CN113514584A - Method for qualitatively and quantitatively detecting torasemide illegally added in food and application - Google Patents
Method for qualitatively and quantitatively detecting torasemide illegally added in food and application Download PDFInfo
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- NGBFQHCMQULJNZ-UHFFFAOYSA-N Torsemide Chemical compound CC(C)NC(=O)NS(=O)(=O)C1=CN=CC=C1NC1=CC=CC(C)=C1 NGBFQHCMQULJNZ-UHFFFAOYSA-N 0.000 title claims abstract description 142
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Images
Classifications
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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
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- G—PHYSICS
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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
- 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
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- G—PHYSICS
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- G01N30/14—Preparation by elimination of some components
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- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
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- G—PHYSICS
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- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
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- G—PHYSICS
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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
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- G—PHYSICS
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Abstract
The invention provides a method for qualitatively and quantitatively detecting torsemide illegally added in food and application thereof, wherein the method comprises the following steps: preparing a sample to be detected and a standard solution of torasemide; respectively measuring a sample to be measured and a standard solution by adopting a high performance liquid chromatography-tandem mass spectrometer; recording chromatographic retention time of each compound in the food sample and the standard solution, taking the percentage of the strongest ion abundance as the relative abundance of the qualitative ion pair, and recording the relative ion abundance of corresponding components in the sample to be detected and the standard solution with the same concentration; when the chromatographic peak with the same retention time as the chromatographic peak of the torasemide standard substance is detected from the sample to be detected, the corresponding compound and the content thereof detected from the sample to be detected are determined. The detection method disclosed by the invention is simple, convenient and rapid in pretreatment, strong in specificity and high in sensitivity, can be used for simultaneously carrying out qualitative and quantitative analysis on torasemide in food such as tablet candies, biscuits, beverages, oral liquids and tea beverages, and can be applied to screening illegal addition of torasemide in food safety supervision.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for qualitatively and quantitatively detecting torsemide illegally added in food and application thereof.
Background
With the development of modern economic society, the living standard of people is greatly improved, and the obesity phenomenon is gradually highlighted by fast-paced living style and unhealthy eating habits. In order to achieve the purpose of slimming in a short period of time, people tend to buy slimming products, and so-called 'natural' slimming foods are particularly favored by people. In order to achieve the effect of quickly losing weight, many illegal merchants artificially add chemical drugs such as diuretics and the like which can reduce the weight in a short time so as to achieve the illusion of losing weight, and the illegally added chemical drugs are unknown in type and dosage, can generate serious adverse reactions after being taken for a long time in large quantities, and seriously threaten the benefits and health of consumers. Therefore, the sampling inspection monitoring capability and the supervision strength of the commodities are continuously enhanced, and the benefits and the life safety of consumers are practically guaranteed.
The chemical name of the torasemide is 1- [ 4-aminopyridine-3-yl ] sulfonyl-3-isopropyl urea, is a new generation of high-efficiency loop diuretic, and is a newly discovered chemical drug illegally added for losing weight. It may cause dizziness, headache, nausea, weakness, vomiting, hyperglycemia, excessive urination, hyperuricemia, hypokalemia, extreme thirst, insufficient blood volume, impotence, esophageal bleeding, digestive tract disorders, etc. as a prescription drug, it should be used under the guidance of a doctor, if it is taken in a large amount without knowing it, it will cause unknown serious consequences, so it is necessary to enhance the screening power of the component.
In the prior art, high performance liquid chromatography is adopted to detect torasemide, but because a sample only aims at the detection of the torasemide in an injection product, when the method is adopted to detect weight-losing food or health-care food, the sample processing method is ambiguous, so that the detection result is inaccurate, the sensitivity is not high, false negative is detected, how to quickly, accurately and effectively detect the torasemide is urgent to develop a new detection method.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a qualitative and quantitative detection method for illegally added torasemide in food and application thereof, in particular to detection of illegally added torasemide in weight-losing food or health-care food, and a method for simultaneously carrying out qualitative and quantitative analysis on torasemide is realized by using a liquid chromatography-mass spectrometry combined technology, so that the blank of simultaneously carrying out qualitative and quantitative detection on torasemide in food at home and abroad is filled.
In order to achieve the above object, one aspect of the present invention provides a method for qualitatively and quantitatively detecting torsemide illegally added to food, comprising the steps of:
s1, sample pretreatment: if the food sample is solid, taking 1g, and accurately measuring to 0.0001g, if the food sample is liquid, taking 1mL, placing in a 50mL measuring flask, adding 35-45 mL of methanol, shaking, ultrasonically extracting, adding methanol to a constant volume to a scale, shaking up, filtering with a 0.22 μm filter membrane, and taking a subsequent filtrate as a sample solution to be measured;
s2, preparing a standard solution: preparing a standard stock solution of torasemide, diluting the standard stock solution with methanol to obtain a standard use solution, and preparing the standard use solution with methanol into standard series solutions with different concentrations;
s3, qualitative determination of samples: respectively measuring the subsequent filtrate and the standard solution of the food sample by adopting a high performance liquid chromatography-tandem mass spectrometer, recording the chromatographic retention time of each compound in the food sample and the standard solution, taking the percentage of the abundance of the strongest ions as the relative abundance of a qualitative ion pair, and recording the relative ion abundance of corresponding components in the sample with the same concentration and the standard solution; when a chromatographic peak consistent with the chromatographic peak retention time of the torasemide standard solution is detected in the sample, the variation range is within +/-2.5 percent, and the allowable deviation of the relative ion abundance does not exceed the range specified in the following table, determining that the corresponding compound is detected in the sample;
maximum allowable deviation of relative ion abundance
S4, quantitative determination of samples: the method comprises the steps of measuring a standard series solution of torasemide by adopting a liquid chromatography-tandem mass spectrometry method, drawing a standard curve according to the peak area A of the standard solution and the concentration of the standard series solution to obtain a regression equation A which is kC + b, substituting the peak area A of a sample to be measured into the regression equation of the standard curve, calculating to obtain the concentration of the torasemide illegally added into the sample to be measured, and then obtaining the content of the torasemide in the sample.
As with the method described above, the order of operations of steps S1 and S2 can be reversed without affecting the result.
In the detection method as described above, preferably, in step S1, when the food sample is solid, the food needs to be ground and the powder is weighed.
In the detection method as described above, preferably, in step S1, the power of ultrasound is 150W, the frequency is 40KHZ, the time is 30min, and the filtrate is diluted with methanol to the concentration range of the standard series solution for sample detection.
In the detection method as described above, preferably, in step S2, a standard stock solution of 100 μ g/mL is prepared, then methanol is added to the standard stock solution of 100 μ g/mL to prepare a standard use solution a having a torasemide concentration of 5 μ g/mL, then methanol is added to the standard use solution a to prepare a standard use solution B having a torasemide concentration of 1 μ g/mL, and then the standard use solutions B of 0.1mL, 0.2mL, 0.4mL, 1.0mL and 2.0mL are taken respectively, placed in a 20mL volumetric flask, diluted to the scale with methanol, shaken to obtain standard series solutions having concentrations of 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in this order.
The detection method as described above, preferably, the standard stock solution of 100 μ g/mL is prepared by accurately weighing 10.0mg of torasemide standard, accurately weighing to 0.0001g, placing in a 100mL volumetric flask, dissolving and diluting to the scale with methanol, and shaking up.
The detection method as described above, preferably, in steps S3 and S4, the condition of the high performance liquid chromatography is that the chromatographic column is Ziziphora CAPCELL-PAK C18A chromatography column or an Acquity UPLC BEH Amide or one with comparable performance; the mobile phase is ammonium acetate solution with A being 10mmol/L, B is acetonitrile, and A: B is 70: 30; the flow rate is 0.5 mL/min; the column temperature is 20 ℃; the amount of the sample was 10. mu.L.
In the detection method, preferably, in steps S3 and S4, the mass spectrum is performed under the conditions that the ion source is an electrospray ion source, the detection method is multiple reaction monitoring, the scanning method adopts a negative ion mode, and the capillary voltage is a negative ion mode: 4500V, ion source temperature 400 deg.C, dry gas flow 12L/min, atomization gas pressure 70psi, sheath gas temperature 250 deg.C, and sheath gas N2(ii) a The air pressure of the air curtain is 35psi, and the auxiliary air pressure is 60 psi; the nozzle voltage is in positive ion mode: 500V; the negative ion mode was 2000V.
In the detection method as described above, preferably, in the mass spectrometry conditions in steps S3 and S4, the time to peak of torasemide is 7.2min, the quantitative ion pair is 346.8 to 261.7, the qualitative ion pair is 346.8 to 196.0, the fragmentation voltage is 80V, the collision voltage is 24V, and the detection mode is negative ion detection.
In the detection method as described above, preferably, in step S4, the standard series solution is measured to obtain a chromatographic peak area of the corresponding standard series solution, and a standard curve is drawn with the concentration of the standard series solution as an abscissa and the peak area of the chromatographic peak as an ordinate; and according to the chromatographic peak area of the sample obtained by detection, corresponding to the drawn standard curve, obtaining the concentration of the torasemide in the corresponding sample introduction sample.
In the detection method, the content X of torasemide in the food can be calculated by substituting the concentration of torasemide in the sample into the following formula:
wherein:
x-the amount of torasemide in a food sample in milligrams per kilogram (mg/kg) or milligrams per liter (mg/L);
c-the concentration of torasemide in the sample solution injected, read from the standard curve, in nanograms per milliliter (ng/mL);
v is the final volume of the sample solution, and the unit is milliliter (mL);
m-the mass or volume represented by the sample solution in grams (g) or milliliters (mL);
k is the dilution multiple;
the calculation results are expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, with the results remaining in three significant digits.
The invention also provides the application of the method for simultaneously qualitatively and quantitatively detecting the illegally added torasemide in the food to the detection of the illegally added torasemide in the food.
As mentioned above, the method for qualitatively and quantitatively detecting the illegally added torasemide in the food and the application thereof have the following beneficial effects:
the invention provides a high performance liquid chromatography-tandem mass spectrometry analysis method for simultaneously carrying out qualitative and quantitative detection on torasemide illegally added in food. The method uses high performance liquid chromatography as separation equipment, a triple quadrupole mass spectrometer as detection equipment, mass spectrum qualitative ion pairs are used for qualitative analysis, and quantitative ions are used for quantitative detection of an external standard method. The method has the advantages of strong specificity, high sensitivity, simple and efficient operation and high analysis speed, can quickly carry out qualitative confirmation on the torsemide illegally added in the sample, and accurately quantify the content of the torsemide, thereby greatly improving the detection sensitivity and avoiding the generation of missed detection and false negative results. The method is suitable for screening and confirming illegally added torasemide, fills the gap of qualitative and quantitative measurement of torasemide at home and abroad, and can provide effective and comprehensive technical support for a supervision department to fight against fake sales.
The detection method is suitable for food such as tablet candy, biscuit, beverage, oral liquid, tea beverage and the like, and comprises the health-care food in the form of tablets, capsules and the like with similar matrixes.
The liquid chromatography-mass spectrometry method adopted by the method has strong specificity and high sensitivity, can realize simultaneous qualitative and quantitative analysis, is simple and efficient to operate, has high analysis speed, is suitable for screening and confirming illegally added torasemide, carries out quantification simultaneously while being qualitative, and can provide effective and comprehensive technical support for a supervision department to fight against counterfeiting and sell counterfeiting.
Drawings
FIG. 1 is a standard curve of torasemide in example 2 of the present invention.
FIG. 2 is an extracted ion chromatogram of different concentrations of torasemide standard series solutions in example 2 of the present invention.
FIG. 3 is a mass spectrum of qualitative detection of torasemide in example 2 of the present invention.
FIG. 4 is a mass spectrum of the quantitative determination of torasemide in example 2 of the present invention.
FIG. 5 is a total ion current chromatogram of an empty sample in example 2 of the present invention.
FIG. 6 is a total ion current chromatogram of a negative sample in an example of the present invention.
FIG. 7 is a total ion current chromatogram of an actual sample in example 9 of the present invention.
Fig. 8 shows the effect of different extraction times on the extraction effect.
FIG. 9 is a Torasemide UV scanning spectrum.
FIG. 10 is an HPLC chromatogram of a blank solution.
FIG. 11 is an HPLC chromatogram of a torasemide control solution.
FIG. 12 is an HPLC chromatogram of a sample solution (torasemide).
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.
Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, the reagents used in the methods are analytically pure or above.
Example 1
Reagents used in this example: acetonitrile (CH)3CN): chromatographically pure, ammonium acetate (CH)3COONH4): chromatographically pure, methanol (CH)3OH): analytically pure, the water is the primary water specified in GB/T6682.
Preparation of 10mmol/L ammonium acetate aqueous solution: ammonium acetate 1.54g was weighed, diluted to 2000mL with water, and filtered through a 0.45 μm filter for use.
The Chinese name, English name, CAS login number, molecular formula and relative molecular mass of the used torasemide standard are shown in Table 1, and the purity of the standard is more than or equal to 95%.
TABLE 1 Torasemide Standard substance information Table
The standard solution is prepared by the following method:
(1) standard stock solution (100. mu.g/mL): accurately weighing 10.0mg (accurate to 0.0001g) of torasemide standard, placing in a 100mL volumetric flask, dissolving with methanol, diluting to scale, shaking up, and making into standard stock solution with concentration of 100 μ g/mL.
(2) Standard use solution a: accurately sucking 1mL of the torasemide standard stock solution, placing the torasemide standard stock solution in a 20mL volumetric flask, diluting the torasemide standard stock solution to a scale mark with methanol, shaking up to prepare a standard use solution A, wherein the concentration of the torasemide is 5 mu g/mL.
(3) Standard use solution B: accurately sucking 2mL of the standard use solution A, placing the solution in a 10mL volumetric flask, diluting the solution to the scale with methanol, and shaking up to prepare a standard use solution B with the concentration of 1 mu g/mL of torasemide.
(4) Standard series of solutions: 0.1mL, 0.2mL, 0.4mL, 1.0mL and 2.0mL of each of the standard solutions B were accurately aspirated, the solutions were placed in a 20mL volumetric flask, diluted to the scale with methanol, shaken up to give a series of standard series solutions S (1) to S (5), and the concentrations of torasemide were 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in this order.
Preparation of a detection sample:
(a) the following method is adopted for the samples of pressed candies, tea leaves, biscuits, tablets and capsules:
grinding an appropriate amount, weighing 1g (accurate to 0.0001g) of the powder, placing into a 50mL measuring flask, adding 40mL of methanol, shaking, ultrasonically extracting for 30min, cooling, adding methanol to a constant volume to scale, shaking up, filtering with a filter membrane (0.22 μm, organic phase type), collecting the filtrate, diluting to a linear range according to the actual concentration, and analyzing with a liquid chromatograph-mass spectrometer.
(b) The following method is adopted for the beverage and the oral liquid:
accurately sucking 1mL, placing in a 50mL measuring flask, adding 40mL of methanol, shaking, ultrasonically extracting for 30min, cooling, adding methanol to a constant volume to scale, shaking, filtering with a filter membrane (0.22 μm, organic phase type), collecting filtrate, diluting to a linear range according to actual concentration, and analyzing with a liquid chromatography-mass spectrometer.
Detecting the condition of the apparatus used
(1) Conditions of liquid chromatography
a) A chromatographic column: zinshengtang CAPCELL-PAK C18Chromatography column (4.6mm × 150mm,5 μm) or Acquity UPLC BEH Amide or one with comparable performance;
b) mobile phase: a is ammonium acetate solution containing 10mmol/L, B is acetonitrile, A: B is 70: 30;
c) flow rate: 0.5 mL/min;
d) column temperature: 20 ℃;
e) sample introduction amount: 10 μ L.
(2) Conditions of Mass Spectrometry
a) An ion source: electrospray ion source (ESI);
b) the detection mode is as follows: multiple Reaction Monitoring (MRM);
c) the scanning mode is as follows: a negative ion mode;
d) capillary voltage: 4500V;
e) ion source temperature: 400 ℃;
f) flow rate of drying gas: 12L/min;
g) atomizing gas pressure: 70 psi;
h) temperature of sheath gas: sheath gas (N) at 250 ℃2)；
i) Air curtain pressure: 35 psi;
j) auxiliary gas pressure: 60 psi;
k) nozzle voltage: positive ion mode: 500V; negative ion mode: 2000V;
l) other mass spectral parameters are shown in Table 2.
TABLE 2 qualitative, quantitative ion and Mass Spectrometry parameters for torasemide
Example 2
(I) qualitative determination
And (3) measuring the sample and the standard series solution according to the high performance liquid chromatography-tandem mass spectrometry conditions, recording the chromatographic retention time of each compound in the sample and the standard series solution, taking the percentage relative to the strongest ion abundance as the relative abundance of a qualitative ion pair, and recording the relative ion abundance of corresponding components in the sample with the same concentration as that in the standard series solution. When a chromatographic peak corresponding to the retention time of the chromatographic peak of the torasemide standard is detected in the sample (within a variation range of +/-2.5%) and the allowable deviation of the relative ion abundance does not exceed the range specified in table 3, the detection of torasemide in the sample can be determined.
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative confirmation
(II) quantitative detection
In the same manner as in example 1, 10. mu.L of standard solutions of torasemide having concentrations of 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL in this order were injected into HPLC-tandem mass spectrometer and measured.
1. Preparation of Standard Curve
And (4) measuring the standard series of solutions according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding standard solution. And (3) taking the concentration (C) of the standard series solution as an abscissa and taking the peak area (A) of a chromatographic peak as an ordinate, drawing a standard curve, and obtaining a regression equation A which is kC + b.
The standard curve of torasemide is shown in fig. 1, and the regression equation obtained is: a is 2207.9C-1038.2 and r is 0.9998.
The extracted ion chromatogram of torasemide is shown in fig. 2.
The mass spectrum of torasemide for qualitative detection is shown in fig. 3, and the mass spectrum of torasemide for quantitative detection is shown in fig. 4.
2. Measurement of sample solution
And (2) determining the sample solution according to the instrument reference condition by adopting a high performance liquid chromatography-tandem mass spectrometry method to obtain the chromatographic peak area of the corresponding sample solution, substituting the peak area A of the sample to be determined into a standard curve regression equation to obtain the concentration of the torasemide in the solution to be determined, wherein the number of parallel determination is not less than two.
3. Blank test
The same procedure was followed except that no sample was added.
The total ion current chromatogram of the blank sample is shown in FIG. 5.
4. Calculation of results
Substituting the concentration measured by the high performance liquid chromatography-tandem mass spectrometry into the following formula to calculate the content of the torasemide in the sample:
in the formula:
x-the amount of torasemide in milligrams per kilogram (mg/kg) in the sample;
c-the concentration of torasemide in the test solution read from the standard curve in nanograms per milliliter (ng/mL);
v is the final volume of the sample solution, and the unit is milliliter (mL);
m-the mass represented by the sample solution in grams (g);
k is the dilution multiple.
The calculation results are expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, with the results remaining in three significant digits.
The absolute difference between two independent measurements obtained under repetitive conditions must not exceed 15% of the arithmetic mean, and the blank test should be free of interference.
Example 3 sensitivity detection
Weighing 1g (accurate to 0.0001g) of blank solid sample or 1mL of blank liquid sample, accurately adding a reference substance solution with a certain concentration into a 50mL measuring flask, adding 40mL of methanol, shaking, ultrasonically extracting for 30min, cooling, adding methanol to fix the volume to scale, shaking up, filtering with a filter membrane (0.22 μm, organic phase type), collecting the filtrate, analyzing by a liquid chromatography-mass spectrometer, calculating the detection limit according to the concentration of torasemide corresponding to the signal-to-noise ratio of 3:1, and calculating the quantification limit according to the concentration of torasemide corresponding to the signal-to-noise ratio of 10: 1. The method in example 2 was followed to obtain a directly detectable torasemide with a limit concentration of 1.5ng/mL and a minimum detection limit concentration of 0.5 ng/mL.
The sampling amount of the solid and the liquid is 1g or 1mL, when the constant volume is 50mL, the quantitative limit of the torasemide is 75 mug/kg or 75 mug/L, and the detection limit is 25 mug/kg or 25 mug/L.
Example 4 examination of conditions of chromatography-Mass Spectrometry
1. Investigation of chromatographic columns
The sample for the illegal addition experiment is complex, the impurities are more, and the requirements on the separation effect and the durability of the chromatographic column are higher. The experiment respectively considers Acquisty UPLC BEH Amide (2.1 multiplied by 50mm,1.7 mu m) and the senior CAPCELL-PAK C18(4.6mm×150mm,5μm)、TechMate C18-ST (4.6 mm. times.250 mm,5 μm) and TechMate C4Separation efficiency and durability of 4 different columns such as ST (4.6 mm. times.250 mm,5 μm), and the like, results are shown in the TableThe analysis time of the Acquity UPLC BEH Amide is shortest, and the Acquity UPLC BEH Amide and the senior CAPCELL-PAK C18The separation effect of (2) can meet the experimental requirements. Although the analysis time of the ultra-high performance chromatographic column is short, the column pressure of the chromatographic column is easily increased due to a sample with more impurities, and the durability is not ideal. The invention finally adopts a senior CAPCELL-PAK C18The (4.6mm multiplied by 150mm,5 mu m) chromatographic column is used for detecting the illegal addition of the torasemide, the analysis time is short, the separation degree can meet the requirement, and the continuous analysis of hundreds of batches of samples can also well meet the experimental requirement.
2. Investigation of mobile phase systems
The mobile phase is selected not only in consideration of the peak shape and separation effect of the analyte, but also in consideration of the ion response intensity. The polarity of torasemide is larger, and a proper mobile phase needs to be selected so as to separate the torasemide from a polarity interference peak, reduce a matrix effect and enhance the ionic response strength. Tests respectively investigate the mobile phase systems of methanol-water, acetonitrile-water, methanol-ammonium acetate aqueous solution, acetonitrile-ammonium acetate aqueous solution and the like, and the results show that the ammonium acetate aqueous solution can effectively improve the peak shape of the torasemide, and the mobile phase system with acetonitrile as an organic phase is favorable for separating and improving the ionic response strength. The ratio of aqueous phase to organic phase was adjusted to finally determine a 10mmol/L ammonium acetate solution: acetonitrile 70:30 as mobile phase for torasemide analysis.
3. Optimization of mass spectrometry conditions
And taking the torasemide standard intermediate solution A with the concentration of 5 mu g/mL for optimizing the mass spectrum condition. Ionization is carried out by adopting an electrospray ionization source (ESI), full scanning is respectively carried out by adopting a positive ion mode and a negative ion mode, and the result shows that the torasemide is subjected to ESI-The response is best in the mode, so the method adopts the negative ion mode for detection. At ESI-Performing parent ion scanning in a mode with a scanning range of M/z 200-500 to obtain [ M + H ]]-Peaks and the corresponding capillary voltage is adjusted appropriately so that the response is highest for the primary scan of parent ions.
And (3) after determining the parent ions and the capillary voltage thereof, scanning the child ions, and optimizing by an automatic optimization program of instrument secondary mass spectrum parameters to obtain the optimal secondary mass spectrum conditions and further obtain the secondary mass spectrum optimization parameters of the torasemide. And finally, the quantitative and qualitative ion pair and the collision energy obtained after optimization are used as MRM scanning parameters of the torasemide.
Example 5 examination of sample pretreatment method
1. Selection of extraction solvent
The method is simple and convenient, and can meet the requirements of experiments.
2. Investigation of extraction time
A sample of primary plant oligofructose (Qingyuan Longtang pharmaceutical and technology Co., Ltd., Lot No. 20081306) is ground and precisely weighed, each portion is about 1g, the primary plant oligofructose is respectively placed in a 50mL measuring flask, 40mL of methanol is added, shaking is carried out, ultrasonic extraction (power 150W and frequency 40kHz) is carried out for 10, 20, 30, 40, 50 and 60min at room temperature, cooling is carried out, methanol is added for fixing the volume to the scale, shaking is carried out uniformly, a filter membrane (0.22 mu m and organic phase type) is used for filtering, and subsequent filtrate is taken and analyzed by a liquid chromatography-mass spectrometer. By comparing the chromatographic responses of samples after ultrasonic extraction for 10min, 20min, 30min, 40min, 50min and 60min by methanol, the peak area of torasemide reaches the maximum value when the extraction time reaches 30min and does not increase when the ultrasonic time continues to increase by using methanol as an extraction solvent, so that the torasemide in the samples can be completely extracted by selecting the ultrasonic extraction time to be 30 min. The effect of extraction time on extraction effect is shown in fig. 8.
Example 6 repeatability test examination
Accurately weighing 6 parts of ground primary plant oligofructose (20081306, batch number 20081306, Shangyuan Longtang medicine science and technology Co., Ltd., Qingyuan), placing each part of 1.0000g into a 50mL measuring flask, adding 40mL of methanol, shaking, performing ultrasonic extraction at room temperature (power 150W, frequency 40kHz) for 30min, cooling, adding methanol to fix the volume to the scale, shaking up, precisely weighing 1.0mL to 100mL measuring flask of supernatant, adding methanol to dilute to the scale, shaking up, filtering the supernatant with a filter membrane (0.22 μm, organic phase type), and taking the subsequent filtrate. The analysis was performed using the liquid phase and mass spectrometry conditions in example 1. The retention time of the torasemide chromatographic peak is obtained, the content of torasemide in the sample is calculated by adopting the quantitative detection method in the embodiment 2, the result is shown in the table 4, the relative standard deviation of the torasemide retention time is 0.3%, the relative standard deviation of the content is 0.7%, and the result shows that the repeatability is good.
TABLE 4 repeatability survey of torasemide
Example 7 recovery test investigation
Accurately weighing 0.5000g of primary plant oligofructose (Qingyuan Longtang pharmaceutical and technology Co., Ltd., batch No. 20081306) with known content, placing the primary plant oligofructose into a 50mL measuring flask, respectively adding torasemide reference substances (shown in Table 5), parallelly operating 3 parts of each sample, adding 40mL of methanol, shaking, performing ultrasonic extraction (power 150W and frequency 40kHz) for 30min at room temperature, cooling, adding methanol to a constant volume, shaking up, precisely measuring 1.0mL to 100mL of supernatant, adding methanol to a diluted volume, shaking up, precisely measuring 1.0mL to 100mL of supernatant, diluting the supernatant to a constant volume, adding methanol to a diluted volume, shaking up, filtering the supernatant with a filter membrane (0.22 μm, organic phase type), and taking a continuous filtrate. The analysis was performed using the liquid phase and mass spectrometry conditions described in example 1, and the regression equation substituted into example 2 for quantification and the average recovery and Relative Standard Deviation (RSDs) (n 9) calculated, the results are shown in table 5.
The results show that the recovery rate of torasemide is between 98.5 and 101.6 percent, the average recovery rate is 99.4 percent, and the RSD is 1.0 percent, which indicates that the method has good accuracy, and the recovery rate can meet the requirement of content determination
TABLE 5 recovery of torasemide and its RSDs (n ═ 9)
Example 8 stability study
The sample solution in example 6 was taken, analyzed under the liquid phase and mass spectrum conditions described in example 1, and subjected to injection measurement for 0, 2, 4, 8, 10, and 12h, respectively, and the peak area RSD of torasemide was calculated, and the results are shown in table 6, which indicates that the sample solution was stable within 12 h.
TABLE 6 Torasemide stability study
EXAMPLE 9 actual sample determination
Qualitative and quantitative measurements of the illegally added torasemide were carried out on 15 batches of food (including health food) samples using the methods of the present invention in examples 1 and 2, respectively. The sample information for 15 lots is shown in table 7. The 15 batches were tested using this method and analyzed using the liquid phase and mass spectrometry conditions described in example 1 and carried over to the regression equation quantification in example 2, where 2 batches were tested to contain torasemide, with sample numbers 8 and 14, respectively. The total ion current chromatogram of the sample is shown in FIG. 7, and the measurement results are shown in Table 7. The result shows that the detection rate is 13.3 percent when the torsemide is illegally added in 2 batches of samples.
TABLE 7 sample information and measurement results
| Numbering | Sample name | Sample batch number | Dosage forms | Torasemide content/mg/ |
||
| 1 | Konjac green tea carnitine milk tea | 20191210 | Instant granule | Not detected out | ||
| 2 | Fruit and vegetable granules for promoting appetite | 20191223 | Granules | Not detected out | ||
| 3 | L-carnitine coffee powder | 20191103 | Powder preparation | Not detected out | ||
| 4 | Bidi brand Xiuer tea | 20190201 | Tea preparation | Not detected out | ||
| 5 | Perfect Gao Qiao Qian Wei le infusion | 20200707 | Instant granule | Not detected out | ||
| 6 | Lotus leaf bitter gourd seasoning tea | 20200105 | Tea preparation | Not detected out | ||
| 7 | Chuntang cassia seed and | Year | 2020, 4 and 10 | Tea preparation | Not detected out | |
| 8 | Plant peptide probiotic tablet candy | 19101501 | Candy | 32300 | ||
| 9 | Barley grass juice | 2020/09/10 | Tea preparation | Not detected out | ||
| 10 | Qingqingyin (decoction for clearing away heat and toxic materials) | 20200601 | Solid beverage | Not detected out | ||
| 11 | Nafier enzyme jelly | 2020/07/28 | Jelly and its production process | Not detected out | ||
| 12 | Dietary fiber orange-flavored beverage | 2019/06/03 | Liquid beverage | Not detected out | ||
| 13 | Changrun tea | 2020/05/15 | Tea preparation | Not detected out | ||
| 14 | Primary core plant oligomeric candy | 20081306 | Candy | 46200 | ||
| 15 | |
8/12/2020 | Tea preparation | Not detected out |
The experimental results show that the total ion current chromatogram of the negative sample (see figure 6) treated by the method has no obvious impurity peak compared with the total ion current chromatogram of the blank sample (see figure 5), and the method has no interference at the position of torasemide. Moreover, as can be seen from the reproducibility test of example 6, the method has good precision and reproducibility (see table 4); as can be seen from the recovery test of example 7, the process of the invention has good recovery and relative standard deviation (see table 5); from the stability test of example 8, it can be seen that the solution has good stability within 12h (see table 6), which indicates that qualitative and quantitative determination of torasemide in food and health food can be rapidly and effectively carried out by using the method of the present invention.
Comparative example
In addition, the high performance liquid chromatography is also adopted to measure the content of the torasemide in the sample, and the torasemide content is taken as a comparative example to verify the accuracy of the measuring method.
1 materials and methods
1.1 instruments and reagents
An Alliance acquity e2695 HPLC system comprising a quaternary high pressure gradient pump, an autosampler, a 2998 diode array detector and an Empower chromatography workstation (Waters corporation, usa); electronic analytical balance model XP 205 (mettler-toledo instruments, switzerland); AS series ultrasonic cleaning machine (tianjin ott sains instruments ltd); Mili-Q deionized water generators (Millipore, USA).
Methanol (chromatographic purity), potassium dihydrogen phosphate, phosphoric acid (analytical purity) (national drug group, Inc.), and Milli-Q ultrapure water as experimental water.
Torasemide reference substance (purity 100.0%, purchased from China institute for food and drug testing)
1.2 Experimental methods
1.2.1 chromatographic conditions
Using TechMate C18-ST (250mm × 4.6mm, 5 μm) chromatography column; the mobile phase is methanol-20 mmol/L potassium dihydrogen phosphate solution (pH is adjusted to 3.0 by phosphoric acid) (20:80, V: V) isocratic elution; detection wavelength: 280 nm; column temperature: 25 ℃; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.
1.2.2 preparation of solution
Precisely weighing 20.10mg of torasemide as reference substance, placing in 20mL volumetric flask, adding methanol to dissolve, fixing volume to scale, and shaking to obtain reference substance stock solution (1.005 mg/mL);
precisely measuring 5mL of reference substance stock solution, placing in 50mL volumetric flask, adding methanol to dilute to constant volume to scale, and shaking to obtain reference substance solution;
standard series working liquids: the reference substance use solutions of 0.5, 1, 2, 4, 6 and 8mL are precisely measured respectively, placed in a 10mL volumetric flask, diluted to the scale with methanol and shaken up, and the concentrations of the standard series solutions are 5.02, 10.0, 20.1, 40.2, 60.3 and 80.4 mu g/mL respectively.
Test solution: accurately weighing about 2g of uniformly mixed sample powder, placing the sample powder into a 100mL volumetric flask, adding a proper amount of methanol, shaking for dissolution, performing ultrasonic extraction for 30min, cooling, adding methanol for dilution to a scale, shaking up, precisely weighing 1mL of supernatant, placing the supernatant into a 20mL volumetric flask, adding methanol for dilution to a scale, shaking up, and performing liquid phase analysis after passing through a 0.45 mu m filter membrane.
2 methodology examination
2.1 selection of wavelength
The ultraviolet detector is adopted in the experiment, and the compound has the maximum absorption peak at 280nm from the torasemide ultraviolet spectrum scanning chart, so that the wavelength of the detector is selected to be 280nm for measurement, so that the detection sensitivity is improved, and the torasemide ultraviolet scanning spectrum chart is shown in figure 9.
2.2 specificity and System Adaptation test
The theoretical plate numbers of torasemide in the liquid chromatogram of the reference solution and the sample solution are 10214 and 11243 respectively, the peak shapes are symmetrical, the separation of the torasemide peak and other impurity peaks in the sample solution is better, a blank solvent does not interfere with sample determination, the method has good specificity, and the blank solvent chromatogram, the reference solution and the sample solution chromatogram are shown in figures 10-12.
2.3 Linear relationship inspection
And (4) taking the standard series working solution, analyzing according to the chromatographic conditions under the item of '1.2.1', and recording a chromatogram. And (3) performing regression by taking the concentration C (mu g/mL) of torasemide as a horizontal coordinate and the peak area A as a vertical coordinate, and drawing a standard curve, wherein the regression equation of torasemide is as follows: a is 114166C-3781.1(r is 0.9997, n is 6), and the result shows that the concentration of the torasemide is in a good linear relation with the peak area within 5.02-80.4 mu g/mL.
2.4 precision test
mu.L of the torasemide control solution was precisely aspirated, and the sample injection was repeated 6 times under the chromatographic conditions of "1.2.1", and the Relative Standard Deviations (RSD) of the retention time and the peak area of the torasemide chromatographic peak were 0.92% (n-6) and 0.83% (n-6), respectively. The results show that the precision of the instrument is good.
2.5 repeatability test
6 parts of the ground primary plant oligofructose (Qingyuan Longtang pharmaceutical and technology Co., Ltd., No. 20081306) are precisely weighed, a sample solution is prepared according to the method under the item '1.2.2', the chromatographic condition is adopted for determination under the item '1.2.1', and the content of the torasemide is calculated according to an external standard method. The relative standard deviation of the retention time of torasemide in the sample is 0.6%, the relative standard deviation of the content is 1.3%, and the result shows that the repeatability is good.
Repeatability survey of torasemide
2.6 Standard recovery experiment
Accurately weighing 1g of primary-core plant oligofructose (Qingyuan Longtang pharmaceutical and technology Co., Ltd., batch No. 20081306) with known content, placing the primary-core plant oligofructose into a 100mL measuring flask, respectively adding torasemide reference substances (specifically shown in the following table), parallelly operating 3 parts for each sample, preparing a sample solution according to the same method under the item '1.2.2', measuring according to the chromatographic condition under the item '1.2.1', quantifying by adopting an external standard method, and calculating the average recovery rate and the relative standard deviation (n is 9), wherein the results are shown in the following table. The result shows that the recovery rate of the torasemide is between 98.3 and 101.1 percent, the average recovery rate is 99.6 percent, and the Relative Standard Deviation (RSD) is 1.0 percent, which indicates that the method has good recovery rate and the accuracy meets the experimental requirement.
Recovery of torasemide and its RSDs (n ═ 9)
2.7 detection Limit investigation
Weighing 2g of blank sample (sample without detecting the torasemide), adding a reference substance solution with a certain concentration, carrying out sample pretreatment according to the method under the item '1.2.2', measuring according to the chromatographic condition under the item '1.2.1', and calculating the detection limit of the torasemide according to the corresponding reference substance concentration when the signal-to-noise ratio is 3. The results showed that the lowest detected concentration of torasemide was 25 ng/mL.
The sampling amount of the solid and the liquid is 2g or 2mL, and when the constant volume is 50mL, the detection limit of the torasemide is 625 mug/kg or 625 mug/L.
3 determination of actual samples
Qualitative and quantitative measurements of the illegally added torasemide were performed on 15 batches of food (including health food) samples from example 9 using the experimental methods described above. Wherein 2 batches of samples are detected to contain the torasemide component, the sample numbers are respectively 8 and 14, and the content of the torasemide in the samples is respectively 33100mg/kg and 46700 mg/kg.
The results measured by the method of the present invention were 32300mg/kg and 46200mg/kg, respectively, and the relative average deviation of the results compared with the comparative examples was 1.2% and 0.5%, respectively, indicating that the results measured by the two methods are similar. But the method established by the invention has more advantages than the prior art in the aspects of repeatability and precision, the detection limit concentration is reduced by 50 times compared with the prior art, the RSD of repeatability is lower, and the method is more beneficial to qualitative and quantitative determination of illegally added torasemide in the product.
In conclusion, the invention provides a liquid chromatography-tandem mass spectrometry analysis method for simultaneously carrying out qualitative and quantitative detection on illegally added torasemide in food (including health food). The method uses a high performance liquid chromatograph as separation equipment, a triple quadrupole mass spectrometer as a detector, and uses mass spectrum qualitative ion pairs for qualitative analysis, and quantitative ions for quantitative detection of an external standard method. The method has the advantages of simple pretreatment, high sensitivity, strong specificity and high efficiency, can simultaneously carry out qualitative and quantitative analysis on illegally added torasemide in food (including health food), and can be applied to food safety supervision to prevent the illegal addition of the weight-reducing medicine in the food.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for qualitatively and quantitatively detecting torasemide illegally added in food is characterized by comprising the following steps:
s1, sample pretreatment: if the food sample is solid, taking 1g, and accurately measuring to 0.0001g, if the food sample is liquid, taking 1mL, placing in a 50mL measuring flask, adding 35-45 mL of methanol, shaking, ultrasonically extracting, adding methanol to a constant volume to a scale, shaking up, filtering with a 0.22 μm filter membrane, and taking a subsequent filtrate as a sample solution to be measured;
s2, preparing a standard solution: preparing a standard stock solution of torasemide, diluting the standard stock solution with methanol to obtain a standard use solution, and preparing the standard use solution with methanol into standard series solutions with different concentrations;
s3, qualitative determination of samples: respectively measuring the subsequent filtrate and the standard solution of the food sample by adopting a high performance liquid chromatography-tandem mass spectrometer, recording the chromatographic retention time of each compound in the food sample and the standard solution, taking the percentage of the abundance of the strongest ions as the relative abundance of a qualitative ion pair, and recording the relative ion abundance of corresponding components in the sample with the same concentration and the standard solution; when a chromatographic peak consistent with the chromatographic peak retention time of the torasemide standard solution is detected in the sample, the variation range is within +/-2.5 percent, and the allowable deviation of the relative ion abundance does not exceed the range specified in the following table, determining that the corresponding compound is detected in the sample;
maximum allowable deviation of relative ion abundance
S4, quantitative determination of samples: the method comprises the steps of measuring a standard series solution of torasemide by adopting a liquid chromatography-tandem mass spectrometry method, drawing a standard curve according to the peak area A of the standard solution and the concentration of the standard series solution to obtain a regression equation A which is kC + b, substituting the peak area A of a sample to be measured into the regression equation of the standard curve, calculating to obtain the concentration of the torasemide illegally added into the sample to be measured, and then obtaining the content of the torasemide in the sample.
2. The detection method according to claim 1, wherein in step S1, when the food sample is solid, the food is ground and the powder is weighed.
3. The method of claim 1, wherein the sample is applied in step S1 by ultrasonic at a power of 150W and a frequency of 40KHZ for 30min, and the filtrate is diluted with methanol to a concentration within the range of the standard series solution.
4. The detection method according to claim 1, wherein in step S2, a standard stock solution of 100 μ g/mL is prepared, then methanol is added to the standard stock solution of 100 μ g/mL to prepare a standard use solution a with a torasemide concentration of 5 μ g/mL, then methanol is added to the standard use solution a to prepare a standard use solution B with a torasemide concentration of 1 μ g/mL, and then 0.1mL, 0.2mL, 0.4mL, 1.0mL and 2.0mL of the standard use solution B are taken respectively, placed in a 20mL volumetric flask, diluted to the scale with methanol, shaken to obtain a standard series solution with a concentration of 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100 ng/mL.
5. The assay of claim 4, wherein the 100 μ g/mL standard stock solution is prepared by accurately weighing 10.0mg of torasemide standard to 0.0001g, placing in a 100mL volumetric flask, dissolving and diluting to the scale with methanol, shaking.
6. The detection method according to claim 1, wherein in steps S3 and S4, the condition of the high performance liquid chromatography is that the chromatographic column is Ziziphora CAPCELL-PAK C18A chromatography column or an Acquity UPLC BEH Amide or one with comparable performance; the mobile phase is ammonium acetate solution with A being 10mmol/L, B is acetonitrile, and A: B is 70: 30; the flow rate was 0.5mL/min, the column temperature was 20 ℃ and the amount of sample was 10. mu.L.
7. The detection method according to claim 1, wherein in steps S3 and S4, the condition of the mass spectrum is that the ion source is an electrospray ion source, the detection mode is multiple reaction monitoring, the scanning mode adopts a negative ion mode, the capillary voltage is a negative ion mode: 4500V, ion source temperature 400 deg.C, dry gas flow 12L/min, atomization gas pressure 70psi, sheath gas temperature 250 deg.C, and sheath gas N2(ii) a The air pressure of the air curtain is 35psi, and the auxiliary air pressure is 60 psi; the nozzle voltage is in positive ion mode: 500V; the negative ion mode was 2000V.
8. The detection method according to claim 7, characterized in that: in the mass spectrometry conditions, the peak-off time, the quantitative ion pair, the qualitative ion pair, the fragmentation voltage, the collision voltage and the detection mode of torasemide are respectively 7.2min, 346.8-261.7, 346.8-196.0, 80V, 24V and negative ions.
9. The detection method according to claim 1, wherein in step S4, the standard series solution is measured to obtain a chromatographic peak area of the corresponding standard series solution, and a standard curve is drawn with the concentration of the standard series solution as abscissa and the peak area of the chromatographic peak as ordinate; and according to the chromatographic peak area of the sample obtained by detection, corresponding to the drawn standard curve, obtaining the concentration of the torasemide in the corresponding sample introduction sample.
10. The detection method according to claim 9, wherein the concentration of torasemide in the sample is substituted into the following formula to calculate the content X of torasemide in the food:
wherein: x-the amount of torasemide in a food sample in milligrams per kilogram (mg/kg) or milligrams per liter (mg/L);
c-the concentration of torasemide in the sample solution injected, read from the standard curve, in nanograms per milliliter (ng/mL);
v is the final volume of the sample solution, and the unit is milliliter (mL);
m-the mass or volume represented by the sample solution in grams (g) or milliliters (mL);
k is the dilution multiple;
the calculation results are expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, with the results remaining in three significant digits.
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