A method for detecting chemical components in torrefied mace based on UPLC-QTOF-MS

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting chemical components in torrefied mace based on UPLC-QTOF-MS, which comprises the following steps: (1) mixing a torrefied mace sample with a solvent, performing extraction, and obtaining a liquid sample to be detected after solid-liquid separation; (2) performing mass spectrometry on the sample to be detected obtained in step (1) by using ultra-high performance liquid chromatography, obtaining component substance peaks, and performing qualitative analysis on the component substance peaks by using analysis software. The method provided by the application has the characteristics of high efficiency, rapidness and accuracy, and is simple to operate, simplifies the steps of complete separation and purification and one-by-one identification, and reduces the cost; 15 chemical components are identified in the application, which provides a basis for further research on the medicinal material basis and comprehensive quality control of torrefied mace.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting chemical components in torrefied mace based on UPLC-QTOF-MS. BACKGROUND

[0002] Mace is the dried mature seed kernel of Myristica fragrans Houtt. of Myristicaceae Myristica plant, also known as gajule, doukou, rouchou, dingtourou, etc. Mace has the functions of warming middle and regulating qi, and astringing intestine and stopping diarrhea, and is used for treating spleen-stomach deficiency, chronic diarrhea, abdominal distention and pain, food aversion and vomiting, etc. There are eight processing methods of mace, including surface coating torrefaction, bran torrefaction, talc torrefaction, steaming, clam powder preparation, frying, soil preparation and frost preparation, etc. At present, bran torrefaction is mainly used, and is included in Chinese Pharmacopoeia 2020 edition. According to traditional Chinese medicine theory, the role of mace processing is to reduce toxicity and increase efficacy (Zhao Guangyun, Wang Xiaoxia, Gao Huizhuan. Separation and identification of chemical components of mace processed products [J]. Chinese Modern Chinese Medicine, 2011, 13 (11).).

[0003] In addition, it is found that mace volatile oil has obvious toxicity (mainly manifested as central nervous system toxicity), and the main toxic component may be volatile components. Jia Tianzhu et al. compared the acute toxicity of different processed products of mace, and proved that the antidiarrheal effect of mace after processing was enhanced, and the toxicity was reduced, which had the dual significance of reducing toxicity and increasing efficacy; Wang Jing et al. used urine metabolomics method to study the difference in long-term toxicity mechanism of mace before and after bran torrefaction, and the results showed that long-term administration of mace raw product group could cause pantothenic acid, carnitine C2:O and amino acid metabolism abnormality, causing liver damage, and had certain hepatotoxicity. After bran torrefaction, these metabolic abnormalities were adjusted to a certain extent, reducing liver damage and hepatotoxicity, and playing a certain liver protection role. The creatinine content in the urine sample of the raw product group was significantly lower than that of the normal group, and after bran torrefaction of mace, the creatinine content in the urine sample had no significant difference with that of the normal group, indicating that mace after bran torrefaction could reduce kidney toxicity.

[0004] Myristicae fructus is rich in volatile oil, fatty oil, lignans and other components. Domestic and foreign scholars have studied the volatile components of raw and processed products of Myristicae fructus to varying degrees. Zhang Genrong et al. used gas chromatography-mass spectrometry (GC-MS) to study the volatile components of Myristicae fructus, and 80 chemical components were separated (Zhang Genrong, Hu Jing, Ding Pi, et al. Analysis of the volatile components of Myristicae fructus by gas chromatography / mass spectrometry [J]. Times Journal of Traditional Chinese Medicine and Pharmacy, 2016, 27(11): 3.); Lai Wenling et al. used GC-MS-DS to study the chemical components of the volatile oil of raw Myristicae fructus, and identified 29 compounds (Lai Wenling, Zeng Zhi, Chen Yixin, et al. Chemical constituents of the back group of traditional Chinese medicine compound (II) volatile oil of Myristicae fructus [J]. Chinese Herbal Drugs, 2002, 33(7): 3.); Schem K.H.P et al. used gas chromatography to analyze the volatile oil of Myristicae fructus in detail, and obtained 62 components, including monoterpene hydrocarbons, sesquiterpenes, aromatic ethers, monoterpene alcohols, esters, etc. (Schenk H P, Lamparsky D. Analysis of nutmeg oil using chromatographic methods [J]. Journal of Chromatography A, 1981, 204: 391-395.).

[0005] However, these technical means and methods are difficult to comprehensively and systematically characterize the chemical component profile and control the intrinsic quality of medicinal materials, and are also difficult to characterize the intrinsic quality of processed products of traditional Chinese medicinal materials. Traditional research on crude drugs sometimes has certain subjectivity and has certain limitations in the complex quality control of traditional Chinese medicinal materials; GC-MS method is more suitable for the quality control of traditional Chinese medicinal materials containing volatile components, but it is not suitable for lignans in Myristicae fructus; traditional Chinese medicinal materials have a wide range of origins, are affected by factors such as variety, production area, planting, harvesting and processing, and are difficult to control, and the fingerprints of different medicinal materials have certain differences, and the reproducibility of the fingerprints is also affected by different laboratory conditions and instrument equipment.

[0006] The Chinese Pharmacopoeia (2020 edition) only has a thin-layer identification method for Myristicae fructus, and the content determination index is dehydrodiisoeugenol. There is no clear standard research on the processed product of Myristicae fructus, i.e. roasted Myristicae fructus. At present, the research on the correlation between the changes of these components and the efficacy of Myristicae fructus after processing is also very limited. Most of the analysis of the chemical components in roasted Myristicae fructus uses silica gel column chromatography, solvent method and other separation and purification methods. No scholar has conducted systematic material basis research on the effective components in the processed product of Myristicae fructus, i.e. roasted Myristicae fructus. A simple, rapid, accurate and efficient method for the whole component analysis of the processed product of Myristicae fructus, i.e. roasted Myristicae fructus, is urgently needed to be developed. SUMMARY

[0007] Problem to be solved by the invention

[0008] In order to solve the problem of lacking systematic research on the chemical composition of the torrefied Myristicae Fructus, the application provides a method for rapidly detecting a plurality of chemical components in the torrefied Myristicae Fructus based on UPLC-QTOF-MS technology, which has the characteristics of rapidness, accuracy, batch detection, simple operation, simplification of the steps of complete separation and purification, and cost reduction; in addition, 15 components are identified by the method, which provides a basis for further research on the pharmacodynamic material basis and comprehensive quality control of the torrefied Myristicae Fructus.

[0009] Solution for solving the problem

[0010] [1]. The application provides a method for detecting chemical components in torrefied Myristicae Fructus based on UPLC-QTOF-MS, which comprises the following steps:

[0011] (1) The torrefied Myristicae Fructus sample is mixed with a solvent for extraction, and after solid-liquid separation, a liquid sample to be detected is obtained;

[0012] (2) The sample to be detected obtained in step (1) is subjected to mass spectrometry by ultra-high performance liquid chromatography separation, and component substance peaks are obtained; the component substance peaks are subjected to qualitative analysis by analysis software;

[0013] In step (1), the solvent is selected from one or more of water, methanol or methanol aqueous solution, ethanol or ethanol aqueous solution;

[0014] The conditions for ultra-high performance liquid chromatography separation in step (2) are as follows: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column; the elution system mobile phase is as follows: the mobile phase A is acetonitrile, and the mobile phase B is methanol aqueous solution, and the gradient elution program is as follows:

[0015] Time / min Mobile phase A / % Mobile phase B / % 0～5 39 61 5～10 39→30 61→70 10～15 30→20 70→80 15～18 20 80 18～19 20→39 80→61 19～24 39 61 .

[0016] [2]. The method according to [1], wherein the solvent is a methanol aqueous solution with a volume concentration of methanol of 80% or more.

[0017] [3]. The method according to [1] or [2], wherein the mass / volume ratio of the torrefied Myristicae Fructus sample to the solvent is (0.3-0.8g):(5-30mL).

[0018] [4]. The method according to any one of [1] to [3], wherein the extraction is ultrasonic crushing, the ultrasonic crushing time is 15-60min, the ultrasonic crushing power is 200-250W, and the ultrasonic crushing frequency is 30-50kHz.

[0019] [5]. The method according to any one of [1] to [4], wherein the method further comprises the following steps: after ultrasonic disruption, cooling, re-weighing, and then supplementing the lost weight with the solvent.

[0020] [6]. The method according to any one of [1] to [5], wherein the solid-liquid separation comprises the steps of centrifugation and filtration, wherein the centrifugation is performed at a speed of 11000 to 13000 rpm for 8 to 15 min; and the filtration is performed by membrane filtration, further by microfiltration with a pore size of 0.10 to 0.50 μm.

[0021] [7]. The method according to any one of [1] to [6], wherein the mobile phase B is a 0.05% to 0.1% formic acid aqueous solution by volume concentration, and the conditions for the ultra-high performance liquid chromatography separation further comprise: a filler particle size of 1.6 to 2.2 μm, and a detection wavelength of 250 to 350 nm.

[0022] [8]. The method according to any one of [1] to [7], wherein the qualitative analysis in step (2) comprises the following steps: (a) extracting substance peaks from a mass spectrum total ion current graph to obtain accurate molecular weights; (b) searching a database according to the first mass spectrum ion fragment information of the compound peaks to obtain mass spectrum fragment information in the database that matches the screened substances; and (c) combining and analyzing the adduct ion category, molecular weight error, retention time, related literature, and the mass spectrum fragmentation information obtained in step (b) to obtain the structure of the compound.

[0023] [9]. The method according to any one of [1] to [8], wherein the mass spectrum detection conditions in step (2) comprise: an ion source of electrospray ion source, and a detection mode of positive ion mode; a drying gas temperature of 200 to 400 ℃, and a flow rate of 1 to 20 L / min; and an atomizer pressure of 10 to 50 psi.

[0024]

[10] . The method according to any one of [1] to [9] for use in analyzing a plurality of chemical components in a retorted nutmeg.

[0025] Effects of the invention

[0026] The present application is directed to the chemical properties of various chemical components in the processed product of Myristicae Fructus, Myristicae Fructus torrefacti, for the first time, UPLC-QTOF-MS technology is used to detect and qualitatively analyze various chemical components in Myristicae Fructus torrefacti, and according to the peak information and peak effect of the chromatographic peak, the best gradient elution program is optimized. The method provided by the present application is simple, efficient, rapid and accurate, 15 kinds of chemical components are identified, some volatile components are detected, and lignan components are also detected, which provides a basis for further studying the medicinal material basis and comprehensive quality control of Myristicae Fructus torrefacti. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Mass spectrum for elution time gradient 1 in Example 1

[0028] Figure 2 Mass spectrum for elution time gradient 2 in Example 1

[0029] Figure 3 Mass spectrum for elution time gradient 3 in Example 1

[0030] Figure 4 Mass spectrum for elution time gradient 4 in Example 1

[0031] Figure 5 Total ion current chromatogram (TIC) of the test sample of Myristicae Fructus torrefacti formula granules in Example 2 in positive ion mode

[0032] Figure 6 Extracted ion current chromatogram (EIC) of m / z 345.1698 in Example 2

[0033] Figure 7 Mass spectrum (MS) of m / z 345.1698 in Example 2

[0034] Figure 8 Total ion current chromatogram (TIC) of the test sample of Myristicae Fructus torrefacti formula granules in Example 3 in positive ion mode

[0035] Figure 9 Extracted ion current chromatogram (EIC) of m / z 359.1857 in Example 3

[0036] Figure 10 Mass spectrum (MS) of m / z 359.1857 in Example 3 DETAILED DESCRIPTION

[0037] For a better understanding of the present application, numerous specific details are given in the following detailed description. The person skilled in the art understands that the present application can also be implemented without certain specific details. In some cases, methods, means, devices and steps that are well known to the person skilled in the art are not described in detail in order to highlight the gist of the present application.

[0038] Unless otherwise defined, the technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0039] Unless otherwise indicated, the term "precisely weighed or precisely measured" as used herein means that the weight should be accurate to one thousandth of the weight taken, the term "weighed" means that the weight should be accurate to one hundredth of the weight taken, the term "precisely added" means that the volume should be accurate to one thousandth of the volume taken, and the term "precisely pipetted" means that the sample is accurately measured by a micro pipette.

[0040] Unless otherwise indicated, the term "filtrate" as used herein means the filtrate collected after the initial filtrate is discarded. Compared with the initial filtrate, the filtrate is closer to the true concentration of the sample because the filter medium (such as filter membrane, filter paper, etc.) can adsorb the solute, thus causing the sample concentration in the initial filtrate to be lower; in addition, the filtrate is cleaner because the solute adsorbed by the filter medium can form a filter cake, reducing the filter pore size, thus being able to trap smaller particles.

[0041] Unless otherwise indicated, the term "ultra performance liquid chromatography" (or "UPLC") as used herein means a new technology developed on the basis of high performance liquid chromatography (HPLC), which has the characteristics of small filler particle size, fast detection speed, large analysis flux, and high sensitivity.

[0042] The percentage concentration referred to in the present specification, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added ingredient in the system after the ingredient is added.

[0043] The temperature parameter in the present specification, unless otherwise specified, allows both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0044] In the present specification, the numerical range represented by "numerical value A ~ numerical value B" refers to a range including the end point values A and B.

[0045] In the present specification, the meaning represented by "may" includes both the meaning of performing a certain treatment and the meaning of not performing a certain treatment.

[0046] In the present specification, the terms "one or some specific / preferred embodiments / arrangements", "another or some other specific / preferred embodiments / arrangements", "one or another embodiment / arrangement", "one or another technical solution" and the like mean that the specific elements (for example, features, structures, properties and / or characteristics) described in relation to the embodiments are included in at least one of the embodiments described herein, and can or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0047] The terms "comprise", "comprising", "include", "including" and "includes" in the specification and claims of the present application and the above-described drawings are intended to cover non-exclusive inclusions. For example, a process, method or system, product or device that includes a series of steps or units is not limited to the listed steps or units, but can optionally include steps or units that are not listed, or can optionally include other steps or units inherent to the process, method, product or device.

[0048] It should be understood that the singular forms of the articles "a", "an" and "the" used in the specification and additional claims of the present application include plural referents unless the context clearly dictates otherwise.

[0049] In the present application, the term "about" can mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters in the present application are approximate values, and the relevant values in the specific examples have been presented as accurately as possible. However, any numerical value inherently and unavoidably contains a standard deviation due to the aforementioned testing device or method. Therefore, unless otherwise explicitly stated, all ranges, quantities, numerical values and percentages used in the present application are modified by "about". Here, "about" generally means that the actual value is within ±10%, ±5%, ±1% or ±0.5% of a certain value or range.

[0050] The present application provides a method for detecting chemical components in torrefied nutmeg based on UPLC-QTOF-MS, which comprises the following steps:

[0051] (1) The torrefied nutmeg sample is mixed with a solvent, extracted, and after solid-liquid separation, a liquid sample to be tested is obtained;

[0052] (2) The sample to be tested obtained in step (1) is subjected to mass spectrometry detection by ultra-high performance liquid chromatography, and the component substance peak is obtained, and the component substance peak is subjected to qualitative analysis by analysis software.

[0053] In some specific embodiments of the present application, the torrefied Myristicae sample includes torrefied Myristicae medicinal materials, torrefied Myristicae decoction pieces and torrefied Myristicae formula granules.

[0054] In some specific embodiments of the present application, the torrefied Myristicae sample is made into powder, precisely weighed, then mixed with solvent, weighed, extracted, shaken, and then the liquid sample is obtained after solid-liquid separation.

[0055] Preferably, the torrefied Myristicae sample is dried before being made into powder, for example, using an oven, the drying temperature is preferably 50-70℃, more preferably 50-60℃, and most preferably 60℃; there is no special limitation on the drying time, and the sample is preferably dried to constant weight. In some specific embodiments of the present application, the torrefied Myristicae sample is made into powder by using a powder machine to grind the dried sample into coarse powder, and then passing through a No. 2 sieve.

[0056] In one or more preferred embodiments of the present application, the prepared torrefied Myristicae powder is precisely weighed, placed in a conical flask with a stopper, and then mixed with a solvent, which is water, methanol or a methanol solution, ethanol or an ethanol solution, preferably the solvent is selected from a methanol solution with a volume concentration of 80% or more, according to the solubility of the chemical components of torrefied Myristicae, most preferably the solvent is a 80% methanol solution.

[0057] In one or more preferred embodiments, the mass-volume ratio of the torrefied Myristicae sample to the solvent is (0.3-0.8g):(5-30mL), preferably 0.5g:(10-20mL), and more preferably 0.5g:15mL. The torrefied Myristicae sample is mixed with the solvent to obtain a mixed solution, and the mixed solution is extracted.

[0058] In the present application, there is no special limitation on the method of extraction, and methods known to those skilled in the art can be used, such as ultrasonic crushing, heating reflux or shaking. In order to make the extraction operation more convenient, ultrasonic crushing is preferably used, and the frequency and power of ultrasonic crushing have little effect on the present application. In some specific embodiments of the present application, the ultrasonic crushing time is 15-60min, preferably 15-40min, and more preferably 30min; the ultrasonic crushing power is 200-250W, preferably 250W; and the ultrasonic crushing frequency is 30-50kHz, preferably 40kHz.

[0059] In one or more preferred embodiments of the present application, the method further comprises the following steps: after ultrasonic crushing of the mixed solution, the solution is cooled, the weight is re-weighed, and the lost weight is made up with the solvent in step (1), for example, a 80% methanol solution is used to make up the lost weight.

[0060] In the present application, the method of solid-liquid separation is not particularly limited, and any method known to those skilled in the art can be used. In some specific embodiments, the solid-liquid separation is preferably carried out by centrifugation and filtration. The centrifugation is preferably carried out at a speed of 11000-13000 rpm, preferably 12000 rpm, and the centrifugation time is preferably 8-15 min, preferably 9-13 min, and more preferably 10 min. The filtration is not particularly limited, and preferably carried out by membrane filtration, more preferably by using a microporous filter membrane with a pore size of 0.10-0.50 μm, and further preferably by using a microporous filter membrane with a pore size of 0.22 μm. The filtrate obtained after filtration is the sample to be tested.

[0061] For the liquid sample to be tested obtained in step (1), in some specific embodiments of the present application, the liquid sample to be tested is obtained by filtration. For the sample to be tested, the present application uses ultra-high performance liquid chromatography to separate the sample to be tested, and then uses mass spectrometry to detect the separated components to obtain component peaks. Subsequently, the component peaks are analyzed qualitatively by software. In the present application, the peaks or component peaks refer to peaks that can be detected by mass spectrometry.

[0062] In some specific embodiments of the present application, the ultra-high liquid chromatography conditions include: the chromatographic column used is an octadecylsilane-bonded silica gel chromatographic column, which has excellent stability and is suitable for separating acidic, neutral and basic compounds; the column length of the chromatographic column can be 100-150 mm, and the packing particle size can be 1.6-2.2 μm; the chromatographic column can be selected from an Acclaim™ RSLC 120 C18 liquid chromatographic column with a size of 2.1 mm x 100 mm and a particle size of 2.2 μm, a Syncronis C18 liquid chromatographic column with a size of 2.1 x 100 mm and a particle size of 1.7 μm, and a Poroshell 120 EC-C18 liquid chromatographic column with a size of 2.1 x 100 mm and a particle size of 1.9 μm. In some preferred embodiments, the chromatographic column used in the present application is an Acclaim™ RSLC 120 C18 liquid chromatographic column with a size of 2.1 mm x 100 mm and a particle size of 2.2 μm.

[0063] In one or more preferred embodiments, considering the separation effect of the chromatographic peaks, the elution system mobile phase is: mobile phase A is acetonitrile, and mobile phase B is aqueous formic acid, preferably aqueous formic acid with a volume concentration of 0.02%-0.2%, more preferably aqueous formic acid with a volume concentration of 0.05%-0.1%, and further preferably aqueous formic acid with a volume concentration of 0.1%.

[0064] By optimizing the gradient elution program, comparing the peak time and peak effect of different time gradients, and obtaining the gradient elution program with the most abundant peak information, the following table is obtained:

[0065] Time / min Mobile phase A / % Mobile phase B / % 0～5 39 61 5～10 39→30 61→70 10～15 30→20 70→80 15～18 20 80 18～19 20→39 80→61 19～24 39 61

[0066] In some embodiments of the present application, the flow rate of the mobile phase is 0.1-0.5 mL / min, preferably 0.3 mL / min.

[0067] In some embodiments of the present application, the temperature of the chromatographic column is 35-45℃, preferably 40℃.

[0068] In some embodiments of the present application, the injection volume is 0.5-4 μL, preferably 2 μL.

[0069] In some embodiments of the present application, the detector uses a diode array detector (DAD detector), and the detection wavelength is 250-350 nm, preferably 275 nm.

[0070] In some embodiments of the present application, the mass spectrometry conditions include: the ion source is not particularly limited and can be any common ion source, preferably an electrospray ion source (ESI), atmospheric pressure chemical ionization (APCI), electron impact ionization (EI), chemical ionization (CI) or fast atom bombardment (FAB), preferably an electrospray ion source (ESI); the detection mode is positive ion mode; the drying gas temperature is 200-400℃, preferably the drying gas temperature is 300℃, the flow rate is 1-20 L / min, preferably the flow rate is 8 L / min; the nebulizer pressure is 10-50 psi, preferably the nebulizer pressure is 35 psi.

[0071] In some embodiments of the present application, the mass spectrometry conditions further include: the capillary voltage is 4000 V in positive ion mode; the capillary exit voltage is 175 V; the cone voltage is 65 V; data acquisition is performed in high resolution mode, the mass-to-charge ratio acquisition range is m / z 100-2000, the sampling speed is 1 spectra / s, and the sampling time is 1000 ms / spectra; the positive ion is calibrated in real time by purine (121.050873) and HP-0921 (922.009798) in mass number, and the nebulization pressure is 5 psi.

[0072] In some embodiments of the present application, the analysis software uses Agilent MassHunter Qualitative Analysis B.07.00.

[0073] In some embodiments of the present application, the qualitative analysis step is: (a) extracting the substance peak from the mass spectrum total ion current diagram to obtain the accurate molecular weight; (b) searching the database according to the first mass spectrum ion fragmentation information of the compound peak to obtain the mass spectrum fragmentation information in the database matching the screened substance; (c) combining and analyzing the adduct ion category, molecular weight error, retention time, related literature and the mass spectrum fragmentation information obtained in step (b) to obtain the structure of the compound. In the present application, the database includes TCM-database and self-built data; the adduct ion category includes M+H, M+NH4, M+Na, M+K, M+Cl, M+COOH, etc.; the molecular weight error is less than 10 ppm in absolute value.

[0074] The method for detecting a plurality of chemical components in torrefied mace constructed in the present application is simple, rapid, accurate, efficient, simple in sample pretreatment, simple in experimental steps and low in cost. The present application separates and identifies 15 chemical components, i.e. unknown lignan, macein A2, macein B1, mace lignan, macein C1, (-)-erythro-7-hydroxy-3,4,5,3',5'-pentamethoxy-8-oxo-4'-neolignan, macein D1, (-)-erythro-7-acetoxy-3,4,3',5'-tetramethoxy-8-oxo-4'-neolignan, (+)-5-methoxy dehydrodiisoeugenol, dehydrodiisoeugenol, Malabaricone C, 4-methoxy dehydrodiisoeugenol, Malabaricone B, 4-methoxy-6-{7-methoxy-3-methyl-5-[(1Z)-1-propen-1-yl]-2,3-dihydro-1-benzofuran-2-yl}-1,3-benzodioxole, and lycorine B, which provides a basis for further studying the pharmacodynamic material basis and comprehensive quality control of torrefied mace.

[0075] Embodiment

[0076] In order to more clearly illustrate the technical solutions of the present application, the following further describes the present application with reference to specific embodiments, but the present application should not be limited thereto, and the present application is only some embodiments. Unless otherwise specified, the instruments, reagents, materials, etc. used in the present application can be obtained by conventional commercial means.

[0077] Experimental instruments, reagents and drugs

[0078] Agilent 1290 ultra-high performance liquid chromatograph (Agilent, Mississauga, Ontario, USA) and mass spectrometer Agilent UPLC-QTOF-MS (Agilent, Mississauga, Ontario, USA).

[0079] Acetonitrile (chromatographically pure, Thermo Fisher); water is distilled water from Watsons; formic acid (analytically pure, SIGMA); the sample of torched mustard seed is provided by Tianjiang Research Institute.

[0080] Example 1: Investigation of the gradient elution procedure of the mobile phase

[0081] 1. Preparation of the sample to be tested of the torched mustard seed formula granules

[0082] (1) Take an appropriate amount of torched mustard seed formula granules, grind finely, take about 0.5 g, accurately weigh, and place in a conical flask with a stopper, accurately add 15 mL of 80% methanol, tightly stop, and weigh.

[0083] (2) Ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, and cool down.

[0084] (3) Weigh again, make up the weight loss with 80% methanol, and shake well.

[0085] (4) Centrifuge at 12000 rpm for 10 min, and analyze after passing through a 0.22 μm filter membrane.

[0086] 2. Investigation of the elution time gradient of the mobile phase of ultra-high liquid chromatography

[0087] An Acclaim TM RSLC 120 C18 liquid chromatography column (column length 100 mm, inner diameter 2.1 mm, particle size 2.2 μm) is used; the column temperature is 40 ℃; the elution system mobile phase A is acetonitrile solution, and the mobile phase B is 0.1% formic acid aqueous solution; the flow rate is 0.3 mL / min; the injection volume is 2 μL, and the detection wavelength is 300 nm; gradient elution is carried out according to the provisions in Table 1-Table 4 respectively, and the mass spectrum is recorded Figure 1 to Figure 4 .

[0088] Table 1: Investigation of elution time gradient 1

[0089] Time / min Mobile phase A / % Mobile phase B / % 0～8 10 90 8～13 45→60 55→40 13～15 60→20 40→80 15～18 20 80

[0090] Table 2: Investigation of elution time gradient 2

[0091] Time / min Mobile phase A / % Mobile phase B / % 0～5 15→20 85→80 5～10 20→30 80→70 10～18 30→40 70→60 18～22 40 60

[0092] Table 3: Investigation of elution time gradient 3

[0093] Time / min Mobile phase A / % Mobile phase B / % 0～5 20 80 5～12 20→35 80→65 12～18 35→55 65→45 18～20 35→60 65→40 20～22 60 40

[0094] Table 4: Investigation of elution time gradient 4

[0095] Time / min Mobile phase A / % Mobile phase B / % 0～5 39 61 5～10 39→30 61→70 10～15 30→20 70→80 15～18 20 80 18～19 20→39 80→61 19～24 39 61

[0096] The results show that the number of chromatographic peaks obtained by time gradient 1 and time gradient 3 is less, the chromatographic peak shape obtained by time gradient 2 is poor, the mass spectrum required by the application should display as many chromatographic peaks and chemical components in the torched mace nut as possible, the chromatographic peak information obtained by time gradient 4 is rich, and the chemical components in the torched mace nut can be comprehensively displayed, and therefore, time gradient 4 is finally selected as the gradient elution condition.

[0097] Example 2

[0098] 1. Preparation of the torched mace nut formula granule sample to be tested

[0099] The method is the same as the steps of "preparation of the torched mace nut formula granule sample to be tested" in "Example 1".

[0100] 2. Chromatographic conditions

[0101] An Acclaim TM RSLC 120 C18 liquid chromatography column (100 mm in length, 2.1 mm in diameter, and 2.2 μm in particle size) is used; the column temperature is 40℃; the elution system mobile phase A is acetonitrile solution, and the mobile phase B is 0.1% formic acid aqueous solution; the flow rate is 0.3 mL / min; the injection volume is 2 μL; the detection wavelength is 300 nm; and the gradient elution procedure is carried out according to Table 4.

[0102] 3. Mass spectrometric detection conditions

[0103] The ion source is a double-spray ESI ion source, the drying gas temperature is 300℃, the flow rate is 8 L / min, the atomizer pressure is 35 psi; in the positive ion mode, the capillary voltage is 4000 V, the capillary outlet voltage is 175 V, and the cone hole voltage is 65 V; high-resolution mode is used for data acquisition, the mass-to-charge ratio acquisition range is m / z 100-2000, the sampling speed is 1 spectra / s, and the sampling time is 1000 ms / spectra; in the positive ion mode, purine (121.050873) and HP-0921 (922.009798) are used for mass number real-time calibration, and the atomization pressure is 5 psi.

[0104] 4. Qualitative analysis of multiple chemical components in the torched mace nut formula granule

[0105] The Agilent MassHunter Qualitative Analysis B.07.00 software was used for qualitative analysis of the multiple chemical components in the roasted mace formula granules. A total of 15 chemical components were isolated and identified, including lignans, malabarone derivatives, i.e. diarylnonanes, etc., which were unknown lignans, macein A2, macein B1, macein, macein C1, (-)-erythro-7-hydroxy-3,4,5,3',5'-pentamethoxy-8-oxo-4'-neolignan, macein D1, (-)-erythro-7-acetoxy-3,4,3',5'-tetramethoxy-8-oxo-4'-neolignan, (+)-5-methoxydehydrosyringon, dehydrosyringon, Malabaricone C, 4-methoxydehydrosyringon, Malabaricone B, 4-methoxy-6-{7-methoxy-3-methyl-5-[(1Z)-1-propen-1-yl]-2,3-dihydro-1-benzofuran-2-yl}-1,3-benzodioxole, and lycorine B. The specific results are shown in Table 5. The lignan components are one of the main components, and the lignan components represented by macein A2, macein B1, macein C1, and macein D1 in the present application respond more obviously under the positive electrode. For example, macein A2 is represented by formula (I) Figure 6 , and from the total ion chromatogram Figure 5 , in addition to the monoisotopic mass m / z 345.1698 [M+H] + , the fragment peak m / z 327.1594 [M+H-H2O] + can also be observed Figure 7 . According to the molecular weight, ion fragment information, and compound database, it is speculated that the compound is macein A2, and its molecular formula is C 20 H 24 O5.

[0106] Table 5: Analysis of chemical components in roasted mace granules based on UPLC-QTOF-MS technology

[0107]

[0108] Example 3

[0109] 1. Preparation of roasted mace formula granule sample to be tested

[0110] (1) Take roasted mace formula granules, grind finely, take about 0.5 g, accurately weigh, put into a conical bottle with a plug, tightly seal, and weigh the weight.

[0111] (2) Ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 min, and cool down.

[0112] (3) Re-weigh, make up the weight loss with 80% methanol, shake well.

[0113] (4) Centrifuge at 12000 rpm for 10 min, analyze after 0.22 μm filter membrane.

[0114] 2. Chromatographic conditions

[0115] An Acclaim TM RSLC 120 C18 liquid chromatographic column (100 mm in length, 2.1 mm in diameter, 2.2 μm in particle size) was used; the column temperature was 40 ℃; the elution system mobile phase A was acetonitrile solution, and the mobile phase B was 0.1% formic acid aqueous solution; the flow rate was 0.3 mL / min; the injection volume was 2 μL, and the detection wavelength was 300 nm; and the gradient elution program was carried out according to Table 4.

[0116] 3. Mass spectrometric detection conditions

[0117] The same as "Mass spectrometric detection conditions" in "Example 2"

[0118] 4. Qualitative analysis of multiple chemical components in the torrefied myristicae granules

[0119] Agilent MassHunter Qualitative Analysis B.07.00 software was used for qualitative analysis of multiple chemical components in the torrefied myristicae granules. A total of 15 chemical components were separated and identified, including lignans, malabaricone derivatives, i.e. diarylnonanes, and the like, and the specific results are shown in Table 5. Among them, Malabaricone B and Malabaricone C belong to this class of compounds, and the basic skeleton structure is that one aromatic ring is connected to each end of a carbon chain composed of 9 carbon atoms. Literature research has found that Malabaricone C has significant biological activities, such as nematocidal activity, antioxidant activity and antibacterial activity, etc. (Qiu Jinglei. Total synthesis of natural product Malabaricone C [D]. Sichuan University, 2007). In the present application, Malabaricone B and Malabaricone C have very obvious responses in the positive ion mode. Taking Malabaricone C as an example, m / z 359.1857 ( Figure 8 ) was extracted from the total ion chromatogram ( Figure 9 ). In the positive ion mode, the quasi-molecular ion peak of the first-order mass spectrum was m / z 359.1857 [M+H] + ( Figure 10 ), and the fragment peak m / z 381.1672 [M+Na] + was also observed ( Figure 9). According to the molecular weight, ion fragment information and compound database, the compound is Malabaricone C, and the molecular formula is C 21 H 26 O5。

[0120] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1.A method for detecting chemical components in torrefied Myristicae Fructus based on UPLC-QTOF-MS, comprising the following steps: (1) mixing a torrefied Myristicae Fructus sample with a solvent to perform extraction, and then performing solid-liquid separation to obtain a liquid sample to be detected; (2) performing mass spectrometry on the sample to be detected obtained in step (1) by using ultra-high performance liquid chromatography, and obtaining component peaks, and performing qualitative analysis on the component peaks by using an analysis software, wherein the qualitative analysis comprises the following steps: (a) extracting substance peaks from a total ion current chromatogram to obtain accurate molecular weights; (b) searching a database according to first mass spectrum ion fragment information of the compound peaks to obtain mass spectrum fragment information in the database that matches the screened substances; and (c) combining and analyzing adduct ion categories, molecular weight errors, retention times, related literatures and the mass spectrum fragmentation information obtained in step (b) to obtain a structure of the compound; wherein the solvent in step (1) is selected from one or more of water, methanol or a methanol aqueous solution, ethanol or an ethanol aqueous solution; the conditions for the ultra-high performance liquid chromatography separation in step (2) are as follows: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, and the packing particle size is 1.6-2.2 μm; the elution system mobile phase is as follows: the mobile phase A is acetonitrile, and the mobile phase B is a 0.05%-0.1% formic acid aqueous solution; the detection wavelength is 250-350 nm, and the gradient elution program is as follows: the method for the detection in step (2) comprises the following: the ion source is an electrospray ion source, and the detection mode is a positive ion mode; the dry gas temperature is 200-400 ℃, and the flow rate is 1-20 L / min; and the atomizer pressure is 10-50 psi; and the components include Myristical A2, Myristical B1, Myristical lignan, Myristical C1, (-)-erythro-7-acetoxy-3, 4, 3', 5'-tetramethoxy-8-oxo-4'-neolignan, Myristical D1, (-)-erythro-7-acetoxy-3, 4, 3', 5'-tetramethoxy-8-oxo-4'-neolignan, (+)-5-methoxydehydrodiisoeugenol, dehydrodiisoeugenol, Malabaricone C, 4-methoxydehydrodiisoeugenol, Malabaricone B, 4-methoxy-6-{7-methoxy-3-methyl-5-[(1Z)-1-propen-1-yl]-2, 3-dihydro-1-benzofuran-2-yl}-1, 3-benzodioxole and Licarin B; the solvent is a methanol aqueous solution with a methanol volume concentration of more than 80%; the mass / volume ratio of the torrefied Myristicae Fructus sample to the solvent is (0.3-0.8 g) :(5-30 mL); the extraction mode is ultrasonic crushing, the ultrasonic crushing time is 15-60 min, the ultrasonic crushing power is 200-250 W, and the ultrasonic crushing frequency is 30-50 kHz; and the method further comprises the following step: after ultrasonic crushing, the sample is cooled, weighed again, and then the lost weight is made up with the solvent. ​ ​ ​ ​ ​ ​ ​ 2. The method of claim 1, wherein, ​ 3. The method according to claim 1 or 2, characterized in that, ​ 4. The method according to claim 1 or 2, characterized in that, ​ 5. The method of claim 4, wherein, ​ 6. The method of claim 1 or 2, wherein, The solid-liquid separation comprises the steps of centrifugation and filtration, wherein the rotation speed of the centrifugation is 11000-13000 rpm, the centrifugation time is 8-15 min; the filtration adopts a microporous filter membrane with a pore size of 0.10-0.50 μm.

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