CN110940755A - Method for detecting 4 tadalafil substances in food - Google Patents
Method for detecting 4 tadalafil substances in food Download PDFInfo
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- CN110940755A CN110940755A CN201911311038.XA CN201911311038A CN110940755A CN 110940755 A CN110940755 A CN 110940755A CN 201911311038 A CN201911311038 A CN 201911311038A CN 110940755 A CN110940755 A CN 110940755A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
Abstract
The invention provides a method for detecting 4 tadalafil substances in food, and belongs to the field of substance detection. The invention realizes the simultaneous detection of 4 tadalafil substances by accurately limiting the conditions of the high performance liquid chromatography, has good repeatability, good sample recovery rate and good stability in 100 hours, and the tadalafil substances comprise demethyltadalafil, N-3 hydroxypropyl demethyl tadalafil, 2-hydroxypropyl demethyl tadalafil and 2-hydroxyethyl demethyl tadalafil.
Description
Technical Field
The invention relates to the technical field of substance detection, in particular to a method for detecting 4 kinds of tadalafil substances in food, wherein the tadalafil substances comprise demethyltadalafil, N-3 hydroxypropyl demethyltadalafil, 2-hydroxypropyl demethyltadalafil and 2-hydroxyethyl demethyltadalafil.
Background
Some chemical drugs, especially PDE5 (phosphodiesterase type 5) inhibitors and weight-reducing drugs are illegally added into functional foods or health-care foods, and the illegally added drugs gradually develop towards novel compounds along with the continuous expansion of national supplementary inspection methods. Tadalafil is a selective, reversible inhibitor of cyclic guanosine monophosphate (cGMP) specific PDE5, and when sexual stimulation results in local nitric oxide release, PDE5 is inhibited by tadalafil, resulting in elevated levels of cGMP within the corpora cavernosa, which results in smooth muscle relaxation, blood flow into the penile tissue, and erection. The prior art discloses a detection method of a single tadalafil substance, but no relevant report about simultaneous detection of four novel tadalafil substances (demethyltadalafil, N-3 hydroxypropyl demethyltadalafil, 2-hydroxypropyl demethyltadalafil and 2-hydroxyethyl demethyltadalafil) is available, and addition is found in China.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting 4 tadalafil substances in food. The detection method provided by the invention can realize simultaneous detection of 4 tadalafil substances.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for detecting 4 tadalafil substances in food, which comprises the following steps:
mixing the food to be detected with acetonitrile to obtain a liquid to be detected;
performing high performance liquid chromatography detection on the liquid to be detected to obtain a liquid chromatogram; the detection conditions of the high performance liquid chromatography comprise:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B;
comparing the obtained liquid chromatogram with a preset standard spectrogram to determine the type of the tadalafil substances in the food to be detected;
the tadalafil compounds include demethyltadalafil, N-3 hydroxypropyl-demethyltadalafil, 2-hydroxypropyl-demethyltadalafil and 2-hydroxyethyl-demethyltadalafil.
Preferably, when the food to be detected is a solid sample, grinding the solid sample, weighing 0.1-1g of the ground solid sample, mixing with 3-40mL of acetonitrile, ultrasonically extracting for 10-30min, cooling to room temperature, metering volume to a volumetric flask of 5-50mL with acetonitrile, shaking up, filtering with a 0.45 mu m organic filter membrane, taking a subsequent filtrate, and properly diluting with acetonitrile to a linear range according to actual concentration to obtain a liquid to be detected.
Preferably, when the food to be detected is a liquid sample, placing 5mL of the liquid sample into a 25mL volumetric flask, adding acetonitrile to a constant volume, shaking up, filtering with a 0.45 μm organic filter membrane, taking a subsequent filtrate, and properly diluting with acetonitrile according to actual concentration to a linear range to obtain a liquid to be detected.
Preferably, when the food to be detected is a liquid sample, placing 5mL of the liquid sample into a 25mL volumetric flask, adding acetonitrile to a constant volume, shaking up, filtering with a 0.45 μm organic filter membrane, taking a subsequent filtrate, and properly diluting with acetonitrile according to actual concentration to a linear range to obtain a liquid to be detected.
Preferably, when the food to be detected is a low-protein low-fat sample, if the food to be detected is a solid preparation, after grinding, precisely weighing the dose once, putting the dose into a 50mL volumetric flask, adding 40mL of acetonitrile, carrying out ultrasonic treatment for 15 minutes, putting the dose to room temperature, diluting the dose to a scale with the acetonitrile, shaking the dose evenly, and filtering the dose with a 0.45-micrometer organic filter membrane for later use; and if the preparation is a liquid preparation, shaking up, precisely measuring the dosage once, putting the preparation in a 50mL volumetric flask, adding acetonitrile to 40mL, shaking for 3 minutes, adding acetonitrile to dilute to a scale, shaking up, and filtering with a 0.45-micrometer organic filter membrane for later use.
Preferably, when the protein content in the food to be detected is more than or equal to 3%, precisely weighing a dose of solid sample or sucking 2-5 mL of liquid sample, putting the sample in a 100mL triangular flask, adding 25mL of water, carrying out ultrasonic treatment for 15 minutes, putting the sample to room temperature, adding 5mL of zinc acetate solution and 5mL of potassium ferrocyanide solution while shaking, adding water to scale, uniformly mixing, standing for 30min, filtering the mixture in a 50mL volumetric flask by using dry filter paper, diluting the mixture to scale by using acetonitrile, shaking up, and filtering the mixture by using a 0.45 mu m organic filter membrane for later use, wherein the zinc acetate solution is prepared by the following steps: weighing 21.9g of zinc acetate, adding 3mL of glacial acetic acid, adding water for dissolving, and diluting to 100 mL; the potassium ferrocyanide solution is prepared by the following steps: 10.6g of potassium ferrocyanide are weighed, dissolved in water and diluted to 100 mL.
Preferably, when the fat content in the food to be detected is more than or equal to 2 percent: accurately weighing a solid sample with one dose, leaching with 20mL of diethyl ether or petroleum ether for 3 times, leaching while shaking, removing an ether layer, accurately adding 50mL of acetonitrile into the solid part, performing ultrasonic treatment for 15 minutes, cooling to room temperature, supplementing with acetonitrile, shaking uniformly, and filtering with a 0.45-micrometer organic filter membrane for later use; precisely absorbing 5-10 mL of liquid sample, adding 20mL of diethyl ether or petroleum ether, shaking, standing for layering, diluting a water layer in a 50mL volumetric flask with acetonitrile to a scale, shaking up, filtering with a 0.45-micrometer organic filter membrane for later use
Preferably, the method further comprises the following steps after the liquid chromatogram is obtained: and calculating the content of the 4 tadalafil substances in the food to be detected by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 4 tadalafil substances as an independent variable and a peak area as a dependent variable.
Preferably, the standard equation is obtained by a method comprising the steps of:
preparation of standard stock solution
Standard stock solutions of demethyltadalafil: accurately weighing 0.01291g of demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile for dissolving, diluting and fixing the volume to a scale, and shaking up;
2-hydroxyethyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxyethyl demethyl tadalafil reference substance, placing the reference substance in a 10mL volumetric flask, adding acetonitrile to dissolve and dilute the reference substance to a constant volume to scale, and shaking up;
n-3 hydroxypropyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of N-3 hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile to dissolve, diluting and fixing the volume to a scale, and shaking up;
2-hydroxypropyl-demethyl-tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile for dissolving, diluting to constant volume to scale, and shaking up;
compounding with mixed standard intermediate liquid
Respectively and precisely measuring 1.2mL of 2-hydroxyethyl demethyl tadalafil, N-3 hydroxypropyl demethyl tadalafil and 2-hydroxypropyl demethyl tadalafil standard stock solutions in 10mL volumetric flasks, adding acetonitrile for diluting to a constant volume to scale, and shaking up to obtain a mixed standard intermediate solution 1;
respectively and precisely measuring 3.125mL of each of 2-hydroxyethyl demethyl tadalafil, demethyl tadalafil and N-3 hydroxypropyl demethyl tadalafil standard stock solutions and 6.25mL of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 25mL volumetric flask, adding acetonitrile to dilute to a constant volume to a scale, and shaking up to obtain a mixed standard intermediate solution 2;
respectively and precisely measuring 500 mu L of 2-hydroxyethyl demethyl tadalafil standard stock solution and N-3-hydroxypropyl demethyl tadalafil standard stock solution, 400 mu L of demethyl tadalafil standard stock solution and 200 mu L of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 2mL volumetric flask, adding acetonitrile for diluting to a constant volume to reach a scale, and shaking up to obtain a mixed standard intermediate solution 3;
standard Curve derivation
Precisely measuring 11.0mL of mixed standard intermediate solution, adding the blank matrix solution, fixing the volume to a 2mL volumetric flask, shaking up, and recording as 'standard yeast-1'; precisely measuring 1.0mL of 'standard yeast-1', adding a blank matrix solution to a 2mL volumetric flask, and shaking up to be marked as 'standard yeast-2'; diluting a standard curve according to the method in sequence in equal proportion to obtain 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6', respectively carrying out high performance liquid chromatography detection on the 'standard curve-1', 'standard curve-2', 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6' to obtain liquid chromatogram maps 1-6, and obtaining a standard equation according to the liquid chromatogram maps 1-6, wherein the high performance liquid chromatography detection conditions are as follows:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B.
The invention provides a method for detecting 4 tadalafil substances in food, which comprises the following steps: mixing the food to be detected with acetonitrile to obtain a liquid to be detected; performing high performance liquid chromatography detection on the liquid to be detected to obtain a liquid chromatogram; the detection conditions of the high performance liquid chromatography comprise: a chromatographic column: c18; column temperature: 35 ℃; flow rate: 1.0 mL/min; sample introduction amount: 10 mu L of the solution; detection wavelength: 230 nm; mobile phase: a: acetonitrile, B: 0.1% aqueous trifluoroacetic acid, gradient elution procedure: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B; comparing the obtained liquid chromatogram with a preset standard spectrogram to determine the type of the tadalafil substances in the food to be detected; the tadalafil compounds include demethyltadalafil, N-3 hydroxypropyl-demethyltadalafil, 2-hydroxypropyl-demethyltadalafil and 2-hydroxyethyl-demethyltadalafil. The invention realizes the simultaneous detection of 4 tadalafil substances, and has good repeatability, good sample recovery rate and good stability within 100 hours.
Drawings
FIG. 1 is a liquid chromatogram of soft capsule blank matrix and standard, wherein the chromatogram peak: 1 is 2-hydroxypropyl-desmethyl-tadalafil, 2 is desmethyl-tadalafil, 3 is 2-hydroxyethyl-desmethyl-tadalafil, 4 is N-3 hydroxypropyl-desmethyl-tadalafil;
FIG. 2 is a secondary mass spectrum of demethyltadalafil;
FIG. 3 is a three-level mass spectrum of demethyltadalafil;
FIG. 4 is a secondary mass spectrum of 2-hydroxyethyl-demethyl-tadalafil;
FIG. 5 is a non-tertiary mass spectrum of 2-hydroxyethyl demethyltadalal;
FIG. 6 is a secondary mass spectrum of N-3 hydroxypropyl desmethyl tadalafil;
FIG. 7 is a tertiary mass spectrum of N-3 hydroxypropyl desmethyl tadalafil;
FIG. 8 is a secondary mass spectrum of 2-hydroxypropyl-desmethyl-tadalafil;
FIG. 9 is a tertiary mass spectrum of 2-hydroxypropyl-desmethyl-tadalafil.
Detailed Description
The invention provides a method for detecting 4 tadalafil substances in food, which comprises the following steps:
mixing the food to be detected with acetonitrile to obtain a liquid to be detected;
performing high performance liquid chromatography detection on the liquid to be detected to obtain a liquid chromatogram; the detection conditions of the high performance liquid chromatography comprise:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B;
comparing the obtained liquid chromatogram with a preset standard spectrogram to determine the type of the tadalafil substances in the food to be detected;
the tadalafil compounds include demethyltadalafil, N-3 hydroxypropyl-demethyltadalafil, 2-hydroxypropyl-demethyltadalafil and 2-hydroxyethyl-demethyltadalafil.
The invention mixes the food to be detected with acetonitrile to obtain the liquid to be detected.
In the invention, when the food to be detected is preferably a solid sample, the solid sample is ground and then 0.1-1g of the ground solid sample is weighed to be mixed with 3-40mL of acetonitrile, ultrasonic extraction is carried out for 10-30min, the mixture is cooled to room temperature, acetonitrile is used for fixing the volume to a volumetric flask of 5-50mL, shaking is carried out, after filtration is carried out by using an organic filter membrane of 0.45 mu m, a subsequent filtrate is taken, and the acetonitrile is used for properly diluting the filtrate to a linear range according to the actual concentration to obtain the liquid to be detected.
In the invention, when the food to be detected is preferably a liquid sample, 5mL of the liquid sample is placed in a 25mL volumetric flask, acetonitrile is added for constant volume, the liquid sample is shaken up, after being filtered by a 0.45-micrometer organic filter membrane, a filtrate is taken, and the liquid sample is properly diluted to be in a linear range by acetonitrile according to the actual concentration to obtain a liquid to be detected.
In the specific embodiment of the invention, when the food to be tested is a simple matrix sample (low protein and low fat), if the food to be tested is a solid preparation, the food to be tested is ground, and the solid preparation is precisely weighed, is placed in a 50mL volumetric flask, is added with 40mL of acetonitrile, is subjected to ultrasonic treatment for 15 minutes, is placed to room temperature, is diluted to a scale by the acetonitrile, is shaken up, and is filtered by a microporous filter membrane (0.45 mu m) for standby; if the preparation is a liquid preparation, shaking up, precisely measuring the dosage once, placing in a 50mL volumetric flask, adding acetonitrile to 40mL, shaking for 3 minutes, adding acetonitrile to dilute to the scale, shaking up, and filtering with a microporous filter membrane (0.45 μm) for later use.
In the specific embodiment of the invention, when the food to be tested is a complex matrix sample (a sample with high protein content (protein content is more than or equal to 3%)): precisely weighing a solid sample (or sucking a liquid sample of 2-5 mL) with one dose, placing the solid sample into a 100mL triangular flask, adding 25mL water, carrying out ultrasonic treatment for 15 minutes, placing the mixture to room temperature, slowly adding 5mL zinc acetate solution and 5mL potassium ferrocyanide solution while shaking, adding water to the scale, and uniformly mixing. Standing for 30min, filtering with dry filter paper into a 50mL volumetric flask, diluting to the mark with acetonitrile, shaking up, and filtering with microporous membrane (0.45 μm) for use. The zinc acetate solution is preferably prepared by the following steps: weighing 21.9g of zinc acetate, adding 3mL of glacial acetic acid, adding water for dissolving, and diluting to 100 mL; potassium ferrocyanide solution: 10.6g of potassium ferrocyanide are weighed, dissolved in water and diluted to 100 mL.
In the specific embodiment of the invention, when the food to be tested is a sample with high fat content (fat content is more than or equal to 2%): accurately weighing a solid sample with one dose, leaching with 20mL of diethyl ether or petroleum ether for 3 times, shaking while leaching to remove an ether layer, accurately adding 50mL of acetonitrile into the solid part, performing ultrasonic treatment for 15 minutes, cooling to room temperature, supplementing with acetonitrile, shaking up, and filtering with a microporous filter membrane (0.45 μm) for later use; precisely sucking 5-10 mL of liquid sample, adding 20mL of diethyl ether or petroleum ether, shaking, standing for layering, diluting a water layer in a 50mL volumetric flask with acetonitrile to a scale, shaking up, and filtering with a microporous filter membrane (0.45 mu m) for later use.
After the liquid to be detected is obtained, carrying out high performance liquid chromatography detection on the liquid to be detected to obtain a liquid chromatogram; the detection conditions of the high performance liquid chromatography comprise:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B.
In the present invention, obtaining the liquid chromatogram preferably further comprises the steps of: and calculating the content of the 4 tadalafil substances in the food to be detected by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 4 tadalafil substances as an independent variable and a peak area as a dependent variable.
In the present invention, the standard equation is preferably obtained by a method comprising the steps of:
preparation of standard stock solution
Standard stock solutions of demethyltadalafil: accurately weighing 0.01291g of demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile for dissolving, diluting and fixing the volume to a scale, and shaking up;
2-hydroxyethyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxyethyl demethyl tadalafil reference substance, placing the reference substance in a 10mL volumetric flask, adding acetonitrile to dissolve and dilute the reference substance to a constant volume to scale, and shaking up;
n-3 hydroxypropyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of N-3 hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile to dissolve, diluting and fixing the volume to a scale, and shaking up;
2-hydroxypropyl-demethyl-tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile for dissolving, diluting to constant volume to scale, and shaking up;
compounding with mixed standard intermediate liquid
Respectively and precisely measuring 1.2mL of 2-hydroxyethyl demethyl tadalafil, N-3 hydroxypropyl demethyl tadalafil and 2-hydroxypropyl demethyl tadalafil standard stock solutions in 10mL volumetric flasks, adding acetonitrile for diluting to a constant volume to scale, and shaking up to obtain a mixed standard intermediate solution 1;
respectively and precisely measuring 3.125mL of each of 2-hydroxyethyl demethyl tadalafil, demethyl tadalafil and N-3 hydroxypropyl demethyl tadalafil standard stock solutions and 6.25mL of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 25mL volumetric flask, adding acetonitrile to dilute to a constant volume to a scale, and shaking up to obtain a mixed standard intermediate solution 2;
respectively and precisely measuring 500 mu L of 2-hydroxyethyl demethyl tadalafil standard stock solution and N-3-hydroxypropyl demethyl tadalafil standard stock solution, 400 mu L of demethyl tadalafil standard stock solution and 200 mu L of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 2mL volumetric flask, adding acetonitrile for diluting to a constant volume to reach a scale, and shaking up to obtain a mixed standard intermediate solution 3;
standard Curve derivation
Precisely measuring 11.0mL of mixed standard intermediate solution, adding the blank matrix solution, fixing the volume to a 2mL volumetric flask, shaking up, and recording as 'standard yeast-1'; precisely measuring 1.0mL of 'standard yeast-1', adding a blank matrix solution to a 2mL volumetric flask, and shaking up to be marked as 'standard yeast-2'; diluting a standard curve according to the method in sequence in equal proportion to obtain 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6', respectively carrying out high performance liquid chromatography detection on the 'standard curve-1', 'standard curve-2', 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6' to obtain liquid chromatogram maps 1-6, and obtaining a standard equation according to the liquid chromatogram maps 1-6, wherein the high performance liquid chromatography detection conditions are as follows:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B.
In a specific embodiment of the present invention, it is preferable that the method further comprises a step of mass spectrometry, and the chromatographic conditions of mass spectrometry include: mobile phase: a: 0.01-0.03mol/L ammonium acetate solution containing 0.05-0.2% acetic acid, B: acetonitrile C: methanol, isocratic elution, and the mobile phase ratio is: c is 35-50:20-30: 20-40; flow rate: 0.1-0.3 mL/min; column temperature: 35 ℃; sample introduction amount: 10 μ L. Mass spectrum parameters: an electrospray ionization source (ESI +), and the spraying voltage is 3-4.58 KV; the temperature of the capillary tube is 280-450 ℃; the flow rate of the auxiliary gas is 10-20 arb, and the flow rate of the sheath gas is 20-60 arb; and (3) detecting positive ions by adopting primary full scanning, secondary scanning and tertiary scanning, wherein the mass range is 50-600.
In the present invention, the mass spectrometric confirmation preferably includes a primary mass spectrum, a secondary mass spectrum and a tertiary mass spectrum.
In order to further illustrate the present invention, the following description will be made in detail with reference to examples to detect 4 tadalafil substances in food products provided by the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. The product scheme is as follows:
1.1 Standard substance
Demethyltadalafil, purity: 98%, manufacturer: aladdin, lot number: f1601050
N-3 hydroxypropyl desmethyl tadalafil, purity: 99.4%, manufacturer: TLC, batch No.: 3000-015A4
2-hydroxypropyl-desmethyl-tadalafil, purity: 98.7%, manufacturer: TLC, batch No.: 1516-
2-hydroxyethyl-demethyltadalafil, purity: 100%, manufacturer: TLC, batch No.: 1821-044A2
Acetonitrile, chromatographic purity, manufacturer: dikma, batch number: r141271
Acetonitrile, analytical grade, manufacturer: national chemical reagents ltd, batch No.: 20150105
Trifluoroacetic acid, chromatographic purity, manufacturer: national chemical reagents ltd, batch No.: 20180302
Methanol, pure chromatography, manufacturer: dikma, batch number: r141271
The water is secondary pure water
1.2 sample information
Wine preparation: xx deer and tortoise liquor, batch No. 20171101, xx wine industry Co., Ltd
Coffee: instant coffee, batch 20180803, xx Limited
And (3) tablet preparation: xx maca tablets, lot 2017010, xx Biotech Limited
Beverage preparation: xx functional beverage, batch No. 20180602, xx food group
Hard capsules: xxx capsules, batch 20180522, xx health products Co
Soft capsules: xx Soft capsules, batch HDO77, xx Biotech Ltd
Oral liquid: xx oral liquid, batch No. 20180401, xx science and technology development Co., Ltd
1.3 instrumentation
A high performance liquid chromatograph of Daian U3000,
an aohaos DV215CD electronic balance,
a Mettler MS-204S electronic balance,
a Jining Tianhua ultrasonic cleaner,
2 sample preparation
2.1 solid State test specimens
Taking a proper amount of sample, grinding and mixing uniformly, weighing 0.1-1g of sample (accurate to 0.001g) in a 5-50mL volumetric flask, adding 3-40mL of acetonitrile, carrying out ultrasonic extraction for 10-30min, cooling to room temperature, fixing the volume with acetonitrile, shaking uniformly, filtering with a filter membrane (0.45 mu m, organic phase type), taking a subsequent filtrate, and diluting properly with acetonitrile according to actual concentration to a linear range for later use.
2.2 liquid samples
Taking a proper amount, shaking uniformly, precisely sucking 5mL into a 25mL volumetric flask, adding acetonitrile to a constant volume, shaking uniformly, filtering by using a filter membrane (0.45 mu m, organic phase type), taking a subsequent filtrate, and diluting properly with acetonitrile according to actual concentration to a linear range for later use.
The method comprises the following specific steps:
when the food to be detected is a simple matrix sample (low protein and low fat), if the food to be detected is a solid preparation, grinding, precisely weighing the dose once, putting the dose into a 50mL volumetric flask, adding 40mL of acetonitrile, carrying out ultrasonic treatment for 15 minutes, putting the volumetric flask to room temperature, diluting the dose to a scale with the acetonitrile, shaking up the dose, and filtering the dose with a microporous filter membrane (0.45 mu m) for later use; if the preparation is a liquid preparation, shaking up, precisely measuring the dosage once, placing in a 50mL volumetric flask, adding acetonitrile to 40mL, shaking for 3 minutes, adding acetonitrile to dilute to the scale, shaking up, and filtering with a microporous filter membrane (0.45 μm) for later use.
When the food to be detected is a complex matrix sample (a sample with high protein content (the protein content is more than or equal to 3%)): precisely weighing a solid sample (or sucking a liquid sample of 2-5 mL) with one dose, placing the solid sample into a 100mL triangular flask, adding 25mL water, carrying out ultrasonic treatment for 15 minutes, placing the mixture to room temperature, slowly adding 5mL zinc acetate solution and 5mL potassium ferrocyanide solution while shaking, adding water to the scale, and uniformly mixing. Standing for 30min, filtering with dry filter paper into a 50mL volumetric flask, diluting to the mark with acetonitrile, shaking up, and filtering with microporous membrane (0.45 μm) for use. The zinc acetate solution is preferably prepared by the following steps: weighing 21.9g of zinc acetate, adding 3mL of glacial acetic acid, adding water for dissolving, and diluting to 100 mL; potassium ferrocyanide solution: 10.6g of potassium ferrocyanide are weighed, dissolved in water and diluted to 100 mL.
When the food to be detected is a sample with high fat content (the fat content is more than or equal to 2%): accurately weighing a solid sample with one dose, leaching with 20mL of diethyl ether or petroleum ether for 3 times, shaking while leaching to remove an ether layer, accurately adding 50mL of acetonitrile into the solid part, performing ultrasonic treatment for 15 minutes, cooling to room temperature, supplementing with acetonitrile, shaking up, and filtering with a microporous filter membrane (0.45 μm) for later use; precisely sucking 5-10 mL of liquid sample, adding 20mL of diethyl ether or petroleum ether, shaking, standing for layering, diluting a water layer in a 50mL volumetric flask with acetonitrile to a scale, shaking up, and filtering with a microporous filter membrane (0.45 mu m) for later use.
Blank matrix extracting solution
Weighing or measuring appropriate amount of blank sample under the item of "sample information", and preparing according to the items of "2.1" and "2.2" to obtain blank matrix extract.
3 preparation of Standard solution
3.1 preparation of Standard stock solution
3.1.1 standard stock solutions of demethyltadalafil: 0.01291g of demethyl tadalafil reference substance is precisely weighed, placed in a 10mL volumetric flask, dissolved by adding acetonitrile, diluted to a constant volume to a scale, and shaken up. (concentration 1.2652 mg/mL.)
3.1.22-hydroxyethyl-demethyl-tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxyethyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile to dissolve and dilute to a constant volume to scale, and shaking up. (concentration 1.0000 mg/mL.)
3.1.3N-3 hydroxypropyl-demethyl tadalafil standard stock solutions: accurately weighing 0.0100g of N-3 hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile to dissolve and dilute to a constant volume to scale, and shaking up. (concentration 0.9940 mg/mL.)
3.1.42-hydroxypropyl-demethyl tadalafil standard stock solution: accurately weighing 0.0999g of 2-hydroxypropyl demethyl tadalafil reference substance, placing the reference substance in a 10mL volumetric flask, adding acetonitrile to dissolve, diluting and fixing the volume to a scale, and shaking up. (concentration 0.9860 mg/mL.)
3.2 preparation of Mixed Standard intermediate solution
Precisely measuring 1.2mL of standard stock solutions of 2-hydroxyethyl-demethyl-tadalafil, N-3-hydroxypropyl-demethyl-tadalafil and hydroxypropyl-demethyl-tadalafil under the item of 3.1 in 10mL volumetric flasks respectively, adding acetonitrile for dilution to a constant volume to reach a scale, and shaking up to obtain a mixed standard intermediate solution (1).
Respectively and precisely measuring 3.125mL of 2-hydroxyethyl-demethyl-tadalafil, demethyl-tadalafil and N-3 hydroxypropyl-demethyl-tadalafil standard stock solutions under the item of '3.1' and 6.25mL of hydroxypropyl-demethyl-tadalafil standard stock solutions in a 25mL volumetric flask, adding acetonitrile for diluting to a constant volume to reach a scale, and shaking up to obtain a mixed standard intermediate solution (2).
Respectively and precisely measuring 500ul of 2-hydroxyethyl demethyl tadalafil standard stock solution and N-3 hydroxypropyl demethyl tadalafil standard stock solution under the item of '3.1', 400ul of demethyl tadalafil standard stock solution and 200ul of hydroxypropyl demethyl tadalafil standard stock solution in a 2mL volumetric flask, adding acetonitrile for diluting to a constant volume to scale, and shaking up to obtain a mixed standard intermediate solution (3).
3.3 Standard Curve preparation
Precisely measuring 1.0mL of the mixed standard intermediate solution (1) under the item of '3.2', adding the blank matrix solution to a volumetric flask with a constant volume of 2mL, shaking up, and recording as 'mark yeast-1'; precisely measuring 1.0mL of 'standard yeast-1', adding a blank matrix solution to a 2mL volumetric flask, and shaking up to be marked as 'standard yeast-2'; diluting the standard curve in equal proportion according to the method to obtain the standard curve-3, the standard curve-4, the standard curve-5 and the standard curve-6. The concentrations of the components are shown in Table 1.
TABLE 1 component concentration Table (Unit: ug/mL)
4. High performance liquid chromatography conditions
A chromatographic column: c18 (4.6X 250mm,5 μm)
Column temperature: 35 deg.C
Flow rate: 1.0mL/min
Sample introduction amount: 10 μ L
Detection wavelength: 230nm
Mobile phase: a: acetonitrile, B: 0.1% aqueous trifluoroacetic acid, gradient elution procedure see table 2:
TABLE 2 mobile phase and gradient elution conditions
5. Methodological validation results
Wine, beverage, tablets, capsules, soft capsules, coffee and oral liquid are selected as verification objects to be respectively examined for specificity, linearity, quantitative limit, detection limit, instrument precision, repeatability, stability and recovery rate.
5.1 limit of quantitation and detection
0.25mL of the mixed standard intermediate (2) under "3.2" was added to 1.0g of the blank medium, and prepared under "1". The detection signals of the 4 compounds were analyzed, and the ratio of the signal to the noise (S/N) was used as an index for examining the quantitative limit and the detection limit.
The signal concentration of S/N10 is the limit of quantitation, and the signal concentration of S/N3 is the limit of detection. When the sample amount was 1.0g and the constant volume was 25mL, the detection limit and the quantification limit of desmethyltadalafil, N-3 hydroxypropyl desmethyltadalafil, hydroxypropyl desmethyltadalafil and 2-hydroxyethyl desmethyltadalafil were calculated. The results are shown in tables 3 and 4.
TABLE 3 detection limit, quantitation limit (μ g/g) of liquid sample methods
TABLE 4 detection limit, quantitation limit (μ g/g) of solid state sample methods
5.2 precision of the Instrument
The standard intermediate solution (3) was mixed under 2.2, and sample introduction was performed for 6 times with a sample introduction amount of 1. mu.L, and the results of the instrument precision investigation were shown in Table 5. The results show that: the precision of the instrument is good.
TABLE 5 results of instrumental precision investigation
5.3 specificity
Injecting the blank matrix extracting solution under the item '2.3' and the quantitative limiting solution under the item '5.1' into a high performance liquid chromatograph, and inspecting whether substances which interfere with the measured components exist in the matrix.
The results show that the blank matrix does not contain substances interfering with desmethyltadalafil, 2-hydroxyethyl desmethyltadalafil, N-3 hydroxypropyl desmethyltadalafil and 2-hydroxypropyl desmethyltadalafil, and the chromatogram is shown in appendix A.
5.4 Standard Curve
Injecting a standard curve solution prepared under the item '3.3' and a quantitative limiting solution (as the lowest solubility point of the standard curve) under the item '5.1' into a high performance liquid chromatograph, measuring corresponding peak areas, drawing the standard curve by taking the concentration (X) as a horizontal coordinate and taking a peak area (Y) as a vertical coordinate, and listing a linear equation and a linear range of the standard curve. The results are shown in tables 6 to 12. The results show that: the linear relationship is good.
TABLE 6 Linear relationship, correlation coefficient and Linear Range (wine base)
TABLE 7 Linear relationship, correlation coefficient and Linear Range (Lehu beverage base)
TABLE 8 Linear relationship, correlation coefficient and Linear Range (Capsule base)
TABLE 9 Linear relationship, correlation coefficient and Linear Range (tablet base)
TABLE 10 Linear relationship, correlation coefficient and Linear Range (Soft Capsule base)
TABLE 11 Linear relationship, correlation coefficient and Linear Range (coffee base)
TABLE 12 Linear relationship, correlation coefficient and Linear Range (oral liquid base)
5.5 repeatability
According to a sample processing method, 4 components are respectively added into 7 blank matrixes of beverage, wine, tablets, capsules, soft capsules, oral liquid and coffee, 0.5mL of mixed standard intermediate liquid (2) under 3.2 is precisely measured and is metered into a volumetric flask with 25mL by using a blank matrix solution, 6 parts of test solution with the concentration of 2.5 mu g/mL (the concentration of hydroxypropyl-demethyl tadalafil is 5 mu g/mL) is prepared in parallel, and the test solution is substituted into a standard curve to calculate the content and RSD. The results are shown in tables 13 to 19. The results show that: the repeatability is good.
TABLE 13 repeatability test results (. mu.g/g) (wine base)
TABLE 14 repeatability test results (. mu.g/g) (Capsule base)
TABLE 15 repeatability test results (. mu.g/g) (tablet base)
TABLE 16 repeatability test results (. mu.g/g) (beverage base)
TABLE 17 results of the repeatability tests (μ g/g) (coffee base)
TABLE 18 repeatability test results (. mu.g/g) (Soft Capsule base)
TABLE 19 repeatability test results (. mu.g/g) (oral base)
5.6 recovery test
According to a sample processing method, 4 components are respectively added into 7 blank matrixes, 0.25mL, 0.5mL and 1mL of mixed standard intermediate solution (2) under 7.2 are precisely measured and fixed into a 25mL volumetric flask by using a blank matrix solution, low (1.25 mu g/mL, hydroxypropyl demethyl tadalafil concentration is 2.5 mu g/mL), medium (2.5 mu g/mL, hydroxypropyl demethyl tadalafil concentration is 5 mu g/mL), high (12.5 mu g/mL, hydroxypropyl demethyl tadalafil concentration is 25 mu g/mL) test sample solutions are prepared, 6 parts of each horizontal concentration are prepared in parallel, the concentration is calculated according to a standard curve, and the recovery rate and RSD are calculated. The results are shown in tables 20 to 26. The results show that: the sample recovery rate is good.
TABLE 20 sample recovery test (wine base) (n ═ 6)
Table 21 sample addition recovery test (capsule matrix) (n ═ 6)
Table 22 sample recovery test (tablet matrix) (n ═ 6)
Table 23 sample addition recovery test (beverage base) (n ═ 6)
Table 24 sample recovery test (coffee base) (n ═ 6)
Table 25 sample application recovery rate test (oral liquid base) (n ═ 6)
Table 26 sample application recovery rate experiment (soft capsule matrix) (n ═ 6)
5.7 stability Studies
A standard solution of quantitative limit under the term of "5.1" (2-hydroxyethyl-demethyl-tadalafil: 1.25. mu.g/mL, demethyl-tadalafil: 1.582. mu.g/mL, N-3-hydroxypropyl-demethyl-tadalafil: 1.243. mu.g/mL, 2-hydroxypropyl-demethyl-tadalafil: 2.465. mu.g/mL) was injected into the sample of 10. mu.L, and the results were shown in Table 27. The results show that: the 2-hydroxyethyl-demethyl-tadalafil, N-3-hydroxypropyl-demethyl-tadalafil, 2-hydroxypropyl-demethyl-tadalafil have good stability within 100 hours.
Table 27 stability test results
6. Mass spectrometric confirmation
6.1 instrumentation
An electronic balance of the MS-204S,
a Thermo LTQ-XL LC/MS instrument,
a TSQ QUANTUMACCESS SMAX spectrometer,
DM Spursil C183 u 150X 2.1mm serNo: 2201984 chromatographic column
6.2 reagents and reagents
6.2.1 the methanol, acetonitrile, ammonium acetate, glacial acetic acid, test water and nitrogen and argon are high-purity (99.999%) respectively.
6.2.2 Demethyl tadalafil, N-3 hydroxypropyl tadalafil, 2-hydroxyethyl-desmethyl tadalafil and 2-hydroxypropyl-desmethyl tadalafil (all for inclusion determination).
6.3 preparation of test solutions
The sample solutions under "5.5 replicates" were each precisely measured and diluted to 0.01mg/mL of the test sample solution with 50% acetonitrile.
6.4 preparation of control solutions
Each of the control solutions of item 3.1 and the mixed standard intermediate solution (2) of item 3.2 was measured precisely, and diluted with 50% acetonitrile to give a control solution of 0.01 mg/mL.
6.5 chromatographic conditions:
6.5.1 mobile phase: a: 0.01-0.03mol/L ammonium acetate solution B containing 0.05-0.2% acetic acid: acetonitrile C: methanol
Isocratic elution, the mobile phase ratio is: c35-50: 20-30:20-40
Flow rate: 0.1-0.3 ml/min;
column temperature: 35 ℃;
sample introduction amount: 10 μ L.
6.5.2 Mass Spectrometry parameters: an electrospray ionization source (ESI +), and the spraying voltage is 3-4.58 KV; the temperature of the capillary tube is 280-450 ℃; the flow rate of the auxiliary gas is 10-20 arb, and the flow rate of the sheath gas is 20-60 arb; and (3) detecting positive ions by adopting primary full scanning, secondary scanning and tertiary scanning, wherein the mass range is 50-600.
6.6 Mass Spectrometry corroboration results verification results
TABLE 28 Secondary Scan and Collision energies
TABLE 29 three-level Scan and Collision energy
The verification results show that: the method is feasible, and the mass spectrum condition is suitable for the confirmatory judgment of the 4 components.
FIG. 1 is a liquid chromatogram of soft capsule blank matrix and standard, wherein the chromatogram peak: 1 is 2-hydroxypropyl-desmethyl-tadalafil, 2 is desmethyl-tadalafil, 3 is 2-hydroxyethyl-desmethyl-tadalafil, and 4 is N-3 hydroxypropyl-desmethyl-tadalafil.
FIG. 2 is a secondary mass spectrum of desmethyltadalafil, FIG. 3 is a tertiary mass spectrum of desmethyltadalafil, FIG. 4 is a secondary mass spectrum of 2-hydroxyethyldesmethyltadalafil, FIG. 5 is a non-tertiary mass spectrum of 2-hydroxyethyldesmethyltadalafil, FIG. 6 is a secondary mass spectrum of N-3 hydroxypropyldesmethyltadalafil, FIG. 7 is a tertiary mass spectrum of N-3 hydroxypropyldesmethyltadalafil, FIG. 8 is a secondary mass spectrum of 2-hydroxypropyltadalafil, and FIG. 9 is a tertiary mass spectrum of 2-hydroxypropyldesmethyltadalafil.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (8)
1. A method for detecting 4 tadalafil substances in food is characterized by comprising the following steps:
mixing the food to be detected with acetonitrile to obtain a liquid to be detected;
performing high performance liquid chromatography detection on the liquid to be detected to obtain a liquid chromatogram; the detection conditions of the high performance liquid chromatography comprise:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B;
comparing the obtained liquid chromatogram with a preset standard spectrogram to determine the type of the tadalafil substances in the food to be detected;
the tadalafil compounds include demethyltadalafil, N-3 hydroxypropyl-demethyltadalafil, 2-hydroxypropyl-demethyltadalafil and 2-hydroxyethyl-demethyltadalafil.
2. The detection method according to claim 1, wherein when the food to be detected is a solid sample, the solid sample is ground, 0.1-1g of the ground solid sample is weighed and mixed with 3-40mL of acetonitrile, ultrasonic extraction is performed for 10-30min, the mixture is cooled to room temperature, the volume is determined to be 5-50mL of volumetric flask by acetonitrile, shaking is performed, the mixture is filtered by a 0.45 μm organic filter membrane, a filtrate is taken, and the filtrate is properly diluted by acetonitrile according to actual concentration to be within a linear range to obtain a liquid to be detected.
3. The detection method according to claim 1, wherein when the food to be detected is a liquid sample, 5mL of the liquid sample is placed in a 25mL volumetric flask, acetonitrile is added for constant volume, shaking is carried out, after filtration through a 0.45 μm organic filter membrane, a filtrate is taken, and the liquid to be detected is obtained by properly diluting the filtrate with acetonitrile according to actual concentration to a linear range.
4. The detection method according to claim 1, wherein when the food to be detected is a low-protein low-fat sample, if the food to be detected is a solid preparation, after being porphyrized, the food to be detected is precisely weighed once, placed in a 50mL volumetric flask, added with 40mL of acetonitrile, ultrasonically treated for 15 minutes, placed to room temperature, diluted to the scale with acetonitrile, shaken up, and filtered with a 0.45 μm organic filter membrane for later use; and if the preparation is a liquid preparation, shaking up, precisely measuring the dosage once, putting the preparation in a 50mL volumetric flask, adding acetonitrile to 40mL, shaking for 3 minutes, adding acetonitrile to dilute to a scale, shaking up, and filtering with a 0.45-micrometer organic filter membrane for later use.
5. The detection method as claimed in claim 1, wherein when the protein content in the food to be detected is greater than or equal to 3%, a solid sample is precisely weighed once or 2-5 mL of a liquid sample is absorbed, the solid sample is placed in a 100mL triangular flask, 25mL of water is added, ultrasonic treatment is carried out for 15 minutes, the mixture is placed to room temperature while shaking, 5mL of zinc acetate solution and 5mL of potassium ferrocyanide solution are added, water is added to the scale, the mixture is uniformly mixed, the mixture is placed for 30 minutes, the mixture is filtered into a 50mL volumetric flask by dry filter paper, the mixture is diluted to the scale by acetonitrile and is shaken uniformly, and the mixture is filtered by a 0.45 μm organic filter membrane for later use, and the zinc acetate solution is prepared by the following steps: weighing 21.9g of zinc acetate, adding 3mL of glacial acetic acid, adding water for dissolving, and diluting to 100 mL; the potassium ferrocyanide solution is prepared by the following steps: 10.6g of potassium ferrocyanide are weighed, dissolved in water and diluted to 100 mL.
6. The detection method according to claim 1, wherein when the fat content in the food to be detected is more than or equal to 2%: accurately weighing a solid sample with one dose, leaching with 20mL of diethyl ether or petroleum ether for 3 times, leaching while shaking, removing an ether layer, accurately adding 50mL of acetonitrile into the solid part, performing ultrasonic treatment for 15 minutes, cooling to room temperature, supplementing with acetonitrile, shaking uniformly, and filtering with a 0.45-micrometer organic filter membrane for later use; precisely absorbing 5-10 mL of liquid sample, adding 20mL of diethyl ether or petroleum ether, shaking, standing for layering, diluting a water layer in a 50mL volumetric flask with acetonitrile to a scale, shaking up, and filtering with a 0.45-micrometer organic filter membrane for later use.
7. The detection method according to claim 1, further comprising the following steps after obtaining the liquid chromatogram: and calculating the content of the 4 tadalafil substances in the food to be detected by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 4 tadalafil substances as an independent variable and a peak area as a dependent variable.
8. The detection method according to claim 7, wherein the standard equation is obtained by a method comprising the steps of:
preparation of standard stock solution
Standard stock solutions of demethyltadalafil: accurately weighing 0.01291g of demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile for dissolving, diluting and fixing the volume to a scale, and shaking up;
2-hydroxyethyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of 2-hydroxyethyl demethyl tadalafil reference substance, placing the reference substance in a 10mL volumetric flask, adding acetonitrile to dissolve and dilute the reference substance to a constant volume to scale, and shaking up;
n-3 hydroxypropyl-demethyl tadalafil standard stock solution: accurately weighing 0.0100g of N-3 hydroxypropyl demethyl tadalafil reference substance, placing in a 10mL volumetric flask, adding acetonitrile to dissolve, diluting and fixing the volume to a scale, and shaking up;
2-hydroxypropyl-demethyl-tadalafil standard stock solution: accurately weighing 0.1000g of 2-hydroxypropyl demethyl tadalafil reference substance, placing the reference substance in a 10mL volumetric flask, adding acetonitrile to dissolve, diluting and fixing the volume to a scale, and shaking up;
compounding with mixed standard intermediate liquid
Respectively and precisely measuring 1.2mL of 2-hydroxyethyl demethyl tadalafil, N-3 hydroxypropyl demethyl tadalafil and 2-hydroxypropyl demethyl tadalafil standard stock solutions in 10mL volumetric flasks, adding acetonitrile for diluting to a constant volume to scale, and shaking up to obtain a mixed standard intermediate solution 1;
respectively and precisely measuring 3.125mL of each of 2-hydroxyethyl demethyl tadalafil, demethyl tadalafil and N-3 hydroxypropyl demethyl tadalafil standard stock solutions and 6.25mL of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 25mL volumetric flask, adding acetonitrile to dilute to a constant volume to a scale, and shaking up to obtain a mixed standard intermediate solution 2;
respectively and precisely measuring 500 mu L of 2-hydroxyethyl demethyl tadalafil standard stock solution and N-3-hydroxypropyl demethyl tadalafil standard stock solution, 400 mu L of demethyl tadalafil standard stock solution and 200 mu L of 2-hydroxypropyl demethyl tadalafil standard stock solution in a 2mL volumetric flask, adding acetonitrile for diluting to a constant volume to reach a scale, and shaking up to obtain a mixed standard intermediate solution 3;
standard Curve derivation
Precisely measuring 11.0mL of mixed standard intermediate solution, adding the blank matrix solution, fixing the volume to a 2mL volumetric flask, shaking up, and recording as 'standard yeast-1'; precisely measuring 1.0mL of 'standard yeast-1', adding a blank matrix solution to a 2mL volumetric flask, and shaking up to be marked as 'standard yeast-2'; diluting a standard curve according to the method in sequence in equal proportion to obtain 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6', respectively carrying out high performance liquid chromatography detection on the 'standard curve-1', 'standard curve-2', 'standard curve-3', 'standard curve-4', 'standard curve-5' and 'standard curve-6' to obtain liquid chromatogram maps 1-6, and obtaining a standard equation according to the liquid chromatogram maps 1-6, wherein the high performance liquid chromatography detection conditions are as follows:
a chromatographic column: c18;
column temperature: 35 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 230 nm;
mobile phase: a: acetonitrile, B: 0.1% trifluoroacetic acid in water,
the gradient elution procedure was: 0min 20% A-80% B, 38min 50% A-50% B, 40-45 min 70% A-30% B, 46-60 min 20% A-80% B.
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| CN112394122A (en) * | 2021-01-19 | 2021-02-23 | 潍坊市检验检测中心 | Method for detecting content of 3-hydroxypropyl demethyl tadalafil in health food |
| CN114894931A (en) * | 2022-04-29 | 2022-08-12 | 北京豪思生物科技股份有限公司 | Detection reagent, detection method and application of guanosine cyclophosphate |
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