CN111208231A - Method for simultaneously detecting six illegally added compounds in plant beverage - Google Patents

Abstract

The invention provides a method for simultaneously detecting six illegally added compounds in plant beverage, which comprises the following steps: (1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution; (2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution; (3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection. The method optimizes the optimal chromatographic mass spectrum condition and the optimal extraction and purification condition, has good linear relation among tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole, has good sensitivity, accuracy and precision, and is suitable for qualitative and quantitative detection of six components such as tetracycline in the plant beverage.

Description

Method for simultaneously detecting six illegally added compounds in plant beverage

Technical Field

The invention belongs to the field of detection and analysis, and particularly relates to a method for simultaneously detecting six illegally added compounds in a plant beverage.

Background

The current laws and regulations stipulate that no medicine must be added to the food produced and managed. At present, herbal tea prepared and sold on site is taken as common food management, so that medicines cannot be added. In the current market competition and commercial environment that attracts the return rate of purchases, illegal addition of compounds is often prohibited in on-site herbal tea selling operations.

In 2019, the Guangdong province market supervision and management bureau arranges to carry out special plant beverage (herbal tea) risk monitoring, and tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole, erythromycin and the like are respectively detected from the samples of Zhujia, Guangdong and Yuexi. The six compounds are all brought into the prescription drug management in China, but the national standard for detecting the components in the plant beverage (herbal tea) is still absent.

The risk monitoring results show that the detection rate of the six ingredients in the specially collected herbal tea sample is 7.8% (8/103). The herbal tea is used as an old non-first-line medicine, a psychotropic medicine, a central inhibitory medicine and the like, is illegally added into common food herbal tea, has a large potential safety risk, and has urgent need in supervision, and related detection methods are urgently needed to be supplemented.

Disclosure of Invention

The invention aims to provide a method for simultaneously detecting six illegally added compounds in plant beverage.

The technical scheme adopted by the invention is as follows:

a method for simultaneously detecting six illegally added compounds in plant beverage comprises the following steps:

(1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution;

(2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution;

(3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection.

Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer under the following chromatographic conditions:

stationary phase: a chromatographic column using carbon octadecyl silane bonded silica gel as a filler;

mobile phase: phase A: formic acid solution; phase B: formic acid acetonitrile solution;

flow rate: 0.25-0.40 mL/min;

and (3) an elution mode: gradient elution.

Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer under the following chromatographic conditions:

sample introduction amount: 2-10 μ L;

stationary phase: phenomenex Kinetex C18 chromatographic column with filler particle diameter of 2.6um and diameter and length of 2.1mm and 100mm respectively;

mobile phase: phase A: a formic acid solution with a volume fraction of 0.1%; phase B: 0.1 percent of formic acid acetonitrile solution by volume fraction;

the elution gradient was: 0.0min-6.5min, the volume fraction of the mobile phase A is changed from 95% to 5%, and the volume fraction of the mobile phase B is changed from 5% to 95%; 6.5min-7.5min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 7.6min-10.0min, the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%;

flow rate: 0.3 mL/min;

column temperature: 35-40 ℃.

Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer tandem mass spectrometer under the following mass spectrometry conditions:

an ionization mode: carrying out electrospray ionization;

the scanning mode is as follows: a positive ion;

the detection mode is as follows: detecting multiple reactive ions;

ionization voltage: 4.0kV-5.0 kV;

air curtain pressure: 241.3kPa-310.3 kPa;

collision gas pressure: 48kPa to 69 kPa;

ionization temperature: 400-500 ℃;

auxiliary heating gas pressure: 320kPa to 380 kPa.

Preferably, in the detecting step, the detection is performed by a liquid chromatography-mass spectrometer tandem mass spectrometer under the following mass spectrometry conditions:

an ionization mode: carrying out electrospray ionization;

detection mode: a positive ion;

the detection mode is as follows: detecting multiple reactive ions;

ionization voltage: 4.5 kV;

air curtain pressure: 275.6 kPa;

collision gas pressure: 62 kPa;

ionization temperature: 450 deg.C

Auxiliary heating gas pressure: 345 kPa.

Preferably, in the step of preparing the reference solution, each reference is dissolved in methanol to a constant volume, so as to prepare the reference solution with a mass concentration of 0.5-2.0 mg/mL.

Preferably, in the step of preparing the reference solution, each reference is added to the same container to prepare a mixed reference solution.

Preferably, in the step of preparing the sample solution, the microfiltration membrane is an organic membrane having a diameter of 0.22 μm.

The invention has the beneficial effects that:

(1) the method optimizes the optimal chromatographic mass spectrum condition and the optimal extraction and purification condition, has good linear relation among tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole, has good sensitivity, accuracy and precision, and is suitable for qualitative and quantitative detection of 6 components such as tetracycline in plant beverages.

(2) According to the conditions of the chromatogram and the mass spectrum of the method, the detection limit of the method of the liquid sample is 0.25mg/kg, and the quantitative limit of the method is 0.5 mg/kg; the method detection limit of the solid sample is 2.5mg/kg, the method quantitative limit is 5.0mg/kg, and the requirement of illegal addition inspection can be met.

Drawings

FIG. 1 is a reference chart of a multi-reaction detection scan of tetracycline.

FIG. 2 is a reference chart of a multi-reaction detection scan of oxytetracycline.

Figure 3 is a reference map spectrum of a multiple reaction detection scan for codeine.

FIG. 4 is a reference chart of a multi-reaction detection scan of pentoxyverine citrate.

FIG. 5 is a reference chart of a multi-reaction detection scan of metronidazole.

FIG. 6 is a reference chart of a multi-reaction detection scan of erythromycin.

Fig. 7 is a total ion flow graph of a blank sample.

Fig. 8 is a total ion flow graph of negative sample addition mixed standard intermediate solution.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. The following is a description of preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The apparatus and reagents in the following examples are as follows:

1. the instrument comprises the following steps:

LC-MS (liquid chromatography-mass spectrometer): AB Sciex Triple Quad 5500;

a chromatographic column: phenomenex kinex Kinetex C18, 2.6um, 2.1 x 100 mm);

a water purifier: MILLIPORE Mili-Q Advantage A10;

balance: sartorius BS323S, METTLER MS205 DU;

numerical control ultrasonic cleaner: KQ-500DE, ultrasonic instruments Inc. of Kunshan.

2. Reagent testing:

methanol: carrying out chromatographic purification;

acetonitrile: carrying out chromatographic purification;

formic acid: carrying out chromatographic purification;

the source information of each control is shown in table 1:

TABLE 1 Source information of each control

Name (R)	Manufacturer of the product	Batch number	Content (wt.)
				Tetracycline derivatives	NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL	130306-201419	96.9％
Oxytetracycline	NATIONAL INSTITUTE FOR THE CONTROL OF PHARMACEUTICAL AND BIOLOGICAL PRODUCT	130487-200703	88.8％
				Pentoxyverine citrate	NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL	100432-201803	99.8％
Codeine	A ChemTek Inc	S032785	99.8％
				Erythromycin	NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL	130307-201417	92.8％
Metronidazole	NATIONAL INSTITUTES FOR FOOD AND DRUG CONTROL	100191-201808	100％

3. Conditions of the apparatus

3.1 liquid chromatography conditions:

sample introduction amount: 2 mu L of the solution;

mobile phase: phase A: 0.1% formic acid solution (volume fraction); phase B: 0.1% formic acid acetonitrile solution (volume fraction);

flow rate: 0.3 mL/min;

column temperature: 35 ℃;

the gradient elution procedure is shown in table 2:

table 2 gradient elution procedure (volume fraction,%)

3.2 Mass Spectrometry conditions:

an ionization mode: carrying out electrospray ionization;

detection mode: a positive ion;

the detection mode is as follows: detecting multiple reactive ions;

ionization voltage: 4.5 kV;

air curtain pressure: 275.6 kPa;

collision gas pressure: 62 kPa;

ionization temperature: 450 deg.C

Auxiliary heating gas 1 pressure: 345 kPa;

auxiliary heating gas 2 pressure: 345 kPa.

For comparison, the control solutions and the test solutions in the following examples were prepared as follows:

preparation of control solutions:

standard stock solutions: precisely weighing 10mg of each of tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin, respectively placing the 10mg into a10 mL volumetric flask, dissolving the 10mg into methanol (with chromatographic purity), diluting the solution to a scale, and preparing a standard storage liquid with the concentration of 1mg/mL, and storing the standard storage liquid at the temperature of minus 18 ℃ in a dark place.

Mixing standard intermediate solution: precisely sucking 0.25mL of each of the standard stock solutions (1mg/mL) of tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin, placing the standard stock solutions into a same 25mL volumetric flask, diluting the volumetric flask to a scale with methanol (pure chromatogram), shaking up to prepare a mixed standard solution with the concentration of 10 mu g/mL, and storing the mixed standard solution at 4 ℃ in a dark place.

Preparation of a test solution:

vegetable beverage (liquid): weighing 1.00g (accurate to 0.01g) of plant type beverage, placing the plant type beverage in a 50mL volumetric flask, adding about 40mL of 50% methanol solution in volume fraction, carrying out ultrasonic extraction for 30min, cooling to room temperature, fixing the volume to scale with the 50% methanol solution in volume fraction, shaking up, filtering with a 0.22 mu m organic filter membrane, loading on a machine, diluting properly according to actual concentration to a standard curve linear range, and analyzing by a liquid chromatography-mass spectrometer.

Vegetable beverage (solids): weighing 0.10g (accurate to 0.001g) of plant type beverage, placing the plant type beverage in a 50mL volumetric flask, adding about 40mL of 50% methanol aqueous solution in volume fraction, ultrasonically extracting for 30min, cooling to room temperature, fixing the volume to the scale with the 50% methanol aqueous solution in volume fraction, shaking up, filtering with a 0.22 mu m organic filter membrane, loading on a machine, diluting properly according to actual concentration to the linear range of a standard curve, and analyzing by a liquid chromatography-mass spectrometer.

Under the above chromatographic mass spectrometry conditions, the reference values of mass spectrometry parameters of each compound (solution concentration 50ng/mL) are shown in Table 3:

the reference spectrum of the multi-reaction detection scan of each compound is shown in figures 1-6:

TABLE 3 reference values for mass spectral parameters of the respective compounds

Qualitative detection

And detecting the test solution and the mixed standard solution according to the chromatographic mass spectrometry conditions, and if the mass chromatographic peaks of the compounds in the detected test solution are consistent with those of the mixed standard solution, and in the spectrogram of the test solution with the background subtracted, the relative abundance of each qualitative ion is compared with the spectrogram of the mixed standard solution obtained under the same conditions with the similar concentration, and the error does not exceed the specified range shown in the table 4, determining that the corresponding compounds exist in the sample.

TABLE 4 extent of variation in relative abundance of qualitative ions in test solutions

Relative ion abundance	>50	20-50	10-20	<10
					Allowable relative deviation%	±20	±25	±30	±50

Quantitative detection

Mixing standard solutions: accurately sucking a proper amount of the mixed standard intermediate solution, diluting the mixed standard intermediate solution by using a methanol solution with the volume fraction of 50%, shaking up, and preparing into series of standard solutions with the concentrations of 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL in sequence.

Detecting the mixed standard solution according to the chromatographic mass spectrometry conditions, and drawing a standard working curve by taking the peak area as a vertical coordinate and the concentration of the mixed standard solution as a horizontal coordinate to obtain a table 5; the results show that the compounds have a good linear relationship in the corresponding linear range.

TABLE 5 regression equation, correlation coefficient and Linear Range for six Compounds

And detecting the test solution, and quantifying the test solution by using a standard working curve. The quantitative calculation formula is as follows:

wherein, X is the content of the compound component in the test sample, and the unit is as follows: mg/kg;

c is the concentration of the tested compound component solution obtained on the standard working curve, and the unit is as follows: ng/mL;

v is the constant volume of the test solution, and the unit is as follows: mL;

m is the sample weighing of the sample, and the unit is as follows: g.

blank values should be subtracted from the calculation.

Example (b): detection limit and quantitation limit determination

The mixed standard solution was added to the negative sample (vegetable beverage sample not containing the above six compounds, the same applies hereinafter) stepwise, and the concentration at S/N.gtoreq.3 was selected as the detection limit and the concentration at S/N.gtoreq.10 was selected as the quantification limit. The results of the detection limit and the quantitative limit calculation for each compound component are shown in table 6:

TABLE 6 detection limit and quantitative limit results for each compound component

The results show that the detection limit of the liquid sample by the method is 0.25mg/kg, and the quantitative limit of the method is 0.5 mg/kg; the method detection limit of the solid sample is 2.5mg/kg, the method quantitative limit is 5.0mg/kg, and the requirement of illegal addition inspection can be met. The relationship between the method quantitation limit of the six compound components and the therapeutic dose is detailed in table 6, and the results show that the method quantitation limit of the six compound components can reach 1/100 of the therapeutic dose, and the method has sufficient sensitivity.

Detection example 1: matrix effect detection

There are two main methods for evaluating the matrix effect: (1) post column sampling method established by Bonfiglio et al; (2) the post-extraction labeling method proposed by Matuszewski et al. The method (1) is a method capable of rapidly and qualitatively determining whether a certain substrate has a substrate effect, but has the defect that quantitative evaluation cannot be carried out. And (2) comparing the response values of various compounds in the matrix of the standard-added sample and the blank solution of the standard-added sample, not only can qualitative analysis be carried out, but also the influence value of the matrix effect can be quantitatively analyzed (SSE, the calculation formula is as follows), so that the matrix effect of the plant beverage is evaluated by adopting the method (2). The mixed standard solution is diluted into a sample matrix solution standard curve containing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, erythromycin and metronidazole and a blank solution standard curve with the concentration gradient of 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL by using a negative sample matrix solution and a methanol solution with the volume fraction of 50%, a standard curve is established by an external standard method, and the ratio of the slope of the sample matrix solution standard curve to the slope of the blank solvent standard curve is the matrix effect SSE of the sample. The evaluation results of each substrate are shown in table 7:

TABLE 7 evaluation results of substrate Effect of different substrates

Detection example 2: accuracy and precision detection

Accurately sucking a proper amount of the mixed standard intermediate solution, diluting with a methanol solution with the volume fraction of 50%, shaking up, and preparing into a solution with the concentration of 1 ug/mL.

And (3) liquid matrix recovery detection: respectively weighing 3 parts of 1g of negative sample solution, placing the solution in 3 50mL volumetric flasks, precisely weighing 0.25mL and 0.50mL of the 1ug/mL solution and 0.25mL of the 10ug/mL mixed standard intermediate solution, adding the corresponding volumetric flasks, adding 40mL of 50% methanol solution, performing ultrasonic extraction at 40 ℃ for 30min, cooling to room temperature, diluting the 50% methanol solution to a constant volume, passing through a 0.22 mu m organic filter membrane, taking the filtrate, sequentially preparing solutions with three levels of low concentration, medium concentration and high concentration, performing accuracy detection, introducing samples for 6 times at each concentration level under the condition of the chromatographic mass spectrum, and averaging the detection values, wherein the results are shown in Table 8:

table 8 liquid matrix recovery and relative standard deviation for six compound components

As can be seen from Table 8, the average recovery rate of the six compound components in the liquid matrix is 66.1-108.6%, the precision is 0.0-10.7%, and the method has good accuracy and precision.

And (3) solid matrix recovery detection: respectively weighing 3 parts of 0.2g of negative sample solution, placing the negative sample solution in 3 50mL volumetric flasks, respectively precisely weighing 0.25mL and 0.50mL of the 1ug/mL solution and 0.25mL of the 10ug/mL mixed standard intermediate solution, adding the corresponding volumetric flasks, adding 40mL of 50% methanol solution, performing ultrasonic extraction at 40 ℃ for 30min, cooling to room temperature, fixing the volume of the 50% methanol solution to a scale, passing through a 0.22 mu m organic filter membrane, taking filtrate, sequentially preparing solutions with three levels of low concentration, medium concentration and high concentration for accuracy detection, performing sample injection for 6 times at each concentration level under the condition of the chromatographic mass spectrum, and averaging the detection values, wherein the results are shown in Table 9:

TABLE 9 solid matrix recovery and relative standard deviation for six compound components

As can be seen from Table 9, the results show that the average recovery rate of the six components such as tetracycline in the solid matrix is 71.5-119.7%, the precision is 0.4-14.6%, and the method has good accuracy and precision.

Detection example 3: specificity of the assay

Determination of the addition of mixed standard intermediate solutions to the negative samples: taking a negative sample, placing the negative sample in a 50mL volumetric flask, adding 0.1mL of 10 mug/mL mixed standard intermediate solution, dissolving the negative sample by 40mL of 50% methanol solution in volume fraction, carrying out ultrasonic treatment for 30min, cooling to room temperature, fixing the volume to scale by 50% methanol solution in volume fraction, shaking up, filtering by a 0.22 mu m filter membrane, taking filtrate, and analyzing by a high performance liquid chromatography-tandem mass spectrometer.

Blank negative sample determination: the negative samples were run as described above except that no mixed standard intermediate solution was added.

The total ion flow graph of the negative sample added with the mixed standard intermediate solution and the total ion flow graph of the blank sample are shown in figures 7-8, and the graphs show that the total ion flow graph of the negative sample added with the mixed standard intermediate solution has no interference and the method has good specificity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (8)

1. A method for simultaneously detecting six illegally added compounds in plant beverage comprises the following steps:

(1) preparation of a test solution: extracting the sample with methanol solution, and filtering with microporous membrane to obtain sample solution;

(2) preparation of control solutions: precisely weighing tetracycline, oxytetracycline, codeine, pentoxyverine citrate, metronidazole and erythromycin reference substances, and dissolving with organic solvent to a constant volume to obtain a reference substance solution;

(3) and (3) detection: and respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer for detection.

2. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the detection step, the detection of the liquid chromatography-mass spectrometer is carried out according to the following chromatographic conditions:

stationary phase: a chromatographic column using carbon octadecyl silane bonded silica gel as a filler;

mobile phase: phase A: formic acid solution; phase B: formic acid acetonitrile solution;

flow rate: 0.25-0.40 mL/min;

and (3) an elution mode: gradient elution.

3. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 2, wherein: in the detection step, the detection of the liquid chromatography-mass spectrometer is carried out according to the following chromatographic conditions:

sample introduction amount: 2-10 μ L;

stationary phase: phenomenex KinetexC18 chromatographic column with filler particle diameter of 2.6um and diameter and length of 2.1mm and 100mm respectively;

mobile phase: phase A: a formic acid solution with a volume fraction of 0.1%; phase B: 0.1 percent of formic acid acetonitrile solution by volume fraction;

the elution gradient was: 0.0min-6.5min, the volume fraction of the mobile phase A is changed from 95% to 5%, and the volume fraction of the mobile phase B is changed from 5% to 95%; 6.5min-7.5min, the volume fraction of the mobile phase A is 5%, and the volume fraction of the mobile phase B is 95%; 7.6min-10.0min, the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%;

flow rate: 0.3 mL/min;

column temperature: 35-40 ℃.

4. The method for simultaneously detecting six illegally added compounds in a plant beverage according to any one of claims 1 to 3, wherein: in the detection step, the detection of the liquid chromatogram-mass spectrometer is carried out according to the following mass spectrum conditions:

an ionization mode: carrying out electrospray ionization;

the scanning mode is as follows: a positive ion;

the detection mode is as follows: detecting multiple reactive ions;

ionization voltage: 4.0kV-5.0 kV;

air curtain pressure: 241.3kPa-310.3 kPa;

collision gas pressure: 48kPa to 69 kPa;

ionization temperature: 400-500 ℃;

auxiliary heating gas pressure: 320kPa to 380 kPa.

5. The method for simultaneously detecting six illegally added compounds in plant beverage according to claim 4, wherein: in the detection step, the detection of the liquid chromatogram-mass spectrometer is carried out according to the following mass spectrum conditions:

an ionization mode: carrying out electrospray ionization;

detection mode: a positive ion;

the detection mode is as follows: detecting multiple reactive ions;

ionization voltage: 4.5 kV;

air curtain pressure: 275.6 kPa;

collision gas pressure: 62 kPa;

ionization temperature: 450 deg.C

Auxiliary heating gas pressure: 345 kPa.

6. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the preparation step of the reference substance solution, each reference substance is dissolved by methanol to a constant volume, so as to prepare the reference substance solution with the mass concentration of 0.5-2.0 mg/mL.

7. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 6, wherein the method comprises the following steps: in the preparation step of the reference substance solution, each reference substance is fixed to the same container to prepare a mixed reference substance solution.

8. The method for simultaneously detecting six illegally added compounds in a plant beverage according to claim 1, wherein: in the step of preparing the test solution, the microfiltration membrane is an organic filtration membrane with a diameter of 0.22 μm.

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