Method-internal standard method for determining capillamine in beverage

Abstract

The invention relates to a method, namely an internal standard method, for determining capillamine in a beverage. The invention discloses a method for determining capillamine in a beverage by using an internal standard method, and belongs to the technical field of food detection. The method mainly comprises the following steps: pretreatment of a sample, determination of characteristic ions of a target object and an internal standard object, establishment of a liquid chromatography-tandem mass spectrometry selective reaction monitoring method, determination of a matrix matching standard curve, and monitoring (qualitative) and detection (quantitative) of capillamine in the beverage. The detection method is good in linear correlation, standard addition recovery rate and repeatability, the detection limit is 0.05 ng / mL, the quantitation limit is 0.1 ng / mL, and a technical guarantee can be provided for detection of the capillamine in the beverage.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a method for determining mitragynine in a beverage by using an internal standard method. Background Art

[0002] Mitragyna speciosa, a common tropical jungle tree in Southeast Asia Mitragyna speciosa , belongs to the genus Mitragynaceae. Scientific research has found that about 25 alkaloids can be extracted from Kratom extracts, among which the highest and most important alkaloid is mitragynine, with a maximum content of up to 66%. The activity of mitragynine is mainly mediated by its active metabolite 7-hydroxymitragynine in vivo and in vitro. The in vitro biological activity of 7-hydroxymitragynine is 10 times that of mitragynine. Both are biased mu-opioid receptor (MOR) agonists and are "atypical opioid compounds" that can be used to prevent or relieve withdrawal symptoms in patients with opioid dependence.

[0003] Mitragynine compounds have certain medicinal value, but frequent use can lead to dependence, that is, addiction, and mixing them with other drugs may cause death.

[0004] At present, the detection of mitragynine is mostly for the content detection in the plant beautiful mitragynine or the body fluids (urine, blood, etc.) of humans / animals, and the methods of LC-UV and LC-MS are mostly used. There is no method for detecting the content of mitragynine in the beverage matrix. Compared with plants or body fluids, the composition of the beverage matrix is ​​more complicated. The present invention is mainly aimed at the beverage matrix, and the extraction of mitragynine is optimized in the pretreatment process, and finally a mixed cation exchange solid phase extraction column is selected. Summary of the invention

[0005] The purpose of the present invention is to solve the gap in existing detection technology and establish a method for determining mitragynine in beverages. The method utilizes solid phase extraction and liquid chromatography-triple quadrupole mass spectrometry, uses an internal standard method to detect mitragynine in beverages, and performs qualitative and quantitative analysis on the content of mitragynine therein.

[0006] The technical scheme adopted by the present invention is as follows: The present invention provides a method for determining mitragynine in beverages, comprising the following steps: after adding an isotope internal standard to the beverage, after weak acidification, extracting with a mixed cationic solid phase extraction column to obtain a test solution, and using liquid chromatography-tandem mass spectrometry to perform qualitative and quantitative analysis on the mitragynine component in the beverage.

[0007] Furthermore, the method for determining mitragynine in a beverage specifically comprises the following steps:

[0008] S1 Preparation and storage of standard solutions of mitragynine and its isotope internal standard mitragynine-d3: Use methanol to prepare standard stock solutions of mitragynine and its isotope internal standard mitragynine-d3, dilute the above stock solutions with methanol as standard working solutions of mitragynine and its isotope internal standard mitragynine-d3, and store at low temperature.

[0009] S2 Determination of characteristic ions of mitragynine and its isotope internal standard and establishment of the selective reaction monitoring (LC-MS / MS-SRM) method: The mass spectrometric information of mitragynine and its isotope internal standard mitragynine-d3 was obtained in full scan mode, and ions with higher abundance and larger mass-to-charge ratio (m / z) were selected as parent ions. Different collision energies were input to investigate the effect of collision energy on parent ion fragmentation, and the optimal daughter ions and collision energies were selected. The LC-MS / MS-SRM method was established based on the ion pair information.

[0010] Drawing of S3 standard curve: A series of standard working solutions of mitragynine were injected under the set liquid chromatography-tandem mass spectrometry conditions, and the standard curve of mitragynine was drawn with the peak area ratio of mitragynine to its internal standard mitragynine-d3 as the ordinate and the concentration as the abscissa.

[0011] S4 Detection of Mitragynine in Beverages:

[0012] Pretreatment of S41 beverages: Place the beverage and internal standard working solution in a centrifuge tube, add ammonium acetate aqueous solution containing phosphoric acid, and wait for extraction.

[0013] S42 solid phase extraction: pass all sample solutions through the activated solid phase extraction column, wash it, collect the eluate, concentrate and dry it, re-dissolve it and wait for sampling and detection.

[0014] S43 LC-MS / MS-SRM analysis and detection: After solid phase extraction is completed and sample is injected, detection begins and chromatograms are recorded.

[0015] S44 Qualitative and quantitative analysis of mitragynine in beverages: The mitragynine detected in beverages was determined by the retention time and ion pair information of the standard, and quantified by the internal standard method.

[0016] Furthermore, in the step S1, a standard stock solution of 1.000 mg / mL mitragynine and its isotope internal standard mitragynine-d3 is prepared with methanol, and the stock solution is diluted with methanol to a standard working solution of 0.010 mg / mL mitragynine and isotope internal standard mitragynine-d3, and stored at low temperature.

[0017] Furthermore, in step S3, an appropriate amount of 0.010 mg / mL of mitragynine standard working solution is added to the blank sample residue to prepare a series of matrix-matched standard working solutions.

[0018] Furthermore, the step S41 is specifically as follows: taking 2 mL of sample into a 10 mL centrifuge tube, adding 20 µL of 50 ng / mL isotope internal standard working solution, and then adding 5 mL of 0.1 mol / L ammonium acetate solution containing 2% phosphoric acid, vortexing to mix, and then extracting.

[0019] The step S42 is specifically as follows: the extraction column is activated with 3 mL of methanol and 3 mL of water in sequence, all the sample liquid is passed through the column, eluted with 3 mL of water and 3 mL of methanol in sequence, drained, eluted with 5 mL of 5% ammonia methanol solution, the eluate is collected in a 10 mL centrifuge tube, and dried with nitrogen in a 45°C water bath. 2 mL of methanol is added, the residue is re-dissolved by ultrasonication for 30 s, and filtered through a 0.2 μm microporous filter membrane for sampling and detection.

[0020] The LC conditions in step S43 are as follows: 2 μL injection, flow rate 0.5 mL / min, chromatographic column ShimNexWR C 18 Analytical column, 5 μm, 4.6*100 mm, column temperature 30 ℃, mobile phase A is 0.005% ammonia solution, mobile phase B is acetonitrile, gradient elution program is 0.1 min, 50% B; 1.5 min, 90% B; 9.5 min, 90% B; 11 min, 50% B; 16 min stop.

[0021] The mass spectrometry conditions in step S43 are set as follows: electrospray ionization source (ESI), positive ion scanning, multiple reaction monitoring (MRM), ion source temperature of 150 ° C, capillary voltage of 1.0 kV, cone voltage of 20 V, desolvation temperature of 550 ° C, and the determination parameters of mitragynine and its isotope internal standard mitragynine-d3 are shown in Table 1.

[0022] Furthermore, the target substance is mitragynine; and the isotope internal standard compound is mitragynine-d3.

[0023] Furthermore, in step S3, an appropriate amount of 0.010 mg / mL target standard working solution was added to 7 portions of blank sample residues after extraction and purification, dried by nitrogen in a 45°C water bath, 2 mL of methanol was added, and the residue was redissolved by ultrasonication for 30 s to prepare a series of matrix-matched standard working solutions.

[0024] Furthermore, in step S3, the concentrations of the series of matrix-matched standard working solutions are 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, and 10 ng / mL.

[0025] Furthermore, the solid phase extraction column is a 60 mg / 3 mL mixed cation exchange solid phase extraction column.

[0026] Furthermore, the determination parameters of the mitragynine and its isotope internal standard compound are specifically shown in Table 1 below.

[0027] Table 1 Determination parameters of mitragynine and its isotope internal standard mitragynine-d3

[0028] Where “*” is the quantitative ion.

[0029] The present invention fills the gap in existing detection technology, and its beneficial effects are:

[0030] The present invention provides a method for determining mitragynine in a beverage. By using an isotope internal standard and liquid chromatography-tandem mass spectrometry detection, the mitragynine in the beverage can be accurately detected, and the content thereof can be qualitatively and quantitatively analyzed.

[0031] The present invention establishes a new detection method, which has good selectivity, high sensitivity, and a detection limit of ng / mL level, and is an accurate and reliable analysis method.

[0032] The detection method of the present invention has good linear correlation and spiked recovery rate, and the isotope internal standard triple quadrupole mass spectrometry detection improves the accuracy and sensitivity, has weak matrix interference, has a detection limit of 0.05 ng / mL, and a quantification limit of 0.1 ng / mL, which can provide technical guarantee for the detection of mitragynine in beverages. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the EIC plot of Mitragynine.

[0034] Figure 2 This is the EIC graph of the internal standard mitragynine-d3 standard sample. DETAILED DESCRIPTION

[0035] The present invention is further explained below in conjunction with specific embodiments and drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0036] Example 1 Detection of mitragynine in different types of beverages. (I) Mitragynine and its isotope internal standard Mitragynine-d

[0037] Among them, the target substance detected is mitragynine, and the isotope internal standard is mitragynine-d3.

[0038] S1 Prepare 1.000 mg / mL standard stock solutions of mitragynine and its isotope internal standard mitragynine-d3 with methanol. Dilute the stock solutions to 0.010 mg / mL standard working solutions of mitragynine and its isotope internal standard mitragynine-d3 with methanol and store at -18 ℃. (II) Mitragynine and its isotope internal standard Mitragynine-d

[0039] The mass spectrometric information of mitragynine and its isotope internal standard mitragynine-d3 was obtained in full scan mode. Ions with higher abundance and larger mass-to-charge ratio (m / z) were selected as parent ions. Different collision energies were input to investigate the effect of collision energy on the fragmentation of parent ions. The optimal daughter ions and collision energy were selected, and the LC-MS / MS-SRM method was established based on the ion pair information.

[0040] The quantitative ion pairs, qualitative ion pairs, and corresponding collision energy mass spectrum information of mitragynine and its isotope internal standard mitragynine-d3 are shown in Table 1.

[0041] Table 1 Quantitative ion pairs, qualitative ion pairs and their corresponding collision energy mass spectrometry information of mitragynine and isotope internal standard mitragynine-d3

[0042] Where “*” is the quantitative ion. (III) Drawing of standard curve

[0043] An appropriate amount of 0.010 mg / mL mitragynine working solution was added to 7 portions of blank sample residues after extraction and purification, dried in a 45 ℃ water bath with nitrogen, and 2 mL of methanol was added. The residue was re-dissolved by ultrasonication for 30 s to prepare a series of matrix-matched standard working solutions. The concentrations of the series of matrix-matched standard working solutions included 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, and 10 ng / mL. The samples were injected under the set liquid chromatography-tandem mass spectrometry conditions, and the standard curve of mitragynine was drawn with the peak area ratio of mitragynine to its internal standard mitragynine-d3 as the ordinate and the concentration as the abscissa.

[0044] After testing, the linear equations and correlation coefficients of mitragynine in different beverages are shown in Table 2.

[0045] Table 2 Linear equations and correlation coefficients of mitragynine in different beverages (IV) Detection of Mitragynine in Beverages

[0046] S1 Pretreatment of beverage samples: Take 2 mL of beer, cocktail, herbal tea, and wine samples and place them in 10 mL centrifuge tubes, add 20 μL of 50 ng / mL internal standard mitragynine-d3 standard working solution, then add 5 mL of 0.1 mol / L ammonium acetate aqueous solution containing 2% phosphoric acid, vortex mix, and wait for extraction.

[0047] S2 Solid phase extraction: The extraction column was activated with 3 mL of methanol and 3 mL of water in turn, all the sample solution in the centrifuge tube was passed through the column, and then 3 mL of water and 3 mL of methanol were used in turn, and the column was drained, and 5 mL of 5% ammonia methanol solution was used to elute, and the eluate was collected in a 10 mL centrifuge tube and dried with nitrogen in a 45°C water bath. 2 mL of methanol was added, and the residue was re-dissolved by ultrasonication for 30 s, and then filtered through a 0.2 μm microporous filter membrane for injection and detection.

[0048] S3 LC-MS / MS-SRM analysis and detection: After solid phase extraction is completed, the sample is injected and tested, and the chromatogram is recorded.

[0049] The LC conditions were set as follows: 2 μL injection, flow rate of 0.5 mL / min, and ShimNex WR C column. 18 Analytical column, 5 μm, 4.6*100 mm, column temperature 30 ℃, mobile phase A is 0.005% ammonia solution, mobile phase B is acetonitrile, gradient elution program is 0.1 min, 50% B; 1.5 min, 90% B; 9.5 min, 90% B; 11 min, 50% B; 16 min stop.

[0050] The mass spectrometry conditions were set as follows: electrospray ionization source (ESI), positive ion scanning, multiple reaction monitoring (MRM), ion source temperature of 150 °C, capillary voltage of 1.0 kV, cone voltage of 20 V, desolvation temperature of 550 °C, and the determination parameters of mitragynine and its isotope internal standard mitragynine-d3 were shown in the mass spectrometry information table in step (ii).

[0051] The mitragynine in the detected beverages was determined by the retention time and qualitative ion pair information of the mitragynine standard and quantified by the internal standard method. (V) Investigation of additive recovery rate

[0052] The sample spike method was used to add 0.1 ng / mL, 0.5 ng / mL, and 1.0 ng / mL of mitragynine to four matrices, and the recovery rate of mitragynine in beverages was determined; and the precision of the quantitative method was investigated by the relative standard deviation of the repeatability experiment. The results showed that the recovery rate of mitragynine in the four beverages was 80.85%~100.91%, and the RSD was 0.27%~5.47%. The recovery rate was high, the accuracy was good, and the precision was good, which could meet the requirements of quantitative detection of mitragynine in actual beverage samples.

[0053] Table 3 Recovery and precision of mitragynine added to four beverages (n=3) (VI) Intra-day and inter-day precision studies

[0054] Beer, cocktail, herbal tea and wine were used as samples, with a spike concentration of 0.5 ng / mL. Samples were injected and tested, and the intra-day precision of the analytical method was obtained by continuous measurement 6 times within 1 day. The inter-day precision of the analytical method was obtained by continuous measurement 3 times a day for 5 days and the average value was taken. The results are shown in Table 4. The intra-day precision of mitragynine was between 1.38% and 1.68%, and the inter-day precision was between 0.98% and 1.64%, which met the requirements of the method.

[0055] Table 4 Intra-day and inter-day precision of mitragynine in four beverages

[0056] The above embodiments describe the implementation methods of the present invention in detail, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the field without departing from the purpose of the present invention. The above description is only a preferred feasible embodiment of the present invention, and does not limit the scope of rights of the present invention. All equivalent structural changes made by using the contents of the present invention specification are included in the scope of rights of the present invention.

Claims

1. A method for determining mitragynine in a beverage, characterized in that, The method comprises the following steps: adding an isotope internal standard to the beverage, weakly acidifying the beverage, extracting the beverage through a solid phase extraction column to obtain a test solution, and using liquid chromatography-tandem mass spectrometry to conduct qualitative and quantitative analysis of the mitragynine component in the beverage, specifically comprising the following steps: S1 Preparation and storage of standard solutions of mitragynine and its isotope internal standard mitragynine-d3: prepare standard stock solutions of mitragynine and its isotope internal standard mitragynine-d3 using methanol, dilute the stock solutions with methanol as standard working solutions of mitragynine and its isotope internal standard mitragynine-d3, and store at low temperature; S2 Determination of characteristic ions of mitragynine and its isotope internal standard mitragynine-d3 and establishment of a selective reaction monitoring (LC-MS / MS-SRM) method: The mass spectrometry information of mitragynine and its isotope internal standard mitragynine-d3 was obtained in full scan mode, and ions with high abundance and large mass-to-charge ratio (m / z) were selected as parent ions. Different collision energies were input to investigate the effect of collision energy on parent ion fragmentation, and the optimal daughter ions and collision energies were selected. The LC-MS / MS-SRM method was established based on the ion pair information. Drawing of S3 standard curve: inject a series of matrix-matched standard working solutions of mitragynine under the set liquid chromatography-tandem mass spectrometry conditions, and draw a matrix-matched standard curve of mitragynine with the peak area ratio of mitragynine to its internal standard mitragynine-d3 as the ordinate and the concentration as the abscissa; S4 Detection of Mitragynine in Beverages: S41 Pretreatment of beverages: Take the beverage and the internal standard working solution and place them in a centrifuge tube, add an aqueous solution of ammonium acetate containing phosphoric acid, and wait for extraction; S42 solid phase extraction: The solid phase extraction column is pre-activated, and the sample solution is completely passed through the column. After elution, the eluate is collected, concentrated and dried, and then re-dissolved before sampling and testing; the solid phase extraction column is a mixed cation exchange solid phase extraction column; S43 Liquid chromatography-tandem mass spectrometry selected reaction monitoring method detection: After solid phase extraction is completed and sample is injected, detection and recording of chromatograms are started, and the LC condition settings include: chromatographic column type, injection volume, flow rate, chromatographic column temperature, mobile phase A, mobile phase B, and gradient elution program; mass spectrometry condition settings include: ion source type, scanning mode, detection mode, ion source temperature, capillary voltage, cone voltage, and desolvation gas temperature; S44 Qualitative and quantitative analysis of mitragynine in beverages: The mitragynine detected in beverages was determined by the retention time and ion pair information of the standard, and quantified by the internal standard method.

2. The method for measuring mitragynine in beverages according to claim 1, wherein The step S1 is specifically as follows: in the step S1, a standard stock solution of 1.000 mg / mL mitragynine and its isotope internal standard mitragynine-d3 is prepared with methanol, and the stock solution is diluted with methanol to a standard working solution of 0.010 mg / mL mitragynine and isotope internal standard mitragynine-d3, and stored at low temperature.

3. The method for measuring mitragynine in beverages according to claim 1, wherein In the step S3, an appropriate amount of 0.010 mg / mL of mitragynine standard working solution is added to the blank sample residue to prepare a series of matrix-matched standard working solutions.

4. The method for measuring mitragynine in beverages according to claim 1, wherein The S41 is as follows: take 2 mL of sample into a 10 mL centrifuge tube, add 20 µL of 50 ng / mL isotope internal standard working solution, then add 5 mL of 0.1 mol / L ammonium acetate solution containing 2% phosphoric acid, vortex mix, and wait for extraction: The specific steps of S42 are as follows: the extraction column is activated with 3 mL of methanol and 3 mL of water in turn, all the sample solution is passed through the column, eluted with 3 mL of water and 3 mL of methanol in turn, drained, eluted with 5 mL of 5% ammonia methanol solution, collected in a 10 mL centrifuge tube, and dried in a 45 ℃ water bath with nitrogen. 2 mL of methanol is added, the residue is re-dissolved by ultrasonication for 30 s, and filtered through a 0.2 μm microporous filter membrane for injection testing; the solid phase extraction column is a 60 mg / 3 mL mixed cation exchange solid phase extraction column; The LC conditions in S43 were set as follows: 2 μL injection, flow rate 0.5 mL / min, and ShimNex WR C column. 18 Analytical column, 5 μm, 4.6*100 mm, column temperature 30 °C, mobile phase A was 0.005% ammonia solution, mobile phase B was acetonitrile, gradient elution program was 0.1 min, 50% B; 1.5 min, 90% B; 9.5 min, 90% B; 11 min, 50% B; 16 min stop; The mass spectrometry conditions in the S43 were set as follows: electrospray ion source (ESI), positive ion scanning, multiple reaction monitoring (MRM) mode, ion source temperature of 150°C, capillary voltage of 1.0 kV, cone voltage of 20 V, and desolvation temperature of 550°C.

5. The method for measuring mitragynine in beverages according to claim 1, wherein The internal standard is: Mitragynine-d3.

6. The method for determining mitragynine in beverages according to claim 3, characterized in that, In step S3, the concentrations of the series of matrix-matched standard working solutions are 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, and 10 ng / mL.

7. The method for determining mitragynine in beverages according to claim 4, wherein The specific determination parameters of mitragynine and its isotope internal standard mitragynine-d3 are shown in the following table: Table 1 Main determination parameters of mitragynine and mitragynine-d3 (* indicates quantitative ions) 8. The method, steps and substances described in claims 1-6, and the application of the determination parameters described in claim 7 in the determination of mitragynine in beverages.

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