阅读说明：本技术 一种保健食品中非法添加化合物的筛查方法 (Screening method for illegally added compounds in health food ) 是由 李先芝 刘洋 钱全全 林露 毛琼丽 严玲 石豪 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种保健食品中非法添加化合物的筛查方法,利用超高效液相色谱—串联四级杆飞行时间质谱仪具有高灵敏度的MSMS功能的特点,能够精确地测定出各化合物的母离子与子离子。收集保健食品中可能存在的非法添加化合物种类,利用超高效液相色谱—串联四级杆飞行时间质谱仪对可能存在的非法添加化合物建立数据库,得到特定条件下的非法添加化合物的保留时间、母离子与子离子信息；对保健食品在特定条件下进行超高效液相色谱—串联四级杆飞行时间质谱分析,得到样品中各化合物的质谱信息,结合已经建立的数据库快速地判断出样品中可能存在的非法添加化合物的种类,为非法添加化合物的快速筛查工作提供了技术手段与方法。(The invention discloses a screening method of illegally added compounds in health food, which can accurately measure parent ions and daughter ions of each compound by utilizing the characteristic that an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer has a high-sensitivity MSMS function. Collecting the types of the illegal addition compounds possibly existing in the health-care food, and establishing a database for the illegal addition compounds possibly existing by using an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer to obtain the retention time of the illegal addition compounds under specific conditions and the information of parent ions and daughter ions; the method comprises the steps of carrying out ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry on health food under specific conditions to obtain mass spectrum information of each compound in a sample, and rapidly judging the type of an illegally added compound possibly existing in the sample by combining with an established database, so that a technical means and a method are provided for rapid screening work of the illegally added compound.)

1. A method for screening an illegally added compound in health food, which is characterized by comprising the following steps:

the illegal addition compounds comprise the following 161 types:

(a) sleep-improving illicit added compounds: chlordiazepoxide, midazolam, nitrazepam, estazolam, oxazepam, alprazolam, lorazepam, clonazepam, triazolam, diazepam, barbital, phenobarbital, secobarbital, amobarbital, clomeprazole, zopiclone, chlorpheniramine maleate, zaleplon, venlafaxine hydrochloride, sinomenine, rotundine;

(b) hypoglycemic illegal addition compounds: tolbutamide, glibenclamide, gliclazide, glipizide, gliquidone, glimepiride, rosiglitazone hydrochloride, repaglinide, pioglitazone hydrochloride, metformin hydrochloride, phenformin hydrochloride, buformin hydrochloride, glibornuride, acarbose, nateglinide;

(c) hypotensive illegally added compounds: losartan potassium, lisinopril, atenolol, clonidine hydrochloride, hydrochlorothiazide, captopril, prazosin hydrochloride, reserpine, nifedipine, amlodipine, nitrendipine, nimodipine, nisoldipine, felodipine, minoxidil, nicardipine hydrochloride;

(d) lipid lowering illegally added compounds: lovastatin, simvastatin, niacin, bezafibrate, gemfibrozil, fenofibrate, fluvastatin sodium, rosuvastatin sodium;

(e) weight-loss illegally added compound: sibutramine hydrochloride, N-monodemethylsibutramine hydrochloride, N-bisdemethylsibutramine hydrochloride, fenfluramine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, phenolphthalein, furosemide, orlistat, and rimonabant hydrochloride;

(f) anti-fatigue illegally added compounds: o-deethylsildenafil, acetamidottadalafil, diketindenafil, N-deethylsildenafil, pyrazol N-desmethylsildenafil, hydroxythioporunit sildenafil, desmethyltalalafil, benseradifenafil, benzothidinalin, N-deethyl-N-methylvardenafil, depiperazinylthiosildenafil, hydroxythiovardenafil, desmethylthiosildenafil, dimethylreddenafil, ketoreddenafil, milrinafil, hydroxyreddenafil, dapoxetine, propoxyphenylsildenafil, N-desmethylsildenafil, vardenafil, N-phenylpropenyltadalafil, propoxyphenylthiomeromolusinafil, N-ethyltadalafil, acetyldinafil, aminosildenafil, thiosildenafil, thioquinafil, thiopiperadenafil, Propoxyphenylthiomorpholinafil, desmethylcarbanafil, sildenafil N-oxide, 2-hydroxyethyl nortadalafil, avanafil, cyclopentafil, N-butyltadalafil, ledenafil carbonate, vardenafil N-oxide, diclofenac, N-t-butoxycarbonyl-N-deethylerythronafil, dithionorcabafenafil, demethylpiperazinylsetafenamic acid, sildenafil dimer impurities, carbadenafil, nafaretic acid, propoxyphenylidenafil, denafil, N-deethylvardenafil, namolindonafil, hydroxypumocelnafil, benzamidonafil, prazolanafil, propoxyphenylhydroxypumodenafil, N-octylnortadalafil, udenafarenafil, talafil methyl chloride, Nitridafil, idenafil, vardenafil piperazinone, 2-hydroxypropyl nortadalafil, devulcanid vardenafil, tadalafil, vardenafil dimer, hydroxychlorodenafil, clodenafil, propoxyphenylthio-sildenafil, decarbed sildenafil, propoxyphenylthio-sildenafil, naerythrodenafil, aminotadalafil, hydroxyvardenafil, thiomeromolesinafil, reddenafil, sildenafil, namolsidenafil, limousinafil, pseudovardenafil, yohimbine hydrochloride, sildenafil impurity 17, sildenafil impurity I, dithio-deethylcarbamazepine, Guidenafil, vardenafil acetyl analogue, sildenafil impurity 12, tadalafil dichlorphenal impurity, sildenafil impurity 14, deethylcarbamazepine, hydroxythiophenadenafil, dithio, dinitro impurity, dinafil impurity, sildenafil impurity, Thiadenafil, diethylamino-pro-tadalafil;

(1) preparation of standard working solutions

Accurately weighing a proper amount of each compound standard substance in a 10mL volumetric flask, dissolving the compound standard substance with methanol or 50% acetonitrile water solution and diluting the compound standard substance to a scale, and preparing a standard stock solution of each compound; dissolving the insoluble compound by using dichloromethane or methanol hydrochloride (1:99) solution, and then fixing the volume to the scale;

standard working solution (1. mu.g/mL): respectively and accurately sucking a proper amount of standard stock solution, placing the standard stock solution into a 10mL volumetric flask, diluting the standard stock solution to a scale with a solvent, and shaking up to prepare a standard working solution; preparing a new preparation for clinical use;

(2) preparation of test solution of sample to be tested

1) Solid or semi-solid samples

Taking a proper amount of solid samples (protein powder, oyster powder, biscuits, candies, coffee, health-care food containing the same matrix as the matrix, tablets, capsules and other dosage forms), uniformly mixing, grinding, or taking a proper amount of semisolid samples (jelly), uniformly mixing, precisely weighing 1g (accurate to 0.001g) and placing in a 50mL volumetric flask, adding a proper amount of methanol, ultrasonically extracting for 15min, cooling to room temperature, metering the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging at 4000 r/min for 5min, filtering supernatant through a microporous membrane, taking subsequent filtrate, and properly diluting with acetonitrile water solution (1+1) according to actual concentration for later use;

2) liquid sample

Shaking a proper amount of sample (beverage, wine), accurately sucking 1mL, placing in a 50mL volumetric flask, adding a proper amount of methanol, ultrasonically extracting for 15min, cooling to room temperature, diluting to constant volume with methanol, filtering with microporous membrane, collecting the filtrate, and diluting with acetonitrile water solution (1+1) according to the actual concentration;

3) lipid matrix (Soft Capsule) sample

Taking a proper amount of a sample (soft capsule), uniformly mixing, precisely weighing 1g (precisely to 0.001g) and placing in a 50mL volumetric flask, adding 5mL of ethyl acetate, shaking to disperse the sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging for 5min at 4000 r/min, filtering the supernatant through a microporous filter membrane, taking a subsequent filtrate, and properly diluting with an acetonitrile aqueous solution (1+1) according to the actual concentration for later use;

4) blank matrix extracting solution

Weighing a proper amount of blank sample, and treating the blank sample by the same method as the sample to prepare a blank matrix extracting solution;

5) blank solution

Processing the sample in the same way without adding the sample to prepare a blank solution;

(3) database establishment

Injecting the standard working solution of the 161 compounds into an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, and acquiring an original data map and a total ion flow map of each compound;

exporting the acquired original data of each compound from masslynx software, importing the original data into unifi data processing software for data processing to obtain the retention time, the parent ion mass spectrogram and the daughter ion mass spectrogram of each compound, and determining the characteristic fragment ions of each compound according to the fragment fracture mechanism and the fracture difficulty of each compound;

summarizing the species, name, molecular formula, retention time and characteristic fragment ion information of each compound, and establishing a 161 compound screening database;

(4) qualitative analysis

1) Performing ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry on the sample to be detected processed by the method in the step (2), comparing the obtained result with data in a database, wherein a blank solvent cannot generate an ion peak identical to that of a standard solution, the retention time of a sample chromatographic peak is consistent with that of the standard solution, the allowable deviation is +/-0.1 min, the accurate mass numbers of parent ions and characteristic fragment ions of the sample are consistent with that of the standard solution, the deviations are less than or equal to 0.05Da, the signal-to-noise ratios (S/N) of the parent ions and the fragment ion chromatographic peaks are all over 3:1, the signal-to-noise ratios are calculated by peak-to-peak (PtP), when the full-scan spectrum is recorded, the relative abundance of the characteristic fragment ions in the sample must exceed 10% of the abundance of the characteristic ion spectrum referred by the standard solution, and screening out a compound meeting qualitative conditions, obtaining a suspected positive sample;

2) for a suspected positive sample, an MSMS method is edited according to retention time and molecular weight of a compound, data are collected, an obtained sub-ion scanning mass spectrogram of the suspected substance is compared with a sub-ion scanning mass spectrogram of a standard solution, and under the same condition and similar concentration, the allowable deviation of the relative abundance of main fragment ions in the sample and the relative abundance of the standard solution does not exceed a specified range, so that the sample can be judged to have a corresponding object to be detected;

the relative abundance (%) of fragment ions is > 50, and the allowable maximum deviation (%) is +/-20; the relative abundance (%) of fragment ions is more than 20-50, and the allowable maximum deviation (%) is +/-25; the relative abundance (%) of fragment ions is more than 10-20, and the allowable maximum deviation (%) is +/-30; the relative abundance (%) of the fragment ions was 10 or less, and the maximum allowable deviation (%) was 50 or less.

2. The method for screening compounds illegally added to health foods according to claim 1, wherein: the liquid chromatographic analysis conditions in the step (3) and the step (4) are as follows: the chromatographic column is a Waters UPLC BEH C18 column (2.1 × 100mm, 1.7 μm), acetonitrile is used as an organic phase mobile phase for gradient elution, and the proportion is slowly changed from the initial 2% to 95% so as to ensure that all compounds can be effectively detected;

in positive ion mode, A is 0.1% formic acid solution, B is acetonitrile, and the gradient elution procedure is as follows (curve 1 is instantaneous change, curve 6 is linear change):

in the case of anion mode collection, a is water and B is acetonitrile, and the gradient elution procedure is shown in the following table (curve 1 is instantaneous change, curve 6 is linear change):

gradient time, min Mobile phase A,% Mobile phase B,% Curve line 0 95 5 Initial 0.50 95 5 6 3.50 70 30 6 6.00 30 70 6 9.00 5 95 6 11.00 5 95 6 14.00 95 5 1

3. The method for screening compounds in health food according to claim 1, wherein: the mass spectrometry conditions in the step (3) and the step (4) are as follows:

parameters of ion source Reference value Reference value Type of ion source ESI+ ESI- Capillary voltage 3.0KV 2.5KV Voltage of taper hole 30 30 Source temperature 150 150 Desolventizing temperature 400 400 Taper hole back-blowing air flow rate 50L/Hr 50L/Hr Desolventizing air flow rate 800L/Hr 800L/Hr

Screening mass spectrometry parameters are as follows:

wherein, the leucine enkephalin is corrected online in real time, the molecular weight is 556.2771 in an ESI + mode, the molecular weight is 554.2615 in an ESI-mode, the scanning time is 0.5s, and the scanning time interval is 10 s.

4. The method for screening compounds illegally added to health foods according to claim 1, wherein: the solvent used in the step (1) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 1: 1.

5. The method for screening compounds illegally added to health foods according to claim 1, wherein: the solvent for dilution in the step (2) is a mixed solution of acetonitrile and water, and the volume ratio of the mixed solution is 1: 1.

Technical Field

The invention relates to a rapid screening method for 161 illegally added compounds in health-care food, in particular to rapid qualitative screening of illegally added compounds in health-care food by using an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry method.

Background

The illegal addition of the health food refers to the addition of varieties which are not declared in the formula in the health food, such as sibutramine, phenolphthalein, furosemide and the like in weight-reducing food. Most chemical drugs are forbidden in health food because they have certain side effects (the specifications of the chemical drugs are marked), and especially after being used in excess amount, serious consequences can be caused, such as drug dependence, organ injury, even organ failure and the like. Driven by market demand and profit, some merchants and lawbreakers, regardless of national legal regulations, add some substances strictly prohibited by the country to health foods to increase their efficacy. These illegal addition substances often have many toxic and side effects, which bring potential risks to human health.

At present, the detection method of illegally added compounds mainly comprises the following methods:

a physical and chemical reaction method: the physical and chemical reaction method is simple to operate, does not need special equipment, is suitable for field operation, and can meet the requirements of basic supervision and inspection. However, the health food has complex matrix, contains one or more traditional Chinese medicine extracts and various auxiliary materials, and has poor physicochemical reaction selectivity, so the health food is usually used for preliminary screening of samples.

Spectroscopic method: the research of the spectroscopy mainly provides a basis for the development of field detection technology. If near-infrared spectroscopy (NIR) is adopted to detect that metformin hydrochloride is illegally added in the hypoglycemic traditional Chinese medicine and the health food, and a mathematical model is established, the functional group characteristics of the target substance are obtained. The method does not need special pretreatment on samples in the detection process, and the samples are not lost in the detection process, but the method is mainly suitable for qualitative detection of specific compounds.

③ chromatography: high performance liquid chromatography has good separation performance and is often used as a method for qualitative analysis and quantitative determination. In a period where liquid chromatography-mass spectrometry is not widespread enough, operations of liquid phase prescreening-liquid mass confirmation-liquid phase quantification are often performed. In actual operation, the retention time of chromatographic peaks of a sample and a reference substance is compared with an ultraviolet spectrogram to perform preliminary qualitative judgment, then a liquid chromatography-mass spectrometry method is adopted, structural information given by mass spectrometry is used for confirmation, and then quantitative determination is performed on liquid chromatography. Compared with a liquid mass method, the liquid phase method has the advantages that the mass spectrum is only used for confirmation, the experiment cost can be reduced, and when the drug addition amount in the sample is higher, the mass spectrum pollution can be reduced by adopting the liquid phase detection mass spectrum for confirmation. However, the disadvantages are that when the content of illegal addition is extremely low, especially close to a trace level (even if the illegal addition is not malicious), the adoption of liquid-phase primary screening can cause a screen leakage phenomenon, the specificity of an ultraviolet spectrogram is not strong, specific structural information cannot be provided, and the health-care food matrix can influence the ultraviolet spectrum. The method has accurate and reliable detection result, and has the limitation that the method only aims at specific compounds for detection, specific detection indexes are required to be determined before detection, and qualitative detection cannot be carried out; furthermore, the sample needs to be subjected to complicated pre-treatments to exclude possible interferences.

And fourthly, a chromatography-mass spectrometry combined method: the chromatography-mass spectrometry combination is a more and more common technology used in illegal addition detection, has the characteristics of high sensitivity and strong specificity, and can obtain the molecular weight and fragment ion information of a target object, thereby realizing the purpose of one-time analysis and structure confirmation. In the case of quantification, a good recovery rate can be obtained. At present, triple quadrupole mass spectrometry is most widely used in laboratories, but determination by combining measurement results with high resolution is also required if necessary. Meanwhile, because the sensitivity of the mass spectrum is very high, the mass spectrum analysis is directly used on the premise that the illegal added medicine amount is not clear enough, instrument pollution is possibly caused, and in addition, the mass spectrum has a matrix effect, so that when the matrix influence is large, the qualitative judgment is also disturbed, and the mass spectrum is required to be used by combining with a necessary pretreatment purification technology.

Disclosure of Invention

The invention aims to solve the problem of illegal addition of health food, and provides a screening method of illegal addition compounds in health food, which comprises the following steps:

the illegal addition compounds comprise the following 161 types:

(a) sleep-improving illicit added compounds: chlordiazepoxide, midazolam, nitrazepam, estazolam, oxazepam, alprazolam, lorazepam, clonazepam, triazolam, diazepam, barbital, phenobarbital, secobarbital, amobarbital, clomeprazole, zopiclone, chlorpheniramine maleate, zaleplon, venlafaxine hydrochloride, sinomenine, rotundine;

(b) hypoglycemic illegal addition compounds: tolbutamide, glibenclamide, gliclazide, glipizide, gliquidone, glimepiride, rosiglitazone hydrochloride, repaglinide, pioglitazone hydrochloride, metformin hydrochloride, phenformin hydrochloride, buformin hydrochloride, glibornuride, acarbose, nateglinide;

(c) hypotensive illegally added compounds: losartan potassium, lisinopril, atenolol, clonidine hydrochloride, hydrochlorothiazide, captopril, prazosin hydrochloride, reserpine, nifedipine, amlodipine, nitrendipine, nimodipine, nisoldipine, felodipine, minoxidil, nicardipine hydrochloride;

(d) lipid lowering illegally added compounds: lovastatin, simvastatin, niacin, bezafibrate, gemfibrozil, fenofibrate, fluvastatin sodium, rosuvastatin sodium;

(e) weight-loss illegally added compound: sibutramine hydrochloride, N-monodemethylsibutramine hydrochloride, N-bisdemethylsibutramine hydrochloride, fenfluramine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, phenolphthalein, furosemide, orlistat, and rimonabant hydrochloride;

(f) anti-fatigue illegally added compounds: o-deethylsildenafil, acetamidottadalafil, diketindenafil, N-deethylsildenafil, pyrazol N-desmethylsildenafil, hydroxythioporunit sildenafil, desmethyltalalafil, benseradifenafil, benzothidinalin, N-deethyl-N-methylvardenafil, depiperazinylthiosildenafil, hydroxythiovardenafil, desmethylthiosildenafil, dimethylreddenafil, ketoreddenafil, milrinafil, hydroxyreddenafil, dapoxetine, propoxyphenylsildenafil, N-desmethylsildenafil, vardenafil, N-phenylpropenyltadalafil, propoxyphenylthiomeromolusinafil, N-ethyltadalafil, acetyldinafil, aminosildenafil, thiosildenafil, thioquinafil, thiopiperadenafil, Propoxyphenylthiomorpholinafil, desmethylcarbanafil, sildenafil N-oxide, 2-hydroxyethyl nortadalafil, avanafil, cyclopentafil, N-butyltadalafil, ledenafil carbonate, vardenafil N-oxide, diclofenac, N-t-butoxycarbonyl-N-deethylerythronafil, dithionorcabafenafil, demethylpiperazinylsetafenamic acid, sildenafil dimer impurities, carbadenafil, nafaretic acid, propoxyphenylidenafil, denafil, N-deethylvardenafil, namolindonafil, hydroxypumocelnafil, benzamidonafil, prazolanafil, propoxyphenylhydroxypumodenafil, N-octylnortadalafil, udenafarenafil, talafil methyl chloride, Nitridafil, idenafil, vardenafil piperazinone, 2-hydroxypropyl nortadalafil, devulcanid vardenafil, tadalafil, vardenafil dimer, hydroxychlorodenafil, clodenafil, propoxyphenylthio-sildenafil, decarbed sildenafil, propoxyphenylthio-sildenafil, naerythrodenafil, aminotadalafil, hydroxyvardenafil, thiomeromolesinafil, reddenafil, sildenafil, namolsidenafil, limousinafil, pseudovardenafil, yohimbine hydrochloride, sildenafil impurity 17, sildenafil impurity I, dithio-deethylcarbamazepine, Guidenafil, vardenafil acetyl analogue, sildenafil impurity 12, tadalafil dichlorphenal impurity, sildenafil impurity 14, deethylcarbamazepine, hydroxythiophenadenafil, dithio, dinitro impurity, dinafil impurity, sildenafil impurity, Thiadenafil, diethylamino-pro-tadalafil;

(1) preparation of standard working solutions

Accurately weighing a proper amount of each compound standard substance in a 10mL volumetric flask, dissolving the compound standard substance with methanol or 50% acetonitrile water solution and diluting the compound standard substance to a scale, and preparing a standard stock solution of each compound; dissolving the insoluble compound by using dichloromethane or methanol hydrochloride (1:99) solution, and then fixing the volume to the scale;

standard working solution (1. mu.g/mL): respectively and accurately sucking a proper amount of standard stock solution, placing the standard stock solution into a 10mL volumetric flask, diluting the standard stock solution to a scale with a solvent, and shaking up to prepare a standard working solution; preparing a new preparation for clinical use;

(2) preparation of test solution of sample to be tested

1) Solid or semi-solid samples

Taking a proper amount of solid samples (protein powder, oyster powder, biscuits, candies, coffee, health-care food containing the same matrix as the matrix, tablets, capsules and other dosage forms), uniformly mixing, grinding, or taking a proper amount of semisolid samples (jelly), uniformly mixing, precisely weighing 1g (accurate to 0.001g) and placing in a 50mL volumetric flask, adding a proper amount of methanol, ultrasonically extracting for 15min, cooling to room temperature, metering the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging at 4000 r/min for 5min, filtering supernatant through a microporous membrane, taking subsequent filtrate, and properly diluting with acetonitrile water solution (1+1) according to actual concentration for later use;

2) liquid sample

Shaking a proper amount of sample (beverage, wine), accurately sucking 1mL, placing in a 50mL volumetric flask, adding a proper amount of methanol, ultrasonically extracting for 15min, cooling to room temperature, diluting to constant volume with methanol, filtering with microporous membrane, collecting the filtrate, and diluting with acetonitrile water solution (1+1) according to the actual concentration;

3) lipid matrix (Soft Capsule) sample

Taking a proper amount of a sample (soft capsule), uniformly mixing, precisely weighing 1g (precisely to 0.001g) and placing in a 50mL volumetric flask, adding 5mL of ethyl acetate, shaking to disperse the sample, adding a proper amount of methanol, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging for 5min at 4000 r/min, filtering the supernatant through a microporous filter membrane, taking a subsequent filtrate, and properly diluting with an acetonitrile aqueous solution (1+1) according to the actual concentration for later use;

4) blank matrix extracting solution

Weighing a proper amount of blank sample, and treating the blank sample by the same method as the sample to prepare a blank matrix extracting solution;

5) blank solution

Processing the sample in the same way without adding the sample to prepare a blank solution;

(3) database establishment

Injecting the standard working solution of the 161 compounds into an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, and acquiring an original data map and a total ion flow map of each compound;

exporting the acquired original data of each compound from masslynx software, importing the original data into unifi data processing software for data processing to obtain the retention time, the parent ion mass spectrogram and the daughter ion mass spectrogram of each compound, and determining the characteristic fragment ions of each compound according to the fragment fracture mechanism and the fracture difficulty of each compound;

summarizing the species, name, molecular formula, retention time and characteristic fragment ion information of each compound, and establishing a 161 compound screening database;

(4) qualitative analysis

1) Performing ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry on the sample to be detected processed by the method in the step (2), comparing the obtained result with data in a database, wherein a blank solvent cannot generate an ion peak identical to that of a standard solution, the retention time of a sample chromatographic peak is consistent with that of the standard solution, the allowable deviation is +/-0.1 min, the accurate mass numbers of parent ions and characteristic fragment ions of the sample are consistent with that of the standard solution, the deviations are less than or equal to 0.05Da, the signal-to-noise ratios (S/N) of the parent ions and the fragment ion chromatographic peaks are all over 3:1, the signal-to-noise ratios are calculated by peak-to-peak (PtP), when the full-scan spectrum is recorded, the relative abundance of the characteristic fragment ions in the sample must exceed 10% of the abundance of the characteristic ion spectrum referred by the standard solution, and screening out a compound meeting qualitative conditions, obtaining a suspected positive sample;

2) for a suspected positive sample, an MSMS method is edited according to retention time and molecular weight of a compound, data are collected, an obtained sub-ion scanning mass spectrogram of the suspected substance is compared with a sub-ion scanning mass spectrogram of a standard solution, and under the same condition and similar concentration, the allowable deviation of the relative abundance of main fragment ions in the sample and the relative abundance of the standard solution does not exceed a specified range, so that the sample can be judged to have a corresponding object to be detected;

the relative abundance (%) of fragment ions is > 50, and the allowable maximum deviation (%) is +/-20; the relative abundance (%) of fragment ions is more than 20-50, and the allowable maximum deviation (%) is +/-25; the relative abundance (%) of fragment ions is more than 10-20, and the allowable maximum deviation (%) is +/-30; the relative abundance (%) of the fragment ions was 10 or less, and the maximum allowable deviation (%) was 50 or less.

Wherein: the liquid chromatographic analysis conditions in the step (3) and the step (4) are as follows: the chromatographic column is a Waters UPLC BEH C18 column (2.1X 100mm, 1.7 μm), acetonitrile is used as an organic phase mobile phase for gradient elution, and the proportion is slowly changed from the initial 2 percent to 95 percent so as to ensure that all compounds can be effectively detected;

in positive ion mode, A is 0.1% formic acid solution, B is acetonitrile, and the gradient elution procedure is as follows (curve 1 is instantaneous change, curve 6 is linear change):

gradient time, min	Mobile phase A,%	Mobile phase B,%	Curve line
				0	95	5	Initial
0.50	95	5	6
				9.00	30	70	6
10.00	30	70	6
				12.00	5	95	6
14.00	5	95	6
				17.00	95	5	1

In the case of anion mode collection, a is water and B is acetonitrile, and the gradient elution procedure is shown in the following table (curve 1 is instantaneous change, curve 6 is linear change):

gradient time, min	Mobile phase A,%	Mobile phase B,%	Curve line
				0	95	5	Initial
0.50	95	5	6
				3.50	70	30	6
6.00	30	70	6
				9.00	5	95	6
11.00	5	95	6
				14.00	95	5	1

Wherein: the mass spectrometry conditions in the step (3) and the step (4) are as follows:

screening mass spectrometry parameters are as follows:

parameter(s)	Fuction 1	Fuction 2
			Scanning mode	MSE	MSE
Time of scan	0～17min	0～14min
			Scanning range	50～1200Da	50～1200Da
Time of scan	0.2s	0.2s
			Energy of collision	5～40V	15V

Leucine enkephalin was corrected online in real time with molecular weight 556.2771 for ESI + mode, 554.2615 for ESI-mode, scan time 0.5s, scan interval 10 s.

Wherein: the solvent used in the step (1) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 1: 1.

Wherein: the solvent for dilution in the step (2) is a mixed solution of acetonitrile and water, and the volume ratio of the mixed solution is 1: 1.

The invention adopts a flight time high-resolution mass spectrometry method to establish an illegal addition compound database to obtain the retention time, parent ion and daughter ion information of each substance under specific conditions, analyzes the sample under specific conditions to obtain the related information of each compound in the sample, brings the sample original data obtained by analysis into software, automatically processes the data by the software to obtain reliable results, has the characteristics of high analysis speed, multiple screened compound types and simple sample pretreatment, and provides a method for screening illegal addition compounds.

Because the flight time high resolution mass spectrum has high sensitivity and can obtain reliable data when the sample amount is small, the compound information of the sample can be obtained without complex pretreatment of the sample in the analysis process. In order to avoid the sample from polluting the instrument, the data acquisition can be carried out in a mode of gradually increasing the sample volume in the sample analysis process.

Drawings

FIG. 1 is a diagram of mass spectrometry for actual sample detection.

Detailed Description

The health food samples comprise solid, semi-solid, liquid and grease matrix (soft capsule) samples.

The 161 illegal addition compounds comprise the following compounds:

(a) sleep-improving illicit added compounds: chlordiazepoxide, midazolam, nitrazepam, estazolam, oxazepam, alprazolam, lorazepam, clonazepam, triazolam, diazepam, barbital, phenobarbital, secobarbital, amobarbital, clomeprazole, zopiclone, chlorpheniramine maleate, zaleplon, venlafaxine hydrochloride, sinomenine, rotundine;

(b) hypoglycemic illegal addition compounds: tolbutamide, glibenclamide, gliclazide, glipizide, gliquidone, glimepiride, rosiglitazone hydrochloride, repaglinide, pioglitazone hydrochloride, metformin hydrochloride, phenformin hydrochloride, buformin hydrochloride, glibornuride, acarbose, nateglinide;

(c) hypotensive illegally added compounds: losartan potassium, lisinopril, atenolol, clonidine hydrochloride, hydrochlorothiazide, captopril, prazosin hydrochloride, reserpine, nifedipine, amlodipine, nitrendipine, nimodipine, nisoldipine, felodipine, minoxidil, nicardipine hydrochloride;

(d) lipid lowering illegally added compounds: lovastatin, simvastatin, niacin, bezafibrate, gemfibrozil, fenofibrate, fluvastatin sodium, rosuvastatin sodium;

(e) weight-loss illegally added compound: sibutramine hydrochloride, N-monodemethylsibutramine hydrochloride, N-bisdemethylsibutramine hydrochloride, fenfluramine hydrochloride, ephedrine hydrochloride, pseudoephedrine hydrochloride, phenolphthalein, furosemide, orlistat, and rimonabant hydrochloride;

(f) anti-fatigue illegally added compounds: o-deethylsildenafil, acetamidottadalafil, diketindenafil, N-deethylsildenafil, pyrazol N-desmethylsildenafil, hydroxythioporunit sildenafil, desmethyltalalafil, benseradifenafil, benzothidinalin, N-deethyl-N-methylvardenafil, depiperazinylthiosildenafil, hydroxythiovardenafil, desmethylthiosildenafil, dimethylreddenafil, ketoreddenafil, milrinafil, hydroxyreddenafil, dapoxetine, propoxyphenylsildenafil, N-desmethylsildenafil, vardenafil, N-phenylpropenyltadalafil, propoxyphenylthiomeromolusinafil, N-ethyltadalafil, acetyldinafil, aminosildenafil, thiosildenafil, thioquinafil, thiopiperadenafil, Propoxyphenylthiomorpholinafil, desmethylcarbanafil, sildenafil N-oxide, 2-hydroxyethyl nortadalafil, avanafil, cyclopentafil, N-butyltadalafil, ledenafil carbonate, vardenafil N-oxide, diclofenac, N-t-butoxycarbonyl-N-deethylerythronafil, dithionorcabafenafil, demethylpiperazinylsetafenamic acid, sildenafil dimer impurities, carbadenafil, nafaretic acid, propoxyphenylidenafil, denafil, N-deethylvardenafil, namolindonafil, hydroxypumocelnafil, benzamidonafil, prazolanafil, propoxyphenylhydroxypumodenafil, N-octylnortadalafil, udenafarenafil, talafil methyl chloride, Nitridafil, idenafil, vardenafil piperazinone, 2-hydroxypropyl nortadalafil, devulcanid vardenafil, tadalafil, vardenafil dimer, hydroxychlorodenafil, clodenafil, propoxyphenylthio-sildenafil, decarbed sildenafil, propoxyphenylthio-sildenafil, naerythrodenafil, aminotadalafil, hydroxyvardenafil, thiomeromolesinafil, reddenafil, sildenafil, namolsidenafil, limousinafil, pseudovardenafil, yohimbine hydrochloride, sildenafil impurity 17, sildenafil impurity I, dithio-deethylcarbamazepine, Guidenafil, vardenafil acetyl analogue, sildenafil impurity 12, tadalafil dichlorphenal impurity, sildenafil impurity 14, deethylcarbamazepine, hydroxythiophenadenafil, dithio, dinitro impurity, dinafil impurity, sildenafil impurity, Thiadenafil, diethylamino-pro-tadalafil.

(1) Preparation of standard working solutions

Accurately weighing a proper amount of each compound standard substance in a 10mL volumetric flask, dissolving the compound standard substance with methanol or 50% acetonitrile water solution and diluting the compound standard substance to a scale, and preparing a standard stock solution of each compound; for the insoluble compound, dichloromethane or methanol hydrochloride (1:99) solution is used to dissolve the insoluble compound, and then the volume is determined to the scale.

Standard working solution (1. mu.g/mL): respectively and accurately sucking a proper amount of standard stock solution, placing the stock solution into a 10mL volumetric flask, diluting the stock solution to a scale with a solvent, and shaking up to prepare a standard working solution. Is used newly.

(2) Preparation of test solution of sample to be tested

1) Solid or semi-solid samples

Taking a proper amount of solid samples (protein powder, oyster powder, biscuits, candies, coffee, health-care food containing the same matrix as the matrix, tablets, capsules and other formulations), uniformly mixing, grinding, or taking a proper amount of semisolid samples (jelly), uniformly mixing, precisely weighing 1g (accurate to 0.001g) and placing in a 50mL volumetric flask, adding a proper amount of methanol, ultrasonically extracting for 15min, cooling to room temperature, metering the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging at 4000 r/min for 5min, filtering supernatant through a microporous membrane, taking subsequent filtrate, and properly diluting with acetonitrile water solution (1+1) according to actual concentration for later use.

2) Liquid sample

Shaking sample (beverage, wine) properly, sucking 1mL accurately, placing in 50mL volumetric flask, adding proper amount of methanol, ultrasonic extracting for 15min, cooling to room temperature, diluting with methanol to constant volume, filtering with microporous membrane, collecting filtrate, and diluting with acetonitrile water solution (1+1) according to actual concentration.

3) Lipid matrix (Soft Capsule) sample

Taking a proper amount of a sample (soft capsule), uniformly mixing, precisely weighing 1g (precisely to 0.001g) and placing in a 50mL volumetric flask, adding 5mL of ethyl acetate, shaking to disperse the mixture, adding a proper amount of methanol, carrying out ultrasonic extraction for 15min, cooling to room temperature, fixing the volume with methanol, transferring to a 50mL centrifugal tube, centrifuging for 5min at 4000 r/min, filtering the supernatant through a microporous filter membrane, taking a subsequent filtrate, and properly diluting with an acetonitrile aqueous solution (1+1) according to the actual concentration for later use.

4) Blank matrix extracting solution

Weighing appropriate amount of blank sample, and processing with the same method to obtain blank matrix extractive solution.

5) Blank solution

The sample was not added, and the same method as the sample was used to prepare a blank solution.

(3) Database establishment

And injecting the standard working solution of 161 compounds into an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer for determination, and acquiring the original data map and the total ion flow map of each compound.

And exporting the acquired original data of each compound from masslynx software, importing the original data into unifi data processing software for data processing to obtain the retention time, the parent ion mass spectrogram and the daughter ion mass spectrogram of each compound, and determining the characteristic fragment ions of each compound according to the fragment fracture mechanism and the fracture difficulty of each compound.

Summarizing the species, name, molecular formula, retention time and characteristic fragment ion information of each compound, and establishing a 161 compound screening database, wherein the database is as follows:

(4) qualitative analysis

1) Performing ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry on the sample to be detected processed by the method in the step (2), comparing the obtained result with data in a database, wherein a blank solvent cannot generate an ion peak identical to that of a standard solution, the retention time of a sample chromatographic peak is consistent with that of the standard solution, the allowable deviation is +/-0.1 min, the accurate mass numbers of parent ions and characteristic fragment ions of the sample are consistent with that of the standard solution, the deviations are less than or equal to 0.05Da, the signal-to-noise ratios (S/N) of the parent ions and the fragment ion chromatographic peaks are all over 3:1, the signal-to-noise ratios are calculated by peak-to-peak (PtP), when the full-scan spectrum is recorded, the relative abundance of the characteristic fragment ions in the sample must exceed 10% of the abundance of the characteristic ion spectrum referred by the standard solution, and screening out a compound meeting qualitative conditions, the sample is a suspected positive sample.

2) And for the suspected positive sample, editing an MSMS method according to the retention time and the molecular weight of the compound, collecting data, comparing the obtained sub-ion scanning mass spectrogram of the suspected substance with the sub-ion scanning mass spectrogram of the standard solution, and judging that the corresponding substance to be detected exists in the sample if the allowable deviation of the relative abundance of the main fragment ions in the sample and the relative abundance of the standard solution does not exceed the range specified in the table 2 under the same conditions and similar concentrations.

TABLE 2 allowable deviation of relative abundance of major fragment ions from that of standard solution

Relative ion abundance (%)	＞50	＞20～50	＞10～20	≤10
					Maximum deviation allowed (%)	±20	±25	±30	±50

The liquid chromatographic analysis conditions in the step (3) and the step (4) are as follows: the chromatographic column was a Waters UPLCBEH C18 column (2.1X 100mm, 1.7 μm), the flow rate was 0.4mL/min, the column temperature was 40 ℃ and the sample volume was 1 μ L, acetonitrile was used as the organic phase mobile phase for gradient elution, the ratio was slowly changed from the initial 2% to 95% to ensure that all compounds were detected efficiently.

In the positive ion mode, A is 0.1% formic acid aqueous solution, B is acetonitrile, and the gradient elution procedure is shown in Table 3 (curve 1 is instant change, curve 6 is linear change);

table 3 positive ion mode gradient elution schedule

Gradient time, min	Mobile phase A,%	Mobile phase B,%	Curve line
				0	95	5	Initial
0.50	95	5	6
				9.00	30	70	6
10.00	30	70	6
				12.00	5	95	6
14.00	5	95	6
				17.00	95	5	1

In the case of anion mode collection, A is water and B is acetonitrile, and the gradient elution procedure is shown in Table 4 (curve 1 is instantaneous change, curve 6 is linear change).

Table 4 anion mode gradient elution schedule

The mass spectrometry conditions in the steps (3) and (4) are shown in tables 5 and 6:

TABLE 5 ion Source parameters

Parameters of ion source	Reference value	Reference value
			Type of ion source	ESI+	ESI-
Capillary voltage	3.0KV	2.5KV
			Voltage of taper hole	30	30
Source temperature	150	150
			Desolventizing temperature	400	400
Taper hole back-blowing air flow rate	50L/Hr	50L/Hr
			Desolventizing air flow rate	800L/Hr	800L/Hr

TABLE 6 screening of Mass Spectrometry method parameters

Parameter(s)	Fuction 1	Fuction 2
			Scanning mode	MSE	MSE
Time of scan	0～17min	0～14min
			Scanning range	50～1200Da	50～1200Da
Time of scan	0.2s	0.2s
			Energy of collision	5～40V	15V

Note: leucine enkephalin was corrected online in real time with molecular weight 556.2771 for ESI + mode, 554.2615 for ESI-mode, scan time 0.5s, scan interval 10 s.

The solvent used in the step (1) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 1: 1. The solvent for dilution in the step (2) is a mixed solution of acetonitrile and water, and the volume ratio of the mixed solution is 1: 1.

The invention discloses a rapid screening method for 161 illegally added compounds in health food, which can accurately determine parent ions and daughter ions of each compound by utilizing the characteristic that an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer has a high-sensitivity MSMS function. Collecting the types of the illegal addition compounds possibly existing in the health-care food, and establishing a database for the illegal addition compounds possibly existing by using an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometer to obtain the retention time of the illegal addition compounds under specific conditions and the information of parent ions and daughter ions; the method comprises the steps of carrying out ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry on health food under specific conditions to obtain mass spectrum information of each compound in a sample, and rapidly judging the type of an illegally added compound possibly existing in the sample by combining with an established database, so that a technical means and a method are provided for rapid screening work of the illegally added compound. The method has the advantages of high sensitivity, strong selectivity, strong qualitative capability and simple and rapid operation (the data acquisition process is completed within a few minutes), retains the complete information of the detected object to the maximum extent, and can be used for rapidly screening illegal additives in health food.

Actual sample detection

Samples such as a mulberry leaf and cassia twig tablet, a glauconin, a hafobei capsule, an astragalus root, rhodiola rosea, chromium yeast soft capsules, soybean isoflavone, a dried orange peel, poria cocos and fructus amomi capsule are collected, screening work of illegally adding compounds is carried out by utilizing the established database, and as a result, the compounds in the database are not detected in the samples, and the detection result is consistent with the result of the Hubei province quality inspection institute.

As shown in fig. 1, a blind sample was prepared, i.e. a nafil-like compound was added to the sample, and the compound was detected in the sample and analyzed by mass spectrometry as sildenafil impurity 14, consistent with the added compound.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.