CN114577928A - Method for detecting Edwardsient in food by using deuterium internal standard substance - Google Patents
Method for detecting Edwardsient in food by using deuterium internal standard substance Download PDFInfo
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Abstract
A method for detecting Edwardsient in food by using deuterium internal standard substance belongs to the field of food detection. Firstly, preparing standard solutions in a standard way, wherein the standard solutions comprise (i) an Edwardsient standard stock solution, (ii) an Edwardsient standard intermediate solution, (iii) an Edwardsient-D3 standard stock solution, (iv) an Edwardsient-D3 standard intermediate solution, and (v) a standard working solution, and the Edwardsient standard intermediate solution is respectively transferred and taken. Secondly, the sample is pretreated, and the reference condition of liquid chromatography-tandem mass spectrometry is determined. And thirdly, sequentially performing qualitative and quantitative determination based on the reference conditions of the liquid chromatography-tandem mass spectrometry and the prepared standard solution. The method has the advantages of accuracy, high efficiency and high speed.
Description
Technical Field
The invention belongs to the field of food detection, relates to a detection method, and particularly relates to a method for detecting Edwardsient in food by using deuterium internal standard.
Background
Edward sweet (Advantame, N- [ 3- (3-hydroxy-4-methoxyphenyl) propyl-L-a-aspartyl ] -L-phenylalanine-1-methyl ester) ] is a very high sweetener, which is a derivative of aspartame. Edwantan has a similar sensory taste to aspartame, but its sweetness is equivalent to 100 times that of aspartame, and its sweetness is 20000 times that of sucrose, and its bitterness and sourness are very slight. Therefore, the Edwardsienta can replace sugars (sucrose, glucose, fructose and the like) to replace the existing high-power sweetener applied to the food industry, and is a novel multipurpose low-energy sweetener.
At present, liquid chromatography and liquid chromatography tandem mass spectrometry are mainly used for relevant detection of the Edwardsient at home and abroad, more research is used for toxicological relevant research, and literature reports for standard detection methods of the Edwardsient in food are reported, but the method adopts an external standard method for quantification.
In GB2760-2014 national food safety standard food additive use Standard, the Edwards sweet limit relates to various foods such as baked food, seasoning, candy and the like, in the detection of liquid chromatography-tandem mass spectrometry, the existence of Matrix Effect (ME) can influence the ionization efficiency of an electrospray ion source, so that the signal intensity of an analyte is enhanced or weakened to different degrees, and the accuracy of an analysis result is influenced. The matrix effect is inspected in frozen drinks, baked foods and compound seasonings, in order to evaluate the matrix effect, different matrix samples are extracted, purified and subjected to constant volume according to a preset pretreatment method to obtain blank matrix extracting solution, then the extracting solution and the constant volume solution are respectively used for preparing standard stock solution of Edwardten sweet into series of standard working solutions of 1 mug/L, 5 mug/L, 20 mug/L, 50 mug/L, 100 mug/L and 250 mug/L, detection is carried out under the same condition, an organic solvent standard curve and a matrix matching standard curve are respectively drawn, and the matrix effect is evaluated by adopting the ratio (K) of the slope of the matrix matching standard curve to the slope of the organic solvent standard curve: if the K value is between 0.9 and 1.1, the matrix effect is not obvious, the matrix enhancement effect is realized when the K is more than 1.1, and the matrix inhibition effect is realized when the K is less than 0.9. The results show that the matrix effect of the Edwardsient is greatly different among different matrixes, and the specific results are shown in figure 1. The matrix effect is different, if an external standard method is adopted, accurate quantification cannot be realized, and if a matrix curve is adopted, the matrix cannot be accurately quantified because the formula of each manufacturer is different, particularly the matrix is not identical due to the fact that the baked food and the seasonings are different.
The deuterium in-band internal standard method well solves the influence of the matrix in the mass spectrum detector so as to meet the detection requirements of different complex matrixes and achieve accurate quantification.
In the domestic and foreign detection standards and documents, no method for detecting the Edwardsient in the food by utilizing the deuterium internal standard is found. Therefore, it is necessary to establish a method for detecting the content of the Edwardsient in the food by using the deuterium internal standard.
Disclosure of Invention
The invention provides a method for detecting Edwardsient in food by utilizing a deuterium internal standard, which has the advantages of accuracy and high efficiency.
The technical scheme adopted by the invention is as follows:
a method for detecting Edwardsienta in food by utilizing deuterium endomarker comprises the following detection steps:
(1) preparing standard solution
Standard stock solutions of edmunda sweet: the standard substance of Edwardsient is weighed at 10mg (accurate to 0.01mg) and dissolved in methanol in a 10mL volumetric flask and diluted to the mark to obtain a standard stock solution of 1 mg/mL. Storing at 4 deg.C in dark place with a shelf life of 6 months. The Edwardsient is used as an external standard.
Standard intermediate solution of edientol: 1.00mL of the standard stock solution of the Edwardsienta is placed in a 100mL volumetric flask, diluted to the scale with methanol to prepare a standard intermediate solution (1) with the concentration of 10 mug/mL, and stored at 4 ℃ in the dark with the retention period of 1 month. 100 mu L of the standard stock solution of the Edwardsienta is placed in a 100mL volumetric flask, diluted to the scale with methanol to prepare a standard intermediate solution (2) with the concentration of 1 mu g/mL, and stored at 4 ℃ in the dark with the retention period of 1 month.
③ standard stock solution of edmuntin-D3: 1mg of the standard substance of Edwardsient D3 was dissolved in 1mL of methanol to give a 1mg/mL standard stock solution. Storing at 4 deg.C in dark place with a shelf life of 6 months. The Edwardsient-D3 is used as a deuterium internal standard.
Edwardsient-D3 standard intermediate solution: 1.00mL of standard stock solution of the Edwardsient D3 is placed in a 100mL volumetric flask, diluted to the scale with methanol to prepare a standard intermediate solution with the concentration of 10 mug/mL, and stored at 4 ℃ in the dark with the preservation period of 1 month.
Fifthly, standard working solutions, namely respectively transferring 10 muL, 100 muL, 500 muL, 1mL, 2mL and 5mL of the standard intermediate solution (2) of the Edwardsient and 10 muL of the standard intermediate solution of the Edwardsient-D3 (taking an internal standard as a reference), putting the standard working solutions into a 10mL volumetric flask for acetonitrile constant volume, and respectively obtaining standard working solutions with the concentrations of 1.0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL of the series of the Edwardsient, wherein each standard working solution also has the Edwardsient-D310 ng/mL.
(2) Sample pretreatment method
Accurately weighing 2g (accurate to 0.01g) of sample, placing in a 50mL centrifuge tube with a plug, adding 200 μ L of standard intermediate solution of Edwardsient-D3 with the concentration of 10 μ g/mL, adding 3mL of water, performing vortex oscillation for 30s, adding 2g of NaCl, adding 20mL of acetonitrile, centrifuging at 12000r/min for 3min, taking 2mL of acetonitrile layer in the centrifuge tube, adding 50mg of C18Vortex for 1min, centrifuge for 5min at 12000r/min, collecting supernatant, filtering with 0.22 μm filter membrane, and analyzing with liquid chromatography-tandem mass spectrometer.
(3) Reference condition for liquid chromatography-tandem mass spectrometry
Chromatographic reference conditions
a) A chromatographic column: c18Column, 4.6mm × 50mm, 2.7 μm;
b) mobile phase: a: water, B: acetonitrile, gradient elution;
c) flow rate: 0.35 mL/min;
d) column temperature: 30 ℃;
e) sample introduction amount: 2 mu L of the solution;
the gradient elution procedure is shown in table 1.
TABLE 1 gradient elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 93 | 7 |
| 0.5 | 93 | 7 |
| 6 | 65 | 35 |
| 7 | 5 | 95 |
| 8 | 5 | 95 |
| 8.1 | 93 | 7 |
| 10 | 93 | 7 |
Mass spectrum reference condition ion source: electrospray ion source (ESI);
the detection mode is as follows: multiple Reaction Monitoring (MRM);
the scanning mode is as follows: a positive ion mode;
ionization voltage (IS): 5500V;
atomizing Gas (Gas 1): 50 psi;
auxiliary Gas (Gas 2): 50 psi;
air curtain pressure (CUR): 35 psi;
atomization Temperature (TEM): at 500 deg.c.
Other mass spectral parameters are shown in table 2.
TABLE 2 qualitative, quantitative ion and Mass Spectrometry parameters for the Compounds
*And (4) quantifying ion pairs.
(5) Qualitative determination
5.1) determining the standard working solution of the Edwardsient series obtained in the step (1) according to the conditions of high performance liquid chromatography-tandem mass spectrometry to obtain the chromatographic retention time of the Edwardsient in the standard working solution and the chromatographic peaks of a quantitative ion pair and a qualitative ion pair, taking the percentage of the abundance of the quantitative ion pair as the relative abundance of the qualitative ion pair, and recording the relative ion abundance of the Edwardsient in the standard working solution.
And 5.2) determining the sample according to the conditions of high performance liquid chromatography-tandem mass spectrometry to obtain a chromatographic peak corresponding to the Edwardsient in the sample. Wherein, the conditions of the high performance liquid chromatography-tandem mass spectrometry in the process of measuring the sample are the same as 5.1).
Chromatographic peaks of quantitative ion pairs and qualitative ion pairs of the Edwardsient are presented in the sample, and the retention time of the chromatographic peaks is consistent with that of the chromatographic peaks of the Edwardsient in the standard solution (the variation range is within +/-2.5 percent); and the deviation of the relative ion abundance and the relative ion abundance ratio of the ions of the standard solution with the equivalent concentration does not exceed the range specified in table 3, the presence of the edmuntam in the sample can be judged.
Table 3 maximum permissible deviation of relative ion abundance in qualitative confirmation
| Relative abundance (%) | Maximum allowable deviation (%) |
| k＞50％ | ±20 |
| 50％≥k＞20％ | ±25 |
| 20％≥k＞10％ | ±30 |
| k≤10％ | ±50 |
(6) Quantitative determination
Determining the content and corresponding chromatographic peak area of the Edwardsient sweet and the Edwardsient sweet-D3 in the standard working solution of the Edwardsient sweet series obtained in the step (1), and obtaining a standard curve of a deuterium internal standard method by taking the concentration ratio of the Edwardsient sweet to the concentration ratio of the Edwardsient sweet-D3 as an abscissa (x axis) and the peak area ratio of the Edwardsient sweet to the concentration ratio of the Edwardsient sweet-D3 as an ordinate (y axis), as shown in Table 4. The content of the Edwardsienta in the sample solution is determined from a standard curve of an internal standard method.
The content of the Edwarden sweet in the sample is calculated according to the formula (1).
In the formula: x represents the content of the Edwardsient in the sample, and the unit is microgram per kilogram (mu g/kg); c represents the concentration of the Edwardsient in the sample solution read from the standard curve of the internal standard method, ng/mL; v represents the volume of the sample extract, mL; m represents the mass of the sample, g.
The calculation is expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, and the calculation retains three significant digits.
Compared with the prior art, the invention has the beneficial effects that: the method has the advantages of accuracy, high efficiency and high speed.
Drawings
FIG. 1 is a graph showing the matrix effect of Edwarden sweet between different matrices in the prior art;
FIG. 2 is a MRM chromatogram of a blank sample of frozen drink without Edwardsient; wherein, (a) is an Edwardsient quantitative ion, (b) is an Edwardsient qualitative ion, and (c) is an Edwardsient-D3 daughter ion;
FIG. 3 is a MRM chromatogram of a blank sample without Edwardsient baked good; wherein, (a) is an Edwardsient quantitative ion, (b) is an Edwardsient qualitative ion, and (c) is an Edwardsient-D3 daughter ion;
FIG. 4 is an MRM chromatogram of a blank sample without Edwarden sweet flavor; wherein, (a) is an Edwardsient quantitative ion, (b) is an Edwardsient qualitative ion, and (c) is an Edwardsient-D3 daughter ion;
FIG. 5 is an MRM chromatogram of a 1ng/mL standard solution of Edwardsient; wherein, (a) is an Edwardsient quantitative ion, (b) is an Edwardsient qualitative ion, and (c) is an Edwardsient-D3 daughter ion;
FIG. 6 is a schematic diagram of a standard curve of the deuterium depleted isotope labeling method.
FIG. 7 is a schematic diagram of the standard curve of the Edwardten sweet external standard method.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1 determination of Edwarden sweetness in frozen beverages-deuterium in-band internal standard method
(1) Sample pretreatment
Accurately weighing 2g (accurate to 0.01g) of sample, placing in a 50mL centrifuge tube with a plug, adding 200 μ L of standard intermediate solution of Edward's-D3 with the concentration of 10 μ g/mL, adding 3mL of water, performing vortex oscillation for 30s, adding 2g of NaCl, adding 20mL of acetonitrile, performing centrifugation for 3min at 12000r/min, taking 2mL of acetonitrile layer in the centrifuge tube, adding 50mg of C18Vortex for 1min, centrifuge for 5min at 12000r/min, collecting supernatant, filtering with 0.22 μm filter membrane, and analyzing with liquid chromatography-tandem mass spectrometer.
(2) Preparation of standard solution and blank solution
Standard stock solutions of edmunda sweet: the standard substance of Edwardsient is weighed at 10mg (accurate to 0.01mg) and dissolved in methanol in a 10mL volumetric flask and diluted to the mark to obtain a standard stock solution of 1 mg/mL. Storing at 4 deg.C in dark place with a shelf life of 6 months.
Standard intermediate solution of edientol: sucking 1.00mL of the standard stock solution of the Edwardsient in a 100mL volumetric flask, diluting the stock solution to a scale with methanol to prepare a standard intermediate solution (1) with the concentration of 10 mug/mL, and storing the intermediate solution at 4 ℃ in a dark place with the storage life of 1 month. Sucking 100 mu L of the Edwardsient sweet standard stock solution into a 100mL volumetric flask, diluting the stock solution to the scale with methanol to prepare a standard intermediate solution (2) with the concentration of 1 mu g/mL, and storing the intermediate solution at 4 ℃ in the dark, wherein the storage life is 1 month.
③ standard stock solution of edmuntin-D3: 1mg of the standard substance of Edwardsient D3 was dissolved in 1mL of methanol to give a 1mg/mL standard stock solution. Storing at 4 deg.C in dark place with a shelf life of 6 months.
Edwardsient-D3 standard intermediate solution: sucking 1.00mL of standard stock solution of the Edwardsient D3 into a 100mL volumetric flask, diluting the stock solution to a scale with methanol to prepare a standard intermediate solution with the concentration of 10 mug/mL, and storing the intermediate solution at 4 ℃ in a dark place with the storage life of 1 month.
Fifthly, standard working solutions, namely respectively transferring 10 muL, 100 muL, 500 muL, 1mL, 2mL and 5mL of the standard intermediate solution of the Edwardsient-D3, and fixing the volume of acetonitrile in a 10mL volumetric flask to obtain standard working solutions of the Edwardsient series with the concentrations of 1.0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL, wherein each standard working solution also comprises the Edwardsient-D310 ng/mL.
(3) Liquid chromatography-Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) determination
Respectively injecting standard working solution and sample solution of the Edwardsienta series into HPLC-MS/MS for determination, establishing a correction curve, and quantifying by an internal standard method.
Chromatographic conditions are as follows:
a) a chromatographic column: c18Column, 4.6mm × 50mm, 2.7 μm;
b) mobile phase: a: water, B: acetonitrile, gradient elution;
c) flow rate: 0.35 mL/min;
d) column temperature: 30 ℃;
e) sample introduction amount: 2 mu L of the solution;
the gradient elution procedure is shown in table 4.
TABLE 4 gradient elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 93 | 7 |
| 0.5 | 93 | 7 |
| 6 | 65 | 35 |
| 7 | 5 | 95 |
| 8 | 5 | 95 |
| 8.1 | 93 | 7 |
| 10 | 93 | 7 |
Reference condition of mass spectrum
An ion source: electrospray ion source (ESI);
the detection mode comprises the following steps: multiple Reaction Monitoring (MRM);
the scanning mode is as follows: a positive ion mode;
ionization voltage (IS): 5500V;
atomizing Gas (Gas 1): 50 psi;
auxiliary Gas (Gas 2): 50 psi;
air curtain pressure (CUR): 35 psi;
atomization Temperature (TEM): at 500 deg.c.
Other mass spectral parameters are shown in table 5.
TABLE 5 qualitative, quantitative ion and Mass Spectrometry parameters for the Compounds
*And (4) quantifying ion pairs.
The obtained calibration curve by the deuterium internal standard method is shown in table 6 and fig. 6.
TABLE 6 correction curve of deuterium in Edwardten sweet by internal standard method
(4) Recovery and repeatability of spiked samples
And respectively adding three levels of standard solution of the Edwardsiene, namely 10 mu g/kg, 20 mu g/kg and 100 mu g/kg, into the frozen beverage without the Edwardsiene, standing at room temperature for 30min, and then carrying out content determination according to the steps. Each addition level was assayed in parallel 6 times. The results of the measurements were compared to the theoretical additive concentrations to obtain the average recovery and relative standard deviation, the results of which are shown in Table 7.
Table 7 recovery and precision of edmuntin in frozen drinks (n ═ 6)
Table 7 shows that the average recovery rate of the three standard-added levels of Edwardsient is 94.0-102.5%, and the relative standard deviation is 6.6-8.7%, which indicates that the recovery rate and the repeatability of the method of the embodiment both meet the requirements.
(5) Sensitivity of the probe
The lowest detection limit of the actually added samples is taken as the detection limit, and in the embodiment, the detection limit of the Edwardsient in the wine is 10 mug/kg.
Example 2 determination of Edwarden sweetness in baked goods-deuterium in-band labeling method
(1) The preparation method of the baked food sample to be measured, the pretreatment method, the preparation method of the standard solution and the measurement method are the same as those in example 1. The calibration curve by the deuterium strip internal standard method is shown in table 6 and fig. 6.
(2) Recovery and repeatability of spiked samples
And respectively adding standard solutions of the Edwatame with the levels of 10 mu g/kg, 20 mu g/kg and 100 mu g/kg into the baked food without the Edwatame, standing for 30min at room temperature, and then carrying out content measurement according to the steps. Each addition level was assayed in parallel 6 times. The results of the measurements were compared to the theoretical additive concentrations to obtain the average recovery and relative standard deviation, the results of which are shown in Table 8.
Table 8 recovery and precision of edmuntin in baked goods (n ═ 6)
Table 8 shows that the average recovery rate of the three standard-added levels of Edwardsient is 92.1% -105.4%, and the relative standard deviation is 5.4% -8.7%, which indicates that the recovery rate and the repeatability of the method of the embodiment both meet the requirements.
(3) Sensitivity of the probe
The detection limit of the actual added sample is the lowest detection limit, and in this example, the detection limit of the Edwardsient in the baked food is 10 mug/kg.
Example 3 measurement of Edwarden in flavors-deuterium in-band labeling method
(1) The pretreatment method, the preparation method of the standard solution and the measurement method of the seasoning sample to be measured were the same as those in example 1. The calibration curve by the deuterium strip internal standard method is shown in table 6 and fig. 6.
(2) Recovery and repeatability of spiked samples
Adding standard solution of Edwardsiene at three levels of 10 mug/kg, 20 mug/kg and 100 mug/kg into the flavoring without Edwardsiene respectively, standing at room temperature for 30min, and performing content determination according to the steps. Each addition level was assayed in parallel 6 times. The results of the measurements were compared to the theoretical additive concentrations to obtain the average recovery and relative standard deviation, the results of which are shown in Table 9.
TABLE 9 recovery and precision of Edwarden sweet in flavor (n ═ 6)
Table 10 shows that the average recovery rate of the three standard-added levels of Edwardsient is 94.6-104.2%, and the relative standard deviation is 6.7-9.1%, which indicates that the recovery rate and the repeatability of the method of the embodiment both meet the requirements.
(3) Sensitivity of the probe
The lowest detection limit of the actually added samples was set as the detection limit, and in this example, the detection limit of edmuntin in the seasoning was set to 10 μ g/kg.
Comparative example 1 determination of Edwarden sweet in frozen drink-external standard method
(1) Sample pretreatment
The procedure was as in example 1 except that "200. mu.L of standard intermediate solution of Edwardsient-D3 at a concentration of 10. mu.g/mL was not added".
The preparation method and measurement method of the standard solution and the obtained calibration curve by the internal standard method are shown in table 6 and fig. 6.
(2) Preparation of standard solution and blank solution
Standard stock solutions of edmunda sweet: the standard substance of Edwardsient is weighed at 10mg (accurate to 0.01mg) and dissolved in methanol in a 10mL volumetric flask and diluted to the mark to obtain a standard stock solution of 1 mg/mL. Storing at 4 deg.C in dark place with a shelf life of 6 months.
Standard intermediate solution of edientol: sucking 1.00mL of the standard stock solution of the Edwardsient in a 100mL volumetric flask, diluting the stock solution to a scale with methanol to prepare a standard intermediate solution (1) with the concentration of 10 mug/mL, and storing the intermediate solution at 4 ℃ in a dark place with the storage life of 1 month. Sucking 100 mu L of the standard stock solution of the Edwardsienta into a 100mL volumetric flask, diluting the stock solution to the scale with methanol to prepare a standard intermediate solution (2) with the concentration of 1 mu g/mL, and storing the intermediate solution at 4 ℃ in the dark with the retention period of 1 month.
(3) Liquid chromatography-Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) determination
Respectively injecting standard working solution and sample solution of the Edwardsienta series into HPLC-MS/MS for determination, establishing a calibration curve, and quantifying by an external standard method.
The chromatographic conditions and mass reference conditions were the same as in example 1.
Other mass spectral parameters are shown in table 10.
TABLE 10 qualitative, quantitative ion and Mass Spectrometry parameters for Eddometant
The calibration curve obtained by the external standard method is shown in table 11 and fig. 7.
TABLE 11 correction curves of Edwardten sweet external standard method
(4) Recovery and repeatability of spiked samples
And respectively adding three levels of standard solution of the Edwardsiene, namely 10 mu g/kg, 20 mu g/kg and 100 mu g/kg, into the frozen beverage without the Edwardsiene, standing at room temperature for 30min, and then carrying out content determination according to the steps. Each addition level was assayed in parallel 6 times. The results of the measurements were compared to the theoretical additive concentrations to obtain the average recovery and relative standard deviation, the results of which are shown in Table 12.
TABLE 12 recovery and precision of Edwardten sweet in frozen drinks (n ═ 6)
Table 12 shows that the average recovery rate range of the three standard-added levels of the Edwardsient in the external standard method is 67.0-80.2%, and the relative standard deviation range is 5.7-9.3%, which shows that the recovery rate of the Edwardsient in the external standard method is far lower than the recovery rate of the Edwardsient in the deuterium internal standard method which is 94.0-102.5%, and the deuterium internal standard method is more suitable for the determination of the Edwardsient in the sample with complex matrix.
Appendix A information on Compounds
TABLE A.1 Compound Chinese name, English name, CAS number, molecular formula, relative molecular mass
| Serial number | Name of Chinese | English name | CAS number | Molecular formula | |
| 1 | Edward sweet | Advantame | 714229-20-6 | C24H30N2O7·H2O | 476.52 |
| 2 | Edward Wan sweet-D3 | Advantame-D3 | C24H27D3N2O7 | 461.53 |
Appendix A illustrates: CAS number, molecular formula, relative molecular mass of Edwardsient and Edwardsient-D3.
The above-mentioned embodiments only express the embodiments of the present invention, but not should be understood as the limitation of the scope of the invention patent, it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the concept of the present invention, and these all fall into the protection scope of the present invention.
Claims (2)
1. A method for detecting Edwardsienta in food by utilizing deuterium internal standard substance is characterized by comprising the following steps:
(1) preparing standard solution
Standard stock solutions of edmunda sweet: weighing the Edwardsienta standard substance in a volumetric flask, dissolving with methanol and diluting to a scale to obtain a standard stock solution of 1 mg/mL; storing at 4 ℃ in the dark, wherein the Edwardsient is used as an external standard;
standard intermediate solution of edientol: placing the standard stock solution of the Edwardsienta into a volumetric flask, diluting the stock solution to a scale with methanol to prepare a standard intermediate solution (1) with the concentration of 10 mu g/mL, and storing the intermediate solution at 4 ℃ in a dark place; in addition, the standard stock solution of the Edwardsienta is placed in a volumetric flask, diluted to the scale with methanol to prepare a standard intermediate solution (2) with the concentration of 1 mug/mL, and stored away from light at 4 ℃;
③ standard stock solution of edmuntin-D3: dissolving the standard substance of the Edwardsient-D3 by using methanol to obtain a standard stock solution of 1 mg/mL; the Edwardsienta-D3 is stored at 4 ℃ in the dark and is used as a deuterium internal standard;
fourthly, standard intermediate solution of Edwardsient sweet-D3: placing the standard stock solution of the Edwardsient-D3 into a volumetric flask, diluting the stock solution to a scale with methanol to prepare a standard intermediate solution with the concentration of 10 mu g/mL, and storing the intermediate solution at 4 ℃ in a dark place;
fifthly, standard working solutions, namely respectively transferring 10 muL, 100 muL, 500 muL, 1mL, 2mL and 5mL of the standard intermediate solution of the Edwardsient sweet (2) and 10 muL of the standard intermediate solution of the Edwardsient sweet-D3, putting the standard working solutions into a volumetric flask, and fixing the volume by adopting acetonitrile to respectively obtain standard working solutions of the Edwardsient sweet series with the concentrations of 1.0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL, wherein each standard working solution also contains the Edwardsient-D310 ng/mL;
(2) sample pretreatment method
Accurately weighing 2g of sample, placing the sample in a 50mL centrifuge tube with a plug, adding 200 μ L of standard intermediate solution of Edwardsient-D3 with the concentration of 10 μ g/mL, adding 3mL of water, carrying out vortex oscillation for 30s, adding 2g of NaCl, adding 20mL of acetonitrile, and separating at 12000r/minTaking 2mL acetonitrile layer in centrifuge tube for 3min, adding 50mg C18Swirling for 1min, centrifuging at 12000r/min for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and analyzing with liquid chromatography-tandem mass spectrometer;
(3) determining reference conditions for liquid chromatography-tandem mass spectrometry
(5) Qualitative determination
5.1) determining the standard working solution of the Edwardsient series obtained in the step (1) according to the conditions of high performance liquid chromatography-tandem mass spectrometry to obtain the chromatographic retention time of the Edwardsient in the standard working solution and the chromatographic peaks of a quantitative ion pair and a qualitative ion pair, taking the percentage of the abundance of the quantitative ion pair as the relative abundance of the qualitative ion pair, and recording the relative ion abundance of the Edwardsient in the standard working solution;
5.2) determining the sample according to the conditions of high performance liquid chromatography-tandem mass spectrometry to obtain a chromatographic peak corresponding to the Edwardsient in the sample; wherein, the conditions of the high performance liquid chromatography-tandem mass spectrometry in the process of measuring the sample are the same as 5.1);
chromatographic peaks of quantitative ion pairs and qualitative ion pairs of the Edwardsient are presented in the sample, the retention time of the chromatographic peaks is consistent with that of the chromatographic peaks of the Edwardsient in the standard solution, and the variation range is within +/-2.5 percent; and the deviation of the relative ion abundance and the ion relative abundance ratio of the standard solution with the equivalent concentration does not exceed the range specified in table 3, the presence of the edmuntame in the sample can be judged;
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative confirmation
(6) Quantitative determination
Determining the contents of the Edwardsient sweet and the Edwardsient sweet-D3 in the standard working solution of the Edwardsient sweet series obtained in the step (1) and the corresponding chromatographic peak areas, taking the concentration ratio of the Edwardsient sweet to the Edwardsient sweet-D3 as an abscissa and the peak area ratio of the Edwardsient sweet to the Edwardsient sweet-D3 as an ordinate, and obtaining an internal standard method standard curve; determining the content of the Edwardsienta in the sample solution from a standard curve of an internal standard method;
calculating the content of the Edwarden sweet in the sample according to the formula (1);
in the formula: x represents the content of the Edwardsient in the sample, and the unit is mu g/kg; c represents the concentration of the Edwardsient in the sample solution read from the standard curve of the internal standard method, ng/mL; v represents the volume of the sample extract, mL; m represents the mass of the sample, g;
the calculation is expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, and the calculation retains three significant digits.
2. The method for detecting Edwardsient in food products by using deuterium internal standard according to claim 1, wherein in step (3), the reference conditions of liquid chromatography-tandem mass spectrometry are as follows:
chromatographic reference conditions
a) A chromatographic column: c18Column, 4.6mm by 50mm, 2.7 μm;
b) mobile phase: a: water, B: acetonitrile, gradient elution;
c) flow rate: 0.35 mL/min;
d) column temperature: 30 ℃;
e) sample introduction amount: 2 mu L of the solution;
the gradient elution procedure is shown in table 1;
TABLE 1
Reference condition of mass spectrum
An ion source: electrospray ion source ESI;
the detection mode is as follows: multiple reaction monitoring, MRM;
the scanning mode is as follows: a positive ion mode;
ionization voltage IS: 5500V;
atomizing Gas 1: 50 psi;
auxiliary Gas 2: 50 psi;
air curtain air pressure CUR: 35 psi;
atomization temperature TEM: at 500 ℃.
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