A method for simultaneous detection of robinin and taxifolin in honey and its application

Abstract

The present invention discloses a method and application for simultaneous detection of robinin and taxifolin in honey, solid phase extraction is used for enrichment, purification and detection using liquid chromatography-mass spectrometer, and robinin and taxifolin are quantified using external standard method. The test results of honey samples show that robinin is detected in acacia honey, and its content is 0.20-2.00mg / kg, and robinin and taxifolin are detected in wolfberry honey at the same time, wherein the robinin content is 0.20-0.80mg / kg, and the taxifolin content is 0.30-1.55mg / kg. Accordingly, when the honey sample is within the above range, it is judged to be the corresponding characteristic honey, otherwise, it is judged to be other honey or adulterated honey. The present invention establishes a method for simultaneous determination of robinin and taxifolin in honey by liquid chromatography-mass spectrometry, and applies it to the identification of single flower honey, which has the characteristics of simple operation, high accuracy and good sensitivity, and has important practical significance for the authenticity evaluation and quality control of acacia honey and wolfberry honey.

Description

Technical Field

[0001] The invention relates to the technical field of food detection and identification, and more particularly to a method for simultaneously detecting robinin and taxifolin in honey and application thereof. Background Art

[0002] Honey is a natural sweet substance that bees collect from plant nectar, secretions or honeydew, combine with their own secretions, transform, dehydrate and store in the honeycomb until mature. Acacia honey is a substance that bees collect from acacia nectar, add their own secretions for biological transformation, and finally store in the honeycomb. It is known as the four famous honeys of China together with jujube honey, vitex honey and litchi honey. It is one of the most stable honey products in my country's major honey sources every year. Acacia honey is a high-quality honey exported by my country and is deeply loved by foreign consumers. It is characterized by light color, fresh taste, sweet but not greasy, unique fragrance of acacia, and not easy to crystallize. Wolfberry honey is brewed by bees collecting wolfberry nectar. Wolfberry honey has both the traditional nutritional and health value of honey and contains some essence of wolfberry. The honey is amber in color and tastes fresh and sweet. Wolfberry honey is a specialty honey in Northwest my country, and its production areas are mainly concentrated in Qinghai, Ningxia and other places.

[0003] Driven by profit, there are problems such as adulteration of honey varieties and confusion of labels in the market. Therefore, honey variety identification is one of the problems that the bee industry needs to solve urgently, and it is also a hot topic of research in recent years.

[0004] Different single-flower honeys have their own characteristic ingredients, such as 4-hydroxyquinoline in jujube honey, longanin in longan honey, malic acid in litchi honey, etc. These unique compounds have been used as characteristic compounds to identify the authenticity of single-flower honey. It has been reported that robinin can be used as a characteristic marker in acacia honey, and taxifolin can be used as a characteristic marker in wolfberry honey. However, experiments have found that there is a certain amount of robinin in wolfberry honey, so there are certain deficiencies in using only one marker to distinguish single-flower honey. At present, there are few literatures that use multiple substances to distinguish single-flower honey. At present, there is no method for simultaneously detecting robinin and taxifolin in honey to distinguish acacia honey and wolfberry honey. Summary of the invention

[0005] In view of this, the present invention first provides a qualitative and quantitative detection method for robinin and taxifolin in a honey sample and applies it to honey detection, by first enriching and purifying and then using a liquid chromatography-mass spectrometer for detection.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] First, the present invention provides a method for simultaneously detecting robinin and taxifolin in honey, comprising the following steps:

[0008] Step 1: Sample processing

[0009] Weigh the honey sample, dissolve it with deionized water and mix well;

[0010] Step 2: Solid Phase Extraction

[0011] Taking the sample solution treated in step 1 and performing solid phase extraction to enrich, purify and elute robinin and taxifolin;

[0012] Step 3: Determination

[0013] Liquid chromatography-mass spectrometry was used to qualitatively and quantitatively detect acaciatin and taxifolin in honey samples, and both were quantified using the external standard method;

[0014] Wherein, the liquid chromatography-mass spectrometry detection conditions are:

[0015] Liquid chromatography conditions:

[0016] Chromatographic column: T3 liquid chromatography column, specification: 150mm×2.1mm, 3μm;

[0017] Flow rate: 0.3 mL / min;

[0018] Injection volume: 2 μL;

[0019] Column temperature: 35 °C;

[0020] Mobile phase A: acetonitrile; Mobile phase B: 0.1vt% formic acid aqueous solution;

[0021] The gradient elution conditions were:

[0022] 0-3.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90;

[0023] 3.0-6.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 90:10;

[0024] 6.0-6.2min, the volume ratio of mobile phase A to mobile phase B changed uniformly to 10:90;

[0025] 6.2-10.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90;

[0026] Ion source: electrospray ionization source ESI;

[0027] Ionization mode: positive ion mode;

[0028] Detection method: multiple reaction monitoring MRM;

[0029] Capillary voltage: 3.0kV;

[0030] Ion source temperature: 120°C;

[0031] Lens voltage: 0.1V;

[0032] Desolvation temperature: 350°C;

[0033] Desolvation gas N2 flow rate: 650L / h;

[0034] Cone gas N2 flow rate: 50L / h;

[0035] Collision gas Ar flow rate: 0.15 mL / min;

[0036] The MRM parameters were set as follows:

[0037]

[0038] Preferably, in step 1, the mass volume ratio of honey to water is 0.2 g / mL.

[0039] Preferably, the honey sample solution in step 1 needs to be vortex mixed at 1000-2000 rpm.

[0040] Preferably, the solid phase extraction step in step 2 is specifically as follows:

[0041] (2.1) Install the extraction column on a vacuum filtration device, activate it with 3 mL of methanol, and then balance it with 6 mL of deionized water;

[0042] (2.2) transferring the sample solution obtained in step 1 to the activated extraction column;

[0043] (2.3) Rinse with 3 mL of deionized water and then dry with a vacuum pump;

[0044] (2.4) Finally, use 3 mL of formic acid and methanol for elution. Blow the eluent to dryness with nitrogen at 30°C, then make up to 2 mL with formic acid and methanol. Vortex mix at 1000-2000 rpm, take 1 mL of the filter membrane and place it in a brown injection bottle for testing.

[0045] Furthermore, the extraction column adopts a PXA solid phase extraction column.

[0046] Preferably, the volume fraction of formic acid methanol is 2%, and the filter membrane is a 0.22 μm nylon filter membrane.

[0047] Preferably, in the external standard method quantification in step 3, the external standard curve equation is:

[0048] Robinin: Y = 0.8371X + 24.607, linear correlation coefficient R 2 =0.9992;

[0049] Taxifolin: Y = 7.98921X + 80.4067, linear correlation coefficient R 2 =0.9991;

[0050] Where X is the concentration of the target in the sample, and Y is the peak area of ​​the target in the sample.

[0051] Preferably, in the method, the detection limit of acaciatin is 0.06 mg / kg, the quantification limit is 0.20 mg / kg, and the recovery rate is >80%; the detection limit of taxolanin is 0.05 mg / kg, the quantification limit is 0.10 mg / kg, and the recovery rate is >80%.

[0052] The present invention also provides an application of the method described in the above technical solution in identifying acacia honey and wolfberry honey.

[0053] Furthermore, acacia honey contains robinin, and wolfberry honey contains robinin and taxifolin. Based on the weight of honey, the robinin content in acacia honey is 0.20-2.00 mg / kg, the robinin content in wolfberry honey is 0.20-0.80 mg / kg, and the taxifolin content in wolfberry honey is 0.30-1.55 mg / kg.

[0054] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for simultaneously detecting robinin and taxifolin in honey and its application, which has the following beneficial effects:

[0055] The invention determines that robinin and taxifolin can be used as characteristic markers of acacia honey and wolfberry honey, and proposes a detection method for robinin and taxifolin in honey by using a liquid chromatography-mass spectrometer. Acacia honey and wolfberry honey can be effectively identified according to their contents. The content of robinin in pure acacia honey is 0.20-2.00 mg / kg, and the contents of robinin and taxifolin in wolfberry honey are 0.20-0.80 mg / kg and 0.30-1.55 mg / kg, respectively.

[0056] The method for detecting robinin and taxifolin in honey provided by the present invention has the advantages of simple and quick operation, strong practicability, high sensitivity, good accuracy and precision, and can realize the effective separation and accurate quantification of robinin and taxifolin in honey. The method is of great significance for the authenticity identification and quality evaluation of honey, and at the same time provides a new indicator for protecting the rights and interests of honey consumers and maintaining the healthy and orderly development of the honey consumption market. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0058] Figure 1 is the linear curve of robinin and taxifolin;

[0059] Figure 2 is the total ion current chromatogram of robinin and taxolacin;

[0060] Figure 3 This is the MRM chromatogram of robinin and taxolacin;

[0061] Figure 4 are the extracted ion chromatograms (EIC) and signal-to-noise ratios of robinin and taxolacin;

[0062] Figure 5 is the mobile phase flow rate optimization diagram;

[0063] Figure 6 This is a graph showing the content of robinin and taxifolin in single flower honey samples. DETAILED DESCRIPTION

[0064] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] The instruments, reagents and materials in the following examples are as follows:

[0066] Liquid chromatography-tandem mass spectrometer: Waters Quattro Premier XE triple quadrupole liquid chromatography-mass spectrometer;

[0067] Column: Atlantis TM T3 liquid chromatography column (150 mm × 2.1 mm, 3 μm);

[0068] Vortex mixer: Vortex-Genie2 (Scientific Industries);

[0069] Electronic analytical balance: XP105 (Mettler Toledo);

[0070] Ultrapure water generator: Mili-Q (Millipore);

[0071] Micro-adjustable pipette: range 20μL, 100μL, 200μL, 1mL, 5mL (ThermoFisher);

[0072] Robinin standard: purity 99.5% (Tianjin Alta Technology Co., Ltd.);

[0073] Taxifolin standard: purity 99.5% (Tianjin Alta Technology Co., Ltd.);

[0074] Volumetric flask: 10mL, 100mL (Tianbo Glass Instrument Co., Ltd.)

[0075] Methanol: chromatographic grade (purity 99.9%, Dima Company);

[0076] Formic acid, hydrochloric acid: chromatographically pure (Sigma-Aldrich, USA)

[0077] Vacuum pump: DOA-P504-BN diaphragm vacuum pump (GAST, USA);

[0078] Ultrapure water was used in all tests.

[0079] The specific measurement steps are as follows:

[0080] Step 1: Draw the standard curve

[0081] (1.1) Preparation of standard stock solution: Accurately weigh 0.01 g of robinin and taxol standard and place them in 10 mL volumetric flasks respectively. Dissolve them in pure methanol and dilute to the mark to prepare 1000 mg / L standard stock solution. Store at 4 °C.

[0082] (1.2) Preparation of standard working solution: Accurately pipette 1 mL of each of the above standard stock solutions into a 10 mL volumetric flask, dilute to the mark with 20% methanol water to prepare a 100 mg / L mixed standard working solution; Accurately pipette 0.1 mL of the above standard stock solutions into a 10 mL volumetric flask, dilute to the mark with 20% methanol water to prepare a 10 mg / L mixed standard working solution, and store at 4°C;

[0083] (1.3) Preparation of blank matrix extract: Weigh 1.00 g of each blank sample and place it in a 50 ml stoppered centrifuge tube and process it in the same solid phase extraction method as described above to serve as the blank matrix extract;

[0084] (1.4) Preparation of a series of standard solutions: Take an appropriate amount of the mixed standard working solution and dilute it with the blank matrix extract to form a series of matrix standard solutions with concentrations of 0.05 mg / L, 0.06 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, and 2.0 mg / L respectively;

[0085] (1.5) With the peak area of ​​the quantitative ions of robinia and tachyphylla as the Y-axis and the concentration of the target component as the X-axis, a standard curve was drawn. The linear curve is shown in the attached Figure 1 The standard curve equations and linear correlation coefficients of robinia and taxolatoin are shown in Table 1:

[0086] Table 1 Linear equations and correlation coefficients of standard curves of robinia and taxolacin

[0087]

[0088] Step 2: Sample processing and measurement

[0089] (2.1) Accurately weigh 1 g (accurate to 0.01 g) of honey sample into a 50 mL centrifuge tube, add 5 mL of deionized water, and vortex mix at 1000-2000 rpm for later use;

[0090] (2.2) Install the PXA solid phase extraction column on the solid phase extraction vacuum filtration device, activate it with 3 mL of methanol, and equilibrate the extraction column with 6 mL of deionized water;

[0091] (2.3) Transfer the honey sample solution in (3.1) above to the activated PXA solid phase extraction column and drip it at a rate of about 1 mL / min. After the loading is completed, rinse it with 3 mL of deionized water and finally dry it with a vacuum pump;

[0092] (2.4) Elute with 3 mL of 2% formic acid in methanol (v / v) solution at a flow rate of about 1 mL / min, blow dry with nitrogen at 30°C, and finally make up to 2 mL with 2% formic acid in methanol (v / v). Vortex mix at 1000-2000 rpm for later use;

[0093] (2.5) Take 1 mL of the above-mentioned fixed-volume solution, filter it through a 0.22 μm nylon filter membrane and transfer it to a brown injection vial. Then perform liquid chromatography-mass spectrometry to obtain the total ion flow diagram of acaciatin and taxolacin. See the attached Figure 2 , where the detection conditions are as follows:

[0094] Liquid chromatography-mass spectrometry was used to qualitatively and quantitatively detect robinin and taxifolin in honey samples, and the external standard method was used for quantification;

[0095] Wherein, the liquid chromatography-mass spectrometry detection conditions are:

[0096] Liquid chromatography conditions:

[0097] Chromatographic column: T3 liquid chromatography column, specification: 150mm×2.1mm, 3μm;

[0098] Flow rate: 0.3 mL / min;

[0099] Injection volume: 2 μL;

[0100] Column temperature: 35 °C;

[0101] Mobile phase A: acetonitrile; Mobile phase B: 0.1vt% formic acid aqueous solution;

[0102] The gradient elution conditions were:

[0103] 0-3.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90;

[0104] 3.0-6.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 90:10;

[0105] 6.0-6.2min, the volume ratio of mobile phase A to mobile phase B changed uniformly to 10:90;

[0106] 6.2-10.0min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90;

[0107] Ion source: electrospray ionization source ESI;

[0108] Ionization mode: positive ion mode;

[0109] Detection method: multiple reaction monitoring MRM;

[0110] Capillary voltage: 3.0kV;

[0111] Ion source temperature: 120°C;

[0112] Lens voltage: 0.1V;

[0113] Desolvation temperature: 350°C;

[0114] Desolvation gas N2 flow rate: 650L / h;

[0115] Cone gas N2 flow rate: 50L / h;

[0116] Collision gas Ar flow rate: 0.15 mL / min;

[0117] The MRM parameters were set as follows:

[0118]

[0119] The chromatograms of acaciatin and taxolatoxin are shown in the attached Figure 3 .

[0120] Optimization and screening of experimental conditions:

[0121] Optimization of mobile phase flow rate: The elution times of robinin and taxifolin on the chromatogram are relatively close, so the mobile phase flow rate is optimized to separate the elution times of robinin and taxifolin. Figure 4 shown.

[0122] Detection limit, quantification limit, recovery and precision of the method:

[0123] The detection limit of the method for robinin is 0.06 mg / kg and the detection limit of the method for taxifolin is 0.05 mg / kg when the sample concentration is not less than 3 times the signal-to-noise ratio (S / N≥3); the quantification limit of the method for robinin is 0.20 mg / kg and the quantification limit of taxifolin is 0.10 mg / kg when the sample concentration is not less than 10 times the signal-to-noise ratio (S / N≥10). The extracted ion chromatograms of the quantitative ions of the two substances are shown in the attached Figure 5 .

[0124] The recovery rate and precision of the present invention were verified by the method of blank sample spike recovery. The specific operation is as follows: standard working solutions of low, medium and high concentration levels (see Table 2) were added to blank honey samples (without robinin and taxifolin), and the analysis and detection were performed according to the above detection method and the recovery rate was calculated. The results are shown in Table 2:

[0125] Table 2 Recovery and precision results at different addition levels (n=6)

[0126]

[0127]

[0128] Table 2 shows that at three concentrations of addition, the recoveries and relative standard deviations of robinia and taxolatoin met the analytical requirements, indicating that this method has high accuracy and precision, and the determination results are true and reliable.

[0129] Example 1

[0130] Establishment and application of robinin and taxifolin as characteristic markers of honey:

[0131] Sample source: The honey samples used in this study were all natural mature honeys collected from the flowering and nectar flow seasons of various nectar-producing plants. All samples were purchased directly from beekeepers and stored at 4°C before testing.

[0132] By using the analytical detection method established by the present invention, honey samples collected from eight different nectar plants were detected, specifically including: 15 batches of acacia honey, 15 batches of wolfberry honey, 15 batches of longan honey, 15 batches of vitex honey, 15 batches of linden honey, 10 batches of jujube flower honey, 10 batches of fennel honey, and 10 batches of rapeseed honey.

[0133] The contents of robinin and taxifolin in all the honey samples mentioned above were tested, compared and analyzed. The results showed that the contents of robinin in acacia honey and wolfberry honey were much higher than those in other honeys. Taxfolin was only found in wolfberry honey. Figure 6 Therefore, it was finally determined that robinin and taxifolin can be used to distinguish and identify acacia honey, wolfberry honey and other honey types.

[0134] According to the test results of the above experimental samples, the identification basis of the following two honey plant sources can be determined as follows:

[0135] Acacia honey

[0136] A) If acacia is detected and the content is within the range of 0.20-2.00 mg / kg, the sample is pure acacia honey;

[0137] B) If acaciatin is not detected, or the detected target substance content is not within the range stated in A, the sample is not pure acacia honey (adulterated or adulterated) or non-acacia honey (honey of other honey species or non-honey).

[0138] Wolfberry Honey

[0139] A) If both robinin and taxifolin are detected, and the content of robinin is within the range of 0.20-0.80 mg / kg, and the content of taxifolin is within the range of 0.30-1.55 mg / kg, then the sample is pure wolfberry honey;

[0140] B) If taxifolin and acaciatin are not detected, or the detected target content is not within the range stated in A, the sample is impure wolfberry honey (adulterated or counterfeit) or non-wolfberry honey (other types of honey or non-honey).

[0141] For further verification, 9 batches of honey samples were purchased from an online e-commerce platform, of which A1 to A6 were marked as acacia honey and B1 to B3 were marked as wolfberry honey. The method of the present invention was used to simultaneously detect acaciatin and taxifolin, and the authenticity of each honey sample was judged using the above-mentioned discrimination basis. The results are shown in Table 3:

[0142] Table 3 Determination results of acaciatin and taxifolin in commercial honey samples and authenticity identification of acacia honey and wolfberry honey

[0143]

[0144]

[0145] a Not detected; b Real acacia honey or pure wolfberry honey; c Not real acacia honey or wolfberry honey.

[0146] The results show that: 4 out of 6 batches of acacia honey samples meet the discrimination criteria of this embodiment and are determined to be authentic acacia honey, and the other 2 batches of samples are identified as inauthentic acacia honey; 1 out of 3 batches of wolfberry honey samples meet the discrimination criteria of this embodiment and are determined to be authentic wolfberry honey, and the other 2 batches of samples are identified as inauthentic wolfberry honey. This result shows that the method of the present invention has good practicality and can be used for authenticity identification of acacia honey and wolfberry honey.

[0147] The embodiments in this specification correspond to the disclosed methods, so the description is relatively simple, and the relevant parts refer to the method part description. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. It will be obvious to professionals and technicians in this field to modify these embodiments, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will meet the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting robinin and taxifolin in honey, characterized in that: The following steps are involved: Step 1: Sample processing Weigh the honey sample, dissolve it with deionized water and mix it well; Step 2: Solid Phase Extraction Taking the sample solution treated in step 1 and performing solid phase extraction to enrich, purify and elute robinin and taxifolin; The solid phase extraction steps are as follows: (2.1) Install the PXA solid phase extraction column on a vacuum filtration device, activate it with 3 mL of methanol, and then balance it with 6 mL of deionized water; (2.2) Transferring the sample solution obtained in step 1 to the activated extraction column; (2.3) Select 3 mL of deionized water for elution, and then use a vacuum pump to dry; (2.4) Finally, use 3 mL of formic acid and methanol for elution. Blow the eluent to dryness with nitrogen at 30°C, then make up to 2 mL with formic acid and methanol. Vortex mix at 1000-2000 rpm, take 1 mL of the filter membrane and place it in a brown injection bottle for testing. Step 3: Determination Liquid chromatography-mass spectrometry was used to qualitatively and quantitatively detect acaciatin and taxifolin in honey samples, and both were quantified using the external standard method; Wherein, the liquid chromatography-mass spectrometry detection conditions are: Liquid chromatography conditions: Chromatographic column: T3 liquid chromatography column, specification: 150 mm×2.1 mm, 3 µm; Flow rate: 0.3 mL / min; Injection volume: 2 µL; Column temperature: 35 °C; Mobile phase A: acetonitrile; Mobile phase B: 0.1vt% formic acid aqueous solution; The gradient elution conditions were: 0-3.0 min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90; 3.0-6.0 min, the volume ratio of mobile phase A to mobile phase B was maintained at 90:10; 6.0-6.2 min, the volume ratio of mobile phase A to mobile phase B changed uniformly to 10:90; 6.2-10.0 min, the volume ratio of mobile phase A to mobile phase B was maintained at 10:90; Ion source: electrospray ionization source ESI; Ionization mode: positive ion mode; Detection method: multiple reaction monitoring MRM; Capillary voltage: 3.0 kV; Ion source temperature: 120 °C; Lens voltage: 0.1 V; Desolvation temperature: 350 °C; Desolvation gas N2 flow rate: 650 L / h; Cone gas N2 flow rate: 50 L / h; Collision gas Ar flow rate: 0.15 mL / min; The MRM parameters were set as follows: 。 2. The method for simultaneous detection of robinin and taxifolin in honey according to claim 1, characterized in that: The mass volume ratio of honey and water in step 1 is 0.2 g / mL.

3. The method for simultaneous detection of robinin and taxifolin in honey according to claim 1, characterized in that: The honey sample solution described in step 1 needs to be vortex mixed at 1000-2000 rpm.

4. The method for simultaneous detection of robinin and taxifolin in honey according to claim 1, characterized in that: The volume fraction of the formic acid methanol is 2%, and the filter membrane is a 0.22 μm nylon filter membrane.

5. The method for simultaneous detection of robinin and taxifolin in honey according to claim 1, characterized in that: In the external standard method quantification described in step 3, the external standard curve equation is: Robinin: Y = 0.8371X +24.607, linear correlation coefficient R 2 =0.9992; Taxifolin: Y = 7.98921X +80.4067, linear correlation coefficient R 2 =0.9991; Where X is the concentration of the target in the sample, and Y is the peak area of ​​the target in the sample.

6. The method for simultaneous detection of robinin and taxifolin in honey according to any one of claims 1 to 5, characterized in that: In the method, the detection limit of acaciatin is 0.06 mg / kg, the quantification limit is 0.20 mg / kg, and the recovery rate is greater than 80%; the detection limit of taxolanin is 0.05 mg / kg, the quantification limit is 0.10 mg / kg, and the recovery rate is greater than 80%.

Images