CN110632194A

CN110632194A - Method for determining aspartame and alitame in food by reversed phase liquid chromatography mixed standard sample adding method

Info

Publication number: CN110632194A
Application number: CN201910878836.4A
Authority: CN
Inventors: 高向阳
Original assignee: Zhengzhou University of Science and Technology
Current assignee: Zhengzhou University of Science and Technology
Priority date: 2019-08-27
Filing date: 2019-09-17
Publication date: 2019-12-31

Abstract

The method comprises the steps of respectively injecting the mixed standard liquid of the alitame and the aspartame and the mixed standard liquid of the alitame and the aspartame added with the liquid to be tested into a liquid chromatograph under the same liquid chromatogram condition, and determining whether the food to be tested contains the alitame or the aspartame according to whether the component signals have increasing changes under the same retention time condition; comparing the peak-out signal of the mixed standard solution with the peak-out signal difference of the mixed standard solution added with the solution to be tested, quantifying, and determining the content of the alitame and the aspartame; the method has the advantages of high working efficiency, low cost, less interference and high accuracy.

Description

Method for determining aspartame and alitame in food by reversed phase liquid chromatography mixed standard sample adding method

Technical Field

The invention relates to the field of food detection, in particular to a method for determining aspartame and alitame in food by a reversed-phase liquid chromatography mixed standard sample adding method.

Background

Aspartame and alitame are commonly added sweeteners in food, and the sweetness is 200 times and 2000 times that of sucrose, respectively. The compound use can realize synergistic effect, reduce cost, improve taste and improve the stability of the sweet taste of the sweetener, and the sweetener has high sweetness, good taste quality, long storage period and no calorie. However, excessive intake of the drug causes adverse effects such as nervous system disorders and destruction of human liver, and therefore, it is one of the indexes commonly measured for food.

At present, methods for measuring aspartame and alitame in food comprise liquid chromatography (GB 5009.263-2016), liquid chromatography-mass spectrometry, ion chromatography and the like, and the methods are usually quantitative analysis by an external standard method, an internal standard method or a normalization method and the like.

The external standard method is to draw a working curve by using 5-7 standard solutions with different concentrations outside a sample to be tested, and then to measure the solution to be tested. And according to the signal value generated by the component to be detected in the liquid to be detected, the quantity of the component to be detected is obtained by searching the working curve or the working curve is substituted into a regression curve equation to obtain the content of the component to be detected. The method mainly has the following defects: (1) 5-7 parts of standard solutions with different concentrations need to be prepared, thereby wasting time and labor and having high cost; (2) the method is characterized in that a plurality of standard solutions, the standard solutions and the solution to be measured are separately measured, simultaneous and synchronous measurement cannot be realized, and when the conditions of an instrument and environmental factors (such as temperature, humidity, atmospheric pressure, voltage, operation factors, instrument working performance and the like) are inevitably fluctuated, certain difference exists, the measurement result is influenced to different degrees, and certain errors are introduced; (3) the background of the standard solution and the background of the solution to be detected are different, the difference is large, and the interference degree or the possible side reaction degree on the detection result are different. The standard solution and the solution to be measured are separately measured, and interference cannot be completely eliminated by a blank deduction method; (4) certain drawing errors exist when the standard curve is drawn by using a graph method; the standard curve needs to be corrected or redrawn frequently, and if a plurality of components to be measured exist in a sample, a plurality of standard curves need to be drawn, so that the workload and the cost input are increased; (5) the standard curve method requires that the sample volumes of the standard liquid and the liquid to be detected are very accurate, the operation of the instrument needs to be strictly controlled, the heights are consistent, and the method is difficult to achieve in actual work.

The internal standard substance selected by the internal standard method is a pure substance which does not exist in the sample, is stable and easily obtained, has physical and chemical properties similar to those of each component to be measured, is uniformly dissolved with the sample, and does not generate any chemical reaction. The operation conditions and sample injection quantity of the internal standard method have little influence on the analysis result, but the relative correction factors of each component to be detected and the internal standard substance are obtained by using the standard solution in advance and can be substituted into the corresponding formula for calculation; moreover, for complex samples, it is sometimes difficult to find a suitable internal standard substance, and it is impossible to determine whether the sample does not contain the used internal standard substance, so that detailed work cannot be carried out in time.

The normalization method is a relative content analysis method, the operation and the sample amount have little influence on the analysis result, but all components in the sample are required to generate peaks and measurement signals, and the relative content of each component in the sample is 100%. This measurement method requires measurement of the respective quantitative correction factor and relative correction factor using a standard solution of each component, requires a large amount of preparation work, and when it is not guaranteed that all components in a sample have peaks or lack a correction factor of an unknown peak, quantitative analysis cannot be performed by a normalization method, and the application is limited.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for qualitatively and quantitatively determining aspartame and alitame in food simultaneously by using a reversed phase liquid chromatography mixing standard sample adding method which is low in cost, high in determination speed, less in interference and wide in application range, aiming at overcoming the defects in the prior art.

The technical scheme of the invention is as follows:

a method for measuring aspartame and alitame in food by a reversed phase liquid chromatography mixed standard sample adding method is characterized in that: the method comprises the following steps of (1) determining whether the food to be detected contains aspartame and alitame by using a reversed-phase liquid chromatography mixed standard sample adding method, and determining the content of aspartame and alitame in the food to be detected at the same time, wherein the method comprises the following steps:

(1) selecting food to be detected;

(2) pretreating the food to be tested to obtain a test solution;

(3) preparing a standard solution: respectively preparing aspartame standard stock solution and alitame standard stock solution, diluting the aspartame standard stock solution and the alitame standard stock solution step by step to respectively obtain aspartame series standard solution and alitame series standard solution, mixing the prepared aspartame series standard solution and the alitame series standard solution to obtain alitame and aspartame mixed standard solution, wherein the concentrations of alitame and alitame in the mixed standard solution are the same;

(4) and (3) chromatographic analysis: taking two identical mixed standard solutions into centrifuge tubes A and B, adding the test solution into the centrifuge tube B, mixing uniformly, and performing chromatographic analysis on the mixed standard solution in the centrifuge tube A under the same chromatographic condition to obtain the retention time t of aspartame_s1And corresponding peak-out signal h_s1Retention time t of alitame_s2And corresponding peak-out signal h_s2(ii) a Carrying out chromatographic analysis on the mixed standard solution in the centrifuge tube B to obtain the retention time t of the aspartame₁And corresponding peak-out signal h_s1+x1Retention time t of alitame₂And corresponding peak-out signal h_s2+x2；

Determination of whether aspartame and alitame are contained:

when t is_s1＝t₁，h_s1+x1-h_s1>0, the food to be detected contains aspartame; when t is_s2＝t₂， h_s2+x2-h_s2>0, the food to be detected contains the alitame;

the method for measuring the content of aspartame and alitame comprises the following steps:

the method for measuring the aspartame content comprises the following steps:

h is to be_s1+x1And h_s1Substituting the mass m of the aspartame in the food to be tested into the following formula_x1And the mass fraction w of aspartame₁：

The method for measuring the content of the alitame comprises the following steps:

h is to be_s2+x2And h_s2Substituting the mass m of the alitame in the food to be detected into the following formula_x2And mass fraction w of alitame₂：

In the formula, m_s1M is the mass of aspartame in the mixed standard solution used_s2The mass of the alitame in the mixed standard liquid is used;

h_s1to correspond to m_s1Measured peak signal, h_s2To correspond to m_s2(iii) the measured peaked signal;

m_x1the mass m of aspartame in the liquid to be tested added in the mixed standard liquid_x2The quality of the alitame in the liquid to be tested is added into the mixed standard liquid;

h_s1+x1is corresponding to (m)_s1+m_x1) (iii) the measured peaked signal; h is_s2+x2Is corresponding to (m)_s2+m_x2) (iii) the measured peaked signal;

m_{sample (A)}Adding the mass of the food to be tested corresponding to the liquid to be tested in the centrifuge tube B;

w₁the mass fraction of the aspartame in the food to be detected is shown; w is a₂The mass fraction of the alitame in the food to be tested is shown.

And (2) selecting the food with the mass fraction range of the alitame and the aspartame being between 0.1mg/kg and 41mg/kg in the step (1).

The food to be detected in the step (1) is selected from any one of coca cola, chrysanthemum crystal, yoghourt, cake, concentrated fruit juice, chocolate products and preserves.

And (2) treating the food to be tested in the step (2) by carrying out vortex or/and ultrasonic treatment, centrifugation and volume fixing on liquid or non-liquid food, and treating the supernatant through a filter membrane to obtain the liquid to be tested.

The concentration of the mixed standard solution of aspartame and alitame in the step (3) is any one of 20 mug/mL, 10 mug/mL, 5 mug/mL, 2.5 mug/mL, 1 mug/mL and 0.5 mug/mL.

And (4) the peak signal in the step (4) is the peak height or the peak area.

In the step (4), the volume ratio of the liquid to be tested to the mixed standard liquid added into the centrifuge tube B is 20: 9.

In the step (4), the chromatographic conditions are that the mobile phase is methanol: water-40: 60, the flow rate is 0.8mL/min, and the wavelength is 200 nm.

The invention has the beneficial effects that: the method comprises the steps of respectively injecting the mixed standard liquid of the alitame and the aspartame and the mixed standard liquid of the alitame and the aspartame added with the liquid to be tested into a liquid chromatograph under the same liquid chromatogram condition, and determining whether the food to be tested contains the alitame or the aspartame according to whether the component signals have increasing changes under the same retention time condition; the peak signal difference of the mixed standard solution and the peak signal difference of the mixed standard solution added with the solution to be tested are compared for quantification, and the content of the alitame and the aspartame is determined, so that (1) the working efficiency is high: the mixed standard solution and multiple components in the food to be measured are subjected to qualitative and quantitative analysis simultaneously under the same system, the same background, the same machine, the same method, the same synchronization and the same conditions, corresponding signals of the components in the solution are only required to be measured, a standard curve is not required to be drawn, quantitative correction factors of the components are not required to be measured, time is saved, the measurement result can be quickly obtained without peaks of all the components and internal standard substance selection, the method is simple and quick, and the working efficiency is greatly improved; (2) the cost is low: the used standard solution, the to-be-detected solution and the reagent amount are less; (3) less interference and high accuracy: the separation analysis and the simultaneous determination of the standard solution and the test solution under the completely same condition are realized, and the interference caused by environmental fluctuation factors and the test solution is reduced; in addition, a blank solution is not required to be measured, blank signals are offset in the measurement process, deduction is basically obtained in a calculation formula, and the accuracy is greatly improved.

Drawings

FIG. 1 is a retention time chromatogram of a mixed standard solution of aspartame and alitame;

FIG. 2 is a chromatogram of a mixed standard solution of aspartame and alitame at 1. mu.g/mL;

FIG. 3 is a chromatogram of a standard solution of aspartame and alitame mixed with a coca-cola test solution;

FIG. 4 is a chromatogram of a test solution to be tested after adding a chrysanthemum crystal into a mixed standard solution of aspartame and alitame;

FIG. 5 is a chromatogram of a mixed standard solution of aspartame and alitame added to a test solution of yogurt;

FIG. 6 is a chromatogram of a mixture of aspartame and alitame mixed standard solution added with a liquid to be tested for cake;

FIG. 7 is a chromatogram of a concentrated juice test solution added to a mixed standard solution of aspartame and alitame;

FIG. 8 is a chromatogram of a chocolate product test solution added with a mixed standard solution of aspartame and alitame;

FIG. 9 is a chromatogram of a mixed standard solution of aspartame and alitame added with a test solution to be tested for sweetmeat;

FIG. 10 is a standard curve for aspartame;

FIG. 11 is a standard curve for alitame.

Detailed Description

Reagents and instruments involved in the experiment are all the prior art, methanol and pure chromatogram are from Shanghai' an spectral experiment science and technology company, Inc.; ethanol, guaranteed reagent, from Shanghai' an spectral laboratory science and technology, Inc., using Yibao purified water (conductivity: 2.23 μ s/cm).

Ethanol-water (2+ 1): 40mL of ethanol was taken and mixed well with 20mL of water.

80% methanol water: 80mL of methanol was taken and made up to 100mL with water.

50% methanol water: 50mL of methanol was taken and made up to 100mL with water.

Aspartame standard substance (C)₁₄H₁₈N ₂O₅，CAThe number S: 22839-47-0) at a concentration of 95% from BePune laboratory science and technology, Inc.

Alitame standard substance (C)₁₄H₂₅N₃O₄S, CAS number: 80863-62-3), the content is 99.2%, and is from Shanghai' an spectral laboratory science and technology Co.

A100250 Multi-tube vortex Mixer, Yuesang science and technology Co., Ltd;

electronic balance (one hundred thousand): MS150/MS205DU, Kailong instruments, Inc. of Changsha;

dynamica V18R desk refrigerated centrifuge, beijing pentazhou eastern scientific and technological development ltd;

SB25-12DTD ultrasonic cleaning machine, Ningbo Xinzhi Biotech Co., Ltd;

high performance liquid chromatograph: utimate3000(2017LH131), saimer fly science ltd; a chromatographic column: hypersil GOLD C18150 mm X4.6 mm (SN: 10567553).

Preparation of aspartame and alitame standard stock solution (0.50 mg/mL): 0.02632g of aspartame (accurate to 0.0001g) is weighed, 0.02520g of alitame (accurate to 0.0001g) is weighed, the aspartame and the alitame are respectively dissolved into a 50mL volumetric flask by water and are subjected to constant volume to scale, aspartame standard stock solution with the concentration of 0.50mg/mL and alitame standard stock solution with the concentration of 0.50mg/mL are respectively obtained and are placed in a refrigerator at about 4 ℃ for storage, and the prepared standard stock solution has the validity period of 90 d.

Preparing a mixed standard solution of aspartame and alitame: diluting the alitame standard stock solution with water step by step to obtain alitame series standard solutions with the concentrations of 150 mu g/mL, 100 mu g/mL, 90 mu g/mL, 80 mu g/mL, 75 mu g/mL, 70 mu g/mL, 60 mu g/mL, 50 mu g/mL, 40 mu g/mL, 30 mu g/mL, 20 mu g/mL, 10 mu g/mL, 5 mu g/mL, 2 mu g/mL, 1 mu g/mL and 0.5 mu g/mL respectively; diluting the aspartame standard stock solution with water step by step to obtain aspartame series standard solutions with the concentrations of 150 mug/mL, 100 mug/mL, 90 mug/mL, 80 mug/mL, 75 mug/mL, 70 mug/mL, 60 mug/mL, 50 mug/mL, 40 mug/mL, 30 mug/mL, 20 mug/mL, 10 mug/mL, 5 mug/mL, 2 mug/mL, 1 mug/mL and 0.5 mug/mL respectively; the alitame series standard solution with the same concentration and the aspartame series standard solution with the same concentration are mixed in equal volume to prepare mixed standard solution with half concentration, namely, the alitame standard solution with 1 volume and 100 mu g/mL are mixed with the alitame standard solution with 1 volume and 100 mu g/mL are mixed to prepare mixed standard solution of alitame and aspartame with 50 mu g/mL. The same method is used to prepare a mixed standard solution of alitame and aspartame, such as 45 mu g/mL, 40 mu g/mL, 35 mu g/mL, 30 mu g/mL, 25 mu g/mL, 20 mu g/mL, 10 mu g/mL, 5 mu g/mL, 2.5 mu g/mL, 1 mu g/mL, 0.5 mu g/mL and the like, and the mixed standard solution is stored in a refrigerator at about 4 ℃ for 30 days.

The method can also be used for uniformly mixing the alitame series standard solution and the aspartame series standard solution with different concentrations and different volumes to obtain the mixed standard solution of the alitame and the aspartame, for example, the alitame standard solution with 1 volume of 150 mu g/mL and the aspartame standard solution with 2 volumes of 75 mu g/mL are uniformly mixed to obtain the mixed standard solution of the alitame and the aspartame with 50 mu g/mL.

Because the concentrations of the alitame and the aspartame in the mixed standard solution of the alitame and the aspartame are the same, the concentration of the mixed standard solution of the alitame and the aspartame is the concentration of the alitame and the aspartame in the mixed standard solution.

Selecting a mobile phase as methanol: water-40: 60, the flow rate was 0.8 mL/min.

Based on the detection sensitivity of aspartame and alitame in an ultraviolet light region, 200nm wavelength is selected for detection, and the retention time of aspartame and alitame is determined as shown in figure 1, so that the aspartame and alitame have good peak appearance.

Selecting the food with the mass fraction of the alitame and the aspartame ranging from 0.1mg/kg to 41 mg/kg. The commercial coca cola, cakes, plain yogurt, chrysanthemum crystal solid beverage, concentrated fruit juice, chocolate products and candied fruits are purchased from Zheng Zhou city, Erqidistrict Mazhai town and many supermarkets.

Example 1

Weighing about 5.000g (accurate to 0.001g) of coca cola carbonated beverage sample in a 50mL beaker, removing carbon dioxide on an ultrasonic oscillator, transferring all the samples into a 25mL volumetric flask, fixing the volume with water, uniformly mixing, enabling the concentration of the measured components to reach the balance, centrifuging for 5min by a 4000r/min centrifuge, enabling the concentrations of aspartame and alitame in a supernatant and a subnatant to be the same, precipitating particle substances possibly existing in a sample by centrifugation, and filtering the supernatant by using a 0.45-micrometer water system filter membrane to obtain the liquid to be tested for chromatographic analysis.

Taking two identical centrifuge tubes A, B, respectively adding 45 mu L of aspartame and alitame mixed standard solution with the concentration of 1 mu g/mL, adding no to-be-tested solution into the centrifuge tube A, and fixing the volume to 1 mL; and adding 100 mu L of coca-cola test solution into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Then, analyzing by chromatograph, diluting to constant volume by centrifugal tube A to obtain mixed standard solution chromatogram map 2, wherein the retention time t of aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; chromatogram of the mixed standard solution and coca cola to be tested obtained by diluting with constant volume in centrifuge tube B, shown in figure 3, wherein the retention time t of aspartame₁3.640min, peak height h_s1+x19.925mAU, retention time t of alitame₂6.527min, peak height h_s2+x26.242 mAU.

From the changes of the peak heights in FIGS. 2 and 3, it was observed that the addition of the coca-cola test solution resulted in an increase in the relative peak heights of aspartame and alitame with respect to the mixed standard solution at the same retention time, within the allowable error range t_s1＝t₁，h_s1+x1-h_s1>0, the coca cola contains aspartame; t is t_s2＝ t₂，h_s2+x2-h_s2>0, the Cola contains alitame, so that the qualitative analysis is carried out.

Let the mass of the alitame or aspartame in the mixed standard solution be m_sThe generated measurement signal is h_sSince m is measured under certain conditions_sAnd h_sIn proportion within a certain range, the following are:

m_s＝k_s×h_s (1)

when the coca cola sample to be tested is added into the mixed standard solution, if the sample does not contain alitame or alitameAspartame with mass of m_sThe generated signal is still h_s(ii) a If the sample contains alitame or aspartame, the mass is set as m_xThe total mass of the alitame or aspartame in the mixed solution is (m)_s+m_x) The measured signal is increased and is set to h_s+xAt the same time have

m_s+m_x＝k_s+x×h_s+x (2)

When measured under the same conditions, k in the formulae (1) and (2) is the same, and thus the division of the formula (1) by the formula (2) is:

therefore, it is not only easy to use

Namely, it is

(3) In the formula, except m_xOuter, m_sIs known, h_s+x、h_sCan be read from the instrument. Therefore, the mass m of the component to be measured in the liquid to be measured can be quickly obtained by measuring the measurement signal, namely the peak height h of the chromatographic peak_xAnd then the mass fraction w of the component in the food sample to be tested is as follows:

as can be seen from the above, when the concentrations of aspartame and alitame in the standard solution added into the centrifuge tube A, B and mixed therein are 1 μ g/mL and the volume thereof is 45 μ L, the mass of aspartame and alitame is 0.045 μ g, i.e., m_s1＝ 0.045μg、m_s20.045 μ g; because 5g of coca cola is pretreated to obtain 25mL of coca cola test solution, 100 muL of test solution m is added into the centrifuge tube B_{Sample (A)}Adding into 100 μ L centrifuge tube BThe total test solution is 25mL to 25000 μ L corresponding to the quality of the food to be tested

Calculate Coca Cola in 100. mu.L of the test solution in centrifuge tube B according to equation (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of aspartame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the coca-cola food according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

the same procedure was followed by 6 replicates and calculations, the results of which are shown in Table 1.

The same procedure was carried out for each of 6 replicates and calculations, with different concentrations of the mixed standard solutions, and the results are shown in table 2: when the concentration of the mixed standard solution is 20 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 9.0 muL, and the volume of the solution to be tested added into the centrifuge tube B is 20.0 muL; when the concentration of the mixed standard solution is 15 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 9.0 muL, and the volume of the solution to be tested added into the centrifuge tube B is 20.0 muL; when the concentration of the mixed standard solution is 10 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 9.0 muL, and the volume of the solution to be tested added into the centrifuge tube B is 20.0 muL; when the concentration of the mixed standard solution is 5 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 18.0 muL, and the volume of the solution to be tested added into the centrifuge tube B is 40.0 muL; when the concentration of the mixed standard solution is 2.5 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 18.0 muL, and the volume of the liquid to be tested added into the centrifuge tube B is 40.0 muL; when the concentration of the mixed standard solution is 1 mug/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 45.0 muL, and the volume of the liquid to be tested added into the centrifuge tube B is 100 muL; when the concentration of the mixed standard solution is 0.5. mu.g/mL, the volume of the mixed standard solution added into the centrifuge tube A, B is 90. mu.L, and the volume of the solution to be tested added into the centrifuge tube B is 200. mu.L.

Example 2

Weighing about 1.000g (accurate to 0.001g) of chrysanthemum crystal sample in a 50mL beaker, adding 10mL of water, carrying out ultrasonic oscillation extraction for 20min, transferring the extracting solution into a 25mL volumetric flask, adding 10mL of water into residues, carrying out ultrasonic extraction for 10min, transferring the two extracting solutions into the same 25mL volumetric flask, carrying out constant volume with water, uniformly mixing to obtain a measured component with balanced concentration, centrifuging for 5min by a 4000r/min centrifuge, wherein the concentrations of aspartame and alitame in a supernatant and a subnatant are the same, centrifuging to precipitate possible granular substances in a sample, taking the supernatant, filtering by a 0.45-micrometer water-based filter membrane to obtain a to-be-measured solution, and carrying out chromatographic analysis.

Taking two identical centrifuge tubes A, B, respectively adding 45 mu L of aspartame and alitame mixed standard solution with the concentration of 1 mu g/mL, adding no to-be-tested solution into the centrifuge tube A, and fixing the volume to 1 mL; and adding 100 mu L of chrysanthemum crystal to-be-tested liquid into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by a chromatograph, and performing constant volume by a centrifugal tube A to obtain a mixed standard liquid chromatogram map 2, t_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; a chromatogram chart of the mixed standard liquid and the chrysanthemum crystal to be tested obtained by the constant volume of the centrifugal tube B, which is shown in figure 4, wherein the retention time t of the aspartame₁3.647min, peak height h_s1+x15.281mAU, retention time t of alitame₂6.540min, peak height h_s2+x22.843 mAU.

From the variation of the peak heights in FIGS. 2 and 4, it is observed that the relative peak heights of aspartame and alitame after adding the chrysanthemum crystal test solution are increased relative to the mixed standard solution under the same retention time, and the retention time is t within the allowable error range_s1＝t₁，h_s1+x1-h_s1>0, the chrysanthemum crystal contains aspartame; t is t_s2＝ t₂，h_s2+x2-h_s2>0, the chrysanthemum crystal contains alitame, so that qualitative analysis is carried out.

In the same manner as in example 1, when the concentrations of aspartame and alitame in the standard solution added to the centrifuge tube A, B were 1. mu.g/mL and the volume was 45. mu.L, the mass of aspartame and alitame was 0.045. mu.g, i.e., m is_s1＝0.045μg、m_s20.045 μ g; because 1g of chrysanthemum crystal is pretreated to obtain 25mL of chrysanthemum crystal test solution, the test solution added into the centrifuge tube B is 100 mu L, m_{Sample (A)}To add the mass of the food to be tested in 100 μ L of the test solution in the centrifuge tube B, the total test solution to be tested is 25mL to 25000 μ L.

Calculating the content of the chrysanthemum crystal in 100 mu L of test solution in the centrifuge tube B according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the chrysanthemum crystal according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

Example 3

Weighing 5.000g (accurate to 0.001g) of yogurt sample into a 50mL centrifuge tube, adding 10mL of ethanol, precipitating protein in the yogurt sample, covering a cover tightly, slightly inverting the centrifuge tube for 5 times (shaking is impossible), uniformly mixing the centrifuge tube, vortexing for 10s, standing for 1min, centrifuging for 5min by a 4000r/min centrifuge, centrifuging to conveniently filter a small amount of filter residue such as protein precipitate, taking supernatant, filtering into a 25mL volumetric flask, washing the filter residue with 8mL of ethanol-water (2+1), centrifuging, transferring the supernatant into the same volumetric flask, then diluting to 25mL with ethanol-water (2+1), uniformly mixing, filtering with a 0.45 mu m organic filter membrane to obtain a solution to be tested, and using the solution to be analyzed by liquid chromatography.

Taking two identical centrifuge tubes A, B, respectively adding 45 mu L of aspartame and alitame mixed standard solution with the concentration of 1 mu g/mL, adding no to-be-tested solution into the centrifuge tube A, and fixing the volume to 1 mL; and adding 100 mu L of yoghourt to-be-tested liquid into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by using a chromatograph, and performing constant volume by using a centrifugal tube A to obtain a mixed standard solution chromatogram map 2, wherein the retention time t of the aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; a chromatogram chart of the mixed standard liquid and the yoghourt to be tested liquid obtained by the constant volume of the centrifugal tube B is shown in figure 5, wherein the retention time t of the aspartame₁3.633min, peak height h_s1+x16.247mAU, retention time t of alitame₂6.497min, peak height h_s2+x22.891 mAU.

From the variation of the peak heights in FIGS. 2 and 5, it is observed that after the yogurt test solution is added, the aspartame and the alitame are increased relative to the mixed standard solution under the same retention time, and within the error range t allowed by the retention time_s1＝t₁，h_s1+x1-h_s1>0, the yoghourt contains aspartame; t is t_s2＝ t₂，h_s2+x2-h_s2>0, the yoghourt contains the alitame, and the qualitative analysis is carried out according to the alitame.

In the same manner as in example 1, when the concentrations of aspartame and alitame in the standard solution added to the centrifuge tube A, B were 1. mu.g/mL and the volume was 45. mu.L, the mass of aspartame and alitame was 0.045. mu.g, i.e., m is_s1＝0.045μg、m_s20.045 μ g; because 5g of yoghourt is pretreated to obtain 25mL of yoghourt to-be-tested solution, the sample solution added into the centrifuge tube B is 100 mu L, m_{Sample (A)}Adding the mixture into a mass of food to be tested corresponding to 100 mu L of liquid to be tested in a centrifuge tube BThe total volume of the test solution to be tested was 25 mL-25000. mu.L.

Calculating the content of the yoghourt in the centrifuge tube B in 100 mu L of the test solution according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the yoghourt according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

Example 4

The cake sample is crushed uniformly by a food processor, 1.000g (accurate to 0.001g) of the cake sample is weighed into a 50mL centrifuge tube, 12mL of 50% methanol aqueous solution is added, the cake sample is swirled uniformly, ultrasonic extraction is carried out for 10min, the cake sample is centrifuged for 5min by a 4000r/min centrifuge, the centrifugation plays a role in filtering solid residues, and the supernatant is transferred into a 25mL volumetric flask. Adding 10mL of 50% methanol aqueous solution into the residue, uniformly mixing by vortex, extracting for 5min by ultrasonic wave, fully extracting the component to be detected, centrifuging for 5min by a 4000r/min centrifuge, centrifuging to filter the solid residue, transferring the supernatant into the same 25mL volumetric flask, completely extracting the component to be detected in the sample to be detected into the supernatant, fixing the volume by water, uniformly mixing, and filtering by a 0.45-micrometer organic system filter membrane to obtain the liquid to be detected for liquid chromatography analysis.

Taking two identical centrifuge tubes A, B, respectively adding 45 mu L of aspartame and alitame mixed standard solution with the concentration of 1 mu g/mL, adding no to-be-tested solution into the centrifuge tube A, and fixing the volume to 1 mL; and adding 100 mu L of cake to-be-tested liquid into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by using a chromatograph, and performing constant volume by using a centrifugal tube A to obtain a mixed standard solution chromatogram map 2, wherein the retention time t of the aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; chromatogram of the mixed standard liquid and the cake to be tested obtained by diluting with constant volume in centrifuge tube B as shown in FIG. 6, wherein the aspartameRetention time t₁3.720min, peak height h_s1+x1At 9.257mAU, the retention time t of alitame₂6.730min, peak height h_s2+x26.495 mAU.

From the changes of the peak heights in FIGS. 2 and 6, it was observed that the relative peak heights of aspartame and alitame after the addition of the pastry test solution to the mixed standard solution were increased at the same retention time, and within the tolerance range t allowed by the retention time_s1＝t₁，h_s1+x1-h_s1>0, the pastry contains aspartame; t is t_s2＝ t₂，h_s2+x2-h_s2>0, the cake contains alitame, so that qualitative analysis is carried out.

In the same manner as in example 1, when the concentrations of aspartame and alitame in the standard solution added to the centrifuge tube A, B were 1. mu.g/mL and the volume was 45. mu.L, the mass of aspartame and alitame was 0.045. mu.g, i.e., m is_s1＝0.045μg、m_s2The sample solution added into the centrifuge tube B is 100 mu L, m, because 1g of cake is pretreated to obtain 25mL of cake sample solution to be tested_{Sample (A)}In order to add 100 μ L of the liquid to be tested to the centrifuge tube B, the total liquid to be tested is 25mL to 25000 μ L.

Calculating the content of 100 mu L of cake in the centrifuge tube B according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the pastry food according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

Example 5

Weighing 2.000g of concentrated fruit juice (accurate to 0.001g) in a 50mL centrifuge tube, adding 10mL of water, carrying out ultrasonic oscillation extraction for 20min, fully extracting components to be detected, transferring the extracting solution into a 25mL volumetric flask, adding 10mL of water into residues, carrying out ultrasonic extraction for 10min, transferring the two extracting solutions into the same 25mL volumetric flask, carrying out constant volume with water, uniformly mixing, leading the components to be detected to reach concentration balance, centrifuging for 5min by a 4000r/min centrifuge, leading the concentrations of aspartame and alitame in supernatant and subnatant to be the same, centrifuging to precipitate possible granular substances in a sample, taking the supernatant, filtering by a 0.45-micrometer water system filter membrane to obtain a liquid to be detected, and carrying out chromatographic analysis.

Taking two identical centrifuge tubes A, B, respectively adding 45 mu L of aspartame and alitame mixed standard solution with the concentration of 1 mu g/mL, adding no to-be-tested solution into the centrifuge tube A, and fixing the volume to 1 mL; and adding 100 mu L of concentrated juice to be tested into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by using a chromatograph, and performing constant volume by using a centrifugal tube A to obtain a mixed standard solution chromatogram map 2, wherein the retention time t of the aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; a chromatogram of the mixed standard liquid and the concentrated juice to be tested obtained by the centrifuge tube B with constant volume is shown in figure 7, wherein the retention time t of the aspartame₁3.647min, peak height h_s1+x116.533mAU, retention time t of alitame₂6.570min, peak height h_s2+x212.489 mAU.

From the changes of the peak heights in FIGS. 2 and 7, it was observed that the relative peak heights of aspartame and alitame after the addition of the concentrated juice to-be-tested solution to the mixed standard solution were increased at the same retention time, and t was within the tolerance range allowed by the retention time_s1＝t₁，h_s1+x1-h_s1>0, the concentrated juice contains aspartame; t is t_s2＝t₂，h_s2+x2-h_s2>0, the concentrated juice contains the alitame, so that qualitative analysis is carried out.

In the same manner as in example 1, when the concentrations of aspartame and alitame in the standard solution added to the centrifuge tube A, B were 1. mu.g/mL and the volume was 45. mu.L, respectively, the mass of aspartame and alitame was 0.045. mu.g, i.e., m is_s1＝0.045μg、m_s2Is 0.045 μ g, 2g of concentrated juice is pretreated to obtain25mL of concentrated juice to be tested, and 100 μ L of sample solution m added into centrifuge tube B_{Sample (A)}In order to add 100 μ L of the test solution to the centrifuge tube B, the total test solution is 25mL to 25000 μ L.

Calculating the concentration of the juice in the centrifuge tube B in 100 mu L of the test solution according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the concentrated fruit juice food according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

Example 6

Homogenizing by a food processing machine according to the mass ratio of the chocolate product to water of 1: 4, weighing 5.000g (accurate to 0.001g) of the chocolate product, homogenizing in a 25mL centrifuge tube, adding 10mL of water, performing ultrasonic oscillation extraction for 20min, standing for 1min, centrifuging for 5min at 4000r/min, transferring the supernatant into a100 mL separating funnel, adding 10mL of water into the centrifuge tube, performing ultrasonic oscillation extraction for 10min, standing and centrifuging, transferring the supernatant into the separating funnel again, adding 15mL of n-hexane into the separating funnel, shaking for 30s, standing for about 5min, making aspartame and alitame insoluble in a fat-soluble solvent, putting the lower-layer water phase into a 25mL volumetric flask, completely extracting the component to be tested in the sample to be tested into the lower-layer water phase, fixing the volume to scale by using a water filter membrane of 0.45 mu m, and filtering to obtain the liquid to be tested, wherein the liquid is used for liquid chromatography analysis.

Taking two identical centrifuge tubes A, B, adding 45 mu L of mixed standard solution of aspartame and alitame at a ratio of 1 mu g/mL, adding no liquid to be tested into centrifuge tube A, and metering to a constant volume of 1 mL; and adding 100 mu L of chocolate product to-be-tested liquid into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by using a chromatograph, and performing constant volume by using a centrifugal tube A to obtain a mixed standard solution chromatogram map 2, wherein the retention time t of the aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; obtaining a mixed standard liquid and a chocolate product to be tested liquid chromatogram map 8 by using a centrifugal tube B to perform constant volume, wherein the retention time t of the aspartame₁3.713min, peak height h_s1+x113.235mAU, retention time t of alitame₂6.730min, peak height h_s2+x29.562 mAU.

From the changes of the peak heights in FIGS. 2 and 8, it is observed that the aspartame and the alitame are increased relative to the mixed standard solution at the respective retention times after the chocolate product to be tested is added, and t is within the tolerance range allowed by the retention time_s1＝t₁，h_s1+x1-h_s1>0, the chocolate product contains aspartame; t is t_s2＝t₂，h_s2+x2-h_s2>0, the chocolate product contains the alitame, so that the qualitative analysis is carried out.

In the same manner as in example 1, when the concentrations of aspartame and alitame in the standard solution added to the centrifuge tube A, B were 1. mu.g/mL and the volume was 45. mu.L, the mass of aspartame and alitame was 0.045. mu.g, i.e., m is_s1＝0.045μg、m_s20.045 μ g, 5g of chocolate product homogenate containing 1g of chocolate product due to the ratio of water to chocolate product being 4 to 1, the sample solution added to the centrifuge tube B being 100 μ L, m_{Sample (A)}In order to add 100 μ L of the test solution to the centrifuge tube B, the total test solution is 25mL to 25000 μ L.

Calculating the content of the chocolate product in the centrifuge tube B in 100 mu L of the test solution according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the chocolate product according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

Example 7

Homogenizing by a food processing machine according to the mass ratio of 1: 2 of preserved fruit to water, weighing 3.000g (accurate to 0.001g) of the preserved fruit homogenate in a 25mL centrifuge tube, adding 10mL of 50% methanol aqueous solution, shaking up, carrying out ultrasonic treatment for 10min, centrifuging at 4000r/min for 5min, transferring the supernatant into a 25mL volumetric flask, adding 10mL of 50% methanol aqueous solution for one time, transferring the supernatant into the same 25mL volumetric flask, completely extracting the component to be detected in the sample to be detected into the supernatant, finally carrying out constant volume by 50% methanol aqueous solution, filtering by a 0.45 mu m organic system filter membrane, and carrying out liquid chromatography.

Taking two identical centrifuge tubes A, B, adding 45 mu L of mixed standard solution of aspartame and alitame at a ratio of 1 mu g/mL, adding no liquid to be tested into centrifuge tube A, and metering to a constant volume of 1 mL; and adding 100 mu L of the succade to-be-tested liquid into the centrifuge tube B, and fixing the volume of the centrifuge tube B to the same scale as that of the centrifuge tube A. Finally, performing chromatographic analysis by using a chromatograph, and performing constant volume by using a centrifugal tube A to obtain a mixed standard solution chromatogram map 2, wherein the retention time t of the aspartame_s13.640min, peak height h_s13.852mAU, the retention time t of alitame_s26.497min, peak height h_s22.654 mAU; chromatogram of the mixed standard liquid and the liquid to be tested obtained by diluting with constant volume in centrifuge tube B is shown in FIG. 9, wherein the retention time t of aspartame₁3.633min, peak height h_s1+x116.510mAU, retention time t of alitame₂6.510min, peak height h_s2+x212.226 mAU.

From the changes of the peak heights in FIGS. 2 and 9, it was observed that the aspartame and the alitame increased relative to the mixed standard solution after the sweetmeat test solution was added for the same retention time, and t was within the tolerance range allowed by the retention time_s1＝t₁，h_s1+x1-h_s1>0, the sweetmeat contains aspartame; t is t_s2＝ t₂，h_s2+x2-h_s2>0, the sweetmeat contains alitame, so that qualitative analysis is carried out.

In the same manner as in example 1, centrifuge tube A, B was added to mix the concentrations of aspartame and alitame in the standard solutionRespectively 1 mug/mL and 45 muL, the mass of aspartame and alitame is 0.045 mug, namely m_s1＝0.045μg、m_s2About 0.045 μ g, 3g of homogenate contained 1g of confectionary since the ratio of water to confectionary was 2 to 1, and the amount of sample solution added to the centrifuge tube B was 100 μ L, m_{Sample (A)}For the mass of food corresponding to 100 μ L of the test solution added to the centrifuge tube B, the total amount of the test solution to be tested is 25mL to 25000 μ L.

Calculating the content of the 100 mu L of the preserved fruit in the centrifuge tube B according to the formula (3):

the quality of aspartame in 100 mu L of test solution is as follows:

the quality of the alitame in 100 mu L of test solution is as follows:

calculating the mass fraction of aspartame and alitame in the preserved fruits according to the formula (4):

the mass fraction of aspartame is as follows:

the mass fraction of the alitame is as follows:

TABLE 1 results of 6 replicates of test foods

As can be seen from Table 1, the results of the 7 examples all had a relative standard deviation RSD of less than 5%, indicating that the precision is good. And through comparison and analysis of the chromatogram and the change of the peak height signal, the content of the component to be detected in the food to be detected can be simply, conveniently and quickly calculated. Meanwhile, through comparison and analysis of the chromatogram, the data of the peak area signal can be substituted into the corresponding formula for calculation.

TABLE 2 measurement of mixed standards at different concentrations

Methodology investigation experiment of the invention

1. Detection limit and quantification limit:

according to Table 1, the detection limit was calculated as 3 times the standard deviation and the quantitation limit was calculated as 10 times the standard deviation. The detection limits and quantitative limits of aspartame and alitame of seven foods to be detected are shown in table 3.

TABLE 3 detection limits and quantitation limits

The detection limit and the quantitative limit of the determination [ S ] of the aspartame and the alitame in the food are known from the national food safety standard GB 5009.263-2016 of Ministry of health, the detection limit of the aspartame and the alitame in the carbonated beverage and the liquid milk beverage is 1mg/kg, the quantitative limit is 3mg/kg, the detection limit of the aspartame and the alitame in the chocolate product, the fruit and the fruit product thereof, the solid beverage and the product thereof is 5mg/kg, and the quantitative limit is 15 mg/kg; the detection limit and the quantification limit of the method are both lower than those in the national food safety standard of Ministry of health.

2. Recovery rate of aspartame and alitame

The recovery rate of the spiked samples was determined using yogurt as a sample, spiking was performed on the samples at three levels, each level was performed for 6 replicates, and the results are shown in table 4.

TABLE 4 determination of aspartame recovery

As can be seen from the measurement results in Table 4, the recovery rate of aspartame in the yogurt measured by the method of the present invention is between 90.2% and 97.2%, and the recovery result is good.

Three levels of spiking recovery tests were performed on the alitame in the samples, respectively, with 6 replicates at each level, and the recovery was calculated as an average, as shown in table 5.

TABLE 5 determination of alitame recovery

As can be seen from the measurement results in Table 5, the recovery rate of the alitame in the yogurt measured by the method of the invention in the sample is between 91.0% and 96.8%, and the results are ideal. The method is particularly suitable for simultaneous qualitative and quantitative analysis of multi-component chromatography, because the components are fully separated in the chromatographic column and do not interfere with each other. In the chromatographic analysis, each component is subjected to peak generation in sequence, and the content can be respectively and rapidly calculated according to the measured value of a peak generation signal; the peak-appearing signal used in the embodiment of the invention is the peak height, and the peak-appearing area can be substituted into a formula for calculation.

3. Comparison experiment by national standard method

3.1 test concentration Range, detection limits and quantitation limits

According to the wavelength and chromatographic conditions specified by the national standard method, the standard mixed use solution of 50 mug/mL, 45 mug/mL, 40 mug/mL, 35 mug/mL, 30 mug/mL, 25 mug/mL, 20 mug/mL, 10 mug/mL, 5 mug/mL, 2.5 mug/mL, 1 mug/mL and 0.5 mug/mL is uniformly injected with 5 muL of sample for determination, the mass concentration of the components is used as an abscissa, and the peak height or the peak area is used as an ordinate to draw a standard curve, so as to obtain a linear regression equation, which is shown in FIG. 10 and FIG. 11. The regression line equation of aspartame is Y-4.6921 x-1.4649, and the coefficient (degree of correlation) R is measured²0.9998, the regression line equation of alitame is Y2.8836 x-1.1944, and the coefficient (degree of correlation) R is measured²＝0.9998。

As known from the national standard method documents, the detection limit of aspartame and alitame in carbonated beverages and liquid milk beverages is 1mg/kg, the quantitative limit is 3mg/kg, the detection limit of aspartame and alitame in solid beverages and products thereof is 5mg/kg, and the quantitative limit is 15 mg/kg.

3.2 recovery and precision of the national Standard method

To examine the relative standard deviation RSD and recovery rate of the national standard method, the same sample was prepared in two identical portions, one portion containing 5mg/kg of standard solution and the other portion containing no standard solution, each sample was subjected to 6 replicates, and the results were averaged as shown in Table 6.

TABLE 6 Standard method normalized recovery and relative standard deviation of the samples

As can be seen from the data in Table 6, the content of the substance to be detected in the sample is quantitatively determined by drawing a standard curve by the national standard method aiming at different substrate characteristics of the food by the external standard method. The results show that the recovery rate of aspartame and alitame is 76.2-92.8%, and the relative standard deviation RSD is 0.45-4.8%.

3.3 results and conclusions of the significance test of the method and the national standard method of the invention

The yogurt is taken as a sample for determination, the method data of the invention is compared with the national standard method data, and the results are shown in Table 7.

TABLE 7 results of the control test

And (3) carrying out variance analysis and T test on a national standard method and a mixed standard sample adding method by using a significance test method, and simultaneously carrying out scientific evaluation. The data and references (Zhang Zhongxin, Doudouqi. food test design and data processing [ M ] are shown in Table 7]Zhengzhou, Zhengzhou university Press 2011.) found that the calculated F value is less than F in the F test chart_0.054.95, showing that no obvious accidental error exists between the mixed standard sample adding method and the national standard method. Thus, with the preconditions and basis for performing the T-test, the data and references from Table 7 (Zhang Zhongxin, Du-Quizi. food test design and data processing [ M ]]Zhengzhou, Zhengzhou university Press 2011.) found that the calculated T value is less than T in the T test chart_0.052.23, the result shows that no significant systematic error exists between the mixed standard sample adding method and the national standard method, and the confidence coefficient of the conclusion is 95%. Therefore, the method has the advantages of scientific and innovative properties, and obvious outstanding advantages and application prospects.

In the above embodiments of the invention, the volume ratio of the added liquid to be tested to the mixed standard liquid is 20:9, and the volume ratio of the added liquid to be tested to the mixed standard liquid can achieve the technical effect of the invention by the above method as long as the peak signals measured by the added liquid to be tested and the mixed standard liquid are obviously different; meanwhile, the method can be applied as long as the component to be measured is above the limit of quantitation.

The method is not only suitable for liquid chromatography, but also suitable for separation analysis methods such as gas chromatography, ion chromatography, supercritical fluid chromatography, capillary electrokinetic chromatography and the like, is not only suitable for separation analysis methods, but also suitable for molecular luminescence analysis methods such as chemiluminescence analysis method, fluorescence analysis method, phosphorescence analysis method, bioluminescence analysis method and the like, is not only suitable for molecular luminescence analysis methods, but also suitable for atomic emission spectrum, atomic absorption spectrum, atomic fluorescence spectrum and ultraviolet and visible molecular absorption spectrum, is not only suitable for determination of aspartame and alitame, is also suitable for determination of other substance components, and is particularly suitable for separation analysis technology. According to different methods, the determination signals can be chromatographic peak height h, chromatographic peak area, relative luminous intensity I, absorbance A and the like, and the feasibility and application fields are very wide.

Claims

1. A method for measuring aspartame and alitame in food by a reversed phase liquid chromatography mixed standard sample adding method is characterized in that: the method comprises the following steps of (1) determining whether the food to be detected contains aspartame and alitame by using a reversed-phase liquid chromatography mixed standard sample adding method, and determining the content of aspartame and alitame in the food to be detected at the same time, wherein the method comprises the following steps:

(1) selecting food to be detected;

(2) pretreating the food to be tested to obtain a test solution;

(4) and (3) chromatographic analysis: taking two identical mixed standard solutions into centrifuge tubes A and B, adding the test solution into the centrifuge tube B, mixing uniformly, and performing chromatographic analysis on the mixed standard solution in the centrifuge tube A under the same chromatographic condition to obtain aspartame preservativeRetention time t_s1And corresponding peak-out signal h_s1Retention time t of alitame_s2And corresponding peak-out signal h_s2(ii) a Carrying out chromatographic analysis on the mixed standard solution in the centrifuge tube B to obtain the retention time t of the aspartame₁And corresponding peak-out signal h_s1+x1Retention time t of alitame₂And corresponding peak-out signal h_s2+x2(ii) a Determination of whether aspartame and alitame are contained:

when t is_s1＝t₁，h_s1+x1-h_s1>0, the food to be detected contains aspartame; when t is_s2＝t₂，h_s2+x2-h_s2>0, the food to be detected contains the alitame;

the method for measuring the aspartame content comprises the following steps:

2. The method for determining aspartame and alitame in food according to claim 1, wherein the method comprises the steps of: selecting the food with the mass fraction of the alitame and the aspartame ranging from 0.1mg/kg to 41mg/kg in the step (1).

3. The method for determining aspartame and alitame in food according to claim 2, wherein the method comprises the steps of: the food to be detected in the step (1) is selected from any one of coca cola, chrysanthemum crystal, yoghourt, cake, concentrated fruit juice, chocolate products and preserves.

4. The method for determining aspartame and alitame in food according to claim 1, wherein the method comprises the steps of: and (2) treating the food to be tested in the step (2) by carrying out vortex or/and ultrasonic treatment, centrifugation and volume fixing on liquid or non-liquid food, and treating the supernatant through a filter membrane to obtain the liquid to be tested.

5. The method for determining aspartame and alitame in food products using reverse phase liquid chromatography spiking method according to claim 1, wherein: the concentration of the mixed standard solution of aspartame and alitame in the step (3) is any one of 20 mug/mL, 10 mug/mL, 5 mug/mL, 2.5 mug/mL, 1 mug/mL and 0.5 mug/mL.

6. The method for determining aspartame and alitame in food products using reverse phase liquid chromatography spiking method according to claim 1, wherein: and (4) the peak signal in the step (4) is the peak height or the peak area.

7. The method for determining aspartame and alitame in food according to claim 1, wherein the method comprises the steps of: and (4) adding the liquid to be tested into the centrifuge tube B in the step (4) and mixing the standard liquid according to the volume ratio of 20: 9.

8. The method for determining aspartame and alitame in food according to claim 1, wherein the method comprises the steps of: the chromatographic conditions in the step (4) are that a mobile phase is methanol: water-40: 60, the flow rate is 0.8mL/min, and the wavelength is 200 nm.