A method for determining pyrrolizidine alkaloids in liquorice

Abstract

The present invention relates to the technical field of quality detection of Chinese medicinal materials, and in particular to a method for determining pyrrolizidine alkaloids in licorice, which uses UPLC-MS / MS to determine cynaroline and its N-oxide, senegiline and its N-oxide, monocrotaline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, senegiline and its N-oxide, euryneline and its N-oxide, heliotropin, senegiline, arugula leaf senegiline and lycopodium N-oxide in licorice. The method for determining pyrrolizidine alkaloids in licorice provided by the present invention can provide technical support for the quality evaluation and safety evaluation of licorice.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection of traditional Chinese medicines, and in particular to a method for determining pyrrolizidine alkaloids in liquorice. Background Art

[0002] Licorice is the root or stem of the leguminous plants Glycyrrhiza uralensis, Glycyrrhiza inflata or Glycyrrhiza glabra. It is a traditional Chinese medicinal material, first recorded in Shennong's Herbal Classic. Licorice tastes sweet and mild, and has the functions of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, and removing phlegm and relieving cough. In recent years, licorice extracts have been widely used as additives in tobacco, medicine, food, cosmetics and other industries. At present, the most studied active ingredients are triterpenoid saponins, glycyrrhizic flavonoids, coumarins, glycyrrhizic polysaccharides and other active ingredients, which show multiple biological activities such as antioxidant, anti-tumor and regulation of glucose and lipid metabolism.

[0003] Pyrrolizidine alkaloids (PAs) are a type of secondary metabolites produced and accumulated by plants in the Asteraceae, Boraginaceae, Leguminosae, Apocynaceae and Orchidaceae families to resist insects and herbivores and pathogenic microorganisms. More than 660 PAs and their nitrogen oxides (PANOs) with different structures have been identified from more than 6,000 plant species. PAs are generally composed of two basic parts: necine (such as necine) and necic acid (such as necic acid). According to the type of necine, they can be divided into retronecine type, otonecine type and platynecine type. Retronecine type and otonecine type PAs form double bonds at the 1st and 2nd carbon atoms and are unsaturated, while platynecine type PAs are saturated. Unsaturated PAs can be divided into three types according to the esterification mode: monoester, open-chain diester or cyclic diester. PAs are biologically and toxicologically inactive protoxins, but when they enter the body, unsaturated PAs will be metabolized to form pyrrole metabolites with an allyl alcohol ester structure, and bind to various nucleophilic enzymes, proteins and DNA, showing health problems such as hepatotoxicity, pulmonary toxicity, genotoxicity, carcinogenicity and developmental toxicity. Due to the widespread presence of PAs in different types of food and their potential risks to human health, their food safety issues have attracted attention, but the properties of Chinese medicinal materials and other food raw materials vary greatly, and there is currently a lack of efficient detection methods for the content of PAs in commonly used Chinese medicinal materials. Therefore, providing a method for determining PAs in licorice, which can simultaneously and accurately determine the content of PAs in licorice, is of great significance for a deep understanding of the safety of licorice. Summary of the invention

[0004] In order to solve the above problems, the purpose of the present invention is to provide a method for determining pyrrolizidine alkaloids in licorice, which uses UPLC-MS / MS to determine echimidine (Em) and its N-oxide (EmN), Jacobine (Jb) and its N-oxide (JbN), Crotaline (Mc) and its N-oxide (McN), Retrorsine (Re) and its N-oxide (ReN), Senecivernine (Sv) and its N-oxide (SvN), and Crotaline (Mc) and its N-oxide (McN) in licorice. Senecionine (Sc) and its N-oxide (ScN), Seneciphylline (Sp) and its N-oxide (SpN), Europine (Eu) and its N-oxide (EuN), Heliotrine (Hn) and its N-oxide (HnN), Intermedine (Im), Senkirkine (Sk), Erucifoline (Er) and Lycopsamine N-oxide (LaN). The detection method provided by the present invention can provide technical support for the quality evaluation and safety evaluation of Licorice.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The present invention provides a method for determining pyrrolizidine alkaloids in liquorice, comprising the following steps:

[0007] (1) Preparation of mixed standards: Dissolve different standards and mix them to obtain mixed standards of different concentrations;

[0008] (2) Pretreatment of the sample to be tested: dissolving the pretreated licorice in an extraction aid and performing ultrasonic extraction, centrifuging and then bathing in water to obtain a solution to be purified; loading the solution to be purified into a pretreated MCX solid phase extraction column, and post-treating to obtain a sample to be re-dissolved; dissolving the sample to be re-dissolved and filtering to obtain a sample to be tested;

[0009] (3) Drawing of standard curve: The mixed standard samples of different concentrations prepared in step (1) are sequentially sent to high performance liquid chromatography and mass spectrometry to draw a standard curve for any standard sample;

[0010] (4) Testing the sample: sending the sample prepared in step (2) to high performance liquid chromatography and mass spectrometry for analysis, and calculating the content of pyrrolizidine alkaloids in licorice according to the standard curve in step (3);

[0011] The pyrrolizidine alkaloids are respectively cymbidium, cymbidium N-oxide, senegiline, senegiline N-oxide, monocrotaline, monocrotaline N-oxide, inverted senegiline, inverted senegiline N-oxide, spring senegiline, spring senegiline N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, heliotropin, heliotropin N-oxide, heliotropin, heliotropin N-oxide, melanin, krisprine, arugula leaf senegiline, and lycopodipine N-oxide.

[0012] In one embodiment of the present invention, in step (1), the standard substance is selected from one of cymbidium, cymbidium N-oxide, seneciopine, seneciopine N-oxide, monocrotaline, monocrotaline N-oxide, inverted seneciopine, inverted seneciopine N-oxide, spring seneciopine, spring seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, euryneline, euryneline N-oxide, heliotropin, heliotropin N-oxide, heliotropin, heliotropin N-oxide, melanin, krispyrine, arugula leaf seneciopine and lycopodipine N-oxide.

[0013] In one embodiment of the present invention, in step (1), the concentration of the mixed standard is 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 ng / mL.

[0014] In one embodiment of the present invention, in step (2), the pre-treatment is to sieve the licorice using a 60-mesh sieve;

[0015] In step (2), the extraction aid is 95% methanol aqueous solution, ethanol and 50% methanol aqueous solution.

[0016] In one embodiment of the present invention, in step (2), during the ultrasonic extraction process, the ultrasonic time is 20 minutes.

[0017] In one embodiment of the present invention, in step (2), during the centrifugation process, the centrifugal speed is 6000 r / min and the centrifugal time is 10 min.

[0018] In one embodiment of the present invention, in step (2), during the water bath process, the water bath temperature is 40° C. and the water bath time is 10-20 min.

[0019] In one embodiment of the present invention, in step (2), the pretreated MCX solid phase extraction column is an MCX solid phase extraction column activated sequentially with methanol and ultrapure water.

[0020] In one embodiment of the present invention, in step (2), the post-treatment is to rinse with ultrapure water and methanol, discard the waste liquid, elute with 5% ammonia methanol solution, and then blow dry the eluent with nitrogen;

[0021] In step (2), filtration is performed using a 0.22 μm filter membrane.

[0022] In one embodiment of the present invention, in steps (3) and (4), the conditions of high performance liquid chromatography are as follows:

[0023] Chromatographic column: Waters Acquity BEH C18; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol; mobile phase elution program: 0.0-0.5 min, 5% B; 0.5-5.0 min, 5%-34.5% B; 5.0-6.0 min, 34.5%-41.1% B; 6.0-7.0 min, 41.1%-100% B; 7.0-7.01 min, 100%-5% B; 7.01-8.5 min, 5% B; column temperature: 40°C; injection volume: 20 μL; flow rate: 0.3 mL / min.

[0024] Preferably, the mobile phase elution program is: 0.0-0.5 min, 5% B; 0.5-5.0 min, 5% B; 5.0-6.0 min, 34.5% B; 6.0-7.0 min, 41.1% B; 7.0-7.01 min, 100% B; 7.01-8.5 min, 5% B; column temperature: 40°C; injection volume: 20 μL; flow rate: 0.3 mL / min.

[0025] In one embodiment of the present invention, in steps (3) and (4), the conditions of mass spectrometry are as follows:

[0026] Ion source: electrospray ion source; Scan mode: positive ion scan; Monitoring method: multiple reaction monitoring (MRM); Capillary voltage: +3500 V; Source temperature: 350°C; Collision gas: helium 0.15 mL / min; Cone gas flow rate: 150 L / h.

[0027] In one embodiment of the present invention, the method for determining pyrrolizidine alkaloids in licorice has a linear relationship for 22 pyrrolizidine alkaloids within a concentration range of 0.63 to 20 μg / L.

[0028] In one embodiment of the present invention, the method for determining pyrrolizidine alkaloids in licorice is to determine the spiked recoveries of licorice samples at 1.25, 12.50, and 62.50 μg / kg levels; the recoveries of 22 pyrrolizidine alkaloids are 74.23% to 106.36%, and the relative standard deviation is 1.98% to 12.11%.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] A method for determining pyrrolizidine alkaloids in licorice of the present invention can simultaneously and rapidly determine 22 pyrrolizidine alkaloids in licorice, and the method can provide technical support for the quality evaluation and safety evaluation of licorice. At the same time, the detection method provided by the present invention is brief and efficient, can rapidly determine pyrrolizidine alkaloids in licorice, has good repeatability and high detection sensitivity, and the detection result is accurate and reliable. The detection results of the method show that licorice, as a traditional Chinese medicine in health food, has different PAs contamination conditions in licorice from different sources, which may be related to the climate, soil and processing process of the place of origin, and there is a certain health risk, but the risk is relatively low. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a multi-reaction ion monitoring diagram of 22 pyrrolizidine alkaloids in Example 1;

[0032] Figure 2 It is a schematic diagram of the standard curve of EmN;

[0033] Figure 3 is a schematic diagram of the standard curve of Em;

[0034] Figure 4 It is a schematic diagram of the standard curve of ReN;

[0035] Figure 5 It is a schematic diagram of the standard curve of JbN;

[0036] Figure 6 It is a schematic diagram of the standard curve of Sk;

[0037] Figure 7 It is a schematic diagram of the standard curve of SvN;

[0038] Figure 8 It is a schematic diagram of the standard curve of Re;

[0039] Fig. 9 It is a schematic diagram of the standard curve of Jb;

[0040] Fig.10 It is a schematic diagram of the standard curve of ScN;

[0041] Fig.11It is a schematic diagram of the standard curve of SpN;

[0042] Fig.12 Schematic diagram of the standard curve of Er;

[0043] Fig.13 It is a schematic diagram of the standard curve of EuN;

[0044] Fig.14 It is a schematic diagram of the standard curve of McN;

[0045] Fig.15 This is a schematic diagram of the standard curve of Sc&Sv;

[0046] Fig.16 It is a schematic diagram of the standard curve of Sp;

[0047] Fig.17 It is a schematic diagram of the standard curve of HnN;

[0048] Fig.18 It is a schematic diagram of the standard curve of Eu;

[0049] Fig.19 It is a schematic diagram of the standard curve of Mc;

[0050] Fig. 20 It is a schematic diagram of the standard curve of LaN;

[0051] Fig.21 It is a schematic diagram of the standard curve of Hn;

[0052] Fig. 22 is a schematic diagram of the standard curve of Im;

[0053] Fig.23 The figure is the distribution and content of pyrrolizidine alkaloids in the four kinds of licorice extracts in Example 1; DETAILED DESCRIPTION

[0054] The present invention provides a method for determining pyrrolizidine alkaloids in liquorice, comprising the following steps:

[0055] (1) Preparation of mixed standards: Dissolve different standards and mix them to obtain mixed standards of different concentrations;

[0056] (2) Pretreatment of the sample to be tested: dissolving the pretreated licorice in an extraction aid and performing ultrasonic extraction, centrifuging and then bathing in water to obtain a solution to be purified; loading the solution to be purified into a pretreated MCX solid phase extraction column, and post-treating to obtain a sample to be re-dissolved; dissolving the sample to be re-dissolved and filtering to obtain a sample to be tested;

[0057] (3) Drawing of standard curve: The mixed standard samples of different concentrations prepared in step (1) are sequentially sent to high performance liquid chromatography and mass spectrometry to draw a standard curve for any standard sample;

[0058] (4) Testing the sample: sending the sample prepared in step (2) to high performance liquid chromatography and mass spectrometry for analysis, and calculating the content of pyrrolizidine alkaloids in licorice according to the standard curve in step (3);

[0059] The pyrrolizidine alkaloids are respectively cymbidium, cymbidium N-oxide, senegiline, senegiline N-oxide, monocrotaline, monocrotaline N-oxide, inverted senegiline, inverted senegiline N-oxide, spring senegiline, spring senegiline N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, senecin, senecin N-oxide, heliotropin, heliotropin N-oxide, heliotropin, heliotropin N-oxide, melanin, krisprine, arugula leaf senegiline, and lycopodipine N-oxide.

[0060] In one embodiment of the present invention, in step (1), the standard substance is selected from one of cymbidium, cymbidium N-oxide, seneciopine, seneciopine N-oxide, monocrotaline, monocrotaline N-oxide, inverted seneciopine, inverted seneciopine N-oxide, spring seneciopine, spring seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, seneciopine, seneciopine N-oxide, euryneline, euryneline N-oxide, heliotropin, heliotropin N-oxide, heliotropin, heliotropin N-oxide, melanin, krispyrine, arugula leaf seneciopine and lycopodipine N-oxide.

[0061] In one embodiment of the present invention, in step (1), the concentration of the mixed standard is 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 ng / mL.

[0062] In one embodiment of the present invention, in step (2), the pre-treatment is to sieve the licorice using a 60-mesh sieve;

[0063] In step (2), the extraction aid is 95% methanol aqueous solution, ethanol and 50% methanol aqueous solution.

[0064] In one embodiment of the present invention, in step (2), during the ultrasonic extraction process, the ultrasonic time is 20 minutes.

[0065] In one embodiment of the present invention, in step (2), during the centrifugation process, the centrifugal speed is 6000 r / min and the centrifugal time is 10 min.

[0066] In one embodiment of the present invention, in step (2), during the water bath process, the water bath temperature is 40° C. and the water bath time is 10-20 min.

[0067] In one embodiment of the present invention, in step (2), the pretreated MCX solid phase extraction column is an MCX solid phase extraction column activated sequentially with methanol and ultrapure water.

[0068] In one embodiment of the present invention, in step (2), the post-treatment is to rinse with ultrapure water and methanol, discard the waste liquid, elute with 5% ammonia methanol solution, and then blow dry the eluent with nitrogen;

[0069] In step (2), filtration is performed using a 0.22 μm filter membrane.

[0070] In one embodiment of the present invention, in steps (3) and (4), the conditions of high performance liquid chromatography are as follows:

[0071] Chromatographic column: Waters Acquity BEH C18; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol; mobile phase elution program: 0.0-0.5 min, 5% B; 0.5-5.0 min, 5%-34.5% B; 5.0-6.0 min, 34.5%-41.1% B; 6.0-7.0 min, 41.1%-100% B; 7.0-7.01 min, 100%-5% B; 7.01-8.5 min, 5% B; column temperature: 40°C; injection volume: 20 μL; flow rate: 0.3 mL / min.

[0072] Preferably, the mobile phase elution program is: 0.0-0.5 min, 5% B; 0.5-5.0 min, 5% B; 5.0-6.0 min, 34.5% B; 6.0-7.0 min, 41.1% B; 7.0-7.01 min, 100% B; 7.01-8.5 min, 5% B; column temperature: 40°C; injection volume: 20 μL; flow rate: 0.3 mL / min.

[0073] In one embodiment of the present invention, in steps (3) and (4), the conditions of mass spectrometry are as follows:

[0074] Ion source: electrospray ion source; Scan mode: positive ion scan; Monitoring method: multiple reaction monitoring (MRM); Capillary voltage: +3500 V; Source temperature: 350°C; Collision gas: helium 0.15 mL / min; Cone gas flow rate: 150 L / h.

[0075] In one embodiment of the present invention, the method for determining pyrrolizidine alkaloids in licorice has a linear relationship for 22 pyrrolizidine alkaloids within a concentration range of 0.63 to 20 μg / L.

[0076] In one embodiment of the present invention, the method for determining pyrrolizidine alkaloids in licorice is to determine the spiked recoveries of licorice samples at 1.25, 12.50, and 62.50 μg / kg levels; the recoveries of 22 pyrrolizidine alkaloids are 74.23% to 106.36%, and the relative standard deviation is 1.98% to 12.11%.

[0077] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0078] Instruments and Materials

[0079] Xevo TQ-S ultra-high performance liquid chromatography-triple quadrupole mass spectrometer, equipped with Mass Lynx chromatography workstation, Waters Company, USA; DG-12D solid phase extraction device, Tianjin Aotesense Company; ultrasonic cleaner; water bath; nitrogen blow dryer; electronic balance; low-temperature high-speed centrifuge.

[0080] PA standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (purity ≥ 95%): Echimidine (Em) and its N-oxide (EmN), Jacobine (Jb) and its N-oxide (JbN), Crotaline (Mc) and its N-oxide (McN), Retrorsine (Re) and its N-oxide (ReN), Senecivernine (Sv) and its N-oxide (SvN), Senecionine (SvN), and , Sc) and its N-oxide (ScN), Seneciphylline (Sp) and its N-oxide (SpN), Europine (Eu) and its N-oxide (EuN), Heliotrine (Hn) and its N-oxide (HnN), Intermedine (Im), Senkirkine (Sk), Erucifoline (Er) and Lycopsamine N-oxide (LaN); chromatographic grade methanol, ammonia water and formic acid, Sigma-Aldrich, USA; mixed cation exchange solid phase extraction column (Oasis MCX, 6cc / 500mg), Waters, USA; 0.22μm organic phase syringe filter, Shanghai Anpu Experiment Technology Co., Ltd.

[0081] The licorice samples came from the domestic Chinese medicinal materials market; among them, LreO came from Huangshan Hualuyuan Biotechnology Co., Ltd.; LreM1 came from Shaanxi Jiahe Biotechnology Co., Ltd.; LreM2 came from Damin Food (Zhangzhou) Co., Ltd.; and LreM3 came from Shandong Kangyuantang Chinese Medicine Pieces Co., Ltd.

[0082] The structural formula of the PAs measured in Example 1 is as follows:

[0083]

[0084]

[0085] Example 1

[0086] This embodiment provides a method for determining pyrrolizidine alkaloids in liquorice, comprising the following steps:

[0087] (1) Preparation of mixed standards

[0088] Accurately weigh 10 mg of each PAs standard (Em, Er, Eu, Hn, Im, Jb, LaN, Mc, ScN, Sv, SpN, EmN, EuN, HnN, McN, JbN, ReN, Sk, SvN, Sp, Re, Sc), dissolve it in methanol and transfer it to a 100 mL volumetric flask to make up to volume, prepare a standard stock solution with a mass concentration of 0.1 mg / mL (Em, Er, Eu, Hn, Im, Jb, LaN, Mc, ScN, Sv, SpN, EmN, EuN, HnN, McN, JbN, ReN, Sk, SvN, Sp, Re, Sc), and store it in a sealed container at -20 °C;

[0089] Pipette 1 mL of each standard stock solution prepared above into a 100 mL volumetric flask, dilute with methanol to obtain a mixed standard stock solution with a mass concentration of 1 μg / mL; dilute the mixed standard stock solution with methanol to obtain a series of standard working solutions, and obtain mixed standards with mass concentrations of 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 ng / mL, respectively (prepared before use).

[0090] (2) Determination

[0091] 1 μL of the mixed standard prepared in step (1) with mass concentrations of 0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 ng / mL was accurately injected into UPLC-MS / MS for determination. The contents of 22 PAs were calculated according to the external standard method. The results are as follows: Figure 1 shown.

[0092] The UPLC-MS / MS assay conditions are as follows:

[0093] Chromatographic parameters: chromatographic column: Waters Acquity BEH C18 (2.1 mm×100 mm, 1.7 μm); flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 20 μL; mobile phase and gradient elution program are shown in Table 1.

[0094] Table 1 Mobile phase gradient elution program

[0095]

[0096] Mass spectrometry parameters: ion source: electrospray ion source (ESI); scanning mode: positive ion scanning; monitoring mode: multiple reaction monitoring (MRM); capillary voltage: +3500V; source temperature: 350°C; collision gas: helium 0.15mL / min; cone gas flow rate: 150L / h. In the case of direct entry into the mass spectrometer without passing through a chromatographic column, the mass spectrometry conditions and parameters such as ion pairs, scanning time, and collision energy for monitoring 22 PAs are shown in Table 2.

[0097] Table 2 Mass spectrometry detection parameters of PAs

[0098]

[0099]

[0100] Methodological investigation and analysis

[0101] Linear relationship investigation and quantitative lower limit determination

[0102] The PAs standard solution was analyzed by the established UPLC-MS / MS method, and the quantitative ion peak area (Y)-concentration (X) calibration curve was established. The regression equation and correlation coefficient were fitted, and the quantification limit was calculated at the mass concentration when the signal-to-noise ratio (S / N) was 10 times (Table 3 and Figure 2-Figure 22 The results showed that the 22 PAs (Em, Er, Eu, Hn, Im, Jb, LaN, Mc, ScN, Sv, SpN, EmN, EuN, HnN, McN, JbN, ReN, Sk, SvN, Sp, Re, Sc) had a good linear relationship in the mass concentration range of 0.1-20 μg / L (r>0.99), and the limit of quantification was 0.02-0.63 μg / L.

[0103] Table 3 Linear relationship and quantification limit of PAs (n=5)

[0104]

[0105] (3) Extraction of licorice samples

[0106] Take the licorice sample LerO and pass it through a 60-mesh sieve, then weigh 2.0 g of the sieved licorice sample LerO into a 250 mL conical flask, add 40 mL of 95% methanol aqueous solution, ethanol, and 50% methanol aqueous solution, respectively, and extract by ultrasonic at room temperature for 20 min, then centrifuge at 6000 r / min for 10 min, transfer the supernatant to a 250 mL beaker, evaporate to 5 mL in a 40°C water bath, and obtain the LerO purification solution;

[0107] Take the licorice sample LreM1 and pass it through a 60-mesh sieve, then weigh 2.0 g of the sieved licorice sample LreM1 into a 250 mL conical flask, add 40 mL of 95% methanol aqueous solution, ethanol, and 50% methanol aqueous solution, respectively, and perform ultrasonic extraction at room temperature for 20 min, then centrifuge at 6000 r / min for 10 min, transfer the supernatant to a 250 mL beaker, evaporate to 5 mL in a 40°C water bath, and obtain the LreM1 purification solution;

[0108] Take the licorice sample LreM2 and pass it through a 60-mesh sieve, then weigh 2.0 g of the sieved licorice sample LreM2 into a 250 mL conical flask, add 40 mL of 95% methanol aqueous solution, ethanol, and 50% methanol aqueous solution, respectively, and ultrasonically extract at room temperature for 20 min, then centrifuge at 6000 r / min for 10 min, transfer the supernatant to a 250 mL beaker, evaporate to 5 mL in a 40°C water bath, and obtain the LreM2 purified solution;

[0109] Take the licorice sample LreM3 and pass it through a 60-mesh sieve, then weigh 2.0 g of the sieved licorice sample LreM3 into a 250 mL conical flask, add 40 mL of 95% methanol aqueous solution, ethanol, and 50% methanol aqueous solution respectively, ultrasonically extract at room temperature for 20 min, then centrifuge at 6000 r / min for 10 min, transfer the supernatant to a 250 mL beaker, evaporate to 5 mL in a 40°C water bath, and obtain the LreM3 purified liquid.

[0110] (4) Purification and re-dissolution of licorice extract samples

[0111] The MCX solid phase extraction column was activated with 5 mL of methanol and 5 mL of ultrapure water in advance, 1 mL of LerO to be purified solution was loaded, and it was eluted twice with 5 mL of ultrapure water and 5 mL of methanol in sequence, the waste liquid was discarded, and 6 mL of 5% ammonia methanol solution was used for elution. The eluate was dried with nitrogen at 40°C to obtain the LerO to be re-dissolved sample; the LerO to be re-dissolved sample was dissolved with 1 mL of 10% methanol aqueous solution, filtered through a 0.22 μm filter membrane, and analyzed by UPLC-MS / MS;

[0112] The MCX solid phase extraction column was activated with 5 mL of methanol and 5 mL of ultrapure water in advance, 1 mL of LreM1 to be purified was loaded, and it was eluted with 5 mL of ultrapure water and 5 mL of methanol in sequence twice, the waste liquid was discarded, and eluted with 6 mL of 5% ammonia methanol solution, and the eluate was dried with nitrogen at 40°C to obtain the LreM1 to be re-dissolved sample; the LreM1 to be re-dissolved sample was dissolved with 1 mL of 10% methanol aqueous solution, filtered through a 0.22 μm filter membrane, and analyzed by UPLC-MS / MS;

[0113] The MCX solid phase extraction column was activated with 5 mL of methanol and 5 mL of ultrapure water in advance, 1 mL of LreM2 to be purified was loaded, and it was eluted with 5 mL of ultrapure water and 5 mL of methanol in sequence twice, the waste liquid was discarded, and 6 mL of 5% ammonia methanol solution was used for elution. The eluate was dried with nitrogen at 40°C to obtain the LreM2 sample to be re-dissolved; the LreM2 sample to be re-dissolved was dissolved with 1 mL of 10% methanol aqueous solution, filtered through a 0.22 μm filter membrane, and analyzed by UPLC-MS / MS;

[0114] The MCX solid phase extraction column was activated with 5 mL of methanol and 5 mL of ultrapure water in advance, 1 mL of LreM3 to be purified was loaded, and it was eluted with 5 mL of ultrapure water and 5 mL of methanol in sequence twice, the waste liquid was discarded, and 6 mL of 5% ammonia methanol solution was used for elution. The eluate was dried with nitrogen at 40°C to obtain the LreM3 sample to be re-dissolved; the LreM3 sample to be re-dissolved was dissolved with 1 mL of 10% methanol aqueous solution, filtered through a 0.22 μm filter membrane, and analyzed by UPLC-MS / MS;

[0115] The PAs of 4 different licorice samples (LerO, LerM1, LerM2, LerM3) were determined by UPLC-MS / MS (the determination method was the same as step (2)). The results are as follows: Fig.23 As shown in the figure, the types and mass concentrations of PAs in the four licorice samples (LerO, LerM1, LerM2, and LerM3) were significantly different.

[0116] Among them, only two PAs (3.75μg / kg SpN and 6.51μg / kg Im) were detected in LreM2; 3.72μg / kg Sk, 2.40μg / kg SpN and 2.64μg / kg LaN were detected in LreM1, and its total PAs content was the lowest; LreO mainly contained 43.16μg / kgSk and 7.49μg / kg SpN, and 3.92μg / kg SvN, 2.85μg / kg Er and 1.34μg / kg ScN were also detected; only 1.99μg / kg Sk was detected in LreM3, which mainly contained 59.97μg / kg Im and 19.74μg / kg Mc, and 4.43μg / kg ReN and 3.60μg / kg LaN were also detected, and its total PAs content was the highest (89.73μg / kg). The above results indicate that licorice from different sources may be contaminated with PAs to different extents, which may be related to the climate, soil and processing of the production area.

[0117] Recovery and precision analysis

[0118] As shown in Table 4, the spiked recoveries of licorice samples were determined at three levels (1.25, 12.50, and 62.50 μg / kg), and each level was measured 5 times in parallel, and the relative standard deviation (RSD) was calculated. The results are shown in Table 4. The recoveries of 22 PAs at different spiked levels were 74.23% to 106.36%, and the relative standard deviation (RSD) was 1.98% to 12.11%. This shows that the UPLC-MS / MS method established in this example has good detection accuracy and high sensitivity for common PAs, and can meet the detection requirements.

[0119] Table 4 Recovery and precision of spiked PAs

[0120]

[0121]

[0122]

[0123] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for determining pyrrolizidine alkaloids in liquorice, characterized in that: The following steps are involved: (1) Preparation of mixed standards: Dissolve different standards and mix them to obtain mixed standards of different concentrations; (2) Pretreatment of the sample to be tested: dissolving the pretreated licorice in an extraction aid for ultrasonic extraction, centrifuging and then immersing in a water bath to obtain a solution to be purified; loading the solution to be purified onto a pretreated MCX solid phase extraction column, and post-treating to obtain a sample to be reconstituted; dissolving the sample to be reconstituted and filtering to obtain a sample to be tested; (3) Drawing of standard curve: The mixed standard samples of different concentrations prepared in step (1) are sequentially sent to high performance liquid chromatography and mass spectrometry to draw a standard curve for any standard sample; (4) Testing the sample: sending the sample prepared in step (2) to high performance liquid chromatography and mass spectrometry for analysis, and calculating the content of pyrrolizidine alkaloids in licorice according to the standard curve in step (3); In step (1), the standard substance is selected from one of cymbidium, cymbidium N-oxide, senepine, senepine N-oxide, monocrotaline, monocrotaline N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, senepine, senepine N-oxide, melanocyte stimulating hormone, senepine, arugula leaf senepine and lycopodipine N-oxide; The pyrrolizidine alkaloids are respectively cymbidium, cymbidium N-oxide, senecine, senecine N-oxide, monocrotaline, monocrotaline N-oxide, inverted senecine, inverted senecine N-oxide, spring senecine, spring senecine N-oxide, senecine, senecine N-oxide, senecine, senecine N-oxide, senecine, senecine N-oxide, senecine, senecine N-oxide, heliotropin, heliotropin N-oxide, heliotropin, heliotropin N-oxide, melanin, krisprine, arugula senecine, and lycopodipine N-oxide; In step (2), the extraction aid is 95% methanol aqueous solution, ethanol and 50% methanol aqueous solution; Among them, the quantification limit of pyrrolizidine alkaloids was 0.02~0.63μg / L; In steps (3) and (4), the conditions of HPLC are as follows: Chromatographic column: Waters Acquity BEH C18; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in methanol; mobile phase elution program: 0.0~0.5min, 5%B; 0.5~5.0min, 5%~34.5%B; 5.0~6.0min, 34.5%~41.1%B; 6.0~7.0min, 41.1%~100%B; 7.0~7.01min, 100%~5%B; 7.01~8.5min, 5%B; column temperature: 40℃; injection volume: 20μL; flow rate: 0.3mL / min; In steps (3) and (4), the mass spectrometry conditions are as follows: Ion source: electrospray ion source; Scan mode: positive ion scan; Monitoring method: multiple reaction monitoring (MRM); Capillary voltage: +3500V; Source temperature: 350℃; Collision gas: helium 0.15 mL / min; Cone gas flow rate: 150 L / h.

2. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: In step (1), the concentrations of the mixed standard were 0.1, 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL.

3. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: In step (2), the pretreatment is to sieve the licorice using a 60-mesh sieve.

4. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: In step (2), the pretreated MCX solid phase extraction column is an MCX solid phase extraction column activated by methanol and ultrapure water in sequence.

5. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: In step (2), the post-treatment is to rinse with ultrapure water and methanol, discard the waste liquid, elute with 5% ammonia methanol solution, and then blow dry the eluent with nitrogen; In step (2), filtration is performed using a 0.22 μm filter membrane.

6. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: The method for determining pyrrolizidine alkaloids in liquorice has a linear relationship for 22 pyrrolizidine alkaloids within a concentration range of 0.63-20 μg / L.

7. A method for determining pyrrolizidine alkaloids in liquorice according to claim 1, characterized in that: The method for determining pyrrolizidine alkaloids in licorice is to determine the spiked recoveries of licorice samples at levels of 1.25, 12.50, and 62.50 μg / kg; the recoveries of 22 pyrrolizidine alkaloids are 74.23% to 106.36%, and the relative standard deviations are 1.98% to 12.11%.

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