A method for determining the residual amount of matrine and oxymatrine in tussah silkworm

Abstract

The invention discloses a method for determining the residual amount of matrine and oxymatrine in tussah silkworm. The method comprises the following steps: ultrasonically extracting a sample with an 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water, purifying the sample through dispersed solid phase extraction, concentrating and re-dissolving the sample, separating the sample with a Kinetex 2.6μm Biphenyl chromatographic column, performing gradient elution using 0.1±0.01% formic acid water and 0.1±0.01% formic acid methanol as mobile phases, performing electrospray ionization, and performing detection under positive ion mode multiple reaction monitoring. The ion pair Q1 / Q3: 249.2 / 148.3 is used as a quantitative ion pair for matrine, and the ion pair Q1 / Q3: 249.2 / 150.3 is used as a qualitative ion pair for oxymatrine. The ion pair Q1 / Q3: 265.2 / 247.3 is used as a quantitative ion pair for oxymatrine, and the ion pair Q1 / Q3: 265.2 / 148.2 is used as a qualitative ion pair for oxymatrine. The method is quantitatively determined using an external standard method. The invention realizes the simultaneous detection of the residual amounts of matrine and oxymatrine in tussah silkworm, the detection limit of matrine is 0.05 μg / kg, and the detection limit of oxymatrine is 0.1 μg / kg.

Description

Technical Field

[0001] The invention relates to a method for determining the residual amounts of matrine and oxymatrine in tussah silkworm, and belongs to the technical field of determining the residual amounts of matrine and oxymatrine in tussah silkworm. Background Art

[0002] my country is the birthplace of tussah silkworms and the world's main tussah silkworm breeding country. The annual output of tussah silkworm cocoons is about 85,000 tons, accounting for more than 90% of the world's total output. Tussah silkworms are raised in wild tussah orchards, which are interspersed with farmland, shelterbelts, orchards, etc. The pesticides used in the prevention and control of agricultural and forestry pests can cause poisoning of tussah silkworms through droplet drift and contaminate tussah leaves, resulting in a serious reduction in tussah silkworm production and huge economic losses to cocoon production. Matrine is a highly efficient broad-spectrum botanical insecticide that has a rapid and persistent toxic effect on the larvae of moths and butterflies. In recent years, due to its advantages of low toxicity and low residue, it has been widely used in the field of preventing and controlling various agricultural and forestry pests such as pine caterpillars, tea caterpillars, and cabbage caterpillars. Tussah silkworms and caterpillars are both Lepidoptera insects. The application of matrine-based pesticides is bound to bring safety risks to tussah silkworm production, and tussah silkworm poisoning incidents occur from time to time.

[0003] Tussah silkworms are animal-derived samples with complex matrices, and a small amount of matrine and oxymatrine can cause poisoning in tussah silkworms. Therefore, although there are a few reports on the determination of matrine residues in the prior art, they are not suitable for the detection of tussah silkworms. For example, the patent with application number 202011608584.2 discloses a high-performance liquid chromatography detection method for matrine pesticide residues in fruits and vegetables, which is used for the detection of fruits and vegetables. The method cannot achieve the extraction and purification of tussah silkworms, nor can it complete the simultaneous detection of matrine and oxymatrine. The detection limit of matrine is 0.1 mg / kg, which is not applicable to tussah silkworms that can be poisoned by a small amount of matrine. There are no reports on the detection of matrine and oxymatrine residues in tussah silkworms, making it difficult to conduct risk assessment of matrine pesticide residues in tussah silkworms. Summary of the invention

[0004] The invention provides a method for determining the residual amounts of matrine and oxymatrine in tussah silkworm, which can realize simultaneous detection of the residual amounts of matrine and oxymatrine in tussah silkworm, with the detection limit of matrine being 0.05 μg / kg and the detection limit of oxymatrine being 0.1 μg / kg.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A method for determining the residual amount of matrine and oxymatrine in tussah silkworm, wherein the sample is extracted by ultrasonic method using 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water, purified by dispersed solid phase extraction, concentrated and re-dissolved, and Kinetex 2.6μmBiphenyl The product was separated on a chromatographic column (100 mm×3.0 mm), with 0.1±0.01% formic acid water and 0.1±0.01% formic acid methanol as the mobile phase for gradient elution. Electrospray ionization (ESI) was used for detection under positive ion mode multiple reaction monitoring (+MRM). Matrine was determined using the ion pair Q1 / Q3: 249.2 / 148.3 as the quantitative ion pair and the ion pair Q1 / Q3: 249.2 / 150.3 as the qualitative ion pair. Matrine oxide was determined using the ion pair Q1 / Q3: 265.2 / 247.3 as the quantitative ion pair and the ion pair Q1 / Q3: 265.2 / 148.2 as the qualitative ion pair. The product was quantified using the external standard method.

[0007] Unless otherwise specified, % in this application refers to volume percentage.

[0008] The above-mentioned 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water refers to the acetonitrile aqueous solution having an acetonitrile volume content of 80±2% and containing 0.2±0.02% ammonia water by volume. It is hereinafter expressed as 80±2% acetonitrile aqueous solution (containing 0.2±0.02% ammonia water). Other similar expressions have similar meanings. 0.1±0.01% formic acid water refers to a formic acid aqueous solution having a formic acid volume content of 0.1±0.01%; 0.1±0.01% formic acid methanol refers to a formic acid methanol solution having a formic acid volume content of 0.1±0.01%. Other similar expressions have similar meanings.

[0009] The above method has high sensitivity, accuracy and reproducibility, and can be used for the determination of matrine and oxymatrine residues in tussah silkworm, and provides a methodological basis for the detection of matrine and oxymatrine residues in related products.

[0010] The inventors found that tussah silkworm is an animal-derived sample with a complex matrix. The extraction rate is extremely low when using existing extractants used for fruits, vegetables, tea, honey, etc., and the detection cannot be completed. However, the extraction efficiency of 80±2% acetonitrile aqueous solution (containing 0.2±0.02% ammonia water) is significantly higher than that of other reagents; ultrasonic extraction combined with anhydrous sodium sulfate as an extraction salt significantly improves the recovery rate compared to not using ultrasound and using other extraction salts. Combined with the purification treatment of this application and the selection of other conditions, the detection of tussah silkworm can be completed accurately and quickly.

[0011] With 0.1±0.01% formic acid water and 0.1±0.01% formic acid methanol as the mobile phase for elution, the separation, peak shape and response value of matrine and oxymatrine are the best, meeting the detection requirements, and it is green, environmentally friendly and simple to prepare.

[0012] In the prior art, Inspire HILIC chromatographic columns (150 mm × 2.1 mm × 3 μm) are mostly used. The HILIC column is a forward chromatographic column, and the solvent used is limited. Only 70% or more acetonitrile can be used, which is difficult to pre-balance, expensive, and has a short service life. The present application uses Kinetex ○ R2.6 μm Biphenyl The column is a core-shell biphenyl chromatographic column. The high performance brought by the core-shell particles combined with the unique stationary phase can achieve reversed-phase retention and enhance the selectivity of polar and aromatic compounds, and can significantly improve the chromatographic peak shape and increase the sensitivity. Its chromatographic separation interaction mechanism: the high-density electron cloud generated by the biphenyl double ring structure can play a role similar to weak cation exchange, thereby enhancing the retention of alkaline analytes.

[0013] The selection of the above-mentioned matrine and oxymatrine ion pair can ensure the response value and stability of the detection.

[0014] The minimum detection limit of matrine in this application is 0.05μg / kg; the minimum detection limit of oxymatrine is 0.1μg / kg. In this application, low-concentration matrine and oxymatrine standard solutions are added to blank tussah silkworm samples to prepare spiked samples, and the extraction and determination are carried out according to the method of this article, and the signal-to-noise ratio is calculated. The spiked amount corresponding to 3 times the signal-to-noise ratio (S / N=3) is taken as the minimum detection limit, which fully considers the interference of the sample matrix and the influence of the recovery rate, and is the method detection limit in the true sense.

[0015] In order to further improve the extraction efficiency, the method of extracting the sample is as follows: after mixing the sample with an 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water, vortex for 1±0.2 min, ultrasonic extraction for 20±2 min, add 5.0 g of anhydrous sodium sulfate to the centrifuge tube, vortex extraction for 2±0.2 min, centrifuge at 4°C and 9500±500 r / min for 5±0.5 min, and the supernatant is to be purified.

[0016] The use of anhydrous sodium sulfate as the extraction salt can significantly improve the recovery rate.

[0017] During the test, the tussah silkworm to be tested is first minced into a homogenous state to obtain a sample, and then the sample is weighed for subsequent operations such as extraction; preferably, the amount of 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water is 4±0.5mL / g sample; the mass amount of anhydrous sodium sulfate is 2±0.2 times the mass of the sample.

[0018] In order to improve the accuracy and effectiveness of the test, the extraction and purification method is as follows: the supernatant obtained from the extracted sample is added to the Agilent QuEChERS reagent purification tube, vortexed for 2±0.2min, and centrifuged at 5000±500r / min at 4°C for 5±0.5min. The supernatant is the purification solution. The Agilent QuEChERS reagent purification tube contains 147.7mg PSA, 15.1mg GCB and 887.2mg magnesium sulfate.

[0019] Purification can be carried out by using a solid phase extraction column or a dispersed solid phase extraction agent to purify the sample to remove impurities such as organic acids, pigments, sugars, fats and phospholipids in the sample matrix, reduce the influence of the matrix effect on the test results, and reduce the adsorption of the target compound as much as possible to meet the test requirements. However, the inventors have found that due to the complexity of the tussah matrix, its extraction and purification is very important, and the effects of different extraction and purification methods and formulas vary greatly. Compared with other methods, the use of QuEChERS purification reagent (containing 147.7 mg PSA, 15.1 mg GCB, 887.2 mg magnesium sulfate) can significantly improve the recovery rate of matrine and oxymatrine, which is simple, fast, and has little background interference.

[0020] In order to reduce the loss rate, the method of concentration and re-dissolution is as follows: the supernatant after extraction and purification is placed in a concentration tube, blown to near dryness with nitrogen in a 40±2°C water bath, and then dissolved by adding a re-dissolution solvent, vortexed for 30±2s, ultrasonicated for 2±0.2min, and vortexed for 1±0.1min, and then the solution is transferred to a centrifuge tube, centrifuged at 13000±500r / min for 5±0.2min, and the supernatant is filtered through a 0.22μm filter membrane before being determined by liquid chromatography-tandem mass spectrometry. The temperature selection during the above concentration is very important, and too high or too low a temperature will increase the loss rate of matrine and oxymatrine.

[0021] In order to further improve the recovery efficiency, the re-dissolution solvent is a 50±1% methanol aqueous solution containing 0.1±0.01% formic acid.

[0022] The choice of 50±1% methanol solution (containing 0.1±0.01% formic acid) as the reconstitution solution has a significantly better recovery rate compared to other reagents.

[0023] Matrine and oxymatrine showed good linear relationship in the range of 0.1-50.0 ng / mL. The regression equation of matrine was y=10399.42713x+191.40500, and the correlation coefficient r was greater than 0.999; the regression equation of oxymatrine was y=11045.75073x+174.69791, and the correlation coefficient r was greater than 0.999. y is the peak area of ​​the ion chromatogram for the quantitative determination of the measured component, and x is the concentration.

[0024] The detection limit of matrine in this application is 0.05μg / kg, and the detection limit of oxymatrine is 0.1μg / kg.

[0025] The technologies not mentioned in the present invention are all referred to the prior art.

[0026] The present application discloses a method for determining the residues of matrine and oxymatrine in tussah silkworms. By improving the chromatographic mass spectrometry conditions and sample pretreatment methods, an ultra-high performance liquid chromatography-tandem mass spectrometry method for determining the residues of matrine and oxymatrine in tussah silkworms was established. The sensitivity, accuracy and precision of this method all meet the requirements of GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods", filling the gap in relevant national testing methods, providing a methodological basis for conducting matrine pesticide residue detection work and risk assessment in tussah silkworms, and providing technical support for studying the toxicity and safety tests of matrine pesticides on tussah silkworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a multiple reaction monitoring (MRM) chromatogram of matrine and oxymatrine standard solutions (10 ng / mL);

[0028] Figure 2 It is the total ion current chromatogram of matrine and oxymatrine standard solution (10 ng / mL);

[0029] Figure 3 The signal-to-noise ratio diagram of the quantitative ion pair 249.2 / 148.3 of matrine (spike amount 0.05 μg / kg);

[0030] Figure 4 The signal-to-noise ratio diagram of the quantitative ion pair 265.2 / 247.3 of oxymatrine (spike amount 0.1 μg / kg); DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0032] Six types of tussah silkworm larvae: healthy tussah silkworm larvae in the blank control group (numbered KB), 4th-instar tussah silkworm larvae poisoned by different concentrations of matrine pesticides (numbered K48-1, K48-2, K96-1), and 5th-instar tussah silkworm larvae (numbered K24-1, K24-2), all provided by Jilin Sericultural Science Research Institute and stored frozen at -18°C.

[0033] Equipment: API4000+ ultra-high performance liquid chromatography-tandem mass spectrometry (ESI ion source, AB SCIEX, USA); 2.6μm Biphenyl Chromatographic column (100 mm × 3.0 mm); vortex mixer (Talboys, USA); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument); KH20R-Ⅱ high-speed refrigerated centrifuge (Hunan Kaida); TTL-DC nitrogen blowing instrument (Beijing Tongtai Lian Technology);

[0034] Reagents: Standard substances: matrine (purity 98.9%, Shanghai Anpu Experimental Technology), oxymatrine (purity 99.9%, Tianjin Alta Technology); chromatographic grade methanol and acetonitrile (Fisher Scientific); chromatographic grade formic acid (CNW); analytical grade concentrated ammonia (28%, Tianjin Zhiyuan Chemical Reagent); analytical grade anhydrous sodium sulfate (Beijing Chemical); QuEChERS reagent purification tube (5982-5256 Agilent): 15mL centrifuge tube containing 147.7mg PSA, 15.1mg GCB, 887.2 mg magnesium sulfate; QuEChERS reagent extraction package (5982-5650 Agilent): containing 4 g magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate, 0.5 g disodium hydrogen citrate; 0.1% formic acid-water solution; 0.1% formic acid-methanol solution; 50% methanol-water solution (containing 0.1% formic acid); 80% acetonitrile-water solution (containing 0.2% ammonia water); 0.2 μm filter membrane (Pall Corporation).

[0035] In each case, unless the temperature is particularly stated, the reaction was carried out at room temperature (15 to 25° C.).

[0036] Example 1

[0037] Preparation of standard solution:

[0038] Matrine standard stock solution: Accurately weigh 10 mg (accurate to 0.1 mg) of matrine into a 10 mL brown volumetric flask, dissolve it in acetonitrile and dilute to the mark to prepare a 1 mg / mL matrine standard stock solution, and store it at -18°C away from light.

[0039] Oxymatrine standard stock solution: Accurately weigh 10 mg (accurate to 0.1 mg) of oxymatrine into a 10 mL brown volumetric flask, dissolve it in acetonitrile and dilute to the mark to prepare a 1 mg / mL oxymatrine standard stock solution, and store it at -18°C away from light.

[0040] Preparation of mixed standard solution (10 μg / mL): Accurately pipette 0.1 mL of matrine and oxymatrine standard stock solutions (1 mg / mL) into 10 mL volumetric flasks, dilute with acetonitrile and make up to 10 mL.

[0041] Preparation of mixed standard solution (100 ng / mL): Accurately pipette 0.1 mL of mixed standard solution (10 μg / mL) into a 10 mL volumetric flask and make up to volume with 50% methanol solution (containing 0.1% formic acid).

[0042] Preparation of mixed standard working solution (10 ng / mL): Accurately pipette 1.0 mL of mixed standard working solution (100 ng / mL) into a 10 mL volumetric flask and make up to volume with 50% methanol solution (containing 0.1% formic acid).

[0043] Preparation of mixed standard working solution series: Pipette 10μL, 50μL, 100μL of 10ng / mL mixed standard solution, and 50μL, 100μL, 200μL, 500μL of 100ng / mL mixed standard solution into 7 sample bottles, dilute to 1.0mL with 50% methanol solution (containing 0.1% formic acid), and mix well. The concentrations of the prepared mixed standard working solutions are 0.1ng / mL, 0.5ng / mL, 1.0ng / mL, 5.0ng / mL, 10.0ng / mL, 20.0ng / mL, 50.0ng / mL.

[0044] Sample processing:

[0045] Extraction: Use a meat grinder to grind the tussah silkworm into a homogenous state to obtain a sample, weigh 2.50 g of the sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 10 mL of 80% acetonitrile aqueous solution (containing 0.2% ammonia water), vortex (2500 r / min) for 1 min, and extract with ultrasound (ultrasonic frequency: 40 KHz) for 20 min. Add 5.0 g of anhydrous sodium sulfate to the centrifuge tube, vortex (2500 r / min) for extraction for 2 min, and centrifuge at 9500 r / min for 5 min (4°C). The supernatant is to be purified.

[0046] Purification: Pipette 5.0 mL of supernatant extract and add it into the Agilent QuEChERS reagent purification tube (15 mL centrifuge tube containing 147.7 mg PSA, 15.1 mg GCB, 887.2 mg magnesium sulfate), vortex for 2 min, centrifuge at 5000 r / min for 5 min (4°C), and the supernatant is the purification solution.

[0047] Accurately pipette 2.00 mL of purified liquid into a 10 mL concentrator tube, blow nitrogen in a 40 ° C water bath until almost dry, accurately add 1.0 mL of 50% methanol solution (containing 0.1% formic acid) to dissolve the residue, vortex for 30 seconds, ultrasonic for 2 minutes, vortex for 1 minute, transfer the solution to a 1.5 mL centrifuge tube, centrifuge at 13000 r / min for 5 minutes, and pass the supernatant through a 0.22 μm filter membrane for liquid chromatography-tandem mass spectrometry determination.

[0048] LC-MS / MS conditions:

[0049] Chromatographic conditions 2.6μm Biphenyl Chromatographic column (100 mm × 3.0 mm); column temperature 40°C; flow rate: 0.4 mL / min; injection volume: 5 μL; mobile phase: 0.1% formic acid-water for pump A, 0.1% formic acid-methanol for pump B. The mobile phase and gradient elution conditions are shown in Table 1.

[0050] Table 1 Mobile phase and gradient elution conditions

[0051]

[0052] Mass spectrometry conditions: ion source: ESI; scanning mode: positive ion mode multiple reaction monitoring (+MRM); ion source parameters: electrospray voltage (IS): 5500V; nebulizer gas pressure (GS1): 413700Pa; auxiliary gas pressure (GS2): 413700Pa; curtain gas pressure (CUR): 206850Pa; ion source temperature (TEM): 550℃; collision gas (CAD): 7mL / min. The parameters of monitored ion pairs, collision gas energy and declustering voltage are shown in Table 2.

[0053] Table 2 Monitoring ion pairs, collision gas energy and declustering voltage parameters

[0054]

[0055] “*” ions are used for quantification. Ionization mode [M+H] + .

[0056] Matrix effect evaluation

[0057] The matrix effect is the main reason affecting the sensitivity and accuracy of liquid chromatography-tandem mass spectrometry. It is caused by the competition between the sample matrix and the target compound when the sample solution is ionized. It is divided into matrix effect enhancement and matrix effect inhibition. In this study, the matrix effect was evaluated by calculating the recovery rate of matrine and oxymatrine in the matrix mixed standard solution. When the recovery rate is 100%, it means there is no matrix effect. When the recovery rate is greater than 100%, it means the matrix effect is enhanced. When the recovery rate is less than 100%, it means the matrix effect is inhibited. The farther the recovery rate deviates from 100%, the more significant the matrix effect is. The difference from 100% within ±20% indicates that the matrix effect is not significant. The results show that the average matrix effect of matrine is -6.8%; the average matrix effect of oxymatrine is +7.4%, both within ±10%, indicating that the matrix effect is small and can be ignored (Table 3).

[0058] Table 3 Evaluation of matrix effect

[0059]

[0060] The values ​​in the table are the differences in recovery rate from 100%.

[0061] Calibration curves and detection limits

[0062] According to the above-mentioned chromatographic mass spectrometry conditions, the mixed standard working solution series was measured, and the peak area of ​​the measured component in the standard solution was used as the ordinate, and the concentration of the measured component in the standard solution was used as the abscissa. The calibration working curve was drawn, and the regression equation and correlation coefficient are shown in Table 4. The results show that matrine and oxymatrine have a good linear relationship in the range of 0.1-50 ng / mL, and the correlation coefficient r is greater than 0.999. In addition, low-concentration matrine and oxymatrine standard solutions were added to blank tussah silkworm samples to prepare spiked samples, which were extracted and measured according to the method in this article, and the signal-to-noise ratio was calculated. The spiked amount corresponding to 3 times the signal-to-noise ratio (S / N=3) was taken as the minimum detection limit. The signal-to-noise ratio of matrine and oxymatrine Figure 3 and Figure 4 It can be seen that the minimum detection limit of matrine is 0.05μg / kg; the minimum detection limit of oxymatrine is 0.1μg / kg.

[0063] Table 4 Regression analysis of calibration curve and method detection limit

[0064]

[0065] y—peak area, x—concentration.

[0066] Precision and spike recovery

[0067] Blank tussah samples were added with matrine and oxymatrine standard solutions at 4 concentrations (1.0ug / kg, 10ug / kg, 40ug / kg, 100ug / kg) to prepare spiked samples. Each spiked level was measured 6 times, and the spiked recovery and relative standard deviation were calculated. The results showed that the relative standard deviation of matrine in tussah was between 1.5% and 3.0%, and the recovery was between 95.4% and 118.5%; the relative standard deviation of oxymatrine was between 1.7% and 3.2%, and the recovery was between 80.2% and 87.8%, which met the requirements of Appendix F for precision and recovery in GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods", indicating that this method is accurate, stable and reliable (Table 5).

[0068] Table 5 Precision and recovery test results (n = 6)

[0069]

[0070] Sample determination

[0071] Six types of tussah silkworm larvae (healthy tussah silkworm larvae in the blank control group (numbered KB), 4th-instar tussah silkworm larvae poisoned by different concentrations of matrine pesticides (numbered K48-1, K48-2, K96-1), and 5th-instar tussah silkworm larvae (numbered K24-1, K24-2)) were tested according to the above method, and the results are shown in Table 6. As can be seen from Table 6, matrine residues were detected in the poisoned silkworm samples, and the matrine residues in some silkworm samples were relatively high. Referring to GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food", it is stipulated that the daily allowable intake (ADI) of matrine is 0.1 mg / kg bw, but the maximum residue limits of matrine and oxymatrine in silkworm-related products have not yet been established. Since the EU has not yet approved the use of matrine and oxymatrine, the maximum residue limit is stipulated to be less than 0.01 mg / kg. In this test, it was found that the matrine content in 2 poisoned silkworm samples far exceeded the maximum residue limit value stipulated by the European Union. Therefore, the abuse of matrine and oxymatrine is bound to lead to food safety risks in silkworm-related products, which makes it imperative to carry out the detection of matrine and oxymatrine residues in tussah silkworms. First, it can guide the concentration and scope of use of matrine and oxymatrine, and provide a reference for the rational and safe use of drugs in silkworm areas and tussah gardens, so as to reduce or eliminate the occurrence of tussah silkworm poisoning incidents; second, it can provide a methodological basis for conducting risk assessment of matrine and oxymatrine residues in silkworm-related products and the formulation of maximum residue limit values.

[0072] Table 6 Test results of tussah samples

[0073]

[0074] In the following comparative examples, all the examples not specifically described are referred to in Example 1.

[0075] Comparative Example 1

[0076] Comparison of ion pairs:

[0077] According to the properties of matrine and oxymatrine compounds, electrospray ionization positive ion mode scanning was adopted. 100 ng / mL standard solution was injected by syringe pump, and m / z: 249.2 [M+H] was obtained when matrine was ionized. + ion as the parent ion; when oxymatrine is ionized, m / z: 265.2 [M+H] +ions as parent ions. The collision energy (CE) value was adjusted to further fragment the parent ions. Matrine selected m / z: 148.3, 150.3, 176.3, 112.2 with relatively high response values ​​as daughter ions; oxymatrine selected m / z: 247.3, 148.2, 136.2, 205.1 as daughter ions. The monitoring ion pairs were set up according to multiple reaction monitoring (+MRM), and the collision gas energy CE and declustering voltage DP parameters were further optimized by Ramp. The ion source parameters and liquid chromatography parameters were set, and the 10 ng / mL mixed standard solution was injected and analyzed to verify the response value and stability of the selected ion pairs. The ion pair Q1 / Q3: 249.2 / 148.3 with the highest response value and stability was finally selected as the quantitative ion pair for matrine, and the ion pair Q1 / Q3: 249.2 / 150.3 with the second highest response value and stability was selected as the qualitative ion pair; the ion pair Q1 / Q3: 265.2 / 247.3 with the highest response value and stability was selected as the quantitative ion pair for oxymatrine. Although the response values ​​of the ion pairs Q1 / Q3: 265.2 / 136.2 and 265.2 / 205.1 were higher than the response value of the ion pair 265.2 / 148.2, the response values ​​of these two ion pairs were unstable and the signal-to-noise ratio was not as high as that of the ion pair 265.2 / 148.2. Therefore, the ion pair Q1 / Q3: 265.2 / 148.2 was selected as the qualitative ion pair for oxymatrine. The optimized mass spectrometry parameters are detailed in Table 2. The multiple reaction monitoring (MRM) chromatograms of matrine and oxidized matrine standard solutions are shown in Figure 1, Figure A is the chromatogram of the matrine ion pair 249.2 / 148.3, Figure B is the chromatogram of the matrine ion pair 249.2 / 150.3, Figure C is the chromatogram of the oxidized matrine ion pair 265.2 / 247.3, and Figure D is the chromatogram of the oxidized matrine ion pair 265.2 / 148.2).

[0078] Comparative Example 2

[0079] Comparison of chromatographic conditions:

[0080] by 2.6μm Biphenyl The chromatographic column (100mm×3.0mm) was used for separation. At a flow rate of 0.4mL / min, three mobile phase systems, 0.1% formic acid water-0.1% formic acid acetonitrile, 0.1% formic acid water-0.1% formic acid methanol, and 0.1% formic acid water-methanol, were used. According to the gradient elution conditions in Table 1, the mixed standard working solution with a concentration of 10ng / mL was detected and analyzed to investigate the influence of the mobile phase on the separation effect, peak shape and response value. The results showed that: when 0.1% formic acid water-0.1% formic acid acetonitrile was used as the mobile phase, the peaks of matrine and oxymatrine appeared too early and the two peaks were not completely separated; when 0.1% formic acid water-methanol was used as the mobile phase, the response values ​​of the two peaks were the smallest in comparison; when 0.1% formic acid water-0.1% formic acid methanol was used as the mobile phase for elution, the separation, peak shape and response value of matrine and oxymatrine were the best, meeting the detection requirements. The total ion current chromatogram of the mixed standard working solution of matrine and oxymatrine is shown in Figure 2 .

[0081] Comparative Example 3

[0082] Comparison of extraction methods:

[0083] Selection of extraction method Based on the fact that matrine and oxymatrine are alkaline compounds, 80% acetonitrile aqueous solution (containing 0.2% formic acid), 80% acetonitrile aqueous solution (containing 0.2% ammonia water), acetonitrile, 0.2% formic acid acetonitrile, and 0.2% ammonia water acetonitrile were selected as extraction agents, and the spiked silkworm samples (spiked amount 10 μg / kg) were extracted to investigate the extraction efficiency of the five solvents for matrine and oxymatrine. The results are shown in Table 7. From the data in Table 7, it can be seen that the five extraction agents all have good recovery rates for matrine; for oxymatrine, only the solvent 80% acetonitrile aqueous solution (containing 0.2% ammonia water) has an extraction rate of 80%, and the recovery rates of the other four solvents are all ≤60%, so 80% acetonitrile aqueous solution (containing 0.2% ammonia water) is selected as the extraction solution.

[0084] Table 7 Recovery rates of different extraction solvents

[0085]

[0086] Anhydrous sodium sulfate and QuEChERS salt reagent were selected as extraction salts, and ultrasonic or non-ultrasonic extraction was performed on the spiked samples. The results are shown in Table 8. As can be seen from Table 8, when anhydrous sodium sulfate was used as the extraction salt and ultrasonic extraction was performed for 20 minutes, the recoveries of matrine and oxymatrine were both above 80%.

[0087] Table 8 Effect of extraction salt and extraction method on recovery rate

[0088]

[0089] Comparative Example 4

[0090] Comparison of purification methods:

[0091] Compare The purification effects of PRIME HLB Plus Short (335 mg) column, QuEChERS purification reagent 1 (containing 149.9 mg PSA, 900.1 mg magnesium sulfate), QuEChERS purification reagent 2 (containing 147.7 mg PSA, 15.1 mg GCB, 887.2 mg magnesium sulfate) and QuEChERS purification reagent 3 (containing 100 mg PSA, 50 mg C18, 20 mg GCB, 400 mg magnesium sulfate) on spiked samples (spiked amount 8 μg / kg) are shown in Table 9. It can be seen from Table 9 that: The RPRIME HLB Plus Short (335mg) column has severe adsorption of matrine, with a recovery rate of 25%; it completely adsorbs oxymatrine, with a recovery rate of 0%; among the three QuEChERS purification reagents, only QuEChERS purification reagent 2 has a recovery rate of more than 80% for matrine and oxymatrine, so QuEChERS purification reagent 2 was selected for sample purification. The low recovery rate of QuEChERS purification reagent 1 may be due to the lack of GCB filler compared to QuEChERS purification reagent 2, and the pigment in the sample was not completely removed, causing matrix inhibition; the low recovery rate of QuEChERS purification reagent 3 may be due to the adsorption of matrine and oxymatrine by the C18 filler added compared to QuEChERS purification reagent 2.

[0092] Table 9 Effect of different solid phase extraction agents on recovery rate

[0093]

[0094]

[0095] Comparative Example 5

[0096] Comparison of concentration and reconstitution methods:

[0097] A 4 ng / mL mixed standard solution was dried with nitrogen in a 40°C water bath and a 50°C water bath, respectively. The results showed that when dried with nitrogen at 50°C, the recoveries of matrine and oxymatrine were between 60% and 70%; when dried with nitrogen at 40°C, the recoveries of matrine and oxymatrine were both greater than 90%, indicating that the loss rate of matrine and oxymatrine was very small when dried with nitrogen at 40°C.

[0098] After the spiked samples (spiked amount 4 μg / kg) were extracted, purified, and dried with concentrated nitrogen, 20% methanol aqueous solution (containing 0.1% formic acid) and 50% methanol aqueous solution (containing 0.1% formic acid) were selected as re-dissolving solutions, and re-dissolved with or without ultrasound. The recovery rates are shown in Table 10. 50% methanol aqueous solution (containing 0.1% formic acid) was used to dissolve the residue, and the samples were re-dissolved with ultrasound, and the recovery rates of matrine and oxymatrine were both above 80%.

[0099] Table 10 Effect of different reconstitution methods on recovery rate

[0100]

Claims

1. A method for determining the residual amount of matrine and oxymatrine in tussah silkworm, characterized in that: The samples were extracted by ultrasonic method with 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water, purified by dispersive solid phase extraction, concentrated and re-dissolved, separated by Kinetex 2.6μm Biphenyl 100Å column, and 0.1±0.01% formic acid water and 0.1±0.01% formic acid methanol were used as mobile phase for gradient elution. Electrospray ionization was used for detection in positive ion mode multiple reaction monitoring. Matrine was detected by ion pair Q1 / Q3: 249.2 / 148.3 as quantitative ion pair and ion pair Q1 / Q3: 249.2 / 150.3 as qualitative ion pair. Oxymatrine was detected by ion pair Q1 / Q3: 265.2 / 247.3 as quantitative ion pair and ion pair Q1 / Q3: 265.2 / 148.2 as qualitative ion pair. External standard method was used for quantification. The extraction cleanup used Agilent QuEChERS reagent cleanup tubes, which contained 147.7 mg PSA, 15.1 mg GCB and 887.2 mg magnesium sulfate.

2. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to claim 1, characterized in that: The method for extracting the sample is as follows: mix the sample with an 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water, vortex for 1±0.2 min, ultrasonically extract for 20±2 min, add 5.0 g of anhydrous sodium sulfate to the centrifuge tube, vortex and extract for 2±0.2 min, centrifuge at 4°C and 9500±500 r / min for 5±0.5 min, and the supernatant is to be purified.

3. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to claim 2, characterized in that: The amount of 80±2% acetonitrile aqueous solution containing 0.2±0.02% ammonia water is 4±0.5 mL / g sample; the mass amount of anhydrous sodium sulfate is 2±0.2 times the mass of the sample.

4. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to any one of claims 1 to 3, characterized in that: The extraction purification method is as follows: adding the supernatant obtained by extracting the sample into the Agilent QuEChERS reagent purification tube, vortexing for 2±0.2min, centrifuging at 5000±500r / min at 4°C for 5±0.5min, and the supernatant is the purification solution.

5. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to any one of claims 1 to 3, characterized in that: The method of concentration and reconstitution is as follows: the supernatant after extraction and purification is placed in a concentrator tube, blown with nitrogen in a 40±2°C water bath until almost dry, add a reconstitution solvent to dissolve, vortex for 30±2s, ultrasonic for 2±0.2min, vortex for 1±0.1min, transfer the solution to a centrifuge tube, centrifuge at 13000±500r / min for 5±0.2min, pass the supernatant through a 0.22μm filter membrane, and then perform liquid chromatography-tandem mass spectrometry determination.

6. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to claim 5, characterized in that: The reconstitution solvent is 50±1% methanol aqueous solution containing 0.1±0.01% formic acid.

7. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to any one of claims 1 to 3, characterized in that: Matrine showed a linear relationship in the range of 0.1-50.0 ng / mL, and the regression equation was: y =10399.42713 x +191.40500, correlation coefficient r Greater than 0.

999.

8. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to any one of claims 1 to 3, characterized in that: Oxymatrine showed a linear relationship in the range of 0.1-50.0 ng / mL, and the regression equation was: y =11045.75073 x +174.69791, correlation coefficient r All are greater than 0.

999.

9. The method for determining the residual amount of matrine and oxymatrine in tussah silkworm according to any one of claims 1 to 3, characterized in that: The detection limits of matrine and oxymatrine were 0.05 μg / kg and 0.1 μg / kg, respectively.

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