Method for determining residual quantity of chlorate and perchlorate in traditional Chinese medicinal materials

Abstract

The invention relates to a method for measuring chlorate and perchlorate residues in traditional Chinese medicinal materials, which comprises the following steps: placing a traditional Chinese medicinal material sample in a dialysis bag, adding a formic acid aqueous solution, placing in the formic acid aqueous solution, and ultrasonically extracting chlorate and perchlorate residues of the sample, the method comprises the following steps: performing dialysis, purifying by a graphitized carbon black solid-phase extraction small column (optional) and an Ag / H ion exchange small column, separating by adopting an AcclaimTMTrinityTMP1 composite ion exchange chromatographic column, determining by adopting ultrahigh pressure liquid chromatography-tandem mass spectrometry, and quantifying by adopting an internal standard method. The method provided by the invention can effectively eliminate macromolecular organic matters such as pigment, saponin, polypeptide, polysaccharide, protein and the like and anions such as Cl <->, NO2 <->, NO3 <->, PO4 < 3->, SO4 < 2->, oxalate and the like in the traditional Chinese medicinal materials, and has the advantages of high sensitivity, good peak pattern, strong response, accurate result and the like.

Description

Technical Field

[0001] The invention relates to detection and analysis of pollutant content in traditional Chinese medicine, in particular to a method for determining the residual amount of chlorate and perchlorate in traditional Chinese medicine. Background Art

[0002] Chlorate is a byproduct of the disinfection process, while perchlorate is an environmental pollutant. Drinking water, milk, vegetables, fruits and meat may be contaminated by this substance. Chlorate and perchlorate are a class of inorganic chlorine compounds that can damage red blood cells, affect the blood's oxygen transport function, and cause mineral loss. The size, ion radius and charge of chlorate ions are very similar to those of iodide ions. They will compete with iodide ions to bind to the sodium-iodide symporter (NIS), inhibit the intake of iodide ions, affect the synthesis and release of triiodothyronine (T3) and tetraiodothyronine (T4), interfere with normal metabolism, affect the normal physiological function of organisms, and induce pathological changes in thyroid tissue, leading to human metabolic function disorders. After chlorate enters the human body, it will cause hematological reactions, damage red blood cells to affect the blood oxygen transport function, and the International Agency for Research on Cancer has listed chlorate as a moderately toxic compound. The National Academy of Sciences of the United States determined that the no-observed-adverse-effect level of perchlorate for inhibiting thyroid iodide uptake was 7 μg / kg·d; the National Academy of Sciences of the United States and the Environmental Protection Agency jointly stipulated that the reference dose of perchlorate should not exceed 0.7 μg / kg·d; in addition, the Environmental Protection Agency has also established a daily intake of perchlorate of 0.37 μg / kg, and stipulated that the maximum permitted concentration in drinking water is 1.0 μg / L. The European Food Safety Authority stipulates that the reference dose of perchlorate is 0.3 μg / kg·bw / d. On May 25, 2020, the European Commission issued Regulation (EU) 2020 / 685, which revised the maximum residue limit of perchlorate in some foods. People often consume Chinese medicinal materials, especially those that are both food and medicine, which are the basis of traditional Chinese medicine food therapy. Therefore, it is of great significance to establish a method for detecting the residual amount of chlorate and perchlorate in Chinese medicinal materials.

[0003] Due to the special characteristics of Chinese medicinal materials, the extracts of Chinese medicinal materials contain a large amount of organic impurities, such as pigments, saponins, polypeptides, polysaccharides, and proteins; the extracts of Chinese medicinal materials also contain a large amount of inorganic ions, such as Cl - 、NO2 - 、NO3 - PO4 3- 、SO4 2-, oxalate, etc. These substances will interfere with the accuracy of the test. How to effectively remove the interference of organic impurities and inorganic ions and separate and detect chlorate and perchlorate is a challenge currently faced by testing staff. If the interference is not removed thoroughly, it will be difficult to obtain accurate test results. In some cases, during the process of eliminating interference, the sample will block the separation equipment, making the experiment impossible to carry out.

[0004] Chinese patent application CN113155986A discloses a method for detecting perchlorate and chlorate in Panax notoginseng, comprising the following steps: (1) preparation of reference solution: preparing an external standard solution with perchlorate solution and chlorate solution; 18 O-labeled perchlorate intermediate solution to prepare internal standard solution, and then use external standard solution and internal standard solution to prepare standard series working solution; (2) Preparation of test solution: Dry and crush Panax notoginseng to obtain Panax notoginseng powder; Mix Panax notoginseng powder, basic alumina, graphite black powder, 18 O-labeled perchlorate intermediate solution and diatomaceous earth are added to the extraction pool, and the extraction solvent is water for rapid extraction. After the extraction and purification, the extract is transferred, fixed to volume with water, shaken, filtered, and subjected to liquid chromatography-tandem mass spectrometry. (3) Detection: Accurately measure the test solution and the reference solution and inject them into the liquid chromatography-tandem mass spectrometer for qualitative and quantitative detection. The specific qualitative detection method is: determine the test solution and the reference solution, and use the retention time and relative abundance of perchlorate and chlorate as qualitative basis to judge the presence of perchlorate and chlorate; the quantitative detection method is: based on the relative abundance of perchlorate and chlorate and the relative abundance of 18 The peak area ratio of O-labeled perchlorate on the internal standard curve is used to calculate the content of perchlorate and chlorate in the sample. This method is suitable for Panax notoginseng, but has general adaptability to other medicinal materials, which limits the scope of detection objects. Summary of the invention

[0005] The purpose of the present invention is to overcome the existing problems in traditional Chinese medicine and provide a method for determining the residual amounts of chlorate and perchlorate in traditional Chinese medicine. The method uses an extract containing formic acid and ultrasonic extraction, and dialyzes with a dialysis bag, which can effectively eliminate interference. On this basis, a graphitized carbon black solid phase extraction column (only required for color-heavy samples) and an activated Ag / H ion exchange column are combined to eliminate the problem of chromatographic column blockage, thereby obtaining a detection result with high sensitivity, good peak shape and strong response, and the method has extremely high practical value.

[0006] The specific plan is as follows:

[0007] A method for determining the residual amount of chlorate and perchlorate in a traditional Chinese medicine comprises the following steps:

[0008] (1) Preparation of standard working solutions: Dilute the mixed standard solution of chlorate and perchlorate, the chlorate isotope internal standard, and the perchlorate isotope internal standard with water to form a series of standard working solutions. Each standard working solution contains chlorate. -18 O3 and Perchlorate -18 O4;

[0009] (2) Preparing Chinese medicinal material samples: grinding the Chinese medicinal material samples with a grinder, and then mixing them to obtain Chinese medicinal material powder;

[0010] (3) Preparation of Chinese herbal medicine sample solution: placing the Chinese herbal medicine powder in a dialysis bag, wherein the dialysis bag contains an extract solution containing a formic acid aqueous solution and a chlorate solution. -18 O3 and Perchlorate -18 O4 internal standard; then the dialysis bag is placed in a glass tube, an aqueous formic acid solution is added into the glass tube, and ultrasonic extraction and dialysis are performed. After completion, the glass tube is taken out and the clear liquid dialyzed from the glass tube is collected; the clear liquid is passed through an activated Ag / H ion exchange column, the effluent is collected, the effluent is filtered through a membrane, and the filtrate is collected as a sample test solution for ultra-high pressure liquid chromatography-tandem mass spectrometry determination;

[0011] (4) Ultra-high pressure liquid chromatography-tandem mass spectrometry:

[0012] Adopt Acclaim TM Trinity TM The sample solution of step (3) is separated by a P1 composite ion exchange chromatography column, and is measured by ultra-high pressure liquid chromatography-tandem triple quadrupole mass spectrometry, and the quantitative ion pair peak area measurement result of the chlorate and perchlorate mixed standard solution after internal standard correction is Y, and the concentration content of the chlorate and perchlorate mixed standard solution is X, and a standard working curve is drawn, and the content of the chlorate and perchlorate to be measured in the sample is calculated by the internal standard method;

[0013] The chromatographic conditions for analyzing chlorate and perchlorate in step (4) are as follows: Chromatographic column: Acclaim TM Trinity TM P1 composite ion exchange chromatography column; flow rate: 0.5mL / min; column temperature: 35℃; injection volume: 3μL; mobile phase: A is ammonium formate solution, B is acetonitrile, gradient elution, elution procedure is as follows:

[0014] Time, minutes Mobile phase A / volume % Mobile phase B / volume % 0.00 40.0 60.0 0.50 40.0 60.0 3.00 10.0 90.0 7.00 10.0 90.0 8.00 40.0 60.0 10.00 40.0 60.0 .

[0015] Further, step (1) comprises: diluting the mixed standard solution of chlorate and perchlorate, the chlorate isotope internal standard and the perchlorate isotope internal standard with water to form a series of standard working solutions, wherein the concentrations of chlorate are 1.0, 2.0, 5.0, 10.0, 50.0 and 100 ng / mL, and the concentrations of perchlorate are 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 ng / mL, and each standard working solution contains 2.0 ng / mL chlorate as the internal standard. -18 O3 and 0.2 ng / mL perchlorate -18 O4 liquid.

[0016] Furthermore, the particle size of the Chinese medicine powder in step (2) is 0.1-100 microns;

[0017] Optionally, the molecular weight cut-off of the dialysis bag in step (3) is 10000-20000 Daltons, which is cut into small pieces of 10 to 20 cm before use, first washed with 40-70 volume % ethanol, 0.001-0.02 mol / L sodium bicarbonate and 0.001-0.005 mol / L EDTA solution, then boiled with distilled water for 20-40 minutes, left at room temperature, and finally rinsed with distilled water before use;

[0018] Preferably, the dialysis bag is recycled after use, and the method is: the dialysis bag is thoroughly washed with distilled water, and then stored in 40-70 volume % ethanol. When in use, one end is tied tightly with a rubber band or rope and washed with distilled water.

[0019] Furthermore, the concentration of formic acid in the extract in step (3) is 0.05-0.2wt%. Preferably, for 1g of the Chinese medicine powder, 10-15mL of 0.1-0.2wt% formic acid aqueous solution is added, and then 40-50μL of 1-2μg / mL chlorate is added. -18 O3 and 0.1-0.2 μg / mL perchlorate -18 O4 mixed internal standard solution, then place the dialysis bag in a glass tube, and add 10-15mL of 0.1-0.2wt% formic acid aqueous solution into the glass tube.

[0020] Furthermore, in step (3), the time of the ultrasonic extraction is 30 minutes to 60 minutes, the time of the dialysis is 60 minutes to 120 minutes, and the temperature of the ultrasound and the dialysis is 20 to 30 degrees Celsius.

[0021] Furthermore, the clear liquid in step (3) is first subjected to a graphitized carbon black solid phase extraction column to remove color, and then subjected to an activated Ag / H ion exchange column, with the sample flow rate controlled to be less than 1 mL / min; the first 6 mL of the effluent is discarded, the effluent is collected in a test tube, 1 mL of the effluent is taken to pass through a 0.20 μm filter membrane, and the filtrate is collected in an injection bottle;

[0022] Preferably, the specifications of the graphitized carbon black solid phase extraction column are 500 mg, 6 mL, and before use, it is activated with 6 mL of methanol and eluted with 6 mL of 0.1 wt% formic acid aqueous solution for equilibrium;

[0023] Preferably, the specification of the Ag / H ion exchange column is 1 g, 2.5 mL, and it is rinsed with 10 mL of pure water before use, and left to stand for activation for 30 minutes before use.

[0024] Furthermore, the Ag / H ion exchange column is reused, and after each use and between reuses, 20 mL of deionized water is drawn with a disposable syringe and pushed through the column at a flow rate of 2 mL / min for cleaning; when the column adsorbs chloride ions, the filler part that adsorbs chloride ions will turn black, and when the black filler extends to 2 / 3, it is no longer reused.

[0025] Furthermore, the mass spectrometry conditions for analyzing chlorate and perchlorate in step (4) are as follows: scanning mode: multiple reaction monitoring MRM, negative ion mode; ion source: electrospray ion source ESI; nebulizer gas 30 mL / min, heating temperature 550° C., ionization voltage -4500 V; quantitative ion pair, qualitative ion pair and collision energy are as follows:

[0026]

[0027] * is the quantitative ion pair.

[0028] Furthermore, the Chinese medicinal materials include at least one of red ginseng, angelica, rehmannia root, coptis root, cooked rehmannia root, radish seed, chuanxiong, pinellia, rhubarb, panax notoginseng, atractylodes, ginseng, scutellaria, eucommia and poria.

[0029] Furthermore, in the method, chlorate has a linear relationship in the range of 1 to 100 ng / mL and perchlorate in the range of 0.05 to 10.0 ng / mL, and the correlation coefficient r 2All of them were greater than 0.99, the detection limit S / N=3 was 6.0μg / kg for chlorate and 1.0μg / kg for perchlorate, the quantification limit S / N≥10 was 18.0μg / kg for chlorate and 3.0μg / kg for perchlorate; the samples of chlorate and perchlorate were subjected to the addition recovery experiment, the recovery rate of chlorate was 70.7%~86.9%, the relative standard deviation was 4.2%~7.4%, the recovery rate of perchlorate was 70.0%~118.3%, the relative standard deviation was 8.6%~16.2%.

[0030] Beneficial effect: The detection accuracy of the method of the present invention is high, and the linear relationship is good in the range of 1-100 ng / mL for chlorate and 0.05-10.0 ng / mL for perchlorate. 2 ) were all greater than 0.99, the detection limits (S / N=3) were 6.0μg / kg for chlorate and 1.0μg / kg for perchlorate, and the quantification limits (S / N≥10) were 18.0μg / kg for chlorate and 3.0μg / kg for perchlorate. The samples of chlorate and perchlorate were subjected to the recovery experiment, and the recovery rate of chlorate was 70.7% to 86.9%, with a relative standard deviation of 4.2% to 7.4%, and the recovery rate of perchlorate was 70.0% to 118.3%, with a relative standard deviation of 8.6% to 16.2%.

[0031] Furthermore, compared with the "National Food Safety Standard for Determination of Chlorate and Perchlorate in Foods" (GB5009.291-2023), the present invention has higher accuracy for Chinese medicinal materials. Taking ginseng samples as an example, according to the "National Food Safety Standard for Determination of Chlorate and Perchlorate in Foods" (GB 5009.291-2023), chlorate in ginseng positive samples cannot be detected, and the perchlorate content is 3.22 mg / kg; while the method of the present invention can detect that the chlorate content in the ginseng positive sample is as high as 35.6 mg / kg, and the perchlorate content is 19.4 mg / kg.

[0032] In conclusion, the method of the present invention can effectively remove macromolecular organic substances such as pigments, saponins, polypeptides, polysaccharides, proteins and Cl - 、NO2 - 、NO3 - PO4 3- 、SO4 2- , oxalic acid and other anions inhibition and interference, the result is accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solution of the present invention, the accompanying drawings will be briefly introduced below. Obviously, the accompanying drawings in the following description only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0034] Figure 1 It is a total ion current diagram of chlorate, perchlorate and internal standard provided in Example 1 of the present invention (perchlorate concentration 5 ng / mL, perchlorate internal standard concentration 0.2 ng / mL, chlorate concentration 100 ng / mL, chlorate internal standard concentration 2 ng / mL);

[0035] Figure 2 is an extracted ion spectrum of a chlorate standard provided in Example 1 of the present invention (chlorate concentration 100 ng / mL);

[0036] Figure 3 It is a chlorate internal standard provided in Example 1 of the present invention. -18 Extracted ion spectrum of O3 standard (chlorate internal standard concentration 2 ng / mL);

[0037] Figure 4 This is an extracted ion spectrum of a perchlorate standard provided in Example 1 of the present invention (perchlorate concentration 5 ng / mL);

[0038] Figure 5 It is a perchlorate internal standard provided in Example 1 of the present invention. -18 Extracted ion spectrum of O4 standard (perchlorate internal standard concentration 0.2 ng / mL);

[0039] Figure 6 It is a total ion flow diagram of chlorate and perchlorate and internal standard detected by the Panax notoginseng powder sample provided in Example 1 of the present invention using this patent;

[0040] Figure 7 This is a total ion flow diagram of a Panax notoginseng powder sample provided in Example 1 of the present invention, in which chlorate and perchlorate detected by this patent and an internal standard are added (the addition level is 1.0 mg / kg for chlorate and 0.1 mg / kg for perchlorate);

[0041] Figure 8 The chlorate working curve after internal standard correction provided in Example 2 of the present invention;

[0042] Fig. 9 This is a perchlorate working curve after internal standard correction provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0043] Regarding the extract, the chlorate and perchlorate measured by the present invention have high solubility and low adsorption, and have high solubility in water. The present invention finds that the extraction efficiency of the target compound can be improved by adding a certain amount of acid, so formic acid aqueous solution is used as the extraction solvent for chlorate and perchlorate.

[0044] Regarding the dialysis bag, the molecular weight cutoff of the dialysis bag is 10000-20000 Dalton, preferably 14000-16000 Dalton. The dialysis bag is preferably a transparent polymer technology material with a diameter of 44mm (DM36). The present invention has no special restrictions on the source of the dialysis bag. The conditions of the ultrasonic extraction are preferably as follows: the ultrasonic time is 30 to 60 minutes, more preferably the ultrasonic time is 60 minutes, and the ultrasonic power is 1200W. The conditions of the dialysis are preferably as follows: the dialysis time is 60 to 120 minutes, more preferably the dialysis time is 120 minutes. The ultrasound and dialysis temperatures are preferably as follows: the temperature is 20 to 30 degrees Celsius, more preferably 30 degrees Celsius. The present invention purifies through dialysis, so that the soluble organic matter of the macromolecules is retained in the dialysis bag, and the target chlorate and perchlorate seep out of the dialysis bag to form a filtrate.

[0045] After obtaining the filtrate, the present invention passes the filtrate through an activated and balanced graphitized carbon black solid phase extraction column (only required for color-heavy samples) and an activated Ag / H composite ion exchange column, and discards the front effluent, and collects the effluent with a test tube. The graphitized carbon black solid phase extraction column is used to remove soluble pigments, and the Ag-type ion exchange column is used to remove high concentrations of Cl in the sample filtrate. - PO4 3- 、SO4 2- , oxalic acid and other anions, the H-type ion exchange column is used to remove heavy metal ions in the sample filtrate and neutralize the alkaline sample. Therefore, after the above treatment, not only can the problem of clogging the chromatographic column be eliminated, but also the interference of the above impurities on the detection target can be avoided, thereby improving the accuracy.

[0046] In the selection of chromatographic columns, chlorate and perchlorate are salts containing strong anions with strong polarity. They are not retained on general C18 chromatographic columns, and have no separation effect on chlorate and perchlorate. The present invention compares the pentafluorophenyl column in the "National Food Safety Standard for Determination of Chlorate and Perchlorate in Food" (GB 5009.291-2023) and the Synergi4uMAX-RP80A C12 column, Acclaim TM Trinity TM P1 composite ion exchange column, these three different types of filler columns, after repeated tests, the results of the comparison of Acclaim TM Trinity TM The P1 composite ion exchange column has good separation effect, sharp and symmetrical peak shape, high sensitivity, and little interference from impurities. TM Trinity TMThe P1 composite ion exchange column can provide reverse phase, cation exchange and anion exchange functions at the same time. The separation can be optimized by adjusting the chromatographic parameters (mobile phase pH, ionic strength and organic strength), avoiding the common mobile phase of NaOH or KOH solution as fluidity. Therefore, it is not necessary to use chemical suppression methods to convert it into H2O before entering the mass spectrometry detection.

[0047] On the basis of determining the chromatographic column, the present invention adopts volatile salt ammonium formate as the mobile phase, repeatedly experiments and optimizes the chromatographic conditions, and the obtained conditions include: when ammonium formate-acetonitrile is used as the mobile phase, the flow rate is 0.5mL / min; the column temperature is 35°C; the injection volume is 3μL; the mobile phase: A is a 20mmol / L ammonium formate solution, B is acetonitrile, and gradient elution can obtain high response of chlorate and perchlorate, sharp and symmetrical peak shape, low baseline noise, and good separation.

[0048] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. In the examples, those without specifying specific techniques or conditions are carried out according to the techniques or conditions described in the literature in this area or according to the product specification. Those without specifying the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially. In the following examples, if not clearly stated, "%" refers to weight percentage.

[0049] Example 1

[0050] Preparation of standard working solutions: Dilute the mixed standard solution of chlorate and perchlorate, the chlorate isotope internal standard and the perchlorate isotope internal standard with water to form a series of standard working solutions. The concentrations of chlorate are 1.0, 2.0, 5.0, 10.0, 50.0 and 100 ng / mL, respectively; the concentrations of perchlorate are 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 ng / mL, respectively. Each standard working solution contains 2.0 ng / mL chlorate as internal standard. -18 O3 and 0.2 ng / mL perchlorate -18 O4 solution.

[0051] Preparation of Chinese medicinal materials samples: Grind the Chinese medicinal materials samples with a grinder and then mix them evenly.

[0052] To determine the residual chlorate and perchlorate in the sample, the steps are as follows:

[0053] Step 1: Extraction and dialysis of residual chlorate and perchlorate in the sample

[0054] Weigh 1g of Chinese medicinal material sample (ground in advance, particle size of 20-40 microns), place the sample in a cleaned dialysis bag, add 10mL of 0.1% formic acid aqueous solution, and then add 40μL of 1μg / mL chlorate -18 O3 and 0.1 μg / mL perchlorate -18 O4 mixed internal standard solution, then place the dialysis bag in a glass tube, add 10mL of 0.1% formic acid aqueous solution into the glass tube. Ultrasonic extraction for 30 to 60 minutes, dialysis for 60 to 120 minutes, the ultrasound and dialysis temperature is 20 to 30 degrees Celsius, then take out the dialyzed clear liquid in the glass tube for purification.

[0055] In this embodiment, the molecular weight cut-off of the dialysis bag is 14,000 Daltons.

[0056] In this embodiment, the dialysis bag is cut into 10 to 20 cm segments before use, first washed with 50% ethanol, 0.01 mol / L sodium bicarbonate and 0.001 mol / L EDTA solution, then boiled with distilled water for 30 minutes, left at room temperature, and finally rinsed with distilled water before use.

[0057] In this embodiment, the dialysis bag is washed and stored in 50% ethanol. When used, one end is tied tightly with a rubber band or a rope and washed with distilled water.

[0058] In this embodiment, the dialysis bag is thoroughly washed with distilled water and stored in 50% ethanol after use. The dialysis bag can be reused if it does not interfere with subsequent experiments.

[0059] In this embodiment, the operation of contacting the dialysis bag requires wearing gloves.

[0060] Step 2: Cleanup of sample solution using graphitized carbon black solid phase extraction cartridge (only required for color-heavy samples) and composite ion exchange column

[0061] Take the dialyzed clear liquid and pass it through an activated and balanced graphitized carbon black solid phase extraction column (only required for color-heavy samples) and an activated Ag / H ion exchange column, and control the sample flow rate to be less than 1 mL / min; discard the first 6 mL of effluent, collect the effluent in a test tube, take 1 mL of the effluent and pass it through a 0.20 μm filter membrane, and collect the filtrate in an injection bottle for determination by ultra-high pressure liquid chromatography-tandem mass spectrometry.

[0062] In this example, the graphitized carbon black solid phase extraction column (500 mg, 6 mL) was activated with 6 mL of methanol and then eluted with 6 mL of 0.1% formic acid aqueous solution for equilibrium before use.

[0063] In this example, the Ag / H ion exchange column (1 g, 2.5 mL) needs to be rinsed with 10 mL of pure water and left to activate for 30 minutes before use.

[0064] In this embodiment, the Ag / H ion exchange column can be reused. After each use and between reuses, 20 mL of deionized water is drawn with a disposable syringe and pushed through the column at a flow rate of 2 mL / min for cleaning. After the column adsorbs chloride ions, the filler part that adsorbs chloride ions will turn black. When the black filler extends to 2 / 3, reuse is not recommended.

[0065] In this embodiment, the microfiltration uses an aqueous filter membrane syringe filter; the pore size of the aqueous filter membrane is preferably 0.20 μm.

[0066] Step 3: Ultra-high pressure liquid chromatography-tandem mass spectrometry

[0067] Adopt Acclaim TM Trinity TM The Chinese medicinal material sample solution obtained in step (2) is separated by a P1 composite ion exchange chromatography column and measured by ultra-high pressure liquid chromatography-tandem triple quadrupole mass spectrometry, and the quantitative ion pair peak area measurement result of the chlorate and perchlorate mixed standard solution after internal standard correction is Y, and the concentration of the chlorate and perchlorate mixed standard solution is X. A standard working curve is drawn, and the content of the chlorate and perchlorate to be measured in the sample is calculated by the internal standard method.

[0068] In this example, the chromatographic conditions for analyzing chlorate and perchlorate are as follows: Chromatographic column: Acclaim TM Trinity TM P1 composite ion exchange chromatography column, 2.1mm x 100mm, 3μm; flow rate: 0.5mL / min; column temperature: 35℃; injection volume: 3μL; mobile phase: A is 20mmol / L ammonium formate solution, B is acetonitrile, gradient elution, elution program is as follows:

[0069] Table 1 Elution schedule

[0070] Time, minutes Mobile phase A / % Mobile phase B / % 0.00 40.0 60.0 0.50 40.0 60.0 3.00 10.0 90.0 7.00 10.0 90.0 8.00 40.0 60.0 10.00 40.0 60.0

[0071] In this embodiment, the mass spectrometry conditions for analyzing chlorate and perchlorate are as follows: scanning mode: multiple reaction monitoring (MRM), negative ion mode; ion source: electrospray ion source (ESI). Nebulizer gas 30mL / min, curtain gas 50, auxiliary heating gas 60, heating temperature 550°C, ionization voltage -4500V. The quantitative ion pair, qualitative ion pair and collision energy are as follows:

[0072] Table 2 Quantitative ion pairs, qualitative ion pairs and collision energy

[0073]

[0074]

[0075] * is the quantitative ion pair.

[0076] Qualitative determination

[0077] For each component to be measured, select 1 parent ion and 2 or more daughter ions. Under the same experimental conditions, the retention time of the substance to be measured in the sample and the retention time of the standard calibration solution of the corresponding concentration in the mixed standard solution should be within ±2.5%; and in the sample spectrum, the relative abundance of the qualifier ions of each component is compared with the relative abundance of the corresponding qualifier ions in the mixed standard solution spectrum with similar concentrations. If the deviation does not exceed the range specified in the table below, it can be determined that the corresponding substance to be measured exists in the sample.

[0078] Table 3 Comparison range table

[0079] Relative ion abundance ＞50％ >20% to 50% >10% to 20% ≦10％ Maximum allowed deviation ±20％ ±25％ ±30％ ±50％

[0080] Quantitative determination

[0081] Under the optimized experimental conditions, UPLC-MS / MS injection detection was performed, and the peak area response values ​​of chlorate and perchlorate quantitative ion pairs after isotope internal standard correction were used as the vertical axis and the corresponding standard solution concentrations were used as the horizontal axis to draw the standard working curve, and the samples were quantified by the internal standard method using the standard working curve.

[0082] When conducting a blank test, follow the above measurement steps except that no sample is added. If the background value is higher than the quantitative limit, replace the reagents, dialysis bags and test vessels until the background value is lower than the quantitative limit.

[0083] Calculation and presentation of results

[0084]

[0085] Where:

[0086] x—residue of the component to be tested in the sample, in micrograms per kilogram (μg / kg);

[0087] C—the concentration of the measured component solution in the sample obtained from the standard working curve after internal standard correction, in nanograms per milliliter (ng / mL);

[0088] C0—the concentration of the measured component solution in the blank sample obtained from the standard working curve after internal standard correction, in nanograms per milliliter (ng / mL);

[0089] V—the final volume of the sample solution, in milliliters (mL);

[0090] m—The mass of the final sample represented by the sample solution, in grams (g)

[0091] The standard spectra of chlorate and perchlorate and their internal standards are shown in the appendix of the instruction manual. Figure 1-5 ,in, Figure 1 is the total ion current of chlorate and perchlorate and internal standard (perchlorate concentration 5 ng / mL, perchlorate internal standard concentration 0.2 ng / mL, chlorate concentration 100 ng / mL, chlorate internal standard concentration 2 ng / mL); Figure 2 This is the extracted ion spectrum of the chlorate standard (chlorate concentration 100 ng / mL); Figure 3 Chlorate internal standard -18 Extracted ion spectrum of O3 standard (chlorate internal standard concentration 2 ng / mL); Figure 4 This is the extracted ion spectrum of the perchlorate standard (perchlorate concentration 5 ng / mL); Figure 5 Is perchlorate internal standard -18 Extracted ion spectrum of O4 standard (perchlorate internal standard concentration 0.2 ng / mL).

[0092] Figure 6 It is a total ion flow diagram of chlorate and perchlorate and internal standard detected by the Panax notoginseng powder sample provided in Example 1 of the present invention using this patent; Figure 7 This is a total ion current diagram of a Panax notoginseng powder sample provided in Example 1 of the present invention, in which chlorate and perchlorate detected by this patent and an internal standard are added (the addition levels are 1.0 mg / kg for chlorate and 0.1 mg / kg for perchlorate).

[0093] from Figure 6 and Figure 7 It can be seen that this method can effectively remove macromolecular organic substances such as pigments, saponins, peptides, polysaccharides, proteins and Cl - 、NO2 - 、NO3 - PO4 3- 、SO4 2- , oxalic acid and other anions inhibition and interference, the result is accurate.

[0094] Example 2 Linear range and linear equation

[0095] Referring to the method in Example 1, the mixed standard solution of chlorate and perchlorate, the chlorate isotope internal standard, and the perchlorate isotope internal standard were diluted with water to form a series of standard working solutions, the concentrations of chlorate were 1.0, 2.0, 5.0, 10.0, 50.0, and 100 ng / mL, and the concentrations of perchlorate were 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 ng / mL, respectively. Each standard working solution contained 2.0 ng / mL chlorate. -18 O3 and 0.2 ng / mL perchlorate -18 O4 liquid.

[0096] Referring to the method in Example 1, Acclaim TM Trinity TM The Chinese medicinal material sample solution obtained in step (2) of separation of the P1 composite ion exchange chromatography column is measured by ultra-high pressure liquid chromatography-tandem triple quadrupole mass spectrometry, and the quantitative ion pair peak area of ​​the mixed standard solution of chlorate and perchlorate after internal standard correction is measured as Y, and the concentration of the mixed standard solution of chlorate and perchlorate is X. A standard working curve is drawn, and the results are shown in FIG. Figure 8 and Fig. 9 .

[0097] from Figure 8 and Fig. 9 It can be seen that chlorate and perchlorate residues are within the corresponding linear range, and there is a good linear relationship between the response peak area of ​​the quantitative ion and the sample concentration.

[0098] Linear equations and correlation coefficients for chlorate and perchlorate residues

[0099] Referring to the method in Example 1, the linear equation of chlorate after internal standard correction is Y=0.284X-0.026, and the linear correlation coefficient is 1.0000; the linear equation of perchlorate after internal standard correction is Y=2.21X+0.125, and the linear correlation coefficient is 0.9999.

[0100] Example 3 Determination of detection limit and quantification limit

[0101] A series of mixed working standard solutions of chlorate and perchlorate with lower mass concentrations were selected, processed according to the method in Example 1 and analyzed and detected under chromatographic and mass spectrometric conditions, and the lower limit of determination was calculated.

[0102] It was found that the detection limit (S / N=3) of the method of the present invention was 6.0 μg / kg for chlorate and 1.0 μg / kg for perchlorate, and the quantification limit (S / N≥10) was 18.0 μg / kg for chlorate and 3.0 μg / kg for perchlorate.

[0103] Example 4 Addition recovery and precision experiment

[0104] Weigh 1.0 g of Chinese herbal medicine samples (rhubarb, notoginseng and atractylodes macrocephala in this example), add 1.0 mg / kg of chlorate and 0.1 mg / kg of perchlorate and mixed standard working solution and the internal standard in Example 1, and make 6 parallel samples for each level. According to the determination steps of the method in Example 1, quantify by internal standard method, and calculate the recovery rate of each sample.

[0105] The results showed that the added recovery rates of chlorate and perchlorate residues in rhubarb samples were 72.0%-77.6% for chlorate and 74.6%-109.3% for perchlorate; the added recovery rates of chlorate and perchlorate residues in Panax notoginseng powder samples were 70.7%-86.5% for chlorate and 70.0%-91.5% for perchlorate; the added recovery rates of chlorate and perchlorate residues in Atractylodes macrocephala samples were 71.8%-86.9% for chlorate and 94.6%-118.3% for perchlorate.

[0106] Example 5 is based on the "National Food Safety Standard Determination of Chlorate and Perchlorate in Food" (GB5009.291-2023).

[0107] The interfering impurities in Chinese medicinal materials include macromolecular organic substances such as pigments, saponins, peptides, polysaccharides, proteins, and Cl - 、NO2 - 、NO3 - PO4 3- 、SO4 2- , oxalic acid and other anions, the "National Food Safety Standard Determination of Chlorate and Perchlorate in Food" (GB 5009.291-2023) and the usual literature methods use graphitized carbon black columns, C18, PLS-A solid phase extraction columns, etc. to adsorb and purify organic substances. However, this embodiment found through comparative experiments that the purification effect of the solid phase extraction column is not ideal for a large amount of water-soluble organic matter in traditional Chinese medicine.

[0108] Traditional Chinese medicine contains a large amount of water-soluble organic matter such as pigments, saponins, peptides, polysaccharides, proteins, etc., which inhibit and interfere with the target substances chlorate and perchloric acid, and also clog the chromatographic column during the detection process.

[0109] For further comparison, this example uses two methods to detect ginseng samples, as follows:

[0110] Weigh 1.0 g of ginseng sample and operate according to the determination steps of the method in Example 1. The internal standard method is used for quantitative determination. The determination results show that the chlorate content is 35.6 mg / kg and the perchlorate content is 19.4 mg / kg.

[0111] The same 1.0g ginseng sample was weighed and operated according to the determination steps of the method in the National Food Safety Standard Determination of Chlorate and Perchlorate in Foods (GB 5009.291-2023). The determination results showed that chlorate was not detected (detection limit 20μg / kg) and the perchlorate content was 3.22mg / kg.

[0112] The comparison results are shown in the table below.

[0113] Table 4 Comparison results

[0114]

[0115] It can be seen that the method of this embodiment can effectively remove macromolecular organic substances such as pigments, saponins, polypeptides, polysaccharides, proteins and Cl - 、NO2 - 、NO3 - PO4 3- 、SO4 2- , oxalic acid and other anions inhibition and interference, the result is accurate.

[0116] Example 6 refers to Chinese patent application CN113155986A

[0117] Referring to Example 1 of Chinese patent application CN113155986A "A method for detecting perchlorate and chlorate in Panax notoginseng", 2 g of Panax notoginseng powder sample was placed in a 34 mL stainless steel extraction cell (a glass fiber filter membrane was previously placed in the extraction cell), and then 1 g of basic alumina, 0.4 g of graphite black powder, and then 100 μL of 18 O-labeled perchlorate intermediate solution (1 μg / mL), and the remaining part is filled with diatomaceous earth; the extraction cell is installed and placed on the extraction disk of the rapid solvent extractor, and quickly extracted and purified online, and then all the liquid is transferred to a 50mL dissolving bottle, fixed to volume with water, and tested on the machine after passing through the membrane.

[0118] The above method uses alkaline alumina and graphite black powder for purification, and has a general purification effect on Chinese medicinal materials containing polysaccharides and polypeptides such as ginseng and scutellaria, and there is a problem of clogging the chromatographic column; and the patent weighs 2g of Panax notoginseng powder and dilutes it to 50mL, which is 25 times diluted. The quantitative limit is 25μg / kg and the detection limit is 10μg / kg, with low sensitivity; in addition, the patent needs to be equipped with an online rapid solvent extractor, which has large operating restrictions.

[0119] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0120] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0121] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials, characterized in that: The following steps are involved: (1) Preparation of standard working solutions: Dilute the mixed standard solution of chlorate and perchlorate, the chlorate isotope internal standard, and the perchlorate isotope internal standard with water to form a series of standard working solutions. Each standard working solution contains chlorate. -18 O3 and Perchlorate -18 O4; (2) Preparing Chinese medicinal material samples: grinding the Chinese medicinal material samples with a grinder, and then mixing them to obtain Chinese medicinal material powder; (3) Preparation of Chinese herbal medicine sample solution: placing the Chinese herbal medicine powder in a dialysis bag containing an extract containing formic acid, water and chlorate -18 O3 and Perchlorate -18 O4 internal standard; then the dialysis bag is placed in a glass tube, an aqueous formic acid solution is added into the glass tube, and ultrasonic extraction and dialysis are performed. After completion, the glass tube is taken out and the clear liquid dialyzed from the glass tube is collected; the clear liquid is passed through an activated Ag / H ion exchange column, the effluent is collected, the effluent is filtered through a membrane, and the filtrate is collected as a sample test solution for ultra-high pressure liquid chromatography-tandem mass spectrometry determination; (4) Ultra-high pressure liquid chromatography-tandem mass spectrometry: Adopt Acclaim TM Trinity TM The sample solution of step (3) is separated by a P1 composite ion exchange chromatography column, and is measured by ultra-high pressure liquid chromatography-tandem triple quadrupole mass spectrometry, and the quantitative ion pair peak area measurement result of the chlorate and perchlorate mixed standard solution after internal standard correction is Y, and the concentration content of the chlorate and perchlorate mixed standard solution is X, and a standard working curve is drawn, and the content of the chlorate and perchlorate to be measured in the sample is calculated by the internal standard method; The chromatographic conditions for analyzing chlorate and perchlorate in step (4) are as follows: Chromatographic column: Acclaim TM Trinity TM P1 composite ion exchange chromatography column; flow rate: 0.5mL / min; column temperature: 35℃; injection volume: 3μL; mobile phase: A is ammonium formate solution, B is acetonitrile, gradient elution, elution procedure is as follows: 。 2. The method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials according to claim 1, characterized in that: Step (1) comprises: diluting a mixed standard solution of chlorate and perchlorate, a chlorate isotope internal standard, and a perchlorate isotope internal standard with water to form a series of standard working solutions, wherein the concentrations of chlorate are 1.0, 2.0, 5.0, 10.0, 50.0, and 100 ng / mL, and the concentrations of perchlorate are 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 ng / mL, and each standard working solution contains 2.0 ng / mL of the internal standard chlorate. -18 O3 and 0.2 ng / mL perchlorate -18 O4 liquid.

3. The method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials according to claim 1, characterized in that: The particle size of the Chinese medicine powder in step (2) is 0.1-100 microns; Optionally, the molecular weight cut-off of the dialysis bag in step (3) is 10000-20000 Daltons, which is cut into small pieces of 10 to 20 cm before use, first washed with 40-70 volume % ethanol, 0.001-0.02 mol / L sodium bicarbonate and 0.001-0.005 mol / L EDTA solution, then boiled with distilled water for 20-40 minutes, left at room temperature, and finally rinsed with distilled water before use; Preferably, the dialysis bag is recycled after use, and the method is: the dialysis bag is thoroughly washed with distilled water, and then stored in 40-70 volume % ethanol. When in use, one end is tied tightly with a rubber band or rope and washed with distilled water.

4. The method for determining the residual amount of chlorate and perchlorate in a Chinese medicinal material according to any one of claims 1 to 3, characterized in that: The concentration of formic acid in the extract in step (3) is 0.05-0.2wt%. Preferably, for 1g of the Chinese medicine powder, 10-15mL of 0.1-0.2wt% formic acid aqueous solution is added, and then 40-50μL of 1-2μg / mL chlorate is added. -18 O3 and 0.1-0.2 μg / mL perchlorate -18 O4 mixed internal standard solution, then place the dialysis bag in a glass tube, and add 10-15mL of 0.1-0.2wt% formic acid aqueous solution into the glass tube.

5. The method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials according to claim 4, characterized in that: The time of the ultrasonic extraction in step (3) is 30 to 60 minutes, the time of the dialysis is 60 to 120 minutes, and the temperature of the ultrasound and the dialysis is 20 to 30 degrees Celsius.

6. The method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials according to claim 4, characterized in that: The clear liquid in step (3) is first subjected to a graphitized carbon black solid phase extraction column to remove color, and then subjected to an activated Ag / H ion exchange column, with the sample flow rate controlled to be less than 1 mL / min; the first 6 mL of the effluent is discarded, the effluent is collected in a test tube, 1 mL of the effluent is filtered through a 0.20 μm filter membrane, and the filtrate is collected in an injection bottle; Preferably, the specifications of the graphitized carbon black solid phase extraction column are 500 mg, 6 mL, and before use, it is activated with 6 mL of methanol and eluted with 6 mL of 0.1 wt% formic acid aqueous solution for equilibrium; Preferably, the specification of the Ag / H ion exchange column is 1 g, 2.5 mL, and it is rinsed with 10 mL of pure water before use, and left to stand for activation for 30 minutes before use.

7. The method for determining the residual amount of chlorate and perchlorate in Chinese medicinal materials according to claim 6, characterized in that: The Ag / H ion exchange column is reused. After each use and at intervals between reuses, 20 mL of deionized water is drawn with a disposable syringe and pushed through the column at a flow rate of 2 mL / min for cleaning. After the column adsorbs chloride ions, the filler part that adsorbs chloride ions will turn black. When the black filler extends to 2 / 3, it is no longer reused.

8. The method for determining the residual amount of chlorate and perchlorate in a Chinese medicinal material according to any one of claims 1 to 3, characterized in that: The mass spectrometry conditions for analyzing chlorate and perchlorate in step (4) are as follows: scanning mode: multiple reaction monitoring MRM, negative ion mode; ion source: electrospray ion source ESI; nebulizing gas 30 mL / min, heating temperature 550° C., ionization voltage -4500 V; quantitative ion pair, qualitative ion pair and collision energy are as follows: * is the quantitative ion pair.

9. The method for determining the residual amount of chlorate and perchlorate in a Chinese medicinal material according to any one of claims 1 to 3, characterized in that: The Chinese medicinal materials include at least one of red ginseng, angelica, rehmannia root, coptis root, cooked rehmannia root, radish seed, chuanxiong, pinellia tuber, rhubarb, notoginseng, atractylodes, ginseng, scutellaria, eucommia bark, and poria.

10. The method for determining the residual amount of chlorate and perchlorate in a Chinese medicinal material according to any one of claims 1 to 3, characterized in that: In the method, chlorate has a linear relationship in the range of 1-100 ng / mL and perchlorate in the range of 0.05-10.0 ng / mL, and the correlation coefficient r 2 All of them were greater than 0.99, the detection limit S / N=3 was 6.0μg / kg for chlorate and 1.0μg / kg for perchlorate, the quantification limit S / N≥10 was 18.0μg / kg for chlorate and 3.0μg / kg for perchlorate; the samples of chlorate and perchlorate were subjected to the addition recovery experiment, the recovery rate of chlorate was 70.7%~86.9%, the relative standard deviation was 4.2%~7.4%, the recovery rate of perchlorate was 70.0%~118.3%, the relative standard deviation was 8.6%~16.2%.

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