CN117169408A - Method for simultaneously detecting 9 glycoside sweeteners in aquatic product - Google Patents

Abstract

The invention belongs to the technical field of glycoside sweetener detection technology, and discloses a method for simultaneously detecting nine kinds of glycoside sweeteners in aquatic products. The nine kinds of glycoside sweeteners are roseoside, stevioside, sweet tree glycoside A, Steviol glycoside B, stevia, steviol glycoside F, steviol glycoside C, steviol glycoside A and steviol glycoside D; the detection method includes S1, preparation of standard solution; S2, sample pretreatment; S3, chromatography and mass spectrometry analysis; S4, methodology verification. The present invention uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to simultaneously detect 9 kinds of glycoside sweeteners in aquatic products. Its detection sensitivity is high, the stability is good, the analysis time is fast, and the degree of automation is high. In order to protect aquatic products Provide technical support for safety and quality.

Description

A method for simultaneous detection of 9 glycoside sweeteners in aquatic products

Technical field

The present invention relates to the technical field of glycoside sweetener detection, specifically a method for simultaneously detecting nine kinds of glycoside sweeteners in aquatic products.

Background technique

Glycoside sweeteners are used in aquatic products to provide sweetness while reducing the calorie content of the product. As sugar substitutes or low-calorie sweeteners, they can help people control their caloric intake while satisfying people's need for sweetness. In order to increase their economic benefits, some aquatic products manufacturers add a variety of sweeteners to aquatic products to improve the taste. However, the amount added directly affects the health of consumers, and the resulting safety issues are increasingly highlighted. Detection and monitoring Sweeteners in aquatic products have become one of the important tasks of the safety monitoring department. Therefore, it is necessary to develop a rapid, accurate and sensitive method for detecting sweeteners in aquatic products to ensure their quality and safety.

At present, common detection methods for sweeteners in aquatic products include high-performance liquid chromatography-ultraviolet detection, capillary gas chromatography, ultraviolet spectrophotometry, and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). ; Among them, capillary gas chromatography and ultraviolet spectrophotometry are generally used to measure a single sweetener or two sweeteners, and the detection sensitivity also needs to be improved; high performance liquid chromatography-ultraviolet detection method has its own limitations. , its detection sensitivity is low, the detection time is long, the separation is poor, and it is susceptible to interference from impurities to produce false positives. It not only increases the workload of experimental operators, but also makes it difficult to adapt to the needs and development trends of simultaneous detection of multiple components.

Compared with the above methods, the ultra-high performance liquid chromatography-tandem mass spectrometry detection method has the advantages of strong separation ability, reliable qualitative analysis results, low detection limit, fast analysis time, and high degree of automation. LC-MS embodies the complementarity of the advantages of chromatography and mass spectrometry, combining the high separation ability of chromatography for complex samples with the high selectivity, high sensitivity and ability of mass spectrometry to provide relative molecular mass and structural information. In recent years, there have been some reports using this method to detect sweeteners in various foods. However, these reports have a single detection parameter. Most of them are for alcoholic products, dairy products, condiments, etc., and for the glycoside roseoside (roseside) in aquatic products, rubusoside), steviolbioside, dulcocoside A, rebaudioside B, stevia, rebaudioside F, rebaudioside C, rebaudioside A The simultaneous detection of nine sweeteners, rebaudioside A) and steviol glycoside D (rebaudioside D), has not been reported yet.

Contents of the invention

The present invention is intended to provide a method for simultaneously detecting 9 kinds of glycoside sweeteners in aquatic products, using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to simultaneously detect 9 kinds of glycoside sweeteners in aquatic products. It has high sensitivity, good stability, fast analysis time and high degree of automation, providing technical support to ensure the safety and quality of aquatic products.

In order to achieve the above objects, the present invention provides the following technical solutions:

A method for simultaneously detecting 9 kinds of glycoside sweeteners in aquatic products. The 9 kinds of glycoside sweeteners are roseoside, steviol glycoside, steviol glycoside A, steviol glycoside B, stevia, steviol glycoside F, and stevia. Glycoside C, Steviol glycoside A and Steviol glycoside D; the detection method includes the following steps:

S1. Prepare standard solution

First, accurately weigh or pipet the above 9 kinds of glycoside sweetener standards, dissolve them in methanol and prepare single standard stock solutions respectively; then accurately pipet equal amounts of the 9 kinds of single standard stock solutions into the same container, and use them again. Dissolve and prepare the mixed standard intermediate solution in methanol; store in the dark at 0 to 8°C;

S2. Sample preprocessing

Accurately weigh the sample to be tested, mix it evenly, add the methanol-water mixed solution to the centrifuge tube, and centrifuge. After the centrifugation is completed, filter it with a filter membrane to obtain the sample to be tested;

S3, chromatography and mass spectrometry analysis

Use a 2.1x100mm, 1.7μm Charged Surface Hybrid C18 chromatographic column to analyze the sample to be tested; first use electrospray ionization, then negative ion scanning, and finally use the multi-reaction monitoring acquisition mode for sampling and analysis;

S4. Methodological validation

First prepare a series of mixed standard solutions with stepwise changes in concentration, and then use UPLC-MS/MS for measurement and analysis. Taking the peak area ratio as the ordinate and the ratio of mass concentration to internal standard concentration as the abscissa, draw a standard working curve and obtain 9 kinds of The linear range, linear regression equation, correlation coefficient, detection limit and quantification limit of glycoside sweeteners; finally, take blank shellfish samples, add appropriate amounts of 9 kinds of glycoside sweetener mixed standard working solutions, and the standard addition levels are respectively With different quantification limits of each glycoside sweetener, multiple test solutions were prepared in parallel at each concentration level, and analyzed and measured to obtain the average recovery rate and relative standard deviation of the 9 glycoside sweeteners; thus completing the analysis of 9 glycoside sweeteners. Detection of glycoside sweeteners.

Further, in S1, the concentration of the single standard stock solution is 1.0 mg/mL, and the concentration of the mixed standard intermediate solution is 10 μg/mL.

Further, in S2, the volume ratio of the methanol-water mixed solution is 1:1, and the centrifugation conditions of the sample in the centrifuge tube are: centrifuge at 5000 r/min for 5 minutes; the filter membrane is a polyethersulfone filter membrane with a pore size of 0.22 μm. .

Further, in S3, the chromatographic conditions are: column temperature: 35°C; flow rate: 0.3mL/min; injection volume: 2μL; mobile phase A is methanol and B is 5mmol/L ammonium acetate solution for gradient elution.

The beneficial effects of the technical solution are:

1. The detection method provided by the present invention can simultaneously detect 9 kinds of roseoside, steviol glycoside, steviol glycoside A, steviol glycoside B, stevia, steviol glycoside F, steviol glycoside C, steviol glycoside A and steviol glycoside D in aquatic products. Glycoside sweeteners; provides technical support to ensure the safety and quality of aquatic products;

2. The detection method provided by the present invention uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry for analysis. It has high sensitivity and selectivity and can accurately determine the presence and content of the target compound. It uses a specific chromatographic column and mass spectrometer. The instrument, combined with advanced collection modes, separates and analyzes samples, which can improve the accuracy and reliability of results;

3. The present invention establishes a standard working curve and related parameters by preparing a standard solution and performing UPLC-MS/MS measurement of a mixed standard solution with multi-level concentration changes. It verifies the linear range, regression equation, correlation coefficient, and detection method of the method. Indicators such as limit limit and quantification limit further improve the stability and reliability of detection;

4. By adding glycoside sweetener mixed standard solutions of different concentrations to the blank sample and conducting parallel analysis, the present invention can calculate the average recovery rate and relative standard deviation, and automatically evaluate the accuracy and precision of the method provided by the present invention. Check.

Description of drawings

Figure 1 is an ion fragment diagram of rubusoside in a specific embodiment of the present invention;

Figure 2 is an ion fragment diagram of steviolbioside in a specific embodiment of the present invention;

Figure 3 is an ion fragment diagram of dulcocoside A in a specific embodiment of the present invention;

Figure 4 is an ion fragment diagram of rebaudioside B in a specific embodiment of the present invention;

Figure 5 is a ion fragment diagram of stevia in a specific embodiment of the present invention;

Figure 6 is a rebaudioside F ion fragment diagram in a specific embodiment of the present invention;

Figure 7 is a fragmentation diagram of rebaudioside C ions in a specific embodiment of the present invention;

Figure 8 is an ion fragment diagram of rebaudioside A in a specific embodiment of the present invention.

Figure 9 is a diagram of rebaudioside D ion fragments in a specific embodiment of the present invention.

Figure 10 is a total ion chromatogram of nine glycoside sweeteners in a specific embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments:

A method for simultaneously detecting 9 glycoside sweeteners in aquatic products. The 9 glycoside sweeteners are roseoside, steviol glycoside, steviol glycoside A, steviol glycoside B, stevia, steviol glycoside F, steviol glycoside C, Steviol glycoside A and Steviol glycoside D; the detection method includes the following steps:

S1. Prepare standard solution

First, accurately weigh or pipet the above 9 kinds of glycoside sweetener standard products, dissolve them in methanol and prepare single standard stock solutions of 1.0mg/mL respectively; then accurately pipet equal amounts of the 9 kinds of single standard standard stock solutions in the same In the container, dissolve again with methanol and prepare a mixed standard intermediate solution of 10 μg/mL; store in the dark at 0 to 8°C;

S2. Sample preprocessing

Accurately weigh the sample to be tested, mix it evenly, add a methanol-water mixed solution with a volume ratio of 1:1 to the centrifuge tube, and centrifuge it for 5 minutes at 5000 r/min. After centrifugation, filter it with polyethersulfone with a pore size of 0.22 μm. Membrane filtration to obtain the sample to be tested;

S3, chromatography and mass spectrometry analysis

Use a 2.1x100mm, 1.7μm Charged Surface Hybrid C18 chromatographic column to analyze the sample to be tested; first use electrospray ionization, then negative ion scanning, and finally use the multiple reaction monitoring acquisition mode for sampling and analysis; among them, the specific chromatographic conditions are: Column Temperature: 35°C; flow rate: 0.3mL/min; injection volume: 2μL; mobile phase A is methanol and B is 5mmol/L ammonium acetate solution for gradient elution;

S4. Methodological validation

First prepare a series of mixed standard solutions with stepwise changes in concentration, and then use UPLC-MS/MS for measurement and analysis. Taking the peak area ratio as the ordinate and the ratio of mass concentration to internal standard concentration as the abscissa, draw a standard working curve and obtain 9 kinds of The linear range, linear regression equation, correlation coefficient, detection limit and quantification limit of glycoside sweeteners; finally, take blank shellfish samples, add appropriate amounts of 9 kinds of glycoside sweetener mixed standard working solutions, and the standard addition levels are respectively With different quantification limits of each glycoside sweetener, multiple test solutions were prepared in parallel at each concentration level, and analyzed and measured to obtain the average recovery rate and relative standard deviation of the 9 glycoside sweeteners; thus completing the analysis of 9 glycoside sweeteners. Detection of glycoside sweeteners.

Specific embodiments

Instruments and reagents

Waters Xevo TQ-XS ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer (Waters Corporation, USA); Dionex ASE 350 accelerated solvent extraction instrument (Thermo Fisher Scientific, USA); AVANTIJ-E large capacity and high speed Refrigerated centrifuge (BEAKMAN Company, USA); BSA224S-CW electronic balance (accuracy 0.1mg, Sartorius Scientific Instruments Co., Ltd., Germany); Multi Reax multi-hole vortex oscillator (Haidorf Company, Germany); Milli-Q ultrapure water Instrument (Millipore Corporation, USA).

Methanol was of chromatographic grade and purchased from Merck, Germany.

Preparation of standard solution

First, accurately weigh or pipette the above 9 kinds of glycoside sweetener standard products, dissolve them in methanol and prepare single standard stock solutions of 1.0mg/mL respectively; then accurately pipette 0.1mL of the 9 kinds of single standard standard stock solutions in 10mL. In a volumetric flask, dissolve with methanol and prepare a mixed standard intermediate solution of 10 μg/mL; store in the dark at 0-8°C. The validity period is 3 months.

Sample preparation

Weigh 5.00g of the sample to be tested (accurate to 0.01g) and mix well, add exactly 25mL of methanol-water (1:1/V/V) into the centrifuge tube, centrifuge at 5000 rpm for 5 minutes, and pass 0.22 μmL filter membrane, to be tested.

Chromatographic conditions

Chromatographic column: ACQUITY UPLC CSH C18 (2.1x100mm, 1.7μm); column temperature: 35°C, flow rate: 0.3mL/min; injection volume: 2μL; mobile phase: A is methanol, B is 5mmol/L ammonium acetate solution, specific Gradient elution process, as shown in Table 1:

Table 1 Mobile phase elution procedures for liquid chromatography

Mass spectrometry conditions

The ion source is electrospray ionization (ESI); the scanning mode is negative ion scanning; the acquisition mode is multiple reaction monitoring (MRM). The specific mass spectrometry parameters of 9 glycoside sweeteners are shown in Table 2:

Table 29 Mass spectrometry parameters of glycoside sweeteners

The obtained ion fragment diagrams of the nine glycoside sweeteners are shown in Figures 1 to 9 respectively, and the total ion chromatograms of the nine glycoside sweeteners are shown in Figure 10.

Methodological validation

Prepare a series of mixed standard solutions with stepwise changes in concentration, and use UPLC-MS/MS for measurement and analysis. The peak area ratio is used as the ordinate (Y), and the ratio of the mass concentration to the internal standard concentration is used as the abscissa (X, ng/mL). Using the standard working curve, we obtained the linear range, linear regression equation, correlation coefficient, detection limit, and quantitation limit of 9 kinds of glycoside sweeteners, as shown in Table 3:

Table 39 Linear ranges, linear regression equations, and correlation coefficients of glycoside sweeteners

Take blank shellfish samples and add appropriate amounts of 9 glycoside sweetener mixed standard working solutions. The spike levels are 1 times the quantification limit, 3 times the quantification limit and 10 times the quantitation limit of each glycoside sweetener. Each concentration Six test solutions were prepared horizontally in parallel and analyzed. The results are shown in Table 4; the average recoveries of the nine glycoside sweeteners were 90.16% to 103.69%, and the relative standard deviations were 2.24% to 7.22%.

Table 4 Average recovery rates and relative standard deviations of nine glycoside sweeteners in aquatic product samples

In summary, it can be seen that the method provided by the present invention for the simultaneous detection of 9 kinds of glycoside sweeteners in aquatic products is stable, reliable, has good accuracy and precision, and can meet the requirements for the simultaneous detection of 9 kinds of glycoside sweeteners in aquatic products. odorant needs.

The above are only embodiments of the present invention, and common knowledge such as specific technical solutions or characteristics that are known in the scheme are not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention. These should also be regarded as the protection scope of the present invention and will not affect the implementation of the present invention. The effect and practicality of the patent. The scope of protection claimed in this application shall be based on the content of the claims, and the specific implementation modes and other records in the description may be used to interpret the content of the claims.

Claims (4)

1. A method for simultaneously detecting 9 kinds of glycoside sweeteners in aquatic products, characterized in that the 9 kinds of glycoside sweeteners are roseoside, steviol glycoside, stevia glycoside A, steviol glycoside B, stevia glycoside , Steviol glycoside F, Steviol glycoside C, Steviol glycoside A and Steviol glycoside D; the detection method includes the following steps: S1. Prepare standard solution First, accurately weigh or pipet the above 9 kinds of glycoside sweetener standards, dissolve them in methanol and prepare single standard stock solutions respectively; then accurately pipet equal amounts of the 9 kinds of single standard stock solutions into the same container, and use them again. Dissolve and prepare the mixed standard intermediate solution in methanol; store in the dark at 0 to 8°C; S2. Sample preprocessing Accurately weigh the sample to be tested, mix it evenly, add the methanol-water mixed solution to the centrifuge tube, and centrifuge. After the centrifugation is completed, filter it with a filter membrane to obtain the sample to be tested; S3, chromatography and mass spectrometry analysis Use a 2.1x100mm, 1.7μm Charged Surface Hybrid C18 chromatographic column to analyze the sample to be tested; first use electrospray ionization, then negative ion scanning, and finally use the multi-reaction monitoring acquisition mode for sampling and analysis; S4. Methodological validation First prepare a series of mixed standard solutions with stepwise changes in concentration, and then use UPLC-MS/MS for measurement and analysis. Taking the peak area ratio as the ordinate and the ratio of mass concentration to internal standard concentration as the abscissa, draw a standard working curve and obtain 9 kinds of The linear range, linear regression equation, correlation coefficient, detection limit and quantification limit of glycoside sweeteners; finally, take blank shellfish samples, add appropriate amounts of 9 kinds of glycoside sweetener mixed standard working solutions, and the standard addition levels are respectively With different quantification limits of each glycoside sweetener, multiple test solutions were prepared in parallel at each concentration level, and analyzed and measured to obtain the average recovery rate and relative standard deviation of the 9 glycoside sweeteners; thus completing the analysis of 9 glycoside sweeteners. Detection of glycoside sweeteners. 2. A method for simultaneously detecting 9 glycoside sweeteners in aquatic products according to claim 1, characterized in that in S1, the concentration of the single standard stock solution is 1.0 mg/mL, and the mixed standard intermediate solution The concentration is 10μg/mL. 3. A method for simultaneously detecting 9 glycoside sweeteners in aquatic products according to claim 1, characterized in that in S2, the volume ratio of the methanol-water mixed solution is 1:1, and the sample is centrifuged The centrifugation conditions in the tube are: centrifuge at 5000r/min for 5 minutes; the filter membrane is a polyethersulfone filter membrane with a pore size of 0.22 μm. 4. A method for simultaneously detecting 9 glycoside sweeteners in aquatic products according to claim 1, characterized in that, in S3, the chromatographic conditions are: column temperature: 35°C; flow rate: 0.3mL/min ; Injection volume: 2 μL; mobile phase A is methanol and B is 5 mmol/L ammonium acetate solution for gradient elution.