CN110988217A - Method for simultaneously determining main high sweetener in dry food packaging paper - Google Patents
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Abstract
The application discloses a method for simultaneously determining a main high sweetener in dry food packaging paper. The method comprises the steps of preparing an internal standard stock solution, preparing a series of standard working solutions, preparing a sample solution, analyzing by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), establishing a standard working curve and calculating a sample result. The method is characterized in that: (1) the silica gel bonded C18 solid phase extraction column is adopted to purify the sample extraction liquid, so that 9 targets can be effectively leached at the same time, and interferents are intercepted in column packing, the influence of interference components in the packing paper on the ionization efficiency of the targets is reduced, the content of 9 high-power sweeteners can be detected at the same time, the detection result is accurate, the interference is less, the sensitivity is high, and the repeatability is good. (2) Considering that the physical differences of 9 targets are large, the double internal standards are used for analysis, the targets are grouped and quantified, and the adverse effect of the targets due to the overlarge peak-appearing time difference is reduced.
Description
Technical Field
The invention relates to the technical field of food additive content determination, in particular to a method for simultaneously determining a main high-power sweetener in dry food packaging paper.
Background
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, particularly functional additives in food 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, various countries adopt a policy of strictly limiting the use of functional additives, and set a large number of standards, which become the key points of investigation in international trade and domestic standards.
Functional additives (acidulants, colorants, sweeteners, etc.) are an important class of food additives to improve the mouthfeel and flavor of food. The application range and the adding limit of functional additives (acidity regulator, colorant and sweetener) which can be used for food processing are definitely specified in national standard GB 2760 food additive use standard. With the increasing requirements for food safety, an important research in the field of food packaging materials focuses on whether additives in the food packaging materials can migrate from the food packaging materials to food and the distribution amount, and provides scientific judgment basis and method for objectively evaluating the intake amount of the additives (functional additives).
Currently, the detection technologies for the common artificially synthesized functional food additives mainly include liquid chromatography (mass spectrometry), ion chromatography, gas chromatography, spectrophotometry, thin layer chromatography, electrochemical methods, capillary electrophoresis, and the like. To date, there has been no report of simultaneous determination of 9 targets using a liquid chromatography-tandem mass spectrometer. In order to ensure the safety of food and related products, the production and use of flavoring agents in paper are enhanced and regulated, so that the development of a simpler, faster and highly sensitive detection method becomes necessary.
Disclosure of Invention
In order to solve the problems, the method has high accuracy and high detection flux.
According to one embodiment of the present application, a method of determining a primary high intensity sweetener in a dry food wrapper, comprises the steps of:
(1) preparing an internal standard stock solution: respectively taking xylitol and warfarin sodium as internal standard substances, and using water as a solvent to prepare internal standard stock solutions;
(2) preparation of a series of standard working solutions: taking standard products of main high sweetener of acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, alitame, neohesperidin, neotame and stevioside as targets, using water as a solvent, preparing a mixed standard stock solution, diluting step by step, and respectively adding an internal standard stock solution to prepare a series of standard working solutions;
(3) preparation of a sample solution: cutting dry food packaging paper, adding a solvent for soaking, performing ultrasonic extraction, shaking uniformly after the ultrasonic extraction is finished, leaching an extract liquor by using a silica gel bonded C18 solid phase extraction column to obtain a purified solution, and concentrating under a nitrogen blowing condition 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.
Typically, in step (3), the sample solution preparation process is: taking dry food packaging paper, cutting a sample with the area of 4 cm multiplied by 4 cm, accurately weighing the sample to the mass of 0.1 mg, cutting the sample into paper sheets with the size of about 1 mm multiplied by 1 mm, completely transferring the paper sheets into a 50 mL conical flask with a plug, accurately adding 500 muL of internal standard stock solution and 10 mL of water of an extraction solvent, infiltrating for 1 h, then performing ultrasonic extraction for 45min under the condition of 60% power, and shaking uniformly after the soaking is finished to obtain an extraction liquid. And purifying the extract by a silica gel bonded C18 solid phase extraction column, namely adding 5 mL of methanol to activate the column, adding 5 mL of water to balance so that less water is reserved on a sieve plate on the column, then transferring 1 mL of extract liquor to sample, discarding the sample liquor, leaching by using 4 mL of 80% methanol aqueous solution in volume ratio, and collecting the leaching liquor. The eluate was concentrated to 1 mL at 60 ℃ under nitrogen blowing, and the resulting solution was regarded as a sample.
Typically, in step (1), accurately weighing 0.0100 g of xylitol and 0.0050 g of warfarin sodium in the same 10 mL volumetric flask to the accuracy of 0.1 mg, dissolving with 10% methanol aqueous solution, fixing the volume, shaking up, and preparing an internal standard stock solution.
Typically, in the step (2), 6 mg of acesulfame potassium, 13mg of saccharin sodium, 5 mg of sodium cyclamate, 15 mg of sucralose, 3mg of aspartame, 3mg of alitame, 12 mg of neohesperidin, 1 mg of neotame and 10 mg of stevioside are accurately weighed respectively, are accurately weighed to 0.1 mg in the same 100 mL volumetric flask, are dissolved by a methanol aqueous solution with the volume concentration of 10% and subjected to constant volume and shake up, are prepared into a mixed standard stock solution, are accurately transferred to 100 muL, 200 muL, 500 muL, 1 mL, 2.5 mL and 5 mL of the mixed standard stock solution and 500 muL of the internal standard stock solution respectively, are mixed in the volumetric flask with water to 10 mL, are prepared into 6-level standard working solutions, and the concentration of each level of the serial solutions is as follows,
typically, in step (3), the silica gel-bonded C18 solid phase extraction column is preferably packed at 500 mg.
Typically, in step (4), HPLC-MS/MS analysis is performed using high performance liquid chromatography conditions: a chromatographic column: XBridge C18 with the specification of 150 mm multiplied by 2.1 mm and the filler particle size of 5 mu m; mobile phase A: methanol, B: 5 mmol/L ammonium acetate in water, column temperature: 40 ℃; sample introduction amount: 5 mu L, gradient elution conditions are as follows,
typically, in step (4), HPLC-MS/MS analysis, wherein the mass spectrometry conditions employed are: 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,
in the step (5), linear regression analysis is performed by taking the ratio of the concentration of each target object to the concentration of the internal standard in the series of standard working solutions as an abscissa and the ratio of the peak area of each target object to the peak area of the internal standard in the chromatogram as an ordinate, so as to obtain a standard working curve of each target object. Substituting the chromatographic peak area of each target object in the sample injection liquid measured under the same condition into a standard working curve, calculating the content of each target object in the sample according to the following formula,
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 silica gel bonded C18 solid phase extraction column is adopted to purify the sample extraction liquid, so that 9 targets can be effectively leached at the same time, and interferents are intercepted in column packing, the influence of interference components in the packing paper on the ionization efficiency of the targets is reduced, the content of 9 high-power sweeteners in the dry food packing paper can be detected at the same time, and the method has the advantages of accurate result, less interference, high sensitivity, good repeatability and the like. (2) Considering that the physical differences of 9 targets are large, the double internal standards are used for analysis, the targets are grouped and quantified, and the adverse effect of the targets due to the overlarge peak-appearing time difference is reduced. (3) The invention has better methodology investigation result, and has the advantages of high sensitivity and good repeatability.
Drawings
FIG. 1 is a standard working solution total ion current chromatogram;
in the figure, 1# is xylitol (retention time is 2.323 min), 2# is acesulfame potassium (retention time is 3.782 min), 3# is saccharin sodium (retention time 6.006 min), 4# is sodium cyclamate (retention time 9.876 min), 5# is sucralose (retention time 15.768 min), 6# is aspartame (retention time 17.779 min), 7# is alitame (retention time 21.386 min), 8# is warfarin sodium (retention time 23.189 min), 9# is neohesperidin (retention time 24.295 min), 10# is neotame (retention time 29.788 min), and 11# is stevioside (retention time 31.184 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
A standard sample of xylitol and warfarin sodium internal standard; standard samples of 9 sweeteners such as acesulfame potassium, saccharin sodium, cyclamate, sucralose, aspartame, alitame, neohesperidin, neotame, stevioside, etc.; methanol, ethanol, ammonium acetate (all chromatographically pure); water (meeting the requirements of first-grade water in GB/T6682).
6460 HPLC-MS/MS mass spectrometer (Agilent, USA); full-automatic quantitative concentration instrument of EVA08
(Beijing Pritaitake instruments Co., Ltd.); KQ-500DE ultrasonic generator (Kunshan ultrasonic instruments Co., Ltd.); electronic balance (METTLER TOLEDO, sensory 0.1 mg); Milli-Q ultrapure water system (Millipore, USA); a500 mg/6 mL silica gel-bonded C18 solid phase extraction column (Waters, USA).
2. Internal standard stock preparation
0.0102 g of xylitol and 0.0055 g of warfarin sodium are accurately weighed respectively, are accurately placed in the same 10 mL volumetric flask to be accurate to 0.1 mg, are dissolved by a methanol water solution with the volume concentration of 10%, and are subjected to constant volume and shaking up to prepare an internal standard stock solution.
3. Preparation of a series of standard working solutions
Accurately weighing 6 mg of acesulfame potassium, 13mg of saccharin sodium, 5 mg of sodium cyclamate, 15 mg of sucralose, 3mg of aspartame, 3mg of alitame, 12 mg of neohesperidin, 1 mg of neotame and 10 mg of stevioside respectively, accurately measuring the content of the sucralose in the same 100 mL volumetric flask to 0.1 mg, dissolving the sucralose in a 10% methanol aqueous solution, fixing the volume, shaking up the dissolved sucralose, and preparing a mixed standard storage solution. Respectively and accurately transferring 100 muL, 200 muL, 500 muL, 1 mL, 2.5 mL and 5 mL of mixed standard stock solution and 500 muL of internal standard stock solution respectively, diluting with water to a constant volume of 10 mL volumetric flasks, uniformly mixing, preparing to obtain 6-grade standard working solution, wherein the concentration of each-grade series solution is as follows,
4. sample solution preparation
Taking a commercially available dry food packaging paper No. 1, cutting a sample with the area of 4 cm multiplied by 4 cm, accurately weighing the sample with the mass of 0.1 mg, shearing the sample into paper sheets with the size of about 1 mm multiplied by 1 mm, completely transferring the paper sheets into a 50 mL conical flask with a plug, accurately adding 500 mu L of internal standard stock solution and 10 mL of water of an extraction solvent, infiltrating for 1 h, then carrying out ultrasonic extraction for 45min under the condition of 60% power, and shaking up after the completion to obtain an extraction liquid. And purifying the mixture by a silica gel bonded C18 solid phase extraction column, wherein the filler content is 500mg, namely, adding 5 mL of methanol to activate the column, then adding 5 mL of water to balance so that less water is reserved on a sieve plate on the column, then transferring 1 mL of extract liquor to sample, discarding the sample liquor, then leaching by using 4 mL of 80% methanol aqueous solution by volume ratio, and collecting the leacheate. The eluate was concentrated to 1 mL at 60 ℃ under nitrogen blowing, and the resulting solution was regarded as a sample.
6. HPLC-MS/MS assay
See summary of the invention section, which is not repeated here.
7. Calculation of results
Quantitative analysis is carried out by a double-internal standard method, wherein the first 3 sweeteners of acesulfame potassium, saccharin sodium and sodium cyclamate are quantified by using xylitol, and the last 6 sweeteners of sucralose, aspartame, alitame, neohesperidin, neotame and stevioside are quantified by using warfarin sodium. In the calculation process, linear regression analysis is carried out by taking the ratio of the concentration of each target object to the concentration of the internal standard in the series of standard working solutions as a horizontal ordinate and the ratio of the peak area of each target object to the peak area of the internal standard in the chromatogram as a vertical ordinate to obtain the standard working curve of each target object. Substituting the chromatographic peak area of each target object in the sample injection liquid measured under the same condition into a standard working curve to obtain the content of each target object in the sample, wherein only neotame is detected in the sample No. 1, and the content is 0.68 mg/kg.
The evaluation of the detection method of the present application is as follows:
① detection limit of the method:
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 recovery and reproducibility of the process by spiking:
a standard solution of a sweetener is added to a blank paper sample, which is then subjected to pretreatment and HPLC-MS/MS analysis, respectively, and the recovery rate is calculated according to the addition amount and the measured value.
The working curve (linear coefficient), detection limit, recovery rate and repeatability of each target are as follows,
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 (7)
1. A method for simultaneously measuring main high sweetener in dry food packaging paper is characterized by comprising the following steps:
(1) preparing an internal standard stock solution: respectively taking xylitol and warfarin sodium as internal standard substances, and using water as a solvent to prepare internal standard stock solutions;
(2) preparation of a series of standard working solutions: taking standard products of main high sweetener of acesulfame potassium, saccharin sodium, sodium cyclamate, sucralose, aspartame, alitame, neohesperidin, neotame and stevioside as targets, using water as a solvent, preparing a mixed standard stock solution, diluting step by step, and respectively adding an internal standard stock solution to prepare a series of standard working solutions;
(3) preparation of a sample solution: cutting dry food packaging paper, adding a solvent for soaking, performing ultrasonic extraction, shaking uniformly after the ultrasonic extraction is finished, leaching an extract liquor by using a silica gel bonded C18 solid phase extraction column to obtain a purified solution, and concentrating under a nitrogen blowing condition 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.
2. The method of claim 1, wherein in the step (3), the sample solution is prepared by: taking dry food packaging paper, cutting a sample with the area of 4 cm multiplied by 4 cm, accurately weighing the sample with the mass of 0.1 mg, shearing the sample into paper sheets with the size of about 1 mm multiplied by 1 mm, completely transferring the paper sheets into a 50 mL conical flask with a plug, accurately adding 500 muL of internal standard stock solution and 10 mL of water of an extraction solvent, infiltrating for 1 h, then performing ultrasonic extraction for 45min under the condition of 60% power, shaking uniformly after the completion to obtain extract liquor, then purifying the extract liquor by a silica gel bonded C18 solid phase extraction column, wherein the filler content is 500mg, namely firstly adding 5 mL of methanol to activate the column, then adding 5 mL of water to balance so that a small amount of water is reserved on a sieve plate on the column, then transferring 1 mL of extract liquor to sample, abandoning the sample liquor, then leaching by using 4 mL of 80% methanol water solution in volume ratio, collecting leacheate, concentrating the leacheate under the condition of 60 ℃ to 1 mL, the sample solution is regarded as the sample solution.
3. The method as claimed in claim 1, wherein in step (1), 0.0100 g of xylitol and 0.0050 g of warfarin sodium are accurately weighed respectively, are accurately weighed in the same 10 mL volumetric flask to the accuracy of 0.1 mg, and are dissolved by 10% methanol aqueous solution, and are subjected to volume fixing, shaking and internal standard stock solution preparation.
4. The method according to claim 2 or 3, characterized in that in the step (2), 6 mg of acesulfame potassium, 13mg of saccharin sodium, 5 mg of sodium cyclamate, 15 mg of sucralose, 3mg of aspartame, 3mg of alitame, 12 mg of neohesperidin, 1 mg of neotame and 10 mg of stevioside are accurately weighed respectively, are accurately measured to 0.1 mg in the same 100 mL volumetric flask, are dissolved by a methanol aqueous solution with a volume concentration of 10% and are subjected to constant volume and shaking uniformly to prepare a mixed standard stock solution, are accurately transferred to 100 muL, 200 muL, 500 muL, 1 mL, 2.5 mL and 5 mL of the mixed standard stock solution and 500 muL of each internal standard stock solution respectively, are mixed uniformly by water to 10 mL volumetric flasks, are subjected to constant volume to prepare 6-grade standard working solutions, and the concentrations of series solutions at each level are as follows,
5. 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: XBridge C18 with the specification of 150 mm multiplied by 2.1 mm and the filler particle size of 5 mu m; mobile phase A: methanol, B: 5 mmol/L ammonium acetate in water, column temperature: 40 ℃; sample introduction amount: 5 mu L, gradient elution conditions are as follows,
6. 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,
7. the method according to claim 1, wherein in the step (5), a linear regression analysis is performed with the ratio of the concentration of each target to the concentration of the internal standard in the series of standard working solutions as the abscissa and the ratio of the peak area of each target to the peak area of the internal standard in the chromatogram as the ordinate to obtain a standard working curve for each target, the chromatographic peak areas of each target in the sample introduction solution measured under the same conditions are 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:
w-the amount of each target in the sample in milligrams per kilogram (mg/kg);
a-peak area of the primary high intensity sweetener;
as-peak area of xylitol and warfarin sodium As internal standard, wherein the first 3 sweeteners were quantified using xylitol and the last 6 sweeteners were quantified using warfarin sodium;
ms is the mass of the internal standard added in the sample solution, and the unit is microgram (microgram);
m-the mass of the sample in grams (g).
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| CN116165304A (en) * | 2023-03-20 | 2023-05-26 | 红塔辽宁烟草有限责任公司 | Method for detecting 8 sweeteners in tipping paper for cigarettes by using ultra-high performance liquid chromatography-tandem mass spectrometry |
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Address after: No. 406-3, Zhongshan North Road, Nanjing, Jiangsu 210019 Applicant after: CHINA TOBACCO JIANGSU INDUSTRIAL Co.,Ltd. Address before: Bell tower of technical center, No.29 Xinglong Street, Jianye District, Nanjing City, Jiangsu Province Applicant before: CHINA TOBACCO JIANGSU INDUSTRIAL Co.,Ltd. |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |