Method for simultaneously detecting 14 sweeteners in pre-prepared vegetable

Abstract

The invention provides a method for simultaneously detecting 14 common sweeteners in a prepared vegetable, and belongs to the technical field of chromatographic analysis and detection. According to the method, a methanol aqueous solution is adopted as an extraction solvent, the methanol aqueous solution passes through a Waters Oasis HLB (Hydrophile Lipophile Balance) purification small column after being subjected to rotary evaporation degreasing, methanol is used for elution, and then LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) is used for qualitative and quantitative detection. On the basis of solid phase extraction-high performance liquid chromatography-tandem mass spectrometry, an analysis method for simultaneously determining the 14 common sweeteners in the pre-prepared vegetables is established for the first time, and the method is good in purification effect, simple to operate and high in sensitivity and can be used for rapidly detecting the 14 sweeteners in the pre-prepared vegetables.

Description

Technical Field

[0001] The invention belongs to the technical field of chromatographic analysis and detection, and particularly relates to a method for simultaneously detecting 14 kinds of sweeteners in pre-prepared dishes. Background Art

[0002] Sweeteners are common food additives in pre-prepared dishes, used to enhance the sweetness of food. There are many types of sweeteners, including natural sweeteners and artificial sweeteners. Some studies have shown that long-term and large-scale intake of sweeteners can have negative effects on health, such as metabolic syndrome, cardiovascular disease and kidney damage. It is crucial to ensure that the use of sweeteners in pre-prepared dishes meets safety standards and regulatory requirements and to accurately detect them. At present, existing sweetener detection methods mainly rely on chemical analysis techniques, such as high-performance liquid chromatography and gas chromatography. These methods have some limitations, such as low sensitivity for the detection of specific sweeteners, poor anti-interference ability, and can only measure one or several simple sweetener mixtures, which is difficult to meet the needs of multi-component determination. Although liquid chromatography-tandem mass spectrometry has the advantages of high throughput, high sensitivity, and strong anti-interference ability, it is more suitable for qualitative and quantitative detection of complex matrices. However, pre-prepared food samples contain a large amount of oil, pigment, protein, dietary fiber, sugar and other substances, and the matrix is ​​complex. Due to poor sample processing, there are certain oils and proteins, resulting in inaccurate test results. There are no reports on methods for detecting sweetener content in pre-prepared dishes in existing patents and literature. Since different sweeteners have different chemical properties and absorption spectra, it is difficult to simultaneously detect multiple sweeteners, such as more than 10 sweeteners, in the same batch of samples. Therefore, it is urgent to develop a method that can simultaneously, quickly, accurately and simply determine multiple sweeteners. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for simultaneously detecting 14 kinds of sweeteners commonly used in pre-prepared dishes, including sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A, and glycyrrhizic acid. The method is characterized by simple operation, convenience, speed, and high sensitivity.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for simultaneously detecting 14 commonly used sweeteners in prepared dishes, comprising the following steps:

[0006] S1. Prepare standard solutions of sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A, and glycyrrhizic acid at certain concentrations, test the standard solutions by LC-MS / MS, and draw a standard curve with the concentration of the standard solution as the abscissa and the corresponding peak area as the ordinate;

[0007] S2. First, methanol-water solution is added to the prepared dish to extract the sweetener, and then the methanol is removed by rotary evaporation. The rotary evaporation liquid without methanol is added with n-hexane for fat removal, and the lower layer of liquid is drawn through a Waters Oasis HLB solid phase extraction column, eluted with methanol, and the solution after methanol elution is used for LC-MS / MS test conditions in the same step S1 to measure the peak area of ​​each corresponding sweetener, and the peak area is substituted into the standard curve to obtain its concentration on the machine, and the actual content of the corresponding sweetener in the prepared dish can be calculated by multiplying the obtained concentration on the machine by the dilution factor.

[0008] According to the method described above, preferably, the LC-MS / MS test conditions are: the chromatographic column of the liquid chromatography is an Acquity UPLC HSS T3 column; the column temperature is 40°C; the mobile phase A is an aqueous solution containing a final concentration of 5 mmol ammonium acetate and a volume fraction of 0.1% formic acid, and the mobile phase B is a methanol solution containing a final concentration of 5 mmol ammonium acetate and a volume fraction of 0.1% formic acid; the flow rate is 0.30 mL / min; the gradient elution program is: 0-1.0 min, 10% to 50% mobile phase B; 1.0-3.0 min, 80% mobile phase B; 3.0-4.0 min, 100% mobile phase B; 4.0-4.5 min, 100% to 10% mobile phase B; 4.5-5.5 min, 10% mobile phase B.

[0009] Preferably, the LC-MS / MS test conditions are: mass spectrometry ion source: electrospray ion source ESI; scanning mode: negative ion mode scanning; ion source interface voltage: -1 kV; nebulizing gas: nitrogen 3.0 L / min; drying gas: nitrogen 10 L / min; collision gas: argon; DL temperature: 250°C; heating module temperature: 400°C; scanning mode: multiple reaction monitoring (MRM); dwell time: 15 ms; delay time: 3 ms.

[0010] As described above, preferably, in step S2, the methanol aqueous solution is a methanol aqueous solution with a volume ratio of 40% to 80%, ultrasonic extraction is used to extract the sweetener, and the upper methanol aqueous solution is taken after centrifugation, and the above operation is repeated for secondary extraction; the upper methanol aqueous solutions after two centrifugations are combined.

[0011] In the method described above, preferably, the ultrasonic time is 10 minutes, the centrifugation condition is 4°C 8000r / min centrifugation for 5 minutes; the pH value of the 80% methanol aqueous solution is 3. By lowering the pH value of the solution and promoting the formation of the non-polar form of the acidic sweetener, the recovery rate of acesulfame potassium, saccharin sodium and sodium cyclamate can be improved. When the ionization of the sweetener is suppressed, the recovery rate of the sweetener can be improved. The preparation method of the 80% methanol aqueous solution with a pH value of 3 is to mix methanol and water in a volume ratio of 4:1, and then adjust the pH to 3 with sulfuric acid with a mass fraction of 98%.

[0012] In the method as described above, preferably, in step S2, the amount of n-hexane added is 5 mL, 5 to 10 mL of methanol is added during methanol elution, and the solution after methanol elution is diluted with methanol before LC-MS / MS testing.

[0013] In the method described above, preferably, the solution to be tested by LC-MS / MS is first filtered with a 0.22 μm polytetrafluoroethylene (PTFE) filter membrane before being sampled for testing.

[0014] In the above method, preferably, in step S2, the multiple reaction monitoring parameters of the 14 sweeteners are performed with reference to the following table:

[0015]

[0016]

[0017] Among them, the * in the table is a quantitative ion; the retention time obtained by detecting the pre-prepared dishes to be tested corresponds to the retention time in the table, which specific sweetener can be qualitatively determined.

[0018] In the method as described above, preferably, in step S2, the methanol aqueous solution is a methanol aqueous solution with a volume ratio of 80%, and 10 mL of methanol is added during methanol elution.

[0019] The purpose of adopting 80% methanol aqueous solution for low-temperature centrifugation in the present invention is to remove protein interference. High concentration of methanol can more effectively promote protein precipitation and avoid the generation of flocs, thereby improving the accuracy and reliability of subsequent detection.

[0020] The purpose of adding n-hexane to the liquid without methanol after rotary evaporation is to remove grease. Both grease and n-hexane are non-polar molecules. Using the principle of like dissolves like, n-hexane can remove grease in liquid. The present invention has found through a large number of experimental studies that the use of mobile phase A as an aqueous solution containing a final concentration of 5mmol ammonium acetate and a volume fraction of 0.1% formic acid, and mobile phase B as a methanol solution containing a final concentration of 5mmol ammonium acetate and a volume fraction of 0.1% formic acid as mobile phases can make the separation and response of 14 sweeteners the best. Under this condition, the peaks of the 14 sweeteners are sharp and the sensitivity is high; when 80% methanol aqueous solution (pH=3) is used as the extraction solvent, the extraction efficiency of the 14 sweeteners is greater than 80%, and the supernatant is clear after centrifugation; when Waters HLB solid phase extraction cartridge is used as a purification cartridge, the recovery rate of the 14 sweeteners is higher than 80%. Therefore, the method of the present invention can simply and quickly remove the interference of impurities in the analysis of sweeteners.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a method for simultaneously detecting 14 kinds of sweeteners commonly used in pre-prepared dishes, which can realize the qualitative and quantitative detection of sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A and glycyrrhizic acid at the same time. The method can simply and quickly remove the interference of impurities in the analysis of sweeteners, overcomes the technical difficulty that multiple sweeteners cannot be detected simultaneously in the prior art, and has the advantages of good purification effect, simple operation and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Quantitative ion chromatograms for 14 sweeteners;

[0024] Figure 2 is the extraction efficiency of different extractants;

[0025] Figure 3 The effects of different protein precipitants on the recovery rates of 14 sweeteners;

[0026] Figure 4 The effect of different solid phase extraction columns on the recovery of 14 sweeteners;

[0027] Figure 5 The effect of different solid phase extraction methods on the recovery rates of 14 sweeteners. DETAILED DESCRIPTION

[0028] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0029] If not otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents used in the methods of the present invention are all analytically pure or above. The sources of the reagents and instruments used in the following examples are: HLB solid phase extraction cartridges were purchased from Waters Technology (Shanghai) Co., Ltd. (Waters); sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, sodium cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A, and glycyrrhizic acid standards (purity> 98.0%) were purchased from Shanghai Anpu Experimental Technology Co., Ltd.; Instruments (Shimadzu Corporation, Japan, LC MS 8060NX). Unless otherwise specified, the reagents used in the present invention are all analytically pure or above.

[0030] Example 1

[0031] A method for simultaneously detecting 14 commonly used sweeteners in prepared dishes comprises the following steps:

[0032] S1. Prepare standard solutions of 14 sweeteners including sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A and glycyrrhizic acid with mass concentrations of 1μg / L, 5μg / L, 10μg / L, 25μg / L, 50μg / L, 100μg / L, 250μg / L and 500μg / L. The detection conditions of LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) are as follows: HPLC column: Acquity UPLC HSS T3 column (100mm×2.1mm, 1.8μm, Waters Company, USA); column temperature: 40℃; mobile phase A: aqueous solution containing a final concentration of 5mmol ammonium acetate and a volume fraction of 0.1% formic acid, mobile phase B: methanol solution containing a final concentration of 5mmol ammonium acetate and a volume fraction of 0.1% formic acid; flow rate: 0.30mL / min; injection volume: 2μL; gradient elution program: 0-1.0min, 10%~50% mobile phase B; 1.0-3.0min, 80% mobile phase B; 3.0-4.0min, 100% mobile phase B; 4.0-4.5min, 100%~10% mobile phase B; 4.5-5.5min, 10% mobile phase B.

[0033] Ion source of mass spectrometry: electrospray ion source (ESI); scanning mode: negative ion mode scanning; ion source interface voltage: -1 kV; nebulizer gas: nitrogen 3.0 L / min; drying gas: nitrogen 10 L / min; collision gas: argon; DL temperature: 250 ° C; heating module temperature: 400 ° C; scanning mode: multiple reaction monitoring (MRM); dwell time: 15 ms; delay time: 3 ms.

[0034] With the mass concentration of the standard solution of each standard as the horizontal axis and the peak area as the vertical axis, a standard curve was drawn and the correlation coefficient r was obtained. 2 The results are shown in Table 1. The 14 compounds have good linear relationships within their respective linear ranges, and the correlation coefficients are all greater than 0.999.

[0035] Table 1 Linear range, regression equation, correlation coefficient, detection limit and quantification limit of 14 sweeteners

[0036]

[0037]

[0038] S2. Weigh 2g of the crushed and evenly mixed sample of the pre-prepared dish into a 50mL centrifuge tube, take 20mL of 80% methanol aqueous solution (methanol: water = 8:2, pH = 3) by ultrasonic for 10min to extract the 14 sweeteners in the pre-prepared dish sample, remove the methanol by rotary evaporation for 40min at a pressure of 150mbar and a temperature of 50℃, take 10mL of the liquid without methanol, add 5mL of n-hexane, vortex mix, and stand for stratification. Take 5mL of the lower layer liquid through the Waters Oasis HLB solid phase extraction column, elute with 10mL of methanol, take the eluate and dilute it with methanol, filter it through a 0.22μm filter membrane, and inject it; use it for LC-MS / MS testing, measure the peak area of ​​14 sweeteners, substitute the peak area into the standard curve to obtain its concentration, and multiply the concentration by the dilution factor to calculate the actual content of the sweetener in the pre-prepared dish.

[0039] The above method was used to test the pre-prepared dishes purchased randomly, and the results showed that the glycyrrhizic acid content in the pre-prepared dishes was 18.02 mg / kg; the acesulfame potassium content was 4.05 mg / kg; the aspartame content was 204.55 mg / kg; and the cyclamate content was 40.27 mg / kg.

[0040] Example 2

[0041] Using direct injection, 14 sweetener standard solutions with a concentration of 100 μg / L were fully scanned separately. Each compound to be tested was preliminarily analyzed in positive and negative ion modes to determine the best ionization method and precursor ion. For these selected precursor ions, the voltage was finely adjusted to ensure that the characteristic fragment ion signal was maximized. Based on the optimization results, two ion pairs with high signal intensity and low interference were further selected as the final qualitative basis. The optimized mass spectrometry conditions are shown in Table 2.

[0042] Table 2 Optimized MRM parameters for 14 sweeteners

[0043]

[0044] Note: * indicates quantitative ion.

[0045] Example 3

[0046] In this example, the mobile phase was investigated and optimized. Six mobile phase systems were compared: ① Mobile phase A was an aqueous solution containing a final concentration of 5 mmol ammonium acetate, and mobile phase B was an acetonitrile solution containing a final concentration of 5 mmol ammonium acetate; ② Mobile phase A was an aqueous solution containing a final concentration of 5 mmol ammonium acetate, and mobile phase B was a methanol solution containing a final concentration of 5 mmol ammonium acetate; ③ Mobile phase A was an aqueous solution containing 0.1% formic acid by volume, and mobile phase B was a methanol solution containing 0.1% formic acid by volume; ④ Mobile phase A was an aqueous solution containing 0.2% formic acid by volume, and mobile phase B was a methanol solution containing 0.1% formic acid by volume. The mobile phase A is a methanol solution containing 0.2% formic acid by volume; ⑤ mobile phase A is an aqueous solution containing 5mmol ammonium acetate and 0.1% formic acid by volume, and mobile phase B is a methanol solution containing 5mmol ammonium acetate and 0.1% formic acid by volume; ⑥ mobile phase A is an aqueous solution containing 10mmol ammonium acetate and 0.1% formic acid by volume, and mobile phase B is a methanol solution containing 10mmol ammonium acetate and 0.1% formic acid by volume. The effect on the separation effect and peak effect of 14 sweeteners. Other conditions are the same as in Example 1, and the 14 standard solutions of sweeteners with a concentration of 100μg / L are tested respectively.

[0047] The results showed that when the mobile phase system was ① and ②, there was no peak of mogroside V, and the peak effects of sucralose and aspartame were poor, but the peak effect of methanol was better than that of acetonitrile, so the aqueous solution-methanol solution was selected for subsequent experimental optimization; when the mobile phase system was ③ and ④, there was no peak of mogroside V and sucralose, but the response of the mobile phase system ③ was better than that of the mobile phase system ④, so the mobile phase system ③ was selected for subsequent experimental optimization; when the mobile phase system was ⑤ and ⑥, all 14 compounds peaked, but when the mobile phase system was ⑥, the response of saccharin sodium, acesulfame potassium, and sodium cyclamate was low. The results showed that when acetonitrile was used as an organic solvent, the peaks of mogroside V and sucralose were significantly inhibited. This may be because acetonitrile is unfavorable to the solubility or ionization state of mogroside V and sucralose, resulting in a decrease in their detection sensitivity in mass spectrometry. In contrast, when methanol is used as the organic solvent, although there is a situation where sucralose does not produce a peak, the overall peak effect is better than that of acetonitrile. When formic acid is used as an additive, the peak elution effect at a concentration of 0.1% is better than that at a concentration of 0.2. This is mainly because a high concentration of formic acid in the negative ion mode will inhibit the formation of negative ions, and a low concentration of formic acid can optimize the ionization environment and solution properties of the mobile phase, thereby improving the peak elution effect of the 14 sweeteners. When ammonium acetate is used as an additive, its high concentration has a significant effect on the peak elution effect of saccharin sodium, acesulfame potassium, and cyclamate. This may be because ammonium acetate changes the ionic strength and pH value of the solution, thereby affecting the ionization efficiency of the sweetener, and the ammonium acetate concentration is relatively high, and long-term use will affect the instrument. The invention finally uses mobile phase system ⑤ as the mobile phase. At this time, the separation and response of the 14 sweeteners are the best. Under this condition, the compound peak shape is sharp and the sensitivity is high, which can meet the detection requirements. The results are shown in FIG. Figure 1 .

[0048] Example 4

[0049] This example compares the effects of 40% methanol water (methanol: water = 2:3 by volume), 60% methanol water (methanol: water = 3:2 by volume), 80% methanol water (methanol: water = 4:1 by volume), 50% acetonitrile water (acetonitrile: water = 1:1 by volume), and 70% ethanol water (ethanol: water = 7:3 by volume) as extractants on the recovery rate. A mixed standard working solution with a mass concentration of 1 mg / L was added to the same batch of pre-prepared food samples, and the operation was carried out according to Example 1. The extraction results are shown in FIG. Figure 2As shown, the results show that the overall recovery rate when 80% methanol water is used as the extraction solvent is better than 40% methanol water, 60% methanol water, 50% acetonitrile water, and 70% ethanol water. Considering that the sample of the present invention is a pre-prepared dish with a complex matrix, a high concentration of organic solvent can more effectively promote protein precipitation and avoid the generation of flocs, thereby improving the accuracy and reliability of subsequent detection, so 80% methanol water solution is selected as the extract of the sweetener.

[0050] Example 5

[0051] Since the pre-prepared dishes contain a large amount of protein, if the protein precipitation is not complete during the pre-treatment process, it will have a serious impact on the test results. Referring to the national standard, 10% potassium ferrocyanide solution and 20% zinc acetate solution are added to precipitate the protein. This example compares the effects of potassium ferrocyanide, zinc acetate, zinc acetate, and 80% methanol as protein precipitants on the recovery rates of 14 sweeteners. The specific operations are as follows:

[0052] 1. Accurately weigh 2 g of the crushed and mixed sample, add 20 mL of 80% methanol (methanol: water = 8:2, pH = 3) and ultrasonically extract for 10 min, then add 2 mL of 10% potassium ferrocyanide solution (10 g of potassium ferrocyanide dissolved in 100 mL of water) and 2 mL of 20% zinc acetate solution (20 g of zinc acetate dissolved in 100 mL of water) for protein precipitation; shake well, vortex for one minute, centrifuge at 8000 r / min for 5 min, take the supernatant into a 100 mL volumetric flask, repeat the extraction once, and combine the supernatants (recorded as potassium ferrocyanide + zinc acetate).

[0053] 2. Accurately weigh 2 g of the crushed and mixed sample, add 20 mL of 80% methanol (methanol: water = 8:2, pH = 3), ultrasonically extract for 10 min, add only 2 mL of 20% zinc acetate solution for protein precipitation; shake well, vortex for one minute, centrifuge at 4 ° C 8000 r / min for 5 min, take the supernatant into a 100 mL volumetric flask, repeat the extraction once, and combine the supernatant (recorded as zinc acetate).

[0054] 3. Accurately weigh 2 g of the crushed and mixed sample, add 20 mL of 80% methanol (methanol: water = 8:2, pH = 3), ultrasonically extract for 10 min, shake well, vortex for one minute, centrifuge at 8000 r / min for 5 min, take the supernatant into a 100 mL volumetric flask, repeat the extraction once, and combine the supernatant (recorded as 80% methanol).

[0055] The supernatant of each group was subjected to rotary evaporation for 40 minutes at a pressure of 150 mbar and a temperature of 50°C to remove methanol. 10 mL of the liquid without methanol was accurately aspirated and added with 5 mL of n-hexane, vortexed and mixed, and allowed to stand for stratification. 5 mL of the lower layer liquid was aspirated and passed through a Waters Oasis HLB solid phase extraction column, eluted with 10 mL of methanol, and the eluate was diluted with methanol and filtered through a 0.22 μm filter membrane before being tested on the machine according to the conditions of Example 1. The results are as follows: Figure 3 And as shown in Table 3.

[0056] Table 3 Effect of different protein precipitants on the recovery of 14 sweeteners (n=3)

[0057]

[0058]

[0059] Note: Different lowercase letters indicate significant differences (P<0.05)

[0060] It can be seen from the results that potassium ferrocyanide and zinc acetate have a greater impact on the recovery rate of glycyrrhizic acid, adventitia, and neohesperidin dihydrochalcone when used as protein precipitants; when zinc acetate is used as a protein precipitant, the recovery rate of glycyrrhizic acid is extremely poor. According to investigations, zinc acetate reacts with glycyrrhizic acid, and glycyrrhizic zinc can be obtained through a specific preparation process. When the sample is centrifuged at 4°C in 80% methanol water, the protein precipitation effect is the best, and the recovery rates of 14 sweeteners are all higher than 80% (see Table 3 for details). Compared with the prior art, the present invention is more convenient to operate in the protein precipitation stage while testing more types of sweeteners. Therefore, 80% methanol water is selected as the protein precipitant of the present invention.

[0061] Example 6

[0062] This example compares the effects of two different columns on the recovery rates of 14 sweeteners. 10 mL of a 100 μg / L mixed standard solution of 14 sweeteners was passed through an Agilent Captiva EMR solid phase extraction column and a Waters Oasis HLB solid phase extraction column at a flow rate of less than 5 mL / min, eluted with methanol, and 1 mL of the eluate was filtered through a 0.22 μm filter membrane. The results were tested on the machine according to the conditions of Example 1. Figure 4 And as shown in Table 4.

[0063] Table 4 Effect of different solid phase extraction columns on the recovery of 14 sweeteners (n=3)

[0064]

[0065]

[0066] Note: Different lowercase letters indicate significant differences (P<0.05)

[0067] It can be seen from the results that the recovery rate of the Waters Oasis HLB solid phase extraction column is better than that of the Agilent Captiva EMR solid phase extraction column. The recovery rates of glycyrrhizic acid, saccharin sodium, acesulfame potassium, cyclamate, aspartame and alitame in the Agilent Captiva EMR solid phase extraction column are all lower than 50% (see Table 4 for details). Since the adsorption mechanism of the EMR solid phase extraction column is mainly based on hydrophobic interaction, its adsorption capacity for polar compounds is relatively limited. Glycyrrhizic acid, saccharin sodium, acesulfame potassium, cyclamate, aspartame and alitame may not be effectively retained on the adsorbent, resulting in poor retention. The good retention effect of the Waters Oasis HLB solid phase extraction column on 14 sweeteners is due to its high adsorption capacity, good chemical stability, reversed phase retention mechanism and polar capture groups, and water wettability. Therefore, the Waters Oasis HLB solid phase extraction column was finally selected as the solid phase extraction column of the present invention.

[0068] Example 7

[0069] Four different experiments were set up to compare the effects of different solid phase extraction methods on the recovery rates of 14 sweeteners. In one group, 14 sweeteners were concentrated in 80% methanol aqueous solution (pH=3) with a concentration of 100 μg / L, and then rotary evaporated at a pressure of 150 mbar and a temperature of 50°C for 40 minutes, and then passed through a Waters Oasis HLB solid phase extraction column activated with 5 mL of methanol and 5 mL of water at a flow rate of less than 5 mL / min, and then eluted with 5 mL of methanol; in the second group, 14 sweeteners were concentrated in 80% methanol aqueous solution (pH=3) with a concentration of 100 μg / L, and then directly passed through an activated Waters Oasis HLB solid phase extraction column at a flow rate of less than 5 mL / min without rotary evaporation, and then eluted with 10 mL of methanol; in the third group, 14 sweeteners were concentrated in 80% methanol aqueous solution (pH=3) with a concentration of 100 μg / L, and then directly passed through an activated Waters Oasis HLB solid phase extraction column at a flow rate of less than 5 mL / min without rotary evaporation, and then eluted with 10 mL of methanol. HLB solid phase extraction column, drained and eluted with 10mL methanol; four groups of 14 kinds of sweeteners were 100μg / L 80% methanol aqueous solution (pH=3) after rotary evaporation at a flow rate of less than 5mL / min through the activated Waters Oasis HLB solid phase extraction column, drained and eluted with 10mL methanol; 1mL of eluate was taken and passed through a 0.22μm filter membrane and tested on the machine according to the conditions of Example 1, and the calculated results were as follows Figure 5 And as shown in Table 5.

[0070] Table 5 Effect of different solid phase extraction methods on the recovery of 14 sweeteners (n=3)

[0071]

[0072] Note: Different lowercase letters indicate significant differences (P<0.05)

[0073] Depend on Figure 5 It can be seen that the recovery rate of 15mL methanol elution is lower than that of 10mL methanol elution and there is no significant difference, the recovery rate of 10mL methanol elution is better than 5mL, and the recovery rate of 10mL methanol elution after rotary evaporation is better than the recovery rate of 15mL methanol elution without rotary evaporation (see Table 5 for details). The above results show that after rotary evaporation effectively removes the methanol solvent in the sample, the recovery rate of the sample significantly improves, and this improvement is attributed to the high efficiency of rotary evaporation technology, which can quickly and gently evaporate volatile solvent methanol under reduced pressure, while reducing the thermal degradation that may occur in the sample at high temperature, thereby protecting the integrity of the target compound, and improving the recovery efficiency of the final product. Therefore, the present invention finally selects 10mL methanol elution after the extract rotary evaporation.

[0074] Example 8

[0075] This example further illustrates the reliability of the method of the present invention through a spike recovery experiment, that is, a known mass of sweetener is added to the pre-prepared dish, and pre-treatment is performed according to the method in Example 1 of the present invention, specifically, 2g of the crushed and mixed sample is weighed into a 50mL centrifuge tube, 14 kinds of sweetener mixed standard solutions with mass concentrations of 25.0, 50.0, and 125.0 mg / kg are added, 20mL of 80% methanol water (methanol: water = 8:2, pH = 3) is taken, ultrasonicated for 10min, centrifuged at 4℃ and 8000r / min for 5min, and then the upper methanol aqueous solution is taken, and the extraction is repeated once; the upper extract is combined and rotary evaporated at a pressure of 150mbar and a temperature of 50℃ for 40min; 10mL of the rotary evaporated liquid is taken and 5mL of n-hexane is added, vortexed and mixed, and allowed to stand for stratification; 5mL of the lower liquid is taken and filtered through Waters Oasis HLB solid phase extraction column, eluted with 10mL methanol; the eluate was diluted 10 times with methanol, and then sampled after passing through a 0.22μm polytetrafluoroethylene (PTFE) filter membrane; the sweetener was determined by the LC-MS / MS detection method in Example 1 of the present invention, and qualitative and quantitative analysis was performed by retention time and characteristic ions; the experiment was repeated 6 times a day for 3 consecutive days, and the peak area of ​​the corresponding sweetener was measured. The peak area was substituted into the standard curve to obtain its concentration on the machine, and the actual content of the sweetener in the pre-prepared dish was calculated by multiplying the obtained concentration on the machine by the dilution multiple. The recovery rate and relative standard deviation (RSD) were calculated by comparing the actual content to the known mass, and the results are shown in Table 6.

[0076] Table 6 Recovery and precision of 14 sweeteners (n=6)

[0077]

[0078]

[0079] The results showed that the recovery rates of the samples at three addition levels (25.0, 50.0, 125.0 mg / kg) were between 86.5% and 107.1%, and the relative standard deviations (RSDs) were all less than 10%. The above results showed that the LC-MS / MS quantitative analysis method established by the present invention has good accuracy and precision for the simultaneous detection of 14 sweeteners.

[0080] Example 9

[0081] This example further illustrates the detection effect of the method of the present invention by comparing the method established by the present invention with the national standard, as shown in Table 7 for details.

[0082] Table 7 Sweetener Testing Method Standards

[0083]

[0084]

[0085] It can be seen from Table 7 that the method of the present invention simultaneously detects 14 sweeteners in the sample within 5.5 minutes, which is significantly superior to the national standard of detecting 1 to 6 sweeteners in the sample in a longer period of time, and the detection limit level is significantly lower than most national standards, indicating that the method has the characteristics of high efficiency and high sensitivity. At the same time, the matrix used in the present invention is a pre-prepared dish that is not yet covered in the current national standard. Therefore, the method of the present invention is more comprehensive in terms of matrix selection.

Claims

1. A method for simultaneously detecting 14 commonly used sweeteners in pre-prepared dishes, characterized in that: It includes the following steps: S1. Prepare standard solutions of sucralose, acesulfame potassium, neotame, stevioside, aspartame, mogroside V, cyclamate, alitame, saccharin sodium, advantame, naringin dihydrochalcone, neohesperidin dihydrochalcone, rebaudioside A, and glycyrrhizic acid at certain concentrations, test the standard solutions by LC-MS / MS, and draw a standard curve with the concentration of the standard solution as the abscissa and the corresponding peak area as the ordinate; S2. First, methanol-water solution is added to the prepared dish to extract the sweetener, and then the methanol is removed by rotary evaporation. The rotary evaporation liquid without methanol is added with n-hexane for fat removal, and the lower layer of liquid is drawn through a Waters Oasis HLB solid phase extraction column, eluted with methanol, and the solution after methanol elution is used for LC-MS / MS test conditions in the same step S1 to measure the peak area of ​​each corresponding sweetener, and the peak area is substituted into the standard curve to obtain its concentration on the machine, and the actual content of the corresponding sweetener in the prepared dish can be calculated by multiplying the obtained concentration on the machine by the dilution factor.

2. The method according to claim 1, characterized in that The LC-MS / MS test conditions are as follows: the chromatographic column of the liquid chromatography is an Acquity UPLC HSS T3 column; the column temperature is 40° C.; the mobile phase A is an aqueous solution containing a final concentration of 5 mmol ammonium acetate and a volume fraction of 0.1% formic acid, and the mobile phase B is a methanol solution containing a final concentration of 5 mmol ammonium acetate and a volume fraction of 0.1% formic acid; the flow rate is 0.30 mL / min; and the gradient elution program is: 0-1.0 min, 10% to 50% mobile phase B; 1.0-3.0 min, 80% mobile phase B; 3.0-4.0 min, 100% mobile phase B; 4.0-4.5 min, 100% to 10% mobile phase B; 4.5-5.5 min, 10% mobile phase B.

3. The method according to claim 1, characterized in that The LC-MS / MS test conditions are as follows: mass spectrometry ion source: electrospray ion source ESI; scanning mode: negative ion mode scanning; ion source interface voltage: -1 kV; nebulizing gas: nitrogen 3.0 L / min; drying gas: nitrogen 10 L / min; collision gas: argon; DL temperature: 250°C; heating module temperature: 400°C; scanning mode: multiple reaction monitoring; dwell time: 15 ms; delay time: 3 ms.

4. The method according to claim 1, characterized in that: In step S2, the methanol aqueous solution is a methanol aqueous solution with a volume ratio of 40% to 80%, and ultrasonic extraction is used to extract the sweetener. After centrifugation, the upper methanol aqueous solution is taken, and the above operation is repeated for secondary extraction; the upper methanol aqueous solutions after two centrifugations are combined.

5. The method according to claim 4, characterized in that The ultrasonic time was 10 min, the centrifugal conditions were 4°C and 8000 r / min for 5 min; the pH value of the methanol aqueous solution was 3.

6. The method according to claim 1, characterized in that The rotary evaporation conditions are: a pressure of 150 mbar and a temperature of 50° C. for 30 to 50 minutes.

7. The method according to claim 1, characterized in that In step S2, the amount of n-hexane added is 5 mL, 5 to 10 mL of methanol is added during methanol elution, and the solution after methanol elution is diluted with methanol before LC-MS / MS testing.

8. The method according to claim 1, characterized in that The solution to be tested by LC-MS / MS was filtered with a 0.22 μm polytetrafluoroethylene filter membrane before injection for detection.

9. The method according to claim 1, characterized in that: In step S2, the multiple reaction monitoring parameters of 14 sweeteners are performed according to the following table: Among them, the * in the table is a quantitative ion; the retention time obtained by detecting the pre-prepared dishes to be tested corresponds to the retention time in the table, which specific sweetener can be qualitatively determined.

10. The method according to claim 1, characterized in that In step S2, the methanol aqueous solution is a methanol aqueous solution with a volume ratio of 80%, and 10 mL of methanol is added during methanol elution.

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