MRM map of perchlorate quantitative ion pairs.
FIG. 4 is an MRM map of perchlorate qualitative ion pairs.
FIG. 5 is an MRM profile of chlorate quantitative ion pairs.
FIG. 6 is an MRM map of chlorate qualitative ion pairs.
Figure 7 is a total ion flow graph of negative samples.
FIG. 8 is a total ion flow graph of perchlorate and chloric acid added to a negative sample.
FIG. 9 is a statistical chart of the recovery rates obtained for different extraction solvents.
FIG. 10 is a bar graph of recovery for different extraction temperatures.
FIG. 11 is a statistical plot of the recovery results of the first purge optimization test.
Fig. 12 is a statistical graph of the recovery results of the second purge material optimization test.
Figure 13 is a total ion flow graph of perchlorate and chlorate as in example 4.
Figure 14 is a total ion flow graph of perchlorate and chlorate as in example 5.
Figure 15 is a total ion flow graph of perchlorate and chlorate as in example 6.
Figure 16 is a total ion flux of perchlorate and chlorate as in example 7.
Figure 17 is a total ion flow graph of perchlorate and chlorate as in example 8.
Figure 18 is a total ion flux of perchlorate and chlorate of example 9.
Figure 19 is a total ion flux of perchlorate and chlorate of example 10.
Figure 20 is a total ion flux of perchlorate and chlorate in example 11.
Figure 21 is a total ion flow graph of perchlorate and chlorate as in example 12.
Figure 22 is a total ion flow graph of perchlorate and chlorate as in example 13.
Figure 23 is a total ion flow graph of perchlorate and chlorate as in example 14.
Figure 24 is a total ion flow graph of perchlorate and chlorate as in example 15.
Figure 25 is a total ion flow graph of perchlorate and chlorate as in example 16.
Figure 26 is a total ion flow graph of perchlorate and chlorate as in example 17.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
The instruments and reagents used in the examples of the present invention were as follows: high performance liquid chromatography tandem mass spectrometer triple Quad5500 (AB, USA), ASE350 type rapid solvent extraction apparatus (Samor Feishell, USA), sartorius CP225D electronic balance, perchlorate standard solution (0.999 g/ml, inorganic vents, MC-CLOX 663048), chlorate standard solution (0.999 g/ml, inorganic vents, M2-CLOX660055, USA), 18 O-Label perchlorate solution (100. Mu.g/ml, cambridge, USA)Isotope Laboratories SDFF-012), formic acid (chromatographically pure, macklin), acetic acid (chromatographically pure, codow), methanol (chromatically pure, honeywell), acetonitrile (chromatographically pure, honeywell), alumina (shanghai pentatricompound chemical agents ltd), diatomaceous earth (Thermo Scientific), graphite black (shanghai ann spectral), and experimental water were each drones' drinking water.
Example 1
A method for detecting perchlorate and chlorate in pseudo-ginseng, the method comprising the steps of:
(1) Preparation of control solutions:
preparing an external standard solution: precisely sucking 100 mu L of each of the perchlorate standard solution and the chlorate standard solution into a 10mL volumetric flask, adding water to a constant volume to a scale, preparing a 10 mu g/mL mixed solution, and storing at 4 ℃; precisely sucking 1.0mL of the mixed solution into a 10mL volumetric flask, adding water to a constant volume to a scale, preparing a mixed standard intermediate solution with the volume of 1 mu g/mL, and storing at 4 ℃;
preparing an internal standard solution: precise suction 18 Marking 100 mu L of perchlorate solution in a 10mL volumetric flask, adding water to constant volume to scale, and preparing into 1 mu g/mL 18 Marking the perchlorate intermediate solution with O, and storing at 4 ℃; then precisely suck 18 Marking 500 mu L of perchlorate intermediate solution in a 10mL volumetric flask, adding water to constant volume to scale, and preparing into 50ng/mL 18 Marking perchlorate working solution with O, and storing at 4 ℃;
standard series working solutions: sucking a certain amount of mixed standard intermediate liquid 18 Marking perchlorate working solution with O, preparing standard series working solution with concentration of 0.4, 1.0, 2.0, 5.0, 10.0, 15.0, 30.0 and 60.0ng/mL by pure water, wherein 18 The concentration of the O-marked perchlorate solution is 2ng/mL, and the solution is prepared for use;
(2) Preparation of a test solution:
pulverizing dried Notoginseng radix, and sieving with No. 4 sieve; precisely weighing 2g (precisely to 0.01 g) of Notoginseng radix powder sample, placing in 34mL stainless steel extraction cell (a piece of glass fiber filter membrane is placed in the extraction cell in advance), sequentially placing 1g of alkaline alumina, and,0.4g of graphite carbon black powder, then 100. Mu.L of graphite carbon black powder is added 18 Marking the perchlorate intermediate solution (1 mu g/mL) by O, and filling the rest part with diatomite; the extraction pond is placed on an extraction disc of a rapid solvent extraction instrument after being well assembled, rapid extraction and online purification are carried out, and the rapid extraction conditions are as follows: the extraction temperature is 120 ℃, the volume of the flushing solvent is 30%, the heat balance time is 6min, the static extraction time is 5min, the extraction pressure is 1500psi, the cyclic extraction is carried out for 2 times, and the nitrogen purging time is 150s; after extraction and purification are finished, transferring all liquid into a 50mL volumetric flask, performing constant volume with water, shaking up, filtering through a 0.22 mu m polyethersulfone filter membrane, and determining by a liquid chromatography-tandem mass spectrometer;
blank matrix solution: taking a negative pseudo-ginseng sample, and obtaining a solution which is a matrix solution according to the operation; blank test: except that no sample is added, the preparation steps are carried out according to the preparation steps of the test sample;
(3) And (3) detection: precisely measuring the test solution and the reference solution, injecting into a liquid chromatography-tandem mass spectrometer, and performing qualitative detection or quantitative detection;
the liquid chromatography-tandem mass spectrometer has the following liquid chromatography conditions:
a chromatographic column: phenomenex Synergi MAX-RP 80A (4.6 mm. Times.250mm, 4 μm);
mobile phase: performing gradient elution by using 0.5% formic acid aqueous solution as a mobile phase A and using 0.2% acetic acid methanol solution as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0-1 min, A (85%) in mobile phase and B (15%) in mobile phase; 1-5.5 min, A in mobile phase (85% → 40%), B in mobile phase (15% → 60%); 5.5-9.5 min, A (40%) in the mobile phase and B (60%) in the mobile phase; 9.5-10.0 min, A in mobile phase (40% → 10%), B in mobile phase (60% → 90%); 10.0-11.0 min, A (10%) in the mobile phase and B (90%) in the mobile phase; 11.0-11.1 min, A in mobile phase (10% → 85%), B in mobile phase (90% → 15%); 11.1-14.0 min, A (85%) in the mobile phase and B (15%) in the mobile phase;
flow rate: 0.5mL/min;
sample introduction amount: 10 mu L of the solution;
column temperature: 35 ℃;
the mass spectrum conditions of the liquid chromatography-tandem mass spectrometer are as follows:
ionization mode: electrospray ion source, scanning mode: negative ion mode, detection mode: multiple reactive ion monitoring, ionization voltage: 4.5kV, gas curtain gas: 137kPa, collision gas: 48kPa, ionization temperature: 450 ℃, auxiliary heating gas 1:413kPa, auxiliary heating gas 2:482kPa;
under the mass spectrum condition, the internal standard quantitative ion pair comprises 18 O-labeled perchlorate quantitative ion pair, and internal standard qualitative ion pair comprising 18 Marking perchlorate qualitative ion pairs by using O, wherein the target quantitative ion pairs comprise perchlorate quantitative ion pairs and chlorate quantitative ion pairs, and the target qualitative ion pairs comprise perchlorate qualitative ion pairs and chlorate qualitative ion pairs;
the mass-to-charge ratio conditions of the multi-reaction monitoring ion scanning MRM of the internal standard quantitative ion pair and the internal standard qualitative ion pair are as follows: 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate quantitative ion pair is 106.9, and the mass-to-charge ratio of the corresponding daughter ion is 88.9; 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate qualitative ion pair is 108.9, and the mass-to-charge ratio of the corresponding daughter ion is 90.9;
the mass-to-charge ratio conditions of the multi-reaction monitoring ion scanning MRM of the target quantitative ions and the target qualitative ions are as follows: the mass-to-charge ratio of parent ions of the perchlorate quantitative ion pairs is 98.8, and the mass-to-charge ratio of corresponding daughter ions is 83.0; the mass-to-charge ratio of parent ions of the chlorate quantitative ion pairs is 82.9, and the mass-to-charge ratio of corresponding daughter ions is 66.9; the mass-to-charge ratio of parent ions of the perchlorate qualitative ion pairs is 100.8, and the mass-to-charge ratio of corresponding daughter ions is 84.9; the mass-to-charge ratio of the parent ion of the chlorate qualitative ion is 84.9, and the mass-to-charge ratio of the corresponding daughter ion is 68.9;
the qualitative determination is as follows: determining the sample and the standard series working solution according to the above chromatographic mass spectrum conditions, if the retention time of the detected mass spectrum peak is consistent with that of the standard sample, the relative abundance of each qualitative ion is consistent with that of the standard working solution with equivalent concentration, and the allowable deviation of the relative abundance does not exceed the range specified in table 2, determining that the corresponding object exists in the sample,
the quantitative determination was: the response values of the tested object in the standard series working solution and the test sample solution are both in the linear range of the instrument, the equal volume of the standard series working solution and the test sample solution is added into the sample for measurement, the internal standard method is used for quantification, 18 marking perchlorate as an internal standard substance of perchlorate and chlorate by O, and if the content of the measured substance in the sample exceeds the linearity of the working curve, diluting the sample as required and then measuring the sample. Blank values need to be deducted from the calculation result. The liquid chromatogram tandem mass spectrum of the standard solution is shown in figures 1-6.
Example 2 methodological validation
The following methodology was set up and tested on the basis of example 1.
(1) Linear relation
Precisely measuring a proper amount of mixed standard intermediate solution (1 mu g/mL) and 18 the O-labeled perchlorate working solution (50 ng/mL) is subjected to constant volume by pure water, standard series working solutions with the concentrations of 0.4 (detection limit), 1, 2, 5, 10, 15, 30 and 60ng/mL (the concentrations of internal standard substances are all 2 ng/mL) are respectively prepared, 10 mu L of working solution is absorbed, and the solution is determined by adopting a liquid chromatogram-tandem mass spectrometry method. And (3) preparing a standard curve by taking the peak area ratio of each characteristic ion mass chromatographic peak to the corresponding isotope internal standard as a vertical coordinate and the concentration of the reference solution as a horizontal coordinate.
The experimental results are as follows: the linear regression equations obtained after background subtraction of perchlorate and chlorate are y =0.75904x +0.22321 and y =0.15480x +0.02758 respectively, and the correlation coefficient R is 2 0.9988 and 0.9988, respectively. From these results, it can be seen that the perchlorate and chlorate have a good linear relationship in the concentration range of 1.0 to 60ng/mL, and that the correlation coefficient (R) is good 2 ) Are all larger than 0.998. According to the characteristic ion mass chromatographic peak signal-to-noise ratio S/N of more than or equal to 3, the detection limit is 10 mug/kg and the quantitative limit is 25 mug/kg according to related data at home and abroad.
(2) Matrix effect
And (3) evaluating the matrix effect of the established pretreatment method of the HPLC-MS/MS method by adopting a common post-extraction and standard-adding method. By comparing response values of the perchlorate and chlorate in the standard sample matrix and the standard blank solution, not only qualitative analysis can be carried out, but also the influence value (SSE) of matrix effect can be quantitatively analyzed. The procedure of example 1 was followed using the pseudo-ginseng powder reference as a sample to obtain a matrix solution. Pure water and the sample matrix solution are respectively used for diluting into working solution with the concentration being in the same linear relation with the above-mentioned investigation, namely the addition concentration gradients of perchlorate and chlorate are 1, 2, 5, 10, 15, 30 and 60ng/mL (the internal standard solution is 2 ng/mL). And establishing a standard curve by an internal standard method, wherein the ratio of the slope of the standard curve of the sample matrix solution to the slope of the standard curve of the blank solvent is the matrix effect of the sample. The results are shown in Table 3.
TABLE 3
| Compound (I) | Regression equation for matrix solution | Regression equation for blank solution | SSE value% |
| Perchlorate salt | y＝0.74828x+0.66997 | y＝0.75904x+0.22321 | 98.6％ |
| Chlorate salt | y＝0.16098x+0.00288 | y＝0.15480x+0.02758 | 103.9％ |
When the SSE value is between 80% and 120%, the matrix effect is negligible. As can be seen from table 3, the SSE values were substantially in the satisfied range when the perchlorate and chlorate were simultaneously quantitatively detected using the internal standard method.
(3) Accuracy of
After sample pretreatment and optimization of mass spectrum parameter conditions are carried out, a standard addition method is adopted, 3 kinds of perchlorate and chlorate mixed liquor (the concentration of internal standard is 2 ng/mL) with different final concentrations, namely low concentration, medium concentration and high concentration (25, 50 and 250 mu g/kg) are added into a sample to carry out a recovery rate test, each concentration is subjected to a parallel test for 6 times, and the results are shown in a table 4.
And (3) low concentration: a sample solution of low concentration for accuracy test was prepared by precisely aspirating 50. Mu.L of a mixed standard intermediate solution (1. Mu.g/mL) and mixing the resulting solution with 2g of a sample, and the sample recovery rate was calculated by the measurement according to the method in example 1.
Medium concentration: a sample solution of low concentration for accuracy evaluation was prepared by precisely aspirating 100. Mu.L of a mixed standard intermediate solution (1. Mu.g/mL) and mixing the resulting solution with 2g of a sample, and the sample recovery rate was calculated by the following method.
High concentration: a500. Mu.L portion of the mixed standard intermediate solution (1. Mu.g/mL) was precisely aspirated, mixed with 2g of the sample, and subjected to the procedure of example 1 to prepare a low-concentration sample solution for accuracy evaluation.
TABLE 4
(4) Precision degree
And adding the mixed solution of perchlorate and chlorate into the blank extracting solution, wherein the detection limit of perchlorate and chlorate is 0.01mg/kg and the quantification limit is 0.025mg/kg according to the characteristic ion mass chromatographic peak signal-to-noise ratio S/N which is more than or equal to 3 and the quantification limit of S/N which is more than or equal to 10. Under the conditions of sample pretreatment and mass spectrum parameter optimization, the ratio of perchlorate to chlorate is measured for multiple times by selecting the intermediate concentration of 0.25mg/kg, and the result is shown in table 5.
TABLE 5
| Composition (I) | |
|
|
|
|
|
Mean value of | RSD(％) |
| Perchlorate salt | 5.385 | 5.37 | 5.311 | 5.337 | 5.311 | 5.316 | 5.34 | 0.6 |
| Chlorate salt | 1.907 | 1.889 | 1.851 | 1.878 | 1.893 | 1.888 | 1.88 | 1.0 |
(5) Method specificity
The total ion flow graph of the negative sample and the total ion flow graph of the negative sample with perchlorate and chlorate added are respectively shown in fig. 7 and fig. 8. As can be seen from FIGS. 7 and 8, the negative sample did not interfere with the measurement, and the specificity of the method was good.
Example 3 chromatographic Condition optimization
The following conditions were selected and tested on the basis of example 1.
(1) Selection of chromatography columns
This experiment compares IC-Pak TM Anion HR column (4.6 mm. Times.75mm, 6 μm) and Synergi TM MAX-RP 80A chromatography column (4.6 mm. Times.250mm, 4 μm), the mobile phase uses 0.5% formic acid water-0.2% acetic acid methanol. The chromatographic behavior of a mixed solution of perchlorate and chlorate at a concentration of 10ng/ml was compared on different chromatographic columns. As a result, it was found that perchlorate is present in IC-Pak TM The retention time on the Anion HR chromatographic column is about 13min, the sensitivity is low, the peak shape is wide and asymmetric, and the separation degree of the perchlorate peak and the chlorate peak is low. In Synergi TM On the MAX-RP 80A chromatographic column, perchlorate and chlorate are well separated, the peak shape is symmetrical, and the response value is high. Therefore Synergi was used in this experiment TM MAX-RP 80A chromatographic column.
(2) Selection of mobile phase
In the experiment, a 20mmol/L ammonium formate-acetonitrile system, a water-0.05% ammonia water-acetonitrile system and a 0.1% formic acid-methanol system are selected as comparison, and the result shows that when the 20mmol/L ammonium formate-acetonitrile system is used as a mobile phase, the retention time of perchlorate and chlorate is longer, although the peak shape of perchlorate is symmetrical, the peak shape response value of chlorate is smaller, and the separation degree of perchlorate and chlorate is smaller. When a water-0.05% ammonia water acetonitrile system is used as a mobile phase, the peak shapes of perchlorate and chlorate are found to be symmetrical, the retention time is 3.5min, the response values are high, but the separation degrees of the perchlorate and the chlorate are poor, after multiple tests, the mobile phase system is found to have large fluctuation relative to the peak shapes, the response value is high and low, and the retention time is easy to drift. In a 0.1% formic acid-methanol system, the retention time of perchlorate is 11min and the retention time of chlorate is 8min, the peak shapes are symmetrical, the response value is stable and the retention time cannot drift although the response value is not high in a water-0.05% ammonia acetonitrile system. The 0.1% formic acid-methanol system was selected as the mobile phase for comprehensive consideration.
The peak shape can be modified by adding acid in the mobile phase, and in order to make the peak shape more symmetrical, a proper amount of acetic acid can be added in methanol. After determining the formic acid water-methanol system as the mobile phase, different concentrations of formic acid and acetic acid methanol were examined. Experiments compared a 0.2% aqueous formic acid-0.2% acetic acid methanol system, a 0.2% aqueous formic acid-0.5% acetic acid methanol system, and a 0.5% aqueous formic acid-0.2% acetic acid methanol system. The result shows that the retention time of perchlorate and chlorate in a 0.2 percent formic acid water-0.2 percent acetic acid methanol system is respectively 10.4min and 9.3min, the peak shape is symmetrical, and the response value is higher. In the 0.2% aqueous formic acid-0.5% acetic acid methanol system, the peak shape is symmetrical, the retention time is basically the same as that of the 0.2% aqueous formic acid-0.2% acetic acid methanol system, but the response value of the miscellaneous peak is increased. The retention time of perchlorate and chlorate in a 0.5 percent formic acid water-0.2 percent acetic acid methanol system is respectively 8.4min and 7.6min, which is shorter than that of a 0.2 percent formic acid water-0.2 percent acetic acid methanol system, and the system has symmetrical peak shape and higher sensitivity. The 0.5% aqueous formic acid-0.2% methanolic acetic acid system was selected as the mobile phase for this experiment.
(3) Selection of extraction solvent
Perchlorate and chlorate are generally easy to dissolve in water, and according to the properties, 4 extraction solvents of water, 0.2 percent acetic acid water, acetonitrile-water (1:1) and acetonitrile-0.2 percent acetic acid water (1:1) are selected for comparison, and the recovery rate results obtained by extracting samples by the 4 extraction solvents are shown in figure 9, and as can be seen from figure 9, when water is taken as the extraction solvent, the recovery rates of perchlorate and chlorate are optimal, and the method selects water as the extraction solvent.
(4) Selection of temperature for rapid extraction apparatus
Taking the extraction solvent as pure water, the volume of the washing solvent is 30%, the heat balance time is 6min, the static extraction time is 5min, the extraction pressure is 1500psi, the circulation extraction is carried out for 2 times, the fixed conditions are adopted, respectively sampling is carried out, and 1 mu g/mL 500 mu L of mixed standard intermediate solution and 18 o-labeled perchlorate intermediate solution (1. Mu.g/Ml, 100. Mu.l) was mixed with 100. Mu.l of perchlorate (final perchlorate/chlorate concentration: 250. Mu.g/kg), extracted at 80, 100, 120 and 150 ℃ respectively in accordance with the procedure of example 1, and the recovery was calculated by measuring the contents of perchlorate and chlorate in the extract, and the results are shown in Table 6 and FIG. 10.
TABLE 6
| Composition (I) | Temperature (. Degree.C.) | Recovery (%) |
| |
80 | 68 |
| |
80 | 117 |
| |
100 | 82 |
| |
100 | 92 |
| |
120 | 94 |
| |
120 | 91 |
| |
120 | 115 |
| |
120 | 53 |
As can be seen from the table, the recovery rate of perchlorate and chlorate at a certain temperature is increased along with the increase of the extraction temperature, but the recovery rate of chlorate at 150 ℃ is reduced, so that the extraction temperature of 120 ℃ is considered to be the extraction temperature of the experiment.
(5) Selection of purification materials
The method considers four purifying materials of aluminum oxide, C18, PSA and GCB and the combination mode of the purifying materials, 1g of each purifying material is added, the operation is carried out according to 1.4 steps, the result shows that a large amount of white bubbles are generated in an extraction collecting bottle added with the C18, and the on-machine result shows that the test solution chlorate added with the C18 has a bimodal phenomenon; both PSA and GCB are affected by adsorbed pigments, and the liquid to which GCB is added is relatively clear in appearance of the extract, while the liquid to which PSA is added is relatively cloudy, and the recovery results are shown in FIG. 11. Then we have performed a second cleaning material optimization test, selecting two materials of alumina and GCB, and examining the amounts of the cleaning materials, wherein the amounts of alumina added are 1g, 2g and 3g, respectively, and GCB added is 0.4g, 0.8g and 1.2g, and the combination of these amounts is clear in appearance, the recovery results are shown in fig. 12, and we finally select the cleaning mode of alumina 1g and GCB0.4 g.
Example 4
Example 4 differs from example 1 in that: in the step (2), the amount of the notoginseng powder is 0.5g, the amount of the basic alumina is 0.5g, and the amount of the graphite carbon black powder is 0.2g, and the rest methods are the same as those in the example 1. The total ion flow of perchlorate and chlorate in this example is shown in figure 13 and the retention times are shown in table 7.
TABLE 7
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.76(8.08) | 1.023e+04 |
| |
98.8/83.0 | 7.77(8.10) | 1.238e+06 |
| |
100.8/84.9 | 7.77(8.10) | 4.461e+05 |
| |
82.9/66.9 | 7.15(7.44) | 3.087e+05 |
| |
84.9/68.9 | 7.15(7.44) | 1.048e+05 |
Example 5
Example 5 differs from example 1 in that: in the step (2), the amount of the notoginseng powder is 5g, the amount of the basic alumina is 2g, and the amount of the graphite carbon black powder is 1g, and the rest of the method is the same as that of the example 1. The total ion flow of the perchlorate and chlorate in this example is shown in fig. 14, and the retention time is shown in table 8.
TABLE 8
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.83(8.08) | 1.141e+04 |
| |
98.8/83.0 | 7.83(8.10) | 7.373e+05 |
| |
100.8/84.9 | 7.82(8.10) | 2.767e+05 |
| |
82.9/66.9 | 7.19(7.44) | 2.033e+05 |
| |
84.9/68.9 | 7.20(7.44) | 6.812e+04 |
Example 6
Example 6 differs from example 1 in that: in the step (2), the quick extraction conditions are as follows: the extraction temperature was 80 ℃, the volume of the rinsing solvent was 30%, the heat equilibration time was 6min, the static extraction time was 3min, the extraction pressure was 1500psi, the cyclic extraction was performed 2 times, the nitrogen purge time was 100s, and the rest of the procedure was the same as in example 1. The total ion flow of the perchlorate and chlorate in this example is shown in fig. 15, and the retention time is shown in table 9.
TABLE 9
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.91(8.08) | 9.147e+03 |
| |
98.8/83.0 | 7.91(8.10) | 8.341e+05 |
| |
100.8/84.9 | 7.91(8.10) | 3.042e+05 |
| |
82.9/66.9 | 7.27(7.44) | 2.150e+05 |
| |
84.9/68.9 | 7.27(7.44) | 7.462e+04 |
Example 7
Example 7 differs from example 1 in that: in the step (2), the rapid extraction conditions are as follows: the extraction temperature was 200 ℃, the volume of the rinsing solvent was 60%, the heat equilibration time was 6min, the static extraction time was 10min, the extraction pressure was 1500psi, the cyclic extraction was performed 1 time, the nitrogen purge time was 200s, and the rest of the methods were the same as in example 1. The total ion flow of the perchlorate and chlorate in this example is shown in fig. 16, and the retention time is shown in table 10.
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.88(8.08) | 1.253e+04 |
| |
98.8/83.0 | 7.88(8.10) | 9.657e+05 |
| |
100.8/84.9 | 7.88(8.10) | 3.370e+05 |
| |
82.9/66.9 | 7.26(7.44) | 7.682e+04 |
| |
84.9/68.9 | 7.26(7.44) | 2.925e+04 |
Example 8
Example 8 differs from example 1 in that: in the step (3), gradient elution was performed using a 0.1% formic acid aqueous solution as a mobile phase a and a 0.1% acetic acid methanol solution as a mobile phase B, and the rest of the procedure was the same as in example 1. The total ion flow of perchlorate and chlorate in this example is shown in figure 17 and the retention times are shown in table 11.
TABLE 11
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.65(8.08) | 1.056e+02 |
| |
98.8/83.0 | 8.43(8.10) | 1.670e+03 |
| |
100.8/84.9 | 10.58(8.10) | 1.475e+07 |
| |
82.9/66.9 | 9.37(7.44) | 2.897e+06 |
| |
84.9/68.9 | 9.37(7.44) | 1.005e+06 |
Example 9
Example 9 differs from example 1 in that: in step (3), gradient elution was performed using a 1.0% formic acid aqueous solution as mobile phase a and a 0.5% acetic acid methanol solution as mobile phase B, and the rest of the procedure was the same as in example 1. The total ion flow of the perchlorate and chlorate in this example is shown in fig. 18, and the retention time is shown in table 12.
TABLE 12
Example 10
Example 10 differs from example 1 in that: in step (3), the flow rate under liquid chromatography conditions was 0.4mL/min, and the rest of the procedure was the same as in example 1. The total ion flow graph of the perchlorate and chlorate in this example is shown in fig. 19, and the retention time is shown in table 13.
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 8.35(8.08) | 3.300e+01 |
| |
98.8/83.0 | 9.95(8.10) | 1.536e+06 |
| |
100.8/84.9 | 9.95(8.10) | 5.647e+05 |
| |
82.9/66.9 | 9.23(7.44) | 3.202e+05 |
| |
84.9/68.9 | 9.23(7.44) | 1.057e+05 |
Example 11
Example 11 differs from example 1 in that: in step (3), the flow rate under liquid chromatography conditions was 0.6mL/min, and the rest of the procedure was the same as in example 1. The total ion flow of the perchlorate and chlorate in this example is shown in figure 20 and the retention time is shown in table 14.
TABLE 14
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.91(8.08) | 3.302e+01 |
| |
98.8/83.0 | 6.72(8.10) | 1.007e+06 |
| |
100.8/84.9 | 6.72(8.10) | 3.715e+05 |
| |
82.9/66.9 | 6.14(7.44) | 2.009e+05 |
| |
84.9/68.9 | 6.14(7.44) | 6.920e+04 |
Example 12
Example 12 differs from example 1 in that: in the step (1), the raw material is processed, 18 the concentration of the O-labeled perchlorate solution was 1ng/mL, and the rest of the method was the same as in example 1. The total ion flow of perchlorate and chlorate in this example is shown in figure 21, and the retention times are shown in table 15.
Watch 15
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.86(8.08) | 4.377e+03 |
| |
98.8/83.0 | 7.86(8.10) | 9.119e+05 |
| |
100.8/84.9 | 7.86(8.10) | 3.300e+05 |
| |
82.9/66.9 | 7.23(7.44) | 2.580e+05 |
| |
84.9/68.9 | 7.22(7.44) | 8.679e+04 |
Example 13
Example 13 differs from example 1 in that: in the step (1), the step (c), 18 the concentration of the O-labeled perchlorate solution was 4ng/mL, and the rest of the method was the same as in example 1. The total ion flow diagram of the perchlorate and chlorate in this example is shown in FIG. 22, and the retention time is shown in the tableShown at 16.
TABLE 16
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 7.88(8.08) | 2.236e+04 |
| |
98.8/83.0 | 7.88(8.10) | 8.333e+05 |
| |
100.8/84.9 | 7.88(8.10) | 2.946e+05 |
| |
82.9/66.9 | 7.25(7.44) | 2.447e+05 |
| |
84.9/68.9 | 7.24(7.44) | 8.299e+04 |
Example 14
Example 14 differs from example 1 in that: in the step (3), the conditions of the mobile phase gradient elution are as follows: 0-1 min, A (100%) in mobile phase and B (0) in mobile phase; 1-5.5 min, mobile phase A (100% → 60%), mobile phase B (0 → 40%); 5.5-9.5 min, A (60%) in the mobile phase and B (40%) in the mobile phase; 9.5-10.0 min, A in mobile phase (60% → 20%), B in mobile phase (40% → 80%); 10.0-11.0 min, A (20%) in the mobile phase and B (80%) in the mobile phase; 11.0-11.1 min, mobile phase A (20% → 100%), mobile phase B (80% → 0); 11.1 to 14.0min, A (100%) in the mobile phase, B (0) in the mobile phase, and the rest of the procedure was the same as in example 1. The total ion flow graph of the perchlorate and chlorate in this example is shown in fig. 23, and the retention time is shown in table 17.
TABLE 17
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 8.41(8.08) | 2.690e+05 |
| |
98.8/83.0 | 8.41(8.10) | 1.971e+07 |
| |
100.8/84.9 | 8.41(8.10) | 7.236e+06 |
| |
82.9/66.9 | 7.94(7.44) | 1.536e+06 |
| |
84.9/68.9 | 7.94(7.44) | 5.168e+05 |
Example 15
Example 15 differs from example 1 in that: in the step (3), the conditions of the mobile phase gradient elution are as follows: 0-1 min, A (50%) in mobile phase and B (50%) in mobile phase; 1-5.5 min, mobile phase A (50% → 0), mobile phase B (50% → 100%); 5.5-9.5 min, A (0) in the mobile phase and B (100%) in the mobile phase; 9.5-10.0 min, A (0 → 20%) in mobile phase, B (100% → 80%) in mobile phase; 10.0-11.0 min, A (20%) in the mobile phase and B (80%) in the mobile phase; 11.0-11.1 min, A in mobile phase (20% → 50%), B in mobile phase (80% → 50%); 11.1-14.0 min, mobile phase A (50%), mobile phase B (50%), the rest of the method is the same as example 1. The total ion flow of the perchlorate and chlorate in this example is shown in figure 24 and the retention times are shown in table 18.
Watch 18
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 8.38(8.08) | 1.360e+03 |
| |
98.8/83.0 | 6.21(8.10) | 7.114e+06 |
| |
100.8/84.9 | 6.21(8.10) | 2.530e+06 |
| |
82.9/66.9 | 6.20(7.44) | 1.018e+06 |
| |
84.9/68.9 | 6.20(7.44) | 3.469e+05 |
Example 16
Example 16 differs from example 1 in that: in the step (3), the mass spectrum conditions are as follows: ionization mode: electrospray ion source, scanning mode: negative ion mode, detection mode: multiple reactive ion monitoring, ionization voltage: 4.0kV, air curtain gas: 69kPa, collision gas: 34kPa, ionization temperature: 250 ℃, auxiliary heating gas 1:275kPa, auxiliary heating gas 2:275kPa, the rest of the process was the same as in example 1. The total ion flow of perchlorate and chlorate in this example is shown in figure 25 with retention times in table 19.
Watch 19
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 8.05(8.08) | 5.985e+04 |
| |
98.8/83.0 | 8.04(8.10) | 5.182e+06 |
| |
100.8/84.9 | 8.05(8.10) | 1.855e+06 |
| |
82.9/66.9 | 7.41(7.44) | 6.794e+05 |
| |
84.9/68.9 | 7.40(7.44) | 2.279e+05 |
Example 17
Example 17 differs from example 1 in that: in the step (3), the mass spectrum conditions are as follows: ionization mode: electrospray ion source, scanning mode: negative ion mode, detection mode: multiple reactive ion monitoring, ionization voltage: 5.0kV, gas curtain gas: 276kPa, collision gas: 62kPa, ionization temperature: 450 ℃, auxiliary heating gas 1:482kPa, auxiliary heating gas 2:482kPa and the rest of the process was the same as in example 1. The total ion flow of perchlorate and chlorate in this example is shown in fig. 26, with retention times as shown in table 20.
| Target analyte | Mass range | Retention time (min) | Area (cps) |
| GLS IS 2 | 108.9/90.9 | 8.04(8.08) | 5.429e+05 |
| |
98.8/83.0 | 8.04(8.10) | 3.582e+07 |
| |
100.8/84.9 | 8.04(8.10) | 1.392e+07 |
| |
82.9/66.9 | 7.40(7.44) | 3.247e+06 |
| |
84.9/68.9 | 7.40(7.44) | 1.155e+06 |
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A method for detecting perchlorate and chlorate in pseudo-ginseng is characterized by comprising the following steps:
(1) Preparation of control solutions:
preparing external standard solution from perchlorate solution and chlorate solution 18 Marking the perchlorate intermediate solution by O to prepare an internal standard solution, and then preparing a standard series working solution by using the external standard solution and the internal standard solution;
(2) Preparation of a test solution:
drying Notoginseng radix, pulverizing, and sieving to obtain Notoginseng radix powder; mixing Notoginseng radix powder, alkaline aluminum oxide, graphite carbon black powder, 18 Adding the O-labeled perchlorate intermediate solution and diatomite into an extraction tank, taking water as an extraction solvent, carrying out rapid extraction, transferring an extraction solution after extraction and purification are finished, carrying out constant volume with water, shaking up, filtering, and measuring by a liquid chromatography-tandem mass spectrometer;
(3) And (3) detection: precisely measuring the test solution and the reference solution, injecting into a liquid chromatogram-tandem mass spectrometer, and performing qualitative detection and quantitative detection;
the liquid chromatography-tandem mass spectrometer has the following liquid chromatography conditions:
a chromatographic column: a C12 chromatographic column;
mobile phase: performing gradient elution by using 0.5% formic acid water solution as a mobile phase A and 0.2% acetic acid methanol solution as a mobile phase B; the conditions of the mobile phase gradient elution are as follows: 0 to 1min, wherein the content of the mobile phase A is 85 percent, and the content of the mobile phase B is 15 percent; 1 to 5.5min, wherein A in the mobile phase is 85% → 40%, and B in the mobile phase is 15% → 60%;5.5 to 9.5min, wherein the content of A in the mobile phase is 40 percent, and the content of B in the mobile phase is 60 percent; 9.5 to 10.0min, A in the mobile phase is 40% → 10%, B in the mobile phase is 60% → 90%; 10.0-11.0 min, 10% of A in the mobile phase and 90% of B in the mobile phase; 11.0 to 11.1min, 10% of A in the mobile phase is → 85%, and 90% of B in the mobile phase is → 15%; 11.1-14.0 min, wherein A in the mobile phase is 85 percent, and B in the mobile phase is 15 percent;
flow rate: 0.5mL/min;
sample introduction amount: 10. mu L;
column temperature: at 35 deg.c.
2. The method of claim 1, wherein in step (1), the control solution is prepared by:
preparation of external standard solution: preparing a mixed solution, wherein the concentrations of a perchlorate solution and a chlorate solution in the mixed solution are both 10 mu g/mL; then preparing the mixed solution into a mixed standard intermediate solution with the concentration of 1 mu g/mL;
preparation of internal standard solution: the preparation was 10. Mu.g/mL 18 Marking the perchlorate intermediate solution with O, and then adding 18 The O-marked perchlorate intermediate solution is prepared into 50ng/mL 18 Marking perchlorate working solution with O;
standard series working solutions: mixing standard intermediate solution and 18 marking the perchlorate working solution by O, preparing the perchlorate working solution into standard series working solution with the concentration of 0.4, 1.0, 2.0, 5.0, 10.0, 15.0, 30.0, 60.0ng/mL by pure water, wherein, 18 the concentration of the O-labeled perchlorate solution is 1 to 4ng/mL.
3. The method of claim 1, wherein in step (2), the fast extraction conditions are: the extraction temperature is 80 to 200 ℃, the volume of the flushing solvent is 30 to 60 percent, the heat balance time is 6min, the static extraction time is 3 to 10min, the extraction pressure is 1500psi, the circulating extraction is performed for 1~2 times, and the nitrogen purging time is 100 to 200s; the filter membrane used for filtration was a 0.22 μm polyethersulfone filter membrane.
4. The method according to claim 1, wherein in the step (2), 0.5 to 5.0g of pseudo-ginseng powder is put into a 34mL extraction pool, 0.5 to 2.0g of alkaline alumina, 0.2 to 1.0g of graphite carbon black powder and 50 to 200 μ L of 1 μ g/mL of graphite carbon black powder are added 18 Marking the perchlorate intermediate solution by O, and the balance in the extraction tank is diatomite.
5. The method of claim 1, wherein in step (3), the mass spectrometry conditions of the liquid chromatography-tandem mass spectrometer are as follows:
ionization mode: electrospray ion source, scanning mode: negative ion mode, detection mode: multiple reactive ion monitoring, ionization voltage: -4.0 to-5.0 kV, air curtain gas: 69 to 276kPa, collision gas: 34 to 62kPa, ionization temperature: 250 to 450 ℃, auxiliary heating gas 1:275 to 482kPa, auxiliary heating gas 2:275 to 482kPa.
6. The method of claim 1, wherein the liquid chromatography conditions are conditions in which the column is Phenomenex synergy MAX-RP 80A, with a specification of 4.6mm x 250mm,4 μm.
7. The method of claim 5, wherein, in the mass spectrometry condition, the ionization voltage: 4.5kV, air curtain gas: 137kPa, collision gas: 48kPa, ionization temperature: 450 ℃, auxiliary heating gas 1:413kPa, auxiliary heating gas 2:482kPa.
8. The method of claim 5, wherein quantifying ion pairs within the mass spectrometry conditions comprises quantifying ion pairs using an internal standard 18 O-labeled perchlorate quantitative ion pair, and internal standard qualitative ion pair comprising 18 Marking a perchlorate qualitative ion pair by O, wherein the target quantitative ion pair comprises the perchlorate quantitative ion pair and the chlorate quantitative ion pair, and the target qualitative ion pair comprises the perchlorate qualitative ion pair and the chlorate qualitative ion pair;
the mass-to-charge ratio conditions of the internal standard quantitative ion pair and the internal standard qualitative ion pair in the multi-reaction monitoring ion scanning MRM are as follows: 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate quantitative ion pair is 106.9, and the mass-to-charge ratio of the corresponding daughter ion is 88.9; 18 the mass-to-charge ratio of the parent ion of the O-labeled perchlorate qualitative ion pair is 108.9, and the mass-to-charge ratio of the corresponding daughter ion is 90.9;
the mass-to-charge ratio conditions of the multi-reaction monitoring ion scanning MRM of the target quantitative ions and the target qualitative ions are as follows: the mass-to-charge ratio of parent ions of the perchlorate quantitative ion pairs is 98.8, and the mass-to-charge ratio of corresponding daughter ions is 83.0; the mass-to-charge ratio of parent ions of the chlorate quantitative ion pairs is 82.9, and the mass-to-charge ratio of corresponding daughter ions is 66.9; the mass-to-charge ratio of parent ions of the perchlorate qualitative ion pairs is 100.8, and the mass-to-charge ratio of corresponding daughter ions is 84.9; the mass-to-charge ratio of the parent ion of the chlorate qualitative ion is 84.9, and the mass-to-charge ratio of the corresponding daughter ion is 68.9.
9. The method of claim 1, wherein in the step (3), the qualitative detection method comprises: measuring a test solution and a reference solution, and judging the existence of perchlorate and chlorate by taking the retention time and the relative abundance of perchlorate and chlorate as qualitative basis; the quantitative detection method comprises the following steps: according to perchlorates and chlorates and 18 and marking the peak area ratio of the perchlorate on the internal standard curve by O, and calculating the content of the perchlorate and the chlorate in the sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011567975.4A CN113155986B (en) | 2020-12-25 | 2020-12-25 | Method for detecting perchlorate and chlorate in panax notoginseng |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011567975.4A CN113155986B (en) | 2020-12-25 | 2020-12-25 | Method for detecting perchlorate and chlorate in panax notoginseng |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113155986A CN113155986A (en) | 2021-07-23 |
| CN113155986B true CN113155986B (en) | 2023-01-03 |
Family
ID=76878075
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011567975.4A Active CN113155986B (en) | 2020-12-25 | 2020-12-25 | Method for detecting perchlorate and chlorate in panax notoginseng |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113155986B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115480019B (en) * | 2022-10-14 | 2023-08-18 | 国贸食品科学研究院有限公司 | Method for measuring chlorate in Chinese yam |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103822814A (en) * | 2014-03-14 | 2014-05-28 | 北京市疾病预防控制中心 | QuEChERS extraction method for poisonous substance extraction and application of QuEChERS extraction method |
| CN106018608A (en) * | 2016-06-16 | 2016-10-12 | 国家烟草质量监督检验中心 | Method for determining perchlorate in tipping paper for cigarettes through accelerated solvent extraction-liquid chromatography-tandem mass spectrometry method |
-
2020
- 2020-12-25 CN CN202011567975.4A patent/CN113155986B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103822814A (en) * | 2014-03-14 | 2014-05-28 | 北京市疾病预防控制中心 | QuEChERS extraction method for poisonous substance extraction and application of QuEChERS extraction method |
| CN106018608A (en) * | 2016-06-16 | 2016-10-12 | 国家烟草质量监督检验中心 | Method for determining perchlorate in tipping paper for cigarettes through accelerated solvent extraction-liquid chromatography-tandem mass spectrometry method |
Non-Patent Citations (4)
| Title |
|---|
| Approaches to sample pretreatment in the determination of perchlorate in real world samples;R. Slingsby 等;《Analytica Chimica Acta》;20060221(第567期);第26-32页 * |
| Perchlorate and chlorate in dietary supplements and flavor enhancing ingredients;Shane A. Snyder 等;《Analytica Chimica Acta》;20060316(第567期);第57-65页 * |
| 分散固相萃取－超高效液相色谱－串联质谱法测定茶叶中的高氯酸盐;彭西甜 等;《分析科学学报》;20200430;第36卷(第2期);第275-279页 * |
| 高效液相色谱串联质谱法测定牛奶中的高氯酸盐;刘艳英 等;《分析测试学报》;20110731;第30卷(第7期);第821-824页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113155986A (en) | 2021-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Global characterization of neutral saccharides in crude and processed Radix Rehmanniae by hydrophilic interaction liquid chromatography tandem electrospray ionization time-of-flight mass spectrometry | |
| CN111879887B (en) | Radix astragali medicinal material and detection method and application of components in preparation thereof | |
| Li et al. | An evaluation of a single-step extraction chromatography separation method for Sm–Nd isotope analysis of micro-samples of silicate rocks by high-sensitivity thermal ionization mass spectrometry | |
| CN109696499B (en) | High-sensitivity determination method for nitrosamine in water based on high-resolution mass spectrum | |
| CN113295797A (en) | Method for rapidly detecting ethyl carbamate in white spirit based on ultra-high performance liquid chromatography combined high-resolution mass spectrometry | |
| CN113155986B (en) | Method for detecting perchlorate and chlorate in panax notoginseng | |
| CN110455961B (en) | High-flux detection method for multiple components in health-care wine | |
| CN116429943A (en) | Quality analysis method of traditional Chinese medicine radix scutellariae based on dual-wavelength equal-baseline differential fusion map | |
| CN111766317A (en) | Method for measuring GS-441524 content in preparation by using polyethylene glycol and water | |
| CN109632997B (en) | Extraction and determination method of liliflorin B in lily medicinal material | |
| CN116183805B (en) | Method for detecting and evaluating components of mulberry chrysanthemum cold granules | |
| CN110609106B (en) | Method for detecting 11 organophosphorus pesticide residues in white paeony root by using ultra-performance liquid chromatography-mass spectrometry technology | |
| CN108426965B (en) | Method for detecting isomaltose, isomaltotriose, maltose and panose in yellow wine | |
| CN110333308B (en) | Method for simultaneously determining NNAL and cotinine in urine with high sensitivity and accuracy | |
| CN110927269A (en) | Method for quantitatively detecting DL-2-hydroxy-4-methyl ethyl valerate in wine | |
| CN107102087B (en) | Method for detecting contents of various organic acids in coptis chinensis by ion chromatography | |
| CN112903836B (en) | Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder | |
| CN114646690B (en) | Detection method for chemical components in immature bitter orange, longstamen onion bulb and cassia twig decoction and fingerprint establishment method | |
| CN110274980B (en) | New distinguishing and identifying method for mountain under forest to participate in garden ginseng | |
| CN111337611A (en) | Method for detecting malachite green, leucomalachite green, crystal violet and leucocyte crystal violet in aquatic products | |
| Yao et al. | Ultra-sensitive speciation analysis of inorganic platinum-chloride complexes in platinum-based drugs by HPLC-ICP-MS | |
| CN111796035A (en) | LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration | |
| CN112345683A (en) | LC-MS simultaneous determination of ligustrazine and aspirin in blood plasma | |
| CN111413439A (en) | Method for determining metformin in blood plasma by rapid hydrophilic interaction chromatography-tandem mass spectrometry | |
| CN109324139A (en) | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |