CN110824064A - Method for determining main sweetener in edible essence and flavor by adopting HPLC-MS/MS - Google Patents

Abstract

The application discloses a method for determining a main sweetener in edible flavors and fragrances by adopting HPLC-MS/MS. The method comprises the steps of matrix solvent preparation, series of standard working solution preparation, sample solution preparation, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, standard working curve establishment and sample result calculation. The method adopts a matrix solvent-based configuration calibration curve for the first time, and uses an HPLC-MS/MS chromatograph to simultaneously determine 9 main sweeteners such as xylitol, acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, neohesperidin, neotame, stevioside and the like in the edible essence. The method prepares a proper matrix solvent for preparing a standard working curve, eliminates the influence on the target object quantification caused by the matrix effect in the mass spectrum ionization process, can simultaneously detect the content of the main sweetener in the edible essence, and has the advantages of accurate result, less interference, high sensitivity, good repeatability and the like.

Description

Method for determining main sweetener in edible essence and flavor by adopting HPLC-MS/MS

Technical Field

The invention belongs to the technical field of food additive physicochemical index detection, and particularly relates to a method for determining a main sweetener in edible essence and flavor by adopting HPLC-MS/MS.

Background

With the concern of society on food safety, relevant government departments have established reasonable and effective supervision and management and safety evaluation regulation and regulation systems to standardize the production, operation and use of the essences, the spices and relevant additives. Sweeteners are an important food additive for improving the taste and flavor of foods, and among them, non-sugar sweeteners are widely used in the modern food industry as a substitute for sucrose due to their advantages of high sweetness, low calorie, and much less involvement in metabolic processes. In recent years, with the improvement of consumption and cognition level, people pay more attention to food safety problems, and the use of various additives in food, particularly sweeteners, is more and more emphasized. The food additive is harmless to human body when used in a prescribed dosage range, and may cause various forms of toxicity manifestation when used in an excessive amount. Therefore, the amount of the catalyst used must be strictly controlled to exert its advantageous effects and to prevent its adverse effects. At present, the sweetening agents such as saccharin sodium, sodium cyclamate and the like are strictly restricted in use and set a large number of standards in various countries, and the sweetening agents become the key points of investigation in international trade and domestic standards. The national standard GB 2760 'food additive use standard' of China has clear regulations on the application range and the addition limit of sweeteners for food processing, including sodium cyclamate, acesulfame potassium, xylitol, alitame, aspartame and the like.

At present, the measurement of the sweetening agent in food such as dairy products, white spirit, beverages, packaging paper and the like and the measurement of the sweetening agent in essence are reported in documents. The sweetener is determined by ion chromatography, capillary electrophoresis, liquid chromatography/tandem mass spectrometry, etc. Due to the difference of physicochemical property, electrochemical property and spectral property of the artificially synthesized sweetener, the liquid chromatography is difficult to meet the requirement of simultaneously detecting various sweeteners, and the ion chromatography and the capillary electrophoresis are not applied much due to the low sensitivity and the like. Because the liquid chromatography/tandem mass spectrometry has strong selectivity, high sensitivity and strong matrix interference resistance, the method is widely applied to the determination of food additives at present. However, the essence as a food additive has complex components and serious matrix interference, which results in low detection accuracy.

Disclosure of Invention

In order to solve the problems, the method for determining the main sweetener in the edible essence and flavor by adopting HPLC-MS/MS adopts the matrix solvent for proportioning, can effectively eliminate false positive, and improves the detection accuracy and the detection flux.

According to one embodiment of the present application,

a method for determining main sweeteners in edible essence based on high performance liquid chromatography-tandem mass spectrometry comprises the following steps:

(1) preparing a matrix solvent: taking edible essence without a target object as a sample, taking water as a solvent, carrying out ultrasonic extraction on the sample, and preparing a matrix solvent by using an extract through a water-phase filter membrane;

(2) preparation of a series of standard working solutions: respectively weighing a certain amount of 9 sweetener standards, taking a matrix solvent as a standard preparation solvent, and preparing a series of standard working solutions by stepwise dilution;

(3) preparation of a sample solution: accurately weighing a certain amount of sample in a centrifuge tube, accurately transferring into a certain volume of ethanol aqueous solution, uniformly dispersing by using a vortex mixer, performing ultrasonic extraction, and filtering an extract solution through a water-phase filter membrane to obtain a sample injection solution;

(4) performing high performance liquid chromatography-tandem mass spectrometry; respectively detecting the series of standard working solutions and the sample injection solution by using an HPLC-MS/MS chromatograph;

(5) and establishing a standard working curve and calculating a sample result.

In the step (1), the preparation process of the matrix solvent comprises the following steps: selecting edible essence without 9 sweetening agents such as xylitol, acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, neohesperidin, neotame, stevioside and the like, accurately weighing 0.5 g of a sample, adding 10 mL of ethanol aqueous solution with the volume fraction of 10%, performing vortex mixing to disperse the sample, performing ultrasonic extraction for 40min under the condition of 60% power, shaking uniformly after the ultrasonic extraction is finished, standing for a moment, and filtering through a 0.22 mu m water-phase filter membrane to obtain a matrix solvent.

In the step (2), respectively and accurately weighing 10mg of xylitol, 10mg of acesulfame potassium, 10mg of saccharin sodium, 5 mg of sodium cyclamate, 10mg of sucralose, 5 mg of aspartame, 10mg of neohesperidin, 5 mg of neotame and 5 mg of stevioside, accurately measuring the content to 0.1mg in the same 100 mL volumetric flask, dissolving by using a matrix solvent, carrying out constant volume, and preparing a mixed standard storage solution. Accurately transferring 50 muL, 100 muL, 250 muL, 0.5 mL and 1mL of mixed standard stock solution respectively, diluting the mixed standard stock solution to a 10 mL volumetric flask with a matrix solvent, uniformly mixing, preparing to obtain 5-grade standard working solution, wherein the concentration of each-grade series solution is as follows,

series standard working solution meter

。

In the step (3), 0.5 g of essence sample is weighed, the precision is 0.1mg, the essence sample is placed in a 50 mL centrifuge tube with a plug, 1mL of ethanol and 9 mL of ultrapure water are accurately transferred, vortex mixing is carried out to disperse the sample matrix, ultrasonic extraction is carried out for 40min under the condition of 60% power, shaking is carried out after the sample matrix is dispersed, standing is carried out for a moment, and the sample is filtered through a 0.22 mu m water phase filter membrane to obtain sample introduction liquid.

In the step (4), HPLC-MS/MS analysis is carried out, wherein the adopted high performance liquid chromatography conditions are as follows: a chromatographic column: ZORBAXeclipse XDB-C18 with the specification of 150 mm multiplied by 4.6 mm and the filler particle size of 3.5 mu m; mobile phase A: methanol, B: 5 mmol/L ammonium acetate in water, gradient elution is shown in the following table; column temperature: 40 ℃; sample introduction amount: 5 μ L.

Gradient elution schedule

。

In the step (4), performing HPLC-MS/MS analysis, wherein the mass spectrum conditions are as follows: an ion source: electrospray ion source (ESI); ion source temperature: 100 ℃; temperature of the drying gas: 300 ℃; flow rate of drying gas: 9L/min; atomizing gas pressure: 40 psi; capillary voltage: the positive ions are 4000V, and the negative ions are 3500V; the detection mode is as follows: multiple reaction monitoring mode (MRM), the parameters are as follows,

target MRM parameter condition table

。

In the step (5), linear regression analysis is performed by taking the concentration of each target in the series of standard working solutions as an abscissa and the peak area of each target in the chromatogram as an ordinate to obtain a standard working curve of each target. Substituting the chromatographic peak area of each target object in the sample injection liquid measured under the same condition into a standard working curve, and obtaining the content of each target object in the sample according to the following formula:

in the above formula:

x-the amount of each target in the sample in milligrams per kilogram (mg/kg);

c, the concentration of each target object in the sample injection solution read from the standard working curve is milligram per liter (mug/mL);

v-volume of extraction system in milliliters (mL);

m-the mass of the sample in grams (g).

Compared with the prior art, the method for determining the main sweetener in the edible essence based on the high performance liquid chromatography-tandem mass spectrometry has the following advantages: (1) the method simultaneously determines the content of the main sweetener in the edible essence by using HPLC-MS/MS, has strong matrix interference resistance and simple pretreatment method; (2) the extraction solvent used in the invention is ethanol aqueous solution with 10% volume fraction, has no pollution to the environment, and accords with the green development concept; (3) the method adopts the matrix solvent to prepare the standard working curve, eliminates the influence on the quantification of the target object caused by the matrix effect in the ionization process of mass spectrum, can accurately and simultaneously detect the content (4) of the main sweetener in the edible essence, has better methodology investigation result, and has the advantages of high sensitivity and good repeatability.

1. The detection limit of the method is as follows:

the lowest concentration of mixed standard working solution was subjected to HPLC-MS/MS analysis, and the limit of detection (LOD) was calculated as a 3-fold signal-to-noise ratio (S/N = 3).

2. The method has the advantages of standard recovery rate and repeatability:

adding a standard solution of a sweetening agent into a blank essence sample, then respectively carrying out pretreatment and HPLC-MS/MS analysis, and calculating the recovery rate according to the addition amount and the measured value.

The working curve (linear coefficient), detection limit, recovery rate and repeatability of each target are shown in the following table,

methodological inspection of objects

。

Drawings

FIG. 1 is an ion pair chromatogram of a standard working solution extraction of example 1.

In the figure, 1 is xylitol (retention time is 3.74 min), 2 is acesulfame potassium (retention time is 6.03 min), 3 is saccharin sodium (retention time is 9.88 min), 4 is sodium cyclamate (retention time is 16.56 min), 5 is sucralose (retention time is 17.73 min), 6 is aspartame (retention time is 18.14 min), 7 is neohesperidin (retention time is 19.59 min), 8 is neotame (retention time is 27.26 min), and 9 is stevioside (retention time is 28.26 min).

Detailed Description

The following are specific examples of the present application and further describe the technical solutions of the present application, but the present application is not limited to these examples.

Example 1

1. Instruments and reagents

Xylitol, acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, neohesperidin, neotame

And 9 sweeteners such as stevioside; methanol, ethanol, ammonium acetate (all chromatographically pure); meets the requirement of first-grade water in GB/T6682.

6460 HPLC-MS/MS Mass spectrometer (Agilent, USA), TM-1 vortex mixer (tin-free)

Watson instruments ltd); KQ-500DE ultrasonic generator (Kunshan ultrasonic instruments Co., Ltd.); electronic balance (METTLER TOLEDO, sensory 0.1 mg); 50 mL centrifuge tubes (Corning Corp., PP).

2. Preparation of matrix solvent

Selecting a material which does not contain xylitol, acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame,

0.5201 g of sample is accurately weighed, 10 mL of ethanol aqueous solution with the volume fraction of 10% is added, vortex mixing is carried out to disperse the sample, ultrasonic extraction is carried out for 40min under the condition of 60% power, shaking is carried out after the ultrasonic extraction is finished, standing is carried out for a moment, and the matrix solvent is obtained by filtering through a 0.22 mu m water phase filter membrane.

3. Series of standard working solutions: the preparation method is shown in the summary part of the invention and is not repeated here.

4. Sample processing

Weighing 0.5105 g of essence sample No. 1, accurately obtaining 0.1mg, placing the sample No. 1 in a 50 mL centrifuge tube with a plug, accurately transferring 1mL of ethanol and 9 mL of ultrapure water, carrying out vortex mixing to disperse the sample matrix, carrying out ultrasonic extraction for 40min under the condition of 60% power, shaking uniformly after the completion, standing for a moment, and filtering through a 0.22 mu m water-phase filter membrane to obtain a sample injection solution.

5. HPLC-MS/MS assay

6. Calculation of results

And (3) carrying out quantitative analysis by an external standard method, namely carrying out regression analysis on the corresponding concentrations of the quantitative ion pair peak areas of various sweetener standard products by using the quantitative ion pair peak areas to obtain various sweetener standard curves, wherein the correlation coefficients are all more than or equal to 0.995, measuring the extracted sample to obtain the quantitative ion pair peak area of the target object, substituting the quantitative ion pair peak area into the standard curves to obtain the content of the sweetener in the sample, wherein only aspartame is detected in the sample, and the content is 2.35 mg/kg.

The specific embodiments described herein are merely illustrative of the spirit of the application. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the present application as defined by the appended claims.

Claims (6)

1. A method for measuring a main sweetener in edible flavors and fragrances by adopting HPLC-MS/MS is characterized by comprising the following steps:

(1) preparing a matrix solvent: taking edible essence without a target object as a sample, taking water as a solvent, carrying out ultrasonic extraction on the sample, and preparing a matrix solvent by using an extract through a water-phase filter membrane;

(2) preparation of a series of standard working solutions: respectively weighing 9 sweetener standards, taking a matrix solvent as a standard preparation solvent, and preparing a series of standard working solutions by stepwise dilution;

(3) preparation of a sample solution: accurately weighing a predetermined amount of sample in a centrifuge tube, accurately transferring into ethanol aqueous solution with a predetermined volume, uniformly dispersing by using a vortex mixer, performing ultrasonic extraction, and filtering an extract solution through a water-phase filter membrane to obtain sample injection solution;

(4) performing high performance liquid chromatography-tandem mass spectrometry; respectively detecting the series of standard working solutions and the sample injection solution by using an HPLC-MS/MS chromatograph;

(5) establishing a standard working curve and calculating a sample result;

wherein, in the step (1), the preparation process of the matrix solvent comprises the following steps: selecting edible essence which does not contain xylitol, acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, neohesperidin, neotame and stevioside serving as sweeteners, accurately weighing 0.5 g of a sample, adding 10 mL of ethanol aqueous solution with the volume fraction of 10%, performing vortex mixing to disperse the sample, performing ultrasonic extraction for 40min under the condition of 60% power, shaking uniformly after the ultrasonic extraction is finished, standing for a moment, and filtering through a 0.22 mu m water-phase filter membrane to obtain a matrix solvent.

2. The method according to claim 1, characterized in that in the step (2), 10mg of xylitol, 10mg of acesulfame potassium, 10mg of saccharin sodium, 5 mg of sodium cyclamate, 10mg of sucralose, 5 mg of aspartame, 10mg of neohesperidin, 5 mg of neotame and 5 mg of stevioside are accurately weighed respectively, are accurately measured to 0.1mg in the same 100 mL volumetric flask, are dissolved and subjected to volume fixing by using a matrix solvent to prepare a mixed standard stock solution, are accurately transferred to 50 muL, 100 muL, 250 muL, 0.5 mL and 1mL respectively, are subjected to volume fixing by using the matrix solvent to 10 mL volumetric flask, are uniformly mixed to prepare 5-level standard working solutions, and the concentration of each-level series solution is as follows:

series standard working solution meter

。

3. The method according to claim 1, characterized in that in the step (3), 0.5 g of essence sample is weighed to be accurate to 0.1mg, the essence sample is placed in a 50 mL centrifuge tube with a plug, 1mL of ethanol and 9 mL of ultrapure water are accurately removed, vortex mixing is carried out to disperse the sample matrix, ultrasonic extraction is carried out for 40min under the condition of 60% power, shaking is carried out after the ultrasonic extraction is finished, standing is carried out for a moment, and the sample is filtered through a 0.22 mu m water-phase filter membrane to obtain sample feeding liquid.

4. The method of claim 1, wherein in step (4), HPLC-MS/MS analysis is performed under the following high performance liquid chromatography conditions: a chromatographic column: ZORBAX Eclipse XDB-C18 with specification of 150 mm × 4.6 mm and filler particle size of 3.5 m; mobile phase A: methanol, B: 5 mmol/L ammonium acetate in water, gradient elution is shown in the following table; column temperature: 40 ℃; sample introduction amount: the volume of the solution is 5 mu L,

gradient elution schedule

。

5. The method of claim 1, wherein in step (4), HPLC-MS/MS analysis is performed under the following mass spectrometry conditions: an ion source: electrospray ion source (ESI); ion source temperature: 100 ℃; temperature of the drying gas: 300 ℃; flow rate of drying gas: 9L/min; atomizing gas pressure: 40 psi; capillary voltage: the positive ions are 4000V, and the negative ions are 3500V; the detection mode is as follows: multiple reaction monitoring mode (MRM), the parameters are as follows,

target MRM parameter condition table

。

6. The method according to claim 1, wherein in the step (5), a linear regression analysis is performed with the concentration of each target in the series of standard working solutions as abscissa and the peak area of each target in the chromatogram as ordinate to obtain a standard working curve of each target, the chromatographic peak area of each target in the sample solution measured under the same conditions is substituted into the standard working curve, and the content of each target in the sample is determined according to the following formula:

in the above formula:

x-the amount of each target in the sample in milligrams per kilogram (mg/kg);

c, the concentration of each target object in the sample injection solution read from the standard working curve is milligram per liter (mug/mL);

v-volume of extraction system in milliliters (mL);

m-the mass of the sample in grams (g).