Method for detecting lasiocarpa alkaloid and lasiocarpa alkaloid nitric oxide in food

Abstract

The invention provides a method for detecting lasiocarpa alkaloid and lasiocarpa alkaloid nitric oxide in food. The method comprises the following steps: pre-treating a to-be-detected object; the method specifically comprises the following steps: adding a first volume of a methanol aqueous solution into each gram of a to-be-detected substance to obtain a to-be-detected substance solution; performing ultrasonic extraction and centrifugation on the to-be-detected substance solution, taking supernate, and performing water bath nitrogen blowing volatilization to a second volume to obtain a to-be-purified solution; the to-be-purified liquid is introduced into an activated MCX purification small column; eluting the enriched MCX purification small column with an ammonia water methanol solution, collecting an eluent, and carrying out nitrogen blowing volatilization on the eluent to obtain a redissolved sample; mixing the redissolved sample with a methanol aqueous solution to obtain a sample test solution; sampling the sample test solution to obtain an on-machine test solution; the on-machine test solution is detected through a liquid chromatograph-mass spectrometer, accurate determination and detection are achieved, the repeatability and reproducibility are excellent, operation is easy and convenient, the speed is high, used equipment is easy to obtain, and the method has high generalizability.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for detecting pilocarpine and pilocarpine nitrogen oxides in food. Background Art

[0002] Pilocarpine and pilocarpine N-oxide are pyrrolizidine alkaloids (PAs), which are natural toxic alkaloids that can cause hepatic sinusoidal obstruction syndrome (HSOS), and are currently one of the most concerned liver damage problems. Chinese herbal medicines and compound prescriptions containing PAS have always been the subject of strict monitoring. Many medicinal plants contain PAS. The 2015 edition of the Chinese Pharmacopoeia includes 8 Chinese herbal medicines containing PAs, involving hundreds of compound prescriptions, and their drug safety cannot be ignored. At present, there is no detection method for pilocarpine and pilocarpine N-oxide in the existing technology. Health foods may be contaminated by raw materials during the production process. Moreover, pilocarpine and pilocarpine N-oxide are not within the determination range of 9 pyrrolidine alkaloids in animal-derived foods in SN / T5450-2022, and are very easy to be overlooked and cause food safety problems. Summary of the invention

[0003] In view of the above problems, the present application is proposed to provide a method for detecting pilocarpine and pilocarpine nitrogen oxides in food that overcomes the above problems or at least partially solves the above problems, comprising:

[0004] The method for detecting pilocarpine and pilocarpine nitrogen oxides in food comprises the following steps:

[0005] Pre-treating the analyte; specifically, adding a first volume of methanol-water solution to the analyte to obtain a analyte solution; ultrasonically extracting and centrifuging the analyte solution, taking the supernatant and volatilizing it in a water bath with nitrogen to obtain a second volume of a purified solution; passing the purified solution into an activated MCX purification column; eluting the enriched MCX purification column with an ammonia-methanol solution, collecting the eluate, and volatilizing the eluate with nitrogen to obtain a reconstituted sample; mixing the reconstituted sample with a methanol-water solution to obtain a sample test solution;

[0006] Sampling the sample test solution to obtain the test solution for the machine;

[0007] The on-machine test solution is detected by liquid spectrometry.

[0008] Furthermore, the step of detecting the mixed liquid to be detected by gas chromatography-mass spectrometry uses a chromatographic column model of Agilent EclipsePlus C18 RRHD 1.8um 2.1*100mm.

[0009] Furthermore, the first volume is 18-25 mL / g.

[0010] Furthermore, the step of ultrasonically extracting and centrifuging the sample solution, and volatilizing the supernatant in a water bath with nitrogen to obtain a second volume of the solution to be purified comprises:

[0011] Performing ultrasonic extraction on the sample solution at room temperature for 20-25 minutes to obtain an extraction treatment solution;

[0012] Centrifuging the extraction treatment liquid at 6000 r / min for 10-15 min to obtain a centrifuged treatment liquid;

[0013] The supernatant of the centrifuged solution was taken and evaporated to 5 ml by nitrogen blowing in a water bath at 40° C. to obtain a solution to be purified.

[0014] Furthermore, the step of passing the liquid to be purified into the activated MCX purification column further comprises:

[0015] The MCX purification column was eluted sequentially with 5-7 ml ultrapure water and 5-7 ml methanol.

[0016] Furthermore, the step of eluting the enriched MCX column with an ammonia methanol solution, collecting the eluate, and volatilizing the eluate with liquid nitrogen to obtain a reconstituted sample comprises:

[0017] Use 4 ml of 5% ammonia methanol solution to elute the MCX purification column, and receive the eluate through a receiving tube;

[0018] The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.

[0019] Furthermore, the step of mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution comprises:

[0020] Dissolve the reconstituted sample in 10% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;

[0021] The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.

[0022] Furthermore, in the step of detecting the on-machine test solution by liquid spectrometry, the mobile phase is 0.1% formic acid aqueous solution and methanol solution.

[0023] Furthermore, it also includes:

[0024] Prepare the target detection object standard solution;

[0025] Determining the target detection object standard solution and determining a standard curve;

[0026] The test result is determined according to the standard curve.

[0027] Furthermore, the method further includes performing a spike recovery test on the substance to be tested to determine a spike recovery rate.

[0028] This application has the following advantages:

[0029] In the embodiments of the present application, in view of the fact that there is no detection scheme for pilocarpine and pilocarpine nitrogen oxides in food and health products in the prior art, the present application detects samples by liquid chromatography-mass spectrometry, filling the gap in the prior art that there is no detection scheme for pilocarpine and pilocarpine nitrogen oxides. The present application provides a method for detecting pilocarpine and pilocarpine nitrogen oxides in food, comprising the steps of: pre-treating the object to be tested; specifically, adding a first volume of methanol aqueous solution to the object to be tested; Obtain a solution of the substance to be tested; ultrasonically extract and centrifuge the solution of the substance to be tested, take the supernatant and evaporate it in a water bath with nitrogen to a second volume to obtain a solution to be purified; pass the solution to be purified into an activated MCX purification column; elute the enriched MCX purification column with an ammonia methanol solution, collect the eluate, and evaporate the eluate with nitrogen to obtain a reconstituted sample; mix the reconstituted sample with a methanol aqueous solution to obtain a sample test solution; sample the sample test solution to obtain a machine test solution; and detect the machine test solution by a liquid chromatography-mass spectrometry instrument. This application fills the market gap through high performance liquid chromatography-tandem mass spectrometry, and can detect heliotropin and heliotropin nitrogen oxides in food (including health food), meeting the requirements of customers with such detection needs, having excellent repeatability and reproducibility, simple operation, fast speed, and easy to obtain equipment, which is highly scalable. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 It is a flow chart of the steps of a method for detecting pilocarpine and pilocarpine nitrogen oxides in food provided in one embodiment of the present application;

[0032] Figure 2It is an operation flow chart of a method for detecting pilocarpine and pilocarpine nitrogen oxides in food provided in one embodiment of the present application;

[0033] Figure 3 is a standard curve diagram of pilocarpine provided in one embodiment of the present application;

[0034] Figure 4 The standard curve of heliotropin A nitrogen oxide provided in one embodiment of the present application;

[0035] Figure 5 It is a chromatogram of the heliotropin standard curve provided in an embodiment of the present application at a concentration point of 10 μg / L;

[0036] Figure 6 It is a chromatogram of the standard curve of heliotropinine nitrogen oxides provided in an embodiment of the present application at a concentration point of 10 μg / L;

[0037] Figure 7 It is a chromatogram of the heliotropin standard curve provided in an embodiment of the present application at a concentration point of 20 μg / L;

[0038] Figure 8 It is a chromatogram of the standard curve of heliotropinine nitrogen oxides provided in an embodiment of the present application at a concentration point of 20 μg / L;

[0039] Fig. 9 It is a chromatogram of the heliotropin standard curve provided in an embodiment of the present application at a concentration point of 50 μg / L;

[0040] Fig.10 It is a chromatogram of the standard curve of heliotropinine nitrogen oxides provided in an embodiment of the present application at a concentration point of 50 μg / L;

[0041] Fig.11 It is a chromatogram of the heliotropin standard curve provided in an embodiment of the present application at a concentration point of 100 μg / L;

[0042] Fig.12 It is a chromatogram of the standard curve of heliotropinine nitrogen oxides provided in an embodiment of the present application at a concentration point of 100 μg / L;

[0043] Fig.13 It is a chromatogram of the heliotropin standard curve provided in an embodiment of the present application at a concentration point of 200 μg / L;

[0044] Fig.14 It is a chromatogram of the standard curve of heliotropinine nitrogen oxides provided in an embodiment of the present application at a concentration point of 200 μg / L;

[0045] Fig.15This is a chromatogram of a food sample spiked with pilocarpine provided in an embodiment of the present application;

[0046] Fig.16 This is a chromatogram of a food sample spiked with heliotropin nitrogen oxides provided in an embodiment of the present application;

[0047] Fig.17 This is a chromatogram of the content of pilocarpine in a food sample provided in an embodiment of the present application;

[0048] Fig.18 This is a chromatogram of determining the nitrogen oxide content of pilocarpine in a food sample provided in an embodiment of the present application. DETAILED DESCRIPTION

[0049] In order to make the objects, features and advantages of the present application more obvious and understandable, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0050] The inventors have found through analysis of the prior art that: SN / T 5450-2022 is a method for determining 9 kinds of bis-pyrrolidine alkaloids in animal-derived foods. However, this method is only used for the determination of the residues of 9 bis-pyrrolidine alkaloids, including scutellarin, ...

[0051] Reference Figure 1-2 , showing a step flow chart and an operation flow chart of a method for detecting pilocarpine and pilocarpine nitrogen oxides in food provided in an embodiment of the present application:

[0052] The method comprises:

[0053] S110, pre-treating the object to be tested; specifically, adding a first volume of methanol aqueous solution to the object to be tested to obtain an object to be tested solution; performing ultrasonic extraction and centrifugation on the object to be tested solution, taking the supernatant and volatilizing it in a water bath with nitrogen to obtain a second volume of a purified solution; passing the purified solution into an activated MCX purification column; eluting the enriched MCX purification column with an ammonia methanol solution, collecting the eluate, and volatilizing the eluate with nitrogen to obtain a reconstituted sample; mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution;

[0054] S120, sampling the sample test solution to obtain the test solution for the machine;

[0055] S130, testing the on-machine test solution by liquid chromatography-mass spectrometry.

[0056] In the embodiments of the present application, in view of the fact that there is no detection scheme for pilocarpine and pilocarpine nitrogen oxides in food and health products in the prior art, the present application detects samples by liquid chromatography-mass spectrometry, filling the gap in the prior art that there is no detection scheme for pilocarpine and pilocarpine nitrogen oxides. The present application provides a method for detecting pilocarpine and pilocarpine nitrogen oxides in food, comprising the steps of: pre-treating the object to be tested; specifically, adding a first volume of methanol aqueous solution to the object to be tested; Obtain a solution of the substance to be tested; ultrasonically extract and centrifuge the solution of the substance to be tested, take the supernatant and evaporate it in a water bath with nitrogen to a second volume to obtain a solution to be purified; pass the solution to be purified into an activated MCX purification column; elute the enriched MCX purification column with an ammonia methanol solution, collect the eluate, and evaporate the eluate with nitrogen to obtain a reconstituted sample; mix the reconstituted sample with a methanol aqueous solution to obtain a sample test solution; sample the sample test solution to obtain a machine test solution; and detect the machine test solution by a liquid chromatography-mass spectrometry instrument. This application fills the market gap through high performance liquid chromatography-tandem mass spectrometry, and can detect heliotropin and heliotropin nitrogen oxides in food (including health food), meeting the requirements of customers with such detection needs, having excellent repeatability and reproducibility, simple operation, fast speed, and easy to obtain equipment, and is highly scalable.

[0057] Next, a method for detecting vinyl chloride in a beverage in this exemplary embodiment will be further described.

[0058] As described in step S110, the object to be tested is pre-treated; specifically, a first volume of methanol-water solution is added to the object to be tested to obtain an object to be tested solution; the object to be tested solution is ultrasonically extracted and centrifuged, and the supernatant is evaporated to a second volume in a water bath with nitrogen to obtain a liquid to be purified; the liquid to be purified is passed into an activated MCX purification column; the enriched MCX purification column is eluted with an ammonia-methanol solution, the eluate is collected, and the eluate is evaporated with nitrogen to obtain a reconstituted sample; the reconstituted sample is mixed with a methanol-water solution to obtain a sample test solution.

[0059] In one embodiment of the present invention, the specific process of "pre-treating the object to be tested; specifically, adding a first volume of methanol-water solution to the object to be tested to obtain an object to be tested solution; performing ultrasonic extraction and centrifugation on the object to be tested solution, taking the supernatant and volatilizing it in a water bath with nitrogen to obtain a second volume of a liquid to be purified; passing the liquid to be purified into an activated MCX purification column; eluting the enriched MCX purification column with an ammonia-methanol solution, collecting the eluate, and volatilizing the eluate with nitrogen to obtain a reconstituted sample; mixing the reconstituted sample with a methanol-water solution to obtain a sample test solution" can be further described in combination with the following description.

[0060] In one embodiment of the present application, the first volume is 18-25 mL / g, and the test object is dissolved by a methanol aqueous solution, wherein the methanol aqueous solution is a 95% methanol aqueous solution.

[0061] In a specific implementation, 2 g of sample was weighed and extracted by adding 40 mL of 95% methanol.

[0062] In one embodiment of the present application, the step of ultrasonically extracting and centrifuging the sample solution, and volatilizing the supernatant by nitrogen blowing in a water bath to a second volume to obtain a solution to be purified comprises:

[0063] Performing ultrasonic extraction on the sample solution at room temperature for 20-25 minutes to obtain an extraction treatment solution;

[0064] Centrifuging the extraction treatment liquid at 6000 r / min for 10-15 min to obtain a centrifuged treatment liquid;

[0065] The supernatant of the centrifuged solution was taken and evaporated to 5 ml by nitrogen blowing in a water bath at 40° C. to obtain a solution to be purified.

[0066] In one embodiment of the present application, the step of passing the liquid to be purified into an activated MCX purification column further includes:

[0067] The MCX purification column was eluted sequentially with 5-7 ml ultrapure water and 5-7 ml methanol.

[0068] It should be noted that after the liquid to be purified is passed through the activated MCX purification column, the MCX purification column is eluted twice with ultrapure water and methanol, the eluent is discarded, and the component to be detected is enriched in the MCX purification column.

[0069] In one embodiment of the present application, the steps of eluting the enriched MCX column with an ammonia methanol solution, collecting the eluate, and volatilizing the eluate with liquid nitrogen to obtain a reconstituted sample include:

[0070] Use 4 ml of 5% ammonia methanol solution to elute the MCX purification column, and receive the eluate through a receiving tube;

[0071] The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.

[0072] In one embodiment of the present application, the step of mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution comprises:

[0073] Dissolve the reconstituted sample in 10% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;

[0074] The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.

[0075] As described in step S120, the sample test solution is sampled to obtain the on-machine test solution.

[0076] As described in step S130, the on-machine test solution is tested by liquid chromatography-mass spectrometry.

[0077] In an embodiment of the present invention, the specific process of "testing the on-machine test solution by using a liquid chromatography-mass spectrometer" in step S130 can be further explained in combination with the following description.

[0078] The instrument parameters are:

[0079] Chromatographic reference conditions:

[0080] Instrument: HPLC / MSMS;

[0081] Chromatographic column: Agilent EclipsePlus C18 RRHD 1.8um 2.1*100mm, or equivalent;

[0082] Mobile phase: A is 0.1% formic acid aqueous solution, B is methanol solution;

[0083] Flow rate: 0.35 mL / min;

[0084] Column temperature: 30°C;

[0085] Injection volume: 2 μL;

[0086] The flow rate gradient elution program is shown in Table 1:

[0087] Time / min Mobile phase A% Mobile phase B% 0.0 95 5 1.0 95 5 6.0 65.5 34.5 8.0 58.9 41.1 10.0 0 100 11.0 95 5 12.0 95 5

[0088] Table 1

[0089] Mass spectrometry setting conditions:

[0090] Ion source: ESI;

[0091] Detection method: multiple reaction monitoring (MRM);

[0092] Scanning mode: positive ion mode;

[0093] Mass spectrometry conditions:

[0094] Ion source: ESI+;

[0095] Ion temperature: 150℃;

[0096] Desolventizing gas flow rate: 1000L / H;

[0097] Desolventization temperature: 500℃;

[0098] Capillary voltage: 1.0kV;

[0099] Collision gas flow: 0.15;

[0100] Scanning mode: multiple reaction monitoring (MRM);

[0101] Cone hole gas flow: 150L / H;

[0102] Dwell time: 0.02S.

[0103] Related test data:

[0104] Compound name: heliotropin; scanning mode: ESI+; parent ion: 412.5m / z; daughter ions: 120.3m / z, 138.3m / z; cone voltage: 30V; collision voltage: 16V; retention time: 8.56min.

[0105] Compound name: Heliotropinine N-oxide; scanning mode: ESI+; parent ion: 428.5m / z; daughter ions: 118.2m / z, 136.2m / z; cone voltage: 30V; collision voltage: 30V; retention time: 9.22min.

[0106] The linear range of the standard curve was 10-200 μg / L;

[0107] refer to Figure 3 , is the standard curve of heliotropinine determined by high performance liquid chromatography tandem mass spectrometry, Figure 5 , Figure 7 , Fig. 9 , Fig.11 and Fig.13 The chromatograms of pilocarpine at concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L and 200 μg / L are shown respectively;

[0108] refer to Figure 4 , is the standard curve of heliotropinine nitrogen oxides determined by high performance liquid chromatography tandem mass spectrometry, Figure 6 , Figure 8 , Fig.10 , Fig.12 and Fig.14 Chromatograms of pilocarpine at concentrations of 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, and 200 μg / L are shown respectively.

[0109] Figure 17-18 This is the chromatogram of pilocarpine and pilocarpine nitrogen oxides determined in a food sample. Figure 15-16 The sample spiked chromatogram shows that the method can achieve high efficiency separation and stable baseline. The recovery rate is within the satisfactory range of 60%-120% through spiked recovery test on food samples. It has good repeatability and reproducibility and accurate quantification.

[0110] It can be seen from the above embodiments that the present scheme adopts high performance liquid chromatography tandem mass spectrometry, which can detect pilocarpine and pilocarpine nitrogen oxides in food (including health food), has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, and is highly scalable.

[0111] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0112] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.

[0113] The above is a detailed introduction to the detection method of pilocarpine and pilocarpine nitrogen oxides in food provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for detecting heliotropin and heliotropin nitrogen oxides in food, characterized in that: Includes steps: Pre-treating the analyte; specifically, adding a first volume of methanol-water solution to the analyte to obtain a analyte solution; ultrasonically extracting and centrifuging the analyte solution, taking the supernatant and volatilizing it in a water bath with nitrogen to obtain a second volume of a purified solution; passing the purified solution into an activated MCX purification column; eluting the enriched MCX purification column with an ammonia-methanol solution, collecting the eluate, and volatilizing the eluate with nitrogen to obtain a reconstituted sample; mixing the reconstituted sample with a methanol-water solution to obtain a sample test solution; Sampling the sample test solution to obtain the test solution for the machine; The on-machine test solution is detected by liquid spectrometry.

2. The detection method according to claim 1, characterized in that: The step of detecting the mixed liquid to be detected by gas chromatography-mass spectrometry uses a chromatographic column model of Agilent EclipsePlus C18 RRHD 1.8um2.1*100mm.

3. The detection method according to claim 1, characterized in that: The first volume is 18-25 mL / g.

4. The detection method according to claim 1, characterized in that: The step of ultrasonically extracting and centrifuging the sample solution, and volatilizing the supernatant in a water bath with nitrogen to obtain a second volume of the solution to be purified comprises: Performing ultrasonic extraction on the sample solution at room temperature for 20-25 minutes to obtain an extraction treatment solution; Centrifuging the extraction treatment liquid at 6000 r / min for 10-15 min to obtain a centrifuged treatment liquid; The supernatant of the centrifuged solution was taken and evaporated to 5 ml by nitrogen blowing in a water bath at 40° C. to obtain a solution to be purified.

5. The detection method according to claim 1, characterized in that: The step of passing the liquid to be purified into an activated MCX purification column further comprises: The MCX purification column was eluted sequentially with 5-7 ml ultrapure water and 5-7 ml methanol.

6. The detection method according to claim 1, characterized in that: The step of eluting the enriched MCX column with an ammonia methanol solution, collecting the eluate, and volatilizing the eluate with liquid nitrogen to obtain a reconstituted sample comprises: Use 4 ml of 5% ammonia methanol solution to elute the MCX purification column, and receive the eluate through a receiving tube; The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.

7. The detection method according to claim 1, characterized in that: The step of mixing the reconstituted sample with a methanol aqueous solution to obtain a sample test solution comprises: Dissolve the reconstituted sample in 10% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution; The primary sample solution is filtered through a 0.22 mm filter membrane to obtain a sample solution.

8. The detection method according to claim 1, characterized in that: In the step of detecting the test solution on the machine by liquid chromatography-mass spectrometry, the mobile phase is 0.1% formic acid aqueous solution and methanol solution.

9. The detection method according to claim 1, characterized in that: Also includes: Prepare standard solution of target detection object; Determining the target detection object standard solution and determining a standard curve; The test result is determined according to the standard curve.

10. The detection method according to claim 1, characterized in that: The method also includes performing a spike recovery test on the test to be tested to determine a spike recovery rate.

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