Method for detecting pyrrolizidine alkaloid

Abstract

The invention discloses a method for detecting pyrrolizidine alkaloid. The method comprises the following steps: (1) carrying out adsorption treatment on an extracting solution of a solid-state or semi-solid-state sample or a liquid-state sample by adopting magnetic graphene oxide; (2) carrying out solid-liquid separation by using magnetic force, taking a solid precipitate, adding an eluent, and desorbing the pyrrolizidine alkaloid adsorbed on the magnetic graphene oxide; carrying out magnetic separation, and collecting a desorption solution; and (3) carrying out quantitative detection on the pyrrolizidine alkaloid by using the desorption solution. The method disclosed by the invention overcomes the defects of poor impurity removal performance, low magnetism, difficulty in separation and low accuracy of a traditional adsorbent, simplifies an operation procedure, is low in consumption of an organic solvent, has the characteristics of high enrichment efficiency, high sensitivity and high accuracy in the aspect of low-concentration pollutant detection, and has a wide application prospect. The method provides a new thought and technical support for simultaneous and accurate determination of various pyrrolizidine alkaloids in tea beverages or tea-containing food, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis methods and environmental functional materials, and in particular to a method for detecting pyrrolizidine alkaloids. Background Art

[0002] Pyrrolizidine alkaloids and their N-oxides are toxic secondary plant metabolites produced by more than 6,000 plants worldwide and are natural toxins. Saturated pyrrolizidine alkaloids and their N-oxides are weakly toxic or non-toxic, while unsaturated pyrrolizidine alkaloids and their N-oxides are hepatotoxic, developmentally toxic, genotoxic and carcinogenic. Pyrrolizidine alkaloids and their N-oxides, as an exogenous pollutant, have been found to be widely present in various tea products. Tea beverages, as a habitual health drink, have become one of the ways of exposure to pyrrolizidine alkaloids and their N-oxides. Accurate determination of the content of pyrrolizidine alkaloids and their N-oxides in tea beverages is a key link in tea beverages.

[0003] However, in actual tea, beverages, water, honey and other foods or beverages, the detection of pyrrolizidine alkaloids is interfered by complex matrices and trace concentrations of tea beverage samples, which affects the sensitivity and accuracy of the analysis, and requires sample pretreatment. Currently, the commonly used pretreatment methods are liquid-liquid extraction and solid phase extraction. However, they have the disadvantages of consuming a large amount of organic solvents, multi-step separation operations and poor extraction performance.

[0004] The pretreatment methods for pyrrolizidine alkaloid detection methods at home and abroad are mainly solid phase extraction and improved QuEChERS method. The solid phase extraction method uses adsorbents, such as PCX-SPE, SCX-SPE, etc., to selectively adsorb the target compound, separate the pyrrolizidine alkaloid from the interfering substances, and then use reverse elution to collect the target compound. However, conventional adsorbents have limited adsorption performance for pyrrolizidine alkaloids and low recovery rates, resulting in low sensitivity and poor accuracy of the detection results. The QuEChERS method has the defects of complex operation, time-consuming, high cost, and insufficient sensitivity. The improved QuEChERS method uses adsorbents to selectively adsorb interfering substances, uses centrifugation to separate interfering substances from pyrrolizidine alkaloids, and removes organic solvents by nitrogen blowing and other means to enrich pyrrolizidine alkaloids and improve the sensitivity of the method. For example, CN118010877A discloses that the present invention specifically discloses a method for determining pyrrolizidine alkaloids in tea, and adopts the improved QuEChERS method. The improved QuEChERS method has core adsorption materials GCB, PSA and C 18 As an adsorbent, it has low adsorption effect on pyrrolizidine alkaloids and lacks enrichment effect, resulting in insufficient sensitivity of the method and low recovery rate, most of which are between 60% and 80%. At the same time, this method has shortcomings such as poor effect in removing interference from tea matrix and complicated operation. Summary of the invention

[0005] In order to solve the problems of the prior art, the present invention provides a magnetic graphene oxide material with adsorption-desorption for pyrrolizidine alkaloids. By utilizing the selective adsorption of magnetic graphene oxide on pyrrolizidine alkaloids, the purpose of efficient enrichment of pyrrolizidine alkaloids and effective removal of tea matrix is ​​achieved through pH adjustment; the magnet effect of magnetic graphene oxide is utilized to establish a simple and rapid pretreatment technology. The present invention greatly improves the removal effect of interfering substances, enhances the enrichment effect of core materials on pyrrolizidine alkaloids, and improves the accuracy and sensitivity of the pyrrolizidine alkaloid detection method.

[0006] The technical solution adopted by the present invention is:

[0007] A method for detecting pyrrolizidine alkaloids, the method comprising the following steps:

[0008] (1) Using magnetic graphene oxide to adsorb the extract of liquid samples or solid or semi-solid samples;

[0009] (2) using magnetic force to separate solid from liquid, taking solid precipitate and adding eluent to desorb pyrrolizidine alkaloids adsorbed on magnetic graphene oxide; performing magnetic separation and collecting desorption liquid;

[0010] (3) The desorbed liquid is subjected to quantitative detection of pyrrolizidine alkaloids.

[0011] In the step (1), the adsorption treatment is to adsorb the pyrrolizidine alkaloid in the solid, semi-solid sample or liquid sample onto the magnetic graphene oxide.

[0012] The liquid sample refers to a sample whose raw material is liquid, such as a beverage.

[0013] The solid samples refer to tea leaves and various solid food samples containing tea extracts, and the semi-solid samples refer to semi-solid food samples such as honey, ice cream, porridge, yogurt, etc. containing tea extracts. Solid and semi-solid samples need to be extracted to obtain extracts.

[0014] The specific extraction method is generally:

[0015] A solid sample or a semi-solid sample is taken, crushed or mixed, and then the pyrrolizidine alkaloid is extracted with dilute sulfuric acid to obtain an extract, which is then processed in step (1).

[0016] Dilute sulfuric acid generally has a concentration of 0.1 to 0.2 M.

[0017] The extraction can be performed twice, and the supernatants from the two extractions are combined to obtain an extract.

[0018] The volume of dilute sulfuric acid is generally 10 to 15 mL / g based on the mass of the solid sample or semi-solid sample.

[0019] The pyrrolizidine alkaloid is one or more of the following: heliotrine, heliotrine-N-oxide, retrorsine, retrorsine-N-oxide, senecionine, senecionine-N-oxide, jacobine, jacobine-N-oxide, intermedine, intermedine-N-oxide, seneciphylline, seneciphylline N-oxide, europine, europine N-oxide, senkirkine

[0020] In the step (1), the magnetic graphene oxide is prepared by chemical coprecipitation. Specifically, magnetic iron oxide nanoparticles are loaded on graphene oxide by chemical coprecipitation to obtain magnetic graphene oxide.

[0021] Preferably, the magnetic graphene oxide is prepared by the following method:

[0022] Ferric chloride and ferrous chloride are added to the graphene oxide suspension, stirred and mixed at 70-80° C., the pH value is adjusted to 10-11, and stirred and reacted at 70-80° C. to obtain magnetic graphene oxide.

[0023] Preferably, the molar ratio of ferric chloride to ferrous chloride is 2-3:1.

[0024] The mass ratio of the graphene oxide to the iron oxide is 1:0.3-3, preferably 1:3.

[0025] The concentration of the graphene oxide suspension is preferably 1 to 10 mg / mL;

[0026] The pH value can be adjusted by using 20-30% ammonia water.

[0027] The stirring reaction time is preferably 1 to 2 hours.

[0028] After the reaction is completed, magnetic solid-liquid separation is preferably used, and the precipitate is centrifuged, washed and dried to obtain magnetic graphene oxide.

[0029] In the step (1), the amount of magnetic graphene oxide used is 10 to 50 mg / 10 mL, preferably 30 mg / 10 mL, based on the volume of the liquid sample or the solid or semi-solid sample extract;

[0030] In the step (1), the adsorption time is 10 to 60 minutes, preferably 30 minutes, and the adsorption temperature is room temperature.

[0031] Preferably, shaking is performed during adsorption.

[0032] In the step (1), during adsorption, the pH value is adjusted to 3-9, preferably 4. The pH value can be adjusted using phosphoric acid, boric acid, acetic acid, sodium hydroxide, or BR buffer.

[0033] In the step (2), the eluent is a methanol solution containing 0.1 to 5% ammonia by volume, preferably a methanol solution containing 1% ammonia by volume.

[0034] The volume of the eluent is 0.5 to 4 mL / 30 mg, preferably 2 mL / 30 mg, based on the mass of the solid precipitate.

[0035] In the step (2), desorption is preferably carried out under ultrasound, and the ultrasound time is 3 to 15 minutes, preferably 6 minutes.

[0036] In the step (3), the quantitative detection can be performed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS).

[0037] In the step (3), the UHPLC-MS / MS chromatographic conditions are as follows: an ACQUITY UPLC HSS T3 column (100х2.1 mm id, 1.8 μm particle size, Agilent, USA) is used to separate the target compound. Column temperature: 40°C. Methanol is used as mobile phase A (MPA) and water is used as mobile phase B (MPB), and 0.1% formic acid and 1 mM ammonium formate are added as buffer for gradient elution, with a flow rate of 0.25 mL / min. Gradient elution program: 0-1 min, 90% B; 1-4 min, 90%-40% B; 4-7 min, 40%-30% B; 7-7.1 min, 30%-2% B; 7.1-11 min, 2% B; 11-11.1 min, 2%-90% B; 11-14 min, 90% B. Injection volume: 3 μL.

[0038] In step (3), the UHPLC-MS / MS mass spectrometry conditions are: the electrospray ion source positive ion scanning mode (ESI+) is used in programmable multiple reaction monitoring mode (sMRM); the ESI is set to a gas temperature of 500°C and a capillary voltage of 5500 V. The flow rates of the curtain gas (CUR) and the collision gas (CAD) are set to 35 psi and 7 psi, respectively. The nebulizer gas pressure (GS1) and the auxiliary gas (GS2) are both set to 50.0 psi.

[0039] The present invention uses magnetic graphene oxide material as an adsorbent to adsorb various pyrrolizidine alkaloids in a sample. After screening various conventional adsorbents and functional group-modified graphene oxide materials, it is found that the optimal adsorbent is graphene oxide material. The graphene oxide material has a large specific surface area and many surface active sites. It can effectively combine with pyrrolizidine alkaloids through π-π interaction, hydrogen bonding and electrostatic interaction, etc., and can provide a sufficiently large phase interface for the subsequent magnetic dispersion solid phase extraction process, promoting the adsorption of target molecules. And after the graphene oxide material is loaded with iron oxide magnetic nanoparticles, magnetic force can be used for solid-liquid separation, which is conducive to improving the separation efficiency.

[0040] The invention also optimizes the adsorption and desorption conditions. The invention has a recovery rate of 80% to 120% for pyrrolizidine alkaloids, a detection limit as low as 0.01 to 0.5 μg / L, a quantitative limit of 3.0 to 5.0 μg / L, and can accurately measure the trace level of pyrrolizidine alkaloids.

[0041] The present invention adopts magnetic graphene oxide material as magnetic dispersed solid phase extraction material, selectively adsorbs a variety of pyrrolizidine alkaloids in the sample, and achieves the purpose of efficient enrichment of pyrrolizidine alkaloids and effective removal of tea matrix by pH adjustment. In the present invention, the target is adsorbed on the magnetic graphene oxide, and the magnetic effect of the magnetic graphene oxide can be quickly separated, and the operation is simple. Then the solid precipitate is eluted, and the desorption liquid is quantitatively detected. The method of the present invention is completely opposite to the technical principle of the traditional method of using an adsorbent to remove impurities and detecting the supernatant. The method of the present invention overcomes the defects of poor impurity removal performance, low magnetism, difficult separation, and low accuracy of traditional adsorbents, simplifies the operating procedure, consumes less organic solvent, and exhibits high enrichment efficiency, high sensitivity, and high accuracy in the detection of low-concentration pollutants. The present invention greatly improves the effect of removing interfering substances, enhances the enrichment effect of the core material on pyrrolizidine alkaloids, improves the accuracy and sensitivity of the pyrrolizidine alkaloid detection method, reduces the detection limit, and improves the recovery rate, providing new ideas and technical support for the simultaneous and accurate determination of multiple pyrrolizidine alkaloids in tea beverages or tea-containing foods, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a comparison chart of the adsorption rate of pyrrolizidine alkaloids by different adsorbents.

[0043] Figure 2 Fourier transform infrared spectra of graphene oxide, magnetic graphene oxide, and iron oxide nanoparticles.

[0044] Figure 3 This is a comparison of the adsorption rates of pyrrolizidine alkaloids on magnetic graphene oxide with different ratios.

[0045] Figure 4 is the adsorption rate of magnetic graphene oxide at different pH values.

[0046] Figure 5 This is a graph showing the adsorption efficiency of magnetic graphene oxide materials for various pyrrolizidine alkaloids in green tea under optimized adsorption conditions.

[0047] Figure 6 The recovery rate of pyrrolizidine alkaloids eluted from magnetic graphene oxide using eluents of different ammonia concentrations. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and tables. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0049] Example 1

[0050] In order to solve the problems of large consumption of organic solvents and complex operation of pyrrolizidine alkaloids in traditional pretreatment, as well as the problem that adsorbents are difficult to separate in dispersed solid phase extraction, the present invention takes the selective enrichment and separation of pyrrolizidine alkaloids in tea beverages as the primary goal, selects a variety of adsorbents to explore the adsorption effect, and finally determines that graphene oxide material loaded with ferroferric oxide magnetic nanoparticles is used as an adsorbent, and performs magnetic dispersed solid phase extraction treatment on pyrrolizidine alkaloids in tea beverages.

[0051] In the process of magnetic dispersion solid phase extraction, in order to obtain the best performance materials, traditional inorganic materials and organic materials were selected as adsorbents for experiments. The traditional inorganic materials are activated carbon, magnesium silicate, and graphite powder, and the organic materials are graphene oxide materials modified with various functional groups, including sulfonic acid functionalized graphene oxide, imidazole ionic liquid graphene oxide, chitosan functionalized graphene oxide, and unmodified graphene oxide materials.

[0052] In the process of synthesizing graphene oxide materials modified with various functional groups, the improved Hummers method is first used to prepare graphene oxide materials, and then sulfonic acid functionalized graphene oxide, imidazole ionic liquid graphene oxide, and chitosan functionalized graphene oxide are prepared respectively.

[0053] The improved Hummers method for preparing graphene oxide aqueous suspension specifically comprises the following steps:

[0054] ① Add 6g of graphite powder and 3g of sodium nitrate to 138mL of 98% sulfuric acid, keep the temperature at 0℃ and stir, then slowly add 18g of potassium permanganate within 30min;

[0055] ② Transfer the three-necked flask containing the mixed solution to an oil bath at 35°C and stir for 30 minutes. Slowly add 276 mL of deionized water at room temperature, and then transfer it to an environment at 96°C for oxidation for 60 minutes.

[0056] ③ After cooling to room temperature, add 250 mL of deionized water to dilute the mixture, and then add 20 mL of hydrogen peroxide until no bubbles are generated. Filter the mixture, rinse with deionized water to remove residual acid until the pH value reaches neutral, and dry in an oven at 50 ° C to obtain unmodified graphene oxide material.

[0057] Imidazole ionic liquid graphene oxide is prepared according to the following steps:

[0058] 0.3 g of graphene oxide was mixed with 50 ml of acetone, and then 0.3 g of 1-hexyl-3-methylimidazolium tetrafluoroborate was added, stirred for 3 hours, washed with deionized water and anhydrous ethanol, and vacuum freeze-dried for 10 hours to obtain imidazole ionic liquid graphene oxide.

[0059] Chitosan functionalized graphene oxide was prepared according to the following steps:

[0060] Chitosan was dissolved in 1% acetic acid to obtain a chitosan dilute acetic acid solution, and then the graphene oxide dispersion was added to the chitosan solution and ultrasonicated for 60 minutes to form a gray uniform solution. The mixture was hydrothermaled at 100°C for 3 hours and then freeze-dried to obtain chitosan functionalized graphene oxide.

[0061] Sulfonic acid functionalized graphene oxide was prepared according to the following steps:

[0062] Add p-aminobenzenesulfonic acid (0.01 mol) to a three-necked bottle containing 1M HCl (100 mL) at 0-5°C and stir continuously. Then, add 10% excess 1M NaNO2 aqueous solution (11 mL) dropwise to the mixture until it becomes a clear solution, stir for another hour, filter the resulting precipitate and wash with deionized water. Add the resulting precipitate p-aminobenzenesulfonic acid diazotide to 300 mL GO (3 mg / mL) solution and stir. The resulting product is washed with deionized water and anhydrous ethanol and freeze-dried in vacuum to obtain sulfonic acid functionalized graphite oxide.

[0063] The conventional adsorbents (activated carbon, magnesium silicate, graphite powder) and synthetic graphene oxide adsorbents were used to adsorb various pyrrolizidine alkaloids and screen the material with the best adsorption, which specifically includes the following steps:

[0064] ① Weigh 0.2 g of adsorbent, the adsorbents are activated carbon, magnesium silicate, graphite powder, sulfonic acid functionalized graphene oxide, imidazole ionic liquid graphene oxide, chitosan functionalized graphene oxide and unmodified graphene oxide material.

[0065] ② Prepare 15 pyrrolizidine alkaloid solutions with a concentration of 500 μg / L each, with a volume of 10 mL, add various adsorbents under neutral conditions, and perform adsorption at a reaction temperature of 298 K, pH = 7, an oscillation speed of 180 rpm, and a reaction time of 30 min.

[0066] ③After the experiment, the solution was filtered through a 0.22 μm organic microporous filter membrane and analyzed by UHPLC-MS / MS. The concentration changes before and after adsorption were compared, the adsorption rates of 15 pyrrolizidine alkaloids were calculated respectively, and the adsorbent material with the largest adsorption rate was selected.

[0067] UHPLC-MS / MS chromatographic conditions: ACQUITY UPLC HSS T3 column (100х2.1mm id, 1.8μm particle size, Agilent, USA) was used to separate the target compounds. Column temperature: 40°C. Methanol was used as mobile phase A (MPA) and water was used as mobile phase B (MPB), and 0.1% formic acid and 1mM ammonium formate were added as buffer for gradient elution at a flow rate of 0.25mL / min. Gradient elution program: 0-1min, 90% B; 1-4min, 90%-40% B; 4-7min, 40%-30% B; 7-7.1min, 30%-2% B; 7.1-11min, 2% B; 11-11.1min, 2%-90% B; 11-14min, 90% B. Injection volume: 3μL.

[0068] The UHPLC-MS / MS mass spectrometry conditions were as follows: the electrospray ion source was in positive ion scanning mode (ESI+) and programmable multiple reaction monitoring mode (sMRM); the ESI was set to a gas temperature of 500°C and a capillary voltage of 5500 V. The flow rates of the curtain gas (CUR) and collision gas (CAD) were set to 35 psi and 7 psi, respectively. The nebulizer gas pressure (GS1) and auxiliary gas (GS2) were both set to 50.0 psi.

[0069] The results are as follows Figure 1 As shown, Figure 1Pyrrolizidine alkaloids represented by seneciopine (Sp) and pyrrolizidine alkaloid nitrogen oxides represented by seneciopine-N-oxide (SpNO) were selected as targets, and the adsorption rates of five adsorbents for these two targets were listed, MgSiO3 represents magnesium silicate, C represents activated carbon, G represents graphite powder, CS-GO represents chitosan graphene oxide, IL-GO represents imidazole ionic liquid graphene oxide, and S-GO represents mercaptosulfonic acid-based graphene oxide.

[0070] Figure 1 The results showed that the best adsorbent material was unmodified graphene oxide.

[0071] Example 2

[0072] The best adsorbent material, graphene oxide, was optimized and loaded with magnetic iron oxide nanoparticles. Magnetic graphene oxide was prepared by chemical coprecipitation method, and the iron content was optimized as follows:

[0073] The graphene oxide material is dispersed in deionized water at a concentration of 3 mg / mL, and ultrasonically treated for 60 minutes. An appropriate amount of iron (III) chloride hexahydrate and iron (II) chloride tetrahydrate (the ratio of iron (III) chloride hexahydrate to iron (II) chloride tetrahydrate is controlled at 2:1-3:1) is added to the above reaction system. According to the chemical reaction, the two generate iron oxide, and the mass ratio of graphene oxide to iron oxide is controlled to be 1:3, 1:1 and 3:1 respectively. The mixture is stirred at 80°C for 30 minutes, and ammonia water (20-30%) is added to adjust the pH value of the solution to 10-11. The mixture is stirred at 80°C for 60 minutes. After the reaction is completed, the black precipitate is separated by magnetic force, centrifuged, washed and dried to obtain magnetic graphene oxide with different iron contents.

[0074] Fourier transform infrared spectra of graphene oxide (GO), magnetic graphene oxide (MGO), and ferroferric oxide nanoparticles are shown in Figure 2. Figure 2 As shown. In the magnetic graphene oxide material, at 3500cm -1 、1620cm -1 、1230cm -1 、1070cm -1 There are characteristic peaks at 570cm-1 and 570cm-1, which are related to the -OH stretching vibration, C=C stretching vibration peak of the aromatic structure skeleton, COC epoxy group stretching vibration peak, alkoxy CO stretching and Fe-O bond stretching vibration. It also contains characteristic peaks of graphene oxide and iron oxide, which confirms that the composite material loads iron oxide on graphene oxide. Figure 2 The results showed that the successful preparation of the composite material magnetic graphene oxide was confirmed.

[0075] 0.2 g of magnetic graphene oxide material with different iron contents was weighed and added into 10 mL of 15 pyrrolizidine alkaloid solutions with concentrations of 500 μg / L each. The adsorption was carried out at a reaction temperature of 298 K, an oscillation speed of 180 rpm, pH = 7, and a reaction time of 30 min. A magnet was used to separate the solution and the adsorption material, and then the solution was passed through a 0.22 μm organic microporous filter membrane. UHPLC-MS / MS was used for sample analysis, and the concentration changes before and after adsorption were compared to calculate the adsorption rate.

[0076] The results are as follows Figure 3 As shown, Figure 3 The adsorption rates of 15 pyrrolizidine alkaloids by magnetic graphene oxide in different proportions are shown in Figure 3. MGO1, MGO2 and MGO3 represent the mass ratios of graphene oxide and iron oxide of 1:0.3, 1:1 and 1:3 respectively. Combined with the hysteresis loop, MGO3 has the best magnetic properties, and the effects of the three proportions on the adsorption rate of magnetic graphene oxide are within 10%. According to the adsorption rate and magnetic strength, it is finally determined that the material for magnetic dispersed solid phase extraction is a magnetic graphene oxide material with a graphene oxide and iron oxide content of 1:3.

[0077] Example 3

[0078] The adsorption conditions of magnetic graphene oxide for various pyrrolizidine alkaloids in tea beverages were optimized, including material pH, adsorbent addition amount and contact time.

[0079] The specific steps are as follows:

[0080] Fifteen pyrrolizidine alkaloids were added to 10 mL of green tea beverage at a concentration of 0.5 mg / L each. Under the same conditions, the pH was adjusted to 3-9, the oscillation time was 10-60 min, the oscillation speed was 180 rpm, and the amount of magnetic graphene oxide added was 10-50 mg, wherein the mass ratio of graphene oxide to iron oxide was 1:3.

[0081] After the adsorption, a magnet was used to separate the solution and the adsorption material, and then the solution was filtered through a 0.22 μm organic microporous filter membrane and analyzed by UHPLC-MS / MS. The concentration changes before and after adsorption were compared and the adsorption rate was calculated.

[0082] In the adsorption condition optimization experiment, the adsorption rates of seneciopine (Sp) and seneciopine-N-oxide (SpNO) on magnetic graphene oxide at different pH values ​​are shown in the following table: Figure 4 As shown, Figure 4 It shows that the adsorption rate under acidic conditions is better than that under neutral and alkaline conditions, and the adsorption effect is best at pH 4.

[0083] The results of optimization experiments on different adsorption conditions showed that the optimal pH value was 4, the optimal adsorption time was 30 min, and the optimal adsorbent addition amount was 30 mg.

[0084] Under the above-mentioned adsorption optimization conditions, the adsorption rates of various pyrrolizidine alkaloids in green tea by magnetic graphene oxide materials are as follows: Figure 5 As shown, the adsorption rates of 15 pyrrolizidine alkaloids in tea beverages were all greater than 70%.

[0085] The 15 pyrrolizidine alkaloids are: Heliotrine (Heliotrine), Heliotrine-N-oxide (Heliotrine-N-oxide, HeNO), Retrorsine (Re), Retrorsine-N-oxide (ReNO), Senecionine (Sc), Senecionine-N-oxide (ScNO), Jacobine (Jb), Jacobine-N-oxide (JbNO), Intermedine (Im), Intermedine-N-oxide (ImNO), Seneciphylline (Sp), Seneciphylline N-oxide (SpNO), Europine (Eu), Europine N-oxide (EuNO), Senkirkine (Sk).

[0086] Example 4

[0087] The pyrrolizidine alkaloids on the magnetic graphene oxide were desorbed and the desorption conditions were optimized. The specific steps are as follows:

[0088] (1) Fifteen pyrrolizidine alkaloids were added to 10 mL of green tea beverage at a concentration of 0.5 mg / L each. Under the same conditions, the pH was adjusted to 4, the oscillation time was 30 min, the oscillation speed was 180 rpm, and the amount of magnetic graphene oxide added was 30 mg, wherein the mass ratio of graphene oxide to iron oxide was 1:3.

[0089] (2) Using magnetic force to separate solid and liquid, taking the solid precipitate and adding an eluent, desorbing the pyrrolizidine alkaloids adsorbed on the magnetic graphene oxide under the action of ultrasound; collecting the desorbed liquid by magnetic separation; passing the desorbed liquid through a 0.22 μm organic microporous filter membrane, and then analyzing the sample by UHPLC-MS / MS to calculate the recovery rate.

[0090] The eluent is a methanol solution of ammonia water, the volume of the eluent is 0.5-4 mL, the ammonia content in the eluent is 0%-5%, the ultrasonic time is 3-15 min, and the desorption optimization is performed to obtain the best desorption conditions.

[0091] The recovery rate of pyrrolizidine alkaloids eluted from magnetic graphene oxide with different ammonia concentrations Figure 6 shown. Figure 6 The results show that the recovery rate is good when the ammonia concentration is above 1%. However, pyrrolizidine alkaloids will degrade if the ammonia concentration is too high and stored for a long time. Considering that the storage time from desorption to detection may vary in different laboratories, in order to prevent high concentration ammonia from degrading the target compound over a long period of time, the preferred ammonia concentration is 1%.

[0092] Finally, the best desorption optimization conditions were obtained by adjusting the recovery rate of pyrrolizidine alkaloids, specifically, the volume was 2 mL, the eluent was a mixed solution of 1% ammonia water and methanol, and the desorption effect was best under ultrasonic treatment for 6 minutes.

[0093] Example 5

[0094] The quantitative detection steps of Examples 1 to 4 are as follows:

[0095] The reagents used in the detection method are prepared as follows:

[0096] (1) Preparation of standard solutions: Standard stock solutions of each substance were initially prepared in 20 mL volumetric flasks. Solid standard substances (5 mg or 10 mg) were dissolved in methanol to obtain an initial concentration of 250 mg / L or 500 mg / L of pyrrolizidine alkaloid standard substances. These solutions were then stored at -20 °C. Subsequently, the previously prepared standard solutions were diluted to 0.1 mg / L or other concentrations required for specific experimental applications to prepare a mixed solution containing 15 pyrrolizidine alkaloids and a separate standard solution of each pyrrolizidine alkaloid.

[0097] (3) Preparation of standard working solution: Accurately pipette a certain volume of standard solution and dilute it with blank matrix solution into a series of standard working solutions of 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, 100 μg / L, 200 μg / L and 500 μg / L. Prepare them before use.

[0098] The present invention mainly verifies the method by evaluating the linear range, recovery, intra-day / inter-day precision, detection limit (LOD) and quantification limit (LOQ). Table 1 shows the linear equation, correlation coefficient, detection limit and quantification limit of 15 pyrrolizidine alkaloids.

[0099] Table 1 Linear equations, correlation coefficients, detection limits and quantification limits of pyrrolizidine alkaloids

[0100]

[0101] See Table 1. In the standard working curve, the linear range of 15 targets is 1-500 μg / L, and the correlation coefficient of the linear fitting curve is R 2 The values ​​were all greater than 0.99, indicating good linearity. The detection limit was 0.01-0.5 μg / L, and the quantification limit was 3.0-5.0 μg / L, which was sufficient for the determination of trace levels of pyrrolizidine alkaloids.

[0102] The green tea beverage was adsorbed with magnetic graphene oxide according to the optimal adsorption conditions of Example 3, and the specific steps were as follows: the pH of 10 mL of green tea beverage was adjusted to 4, the oscillation time was 30 min, the oscillation speed was 180 rpm, the amount of magnetic graphene oxide added was 30 mg, wherein the mass ratio of graphene oxide to iron oxide was 1:3, and after the adsorption treatment, solid-liquid separation was performed by magnetic force, and the supernatant was taken to obtain a green tea purified liquid.

[0103] Spiking experiments of different concentrations were carried out in green tea purified liquid to evaluate the sensitivity and accuracy. The results showed that the detection limit of 15 target substances was 0.01-0.5 μg / L, and the quantification limit was 3.0-5.0 μg / L. The method of the present invention can accurately determine the trace levels of pyrrolizidine alkaloids.

[0104] For green tea beverage samples, the accuracy and precision were verified at three spike levels (10, 50 and 100 μg / L). The specific steps are as follows:

[0105] (1) Fifteen pyrrolizidine alkaloids were added to 10 mL of green tea beverage at concentrations of 10, 50, and 100 μg / L, respectively. Under the same conditions, the pH was adjusted to 4, the oscillation time was 30 min, the oscillation speed was 180 rpm, and the amount of magnetic graphene oxide added was 30 mg. The mass ratio of graphene oxide to iron oxide was 1:3.

[0106] (2) using magnetic force to separate solid and liquid, taking the solid precipitate and adding 2 mL of eluent, ultrasonicating for 6 min to desorb the pyrrolizidine alkaloids adsorbed on the magnetic graphene oxide; and collecting the desorbed liquid by magnetic separation;

[0107] (3) The desorbed liquid was subjected to UHPLC-MS / MS quantitative analysis, and the recovery rate was calculated. The results are shown in Table 2. Table 2 shows the recovery rate and intra-day / inter-day precision of the three concentrations of pyrrolizidine bio-spiking experiment in green tea beverage.

[0108] Table 2 Recovery and intra-day / inter-day precision of pyrrolizidine biospiked

[0109]

[0110]

[0111] As shown in Table 2, at different spike levels, the recoveries of 15 pyrrolizidine alkaloids ranged from 70% to 100%, and the relative standard deviations ranged from 1% to 12%. These results confirm the reliability of the method for detecting pyrrolizidine alkaloids using the prepared magnetic graphene oxide material, which has great application value.

[0112] Example 6

[0113] 1. Take 20 g of tea sample and grind it with a grinder. The ground dry tea sample (1.0 g) is weighed into a 50 mL centrifuge tube, and then 15 pyrrolizidine alkaloids are added at concentrations of 10, 50 and 100 μg / kg, respectively.

[0114] 2. Add 10 mL of 0.1 M H2SO4 and vortex for 5 min to extract the pyrrolizidine alkaloids.

[0115] 3. The extract was centrifuged at 10000 rpm (11627хg) for 10 min, the supernatant was taken, and the solid precipitate was re-extracted under the same conditions.

[0116] 4. Combine the supernatants obtained from the two extraction methods to a volume of approximately 20 mL, and then use 10 mL of the crude extract as a sample for testing.

[0117] 5.10mL crude extract, adjusted pH to 4, oscillation time 30min, oscillation speed 180rpm, the amount of magnetic graphene oxide added is 30mg, and the mass ratio of graphene oxide to iron oxide is 1:3.

[0118] 6. Use magnetic force to separate solid and liquid, take the solid precipitate and add 2 mL of eluent, sonicate for 6 min to desorb the pyrrolizidine alkaloids adsorbed on the magnetic graphene oxide; collect the desorbed liquid by magnetic separation;

[0119] 7. The desorbed liquid was subjected to UHPLC-MS / MS quantitative analysis and the recovery rate was calculated. The results showed that at different spike levels, the recoveries of 15 pyrrolizidine alkaloids were between 70% and 100%, and the relative standard deviations were between 1% and 16%.

Claims

1. A method for detecting pyrrolizidine alkaloids, characterized in that The method comprises the following steps: (1) Using magnetic graphene oxide to adsorb the extract of solid or semi-solid samples or liquid samples; (2) using magnetic force to separate solid from liquid, taking solid precipitate and adding eluent to desorb pyrrolizidine alkaloids adsorbed on magnetic graphene oxide; performing magnetic separation and collecting desorption liquid; (3) The desorbed liquid is subjected to quantitative detection of pyrrolizidine alkaloids.

2. The method according to claim 1, characterized in that The extracts of solid and semi-solid samples are obtained by the following method: A solid sample or a semi-solid sample is taken, crushed or mixed, and then the pyrrolizidine alkaloid is extracted with dilute sulfuric acid to obtain an extract.

3. The method according to claim 1, characterized in that The pyrrolizidine alkaloids are one or more of the following compounds: heliotropin, heliotropin-N-oxide, sedumine, sedumine-N-oxide, sedumine, sedumine-N-oxide, sedumine, sedumine-N-oxide, melanocyte-stimulating hormone, melanocyte-stimulating hormone-N-oxide, sedumine, sedumine-N-oxide, heliotropin, heliotropin-N-oxide, heliotropin, heliotropin-N-oxide, heliotropin, heliotropin-N-oxide, and heliotropin.

4. The method according to claim 1, characterized in that The magnetic graphene oxide is prepared by a chemical coprecipitation method, in which magnetic iron oxide nanoparticles are loaded on the graphene oxide to obtain the magnetic graphene oxide.

5. The method according to claim 4, characterized in that The mass ratio of graphene oxide to iron oxide is 1:0.3-3, and the iron oxide is prepared by the reaction of ferric chloride and ferrous chloride.

6. The method according to claim 5, characterized in that The mass ratio of the graphene oxide to the iron oxide is 1:

3.

7. The method according to claim 1, characterized in that In the step (1), the amount of magnetic graphene oxide used is 10 to 50 mg / 10 mL based on the volume of the liquid sample or the solid or semi-solid sample extract, the adsorption time is 10 to 60 min, and the pH value is adjusted to 3 to 9 during adsorption.

8. The method according to claim 7, characterized in that In the step (1), the amount of magnetic graphene oxide used is 30 mg / 10 mL based on the volume of the liquid sample or the solid or semi-solid sample extract, the adsorption time is 30 min, and the pH value is adjusted to 4 during adsorption.

9. The method according to claim 1, characterized in that In the step (2), the eluent is a methanol solution containing 0.1 to 5% ammonia water by volume, the volume of the eluent is 0.5 to 4 mL / 30 mg based on the mass of the solid precipitate, and the desorption is carried out under ultrasound for 3 to 15 min.

10. The method according to claim 9, characterized in that In the step (2), the eluent is a methanol solution containing 1% ammonia water by volume, the volume of the eluent is 2 mL / 30 mg based on the mass of the solid precipitate, and the desorption is carried out under ultrasound for 6 minutes.

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