Method for determining food additives in pastries by means of high-performance liquid chromatography / mass spectrometry

Abstract

The present invention belongs to the technical field of food additive detection, and particularly relates to a method for determining food additives in pastries by means of high-performance liquid chromatography / mass spectrometry. The detection method comprises: (1) pre-treating a sample to be detected; (2) determining parameter conditions for high-performance liquid chromatography and mass spectrometry; (3) determining the peak time, the detection ion pair and the quantitative ion pair of each food additive; and (4) testing a sample solution to be detected. The detection method of the present invention involves simple and convenient pre-treatment operation, utilizes high-performance liquid chromatography / mass spectrometry, and also uses mother ions, characteristic fragment ions and peak time as a qualitative basis, such that the obtained linear range in the method is wide, the detection limit is low and the recovery rate is high. The method is suitable for simultaneously detecting five types of food additives in pastries.

Description

A method for determining food additives in cakes by high performance liquid chromatography / mass spectrometry TECHNICAL FIELD

[0001] The present application belongs to the technical field of food additive detection, and particularly relates to a method for determining food additives in cakes by high performance liquid chromatography / mass spectrometry. BACKGROUND

[0002] Cakes are a kind of food processed by steaming, baking, frying, stir-frying and the like from flour, oil, sugar, eggs and the like as main raw materials. Since cakes belong to high-fat, high-protein and high-sugar food, they are easily contaminated by bacteria and molds and thus deteriorate, and therefore, adding appropriate additives in food can slow down the deterioration of food, prolong the shelf life of food and improve economic benefits. However, due to the fact that some enterprises do not strictly control auxiliary materials, and the fact that detection equipment technology is backward, the phenomenon of using food additives beyond the scope and limit in cakes is common.

[0003] At present, the food additives contained in cakes mainly include cyclamate, sodium saccharin, benzoic acid, acesulfame and sorbic acid. However, if the five kinds of food additives are detected at the same time, multiple detection methods need to be used, and the detection period is long.

[0004] Therefore, there is an urgent need to provide a method which is efficient and can simultaneously detect the five kinds of food additives.

[0005] SUMMARY

[0006] To solve the above technical problems, the present application provides a method for determining food additives in cakes by high performance liquid chromatography / mass spectrometry.

[0007] To achieve the above object, the present application provides the following technical scheme:

[0008] One of the technical schemes of the present application is:

[0009] A method for determining food additives in cakes by high performance liquid chromatography / mass spectrometry, characterized in that it comprises the following steps:

[0010] (1) Pretreatment of the sample to be tested: 2 g of the cake sample is weighed and transferred into a 50 mL centrifuge tube, 15 mL of pure water is added, vortexed and mixed, ultrasonic treatment is performed at 50°C for 20 min, a decontaminating agent is added for decontamination, vortexed and mixed, centrifuged at 8000 r / min for 4 min, the water phase is transferred into a 25 mL volumetric flask, 10 mL of pure water is added to the residue, vortexed and mixed, ultrasonic treatment is performed at 50°C for 20 min, centrifuged at 8000 r / min for 4 min, the supernatant is combined, diluted with pure water to 25 mL, and filtered through a 0.22 μm filter membrane to obtain a sample solution to be tested; in addition, for cakes with high oil content, n-hexane can be added to the supernatant, vortexed and mixed, the upper liquid is discarded, and the lower liquid is diluted with pure water to 25 mL and filtered through a 0.22 μm filter membrane;

[0011] (2) Determination of the parameter conditions of high performance liquid chromatography and mass spectrometry;

[0012] (3) Determination of the peak time and monitoring ion pair of each food additive;

[0013] (4) Testing of the sample solution to be tested: the sample to be tested is injected, high performance liquid chromatography-mass spectrometry detection is performed, and quantitative analysis of the sample to be tested is performed according to the peak time and the monitoring ion pair of the food additive.

[0014] Preferably, the food additive includes one or more of acesulfame potassium, benzoic acid, sorbic acid, sodium cyclamate and sodium saccharin. Among them, the five food additives detected by the present application are sodium or potassium salts, which have good water solubility. The cake includes cake, bread, shortcake and biscuit.

[0015] Preferably, the decontamination process in step (1) is: after the water bath ultrasonic treatment, the mixture is cooled to room temperature, and potassium ferrocyanide solution and zinc acetate solution are added for decontamination; wherein the volume-to-mass ratio of the potassium ferrocyanide solution, the zinc acetate solution and the cake sample is 1 mL: 1 mL: 2 g.

[0016] Further, the concentrations of the potassium ferrocyanide solution and the zinc acetate solution are 92 g / L and 183 g / L, respectively.

[0017] Preferably, the parameter conditions of high performance liquid chromatography in step (2) are:

[0018] The chromatographic column is an Agilent 2.1x150 mm C18 column; the mobile phase is methanol and water, and the volume ratio of the two is 40:60; the column temperature is 40°C; the injection amount is 20 μL; the elution mode is isocratic elution; the flow rate is 0.4 mL / min; and the detection time is 2 min.

[0019] Beneficial effects: the detection is carried out by using the above process conditions, isodense elution is adopted, an Agilent C18 column (3 μm, 2.1*150 mm) can be used for separation in a short time, and work efficiency is greatly improved.

[0020] Preferably, the parameter conditions of the mass spectrometry in step (2) are as follows:

[0021] The mass spectrometer is an Agilent G6420A type triple quadrupole mass spectrometer, the ionization mode is an electrospray ion source, the scanning mode is Negalive, the detection mode is MRM, the capillary is -4000 V, the nebulizer is 15 Psi, the gas flow is 11.0 L / min, the gas temperature is 300 DEG C, the fragmentor is 50-130 V, and the collision energy is 0-20 eV.

[0022] Preferably, the specific steps for determining the peak time and monitoring ion pairs of each food additive in step (3) are as follows:

[0023] 2 g of the cake negative sample is weighed, pretreated by the pretreatment in step (1), and the supernatant is taken into a 25 mL volumetric flask; the corresponding standard material solution is added, and the initial mobile phase (methanol and water, the volume ratio is 40:60, the same below) is used for constant volume (using the mobile phase for constant volume can reduce solvent interference and enhance the response value), to obtain a standard working solution; the standard working solution is subjected to parent ion scanning by the mass spectrometer, the monitoring ion pairs of each food additive are determined, and the standard working solution is subjected to liquid chromatography detection, and the peak time of each food additive is determined.

[0024] Further, the standard material solution is as follows:

[0025] The single food additive solution has a concentration of 1000 ng / mL.

[0026] Preferably, the injection volume of the sample solution to be detected in step (4) is 20 μL.

[0027] Compared with the prior art, the method has the following advantages and technical effects:

[0028] The detection method for food additives in cakes provided by the application is a high performance liquid chromatography-triple quadrupole mass spectrometry method under specific process conditions, has the advantages of short time, high efficiency and accurate results. DETAILED DESCRIPTION

[0029] Example 1

[0030] A detection method for food additives in a cake, comprising the following steps:

[0031] Food additives include acesulfame, benzoic acid, sorbic acid, sodium cyclamate, sodium saccharin; each food additive is configured into a standard material solution of 1000 ng / mL;

[0032] (1) Sample processing: 2.0 g of cake sample was weighed into a 50 mL centrifuge tube, 15 mL of pure water was added, vortexed and mixed, ultrasonic was performed at 50℃ for 20 min, cooled to room temperature, 1 mL of potassium ferrocyanide and 1 mL of zinc acetate were added, vortexed and mixed, centrifuged at 8000 r / min for 4 min, the water phase was transferred into a 25 mL volumetric flask, the residue was added with 10 mL of pure water, vortexed and mixed, ultrasonic was performed at 50℃ for 20 min, centrifuged at 8000 r / min for 4 min, the supernatant was combined, diluted with pure water to 25 mL, filtered through a 0.22 μm filter membrane to obtain a sample solution to be tested;

[0033] (2) The conditions of the instrument used were set as follows: the conditions of chromatography included: the liquid chromatograph was an Agilent 1290 type high performance liquid chromatograph; the chromatographic column was an Agilent 2.1*150 mm C18 column; the mobile phase was methanol+water (40 mL+60 mL); the chromatographic column temperature was 40℃; the sample injection amount was 20 μL; the elution mode was isocratic elution; the flow rate was 0.4 mL / min; the detection time was 2 min;

[0034] The conditions of mass spectrometry included: the mass spectrometer was an Agilent G6420A type triple quadrupole mass spectrometer; the ionization mode was an electrospray ion source; the scanning mode was Negalive; the detection mode was MRM; the Capillary was -4000 V; the Nebulizer was 15 Psi; the Gas Flow was 11.0 L / min; the Gas Temp was 300℃; the Fragmentor was 50-130 V; the Collision Energy was 0-20 eV;

[0035] (3) The peak time of each food additive and the determination of the monitoring ion pair:

[0036] 2.0 g of cake negative matrix was weighed into a 25 mL volumetric flask, the corresponding standard material solution was added, diluted with the initial mobile phase to obtain a single standard working solution with a concentration of 500 ng / mL, the standard working solution was scanned by the mass spectrometer to determine the monitoring ion pair of each food additive; the standard working solution was detected by high performance liquid chromatography to determine the peak time of each food additive. The reference peak time (RT) of each food additive was:

[0037] Benzoic acid (0.857 min), sorbic acid (0.708 min), sodium saccharin (1.053 min), sodium cyclamate (0.679 min) and acesulfame (0.611 min).

[0038] (4) Determination of Fragmentor and Collision Energy of each food additive:

[0039] Each single food additive standard working solution with a concentration of 500 ng / mL was subjected to ion optimization scanning under Optimizer mode, and the Fragmentor and Collision Energy of each single food additive were determined under this mode; the relevant test results are shown in Table 1:

[0040] Table 1 Main reference mass spectrum parameters of 5 food additives

[0041] (5) Test of the sample solution to be tested: the sample solution to be tested was taken for injection, the injection volume was 20 μL, and liquid chromatography-mass spectrometry detection was performed, and the sample to be tested was quantitatively analyzed according to the peak time and monitored ion pair.

[0042] Effect verification

[0043] Detection limit and determination limit of the method in Effect Example 1

[0044] When the sample to be tested is determined, if the peak time of the chromatographic peak of the sample to be tested is consistent with that of the standard, and after deducting the background, the relative abundance of each qualitative ion in the sample spectrum is close to that of the standard solution spectrum obtained under the same conditions, and the maximum allowable relative deviation conforms to the range specified in EU 2002 / 657 / EC (Q1 / Q3 ion pair (parent ion / daughter ion) is shown in Table 1), it can be judged that the corresponding component is detected in the sample to be tested.

[0045] However, for benzoic acid and sorbic acid, since only one secondary fragment ion is produced under ESI-detection mode, it is impossible to calculate the relative abundance of the fragment ion, therefore, the peak time combined with one Q1 / Q3 ion pair is used for qualitative analysis in the present application. That is, the method of adding a standard substance solution in a negative matrix, and determining the detection limit (LOD) according to the signal-to-noise ratio (S / N) of the MRM chromatographic peak is greater than 3 times, and the minimum quantitative limit (LOQ) is greater than 10 times. The LOD and LOQ of the 5 target components are shown in Table 2.

[0046] The determination limit of the method is calculated according to the signal-to-noise ratio of the instrument, and the determination limit is generally defined as the concentration when the signal-to-noise ratio (S / N) is 3:1. The calculation formula is: D=3N / S (1)

[0047] In formula (1), N-noise; S-detector sensitivity; D-detection limit; S / N-signal-to-noise ratio

[0048] And the sensitivity formula is: S = I / Q (2)

[0049] In formula (2): S-detection sensitivity; I-signal response value; Q-injection volume

[0050] The signal-to-noise ratio of food additives when the injection volume is 5.0 ng / mL and 10 ng / mL is shown in Table 2 below, and the detection limit can be obtained by substituting formula (1) as shown in Table 2 below, and the determination limit can be calculated according to formula (3) as shown in Table 2 below.

[0051] c1- is the detection limit of benzoic acid, sorbic acid, sodium saccharin, sweetener, acesulfame under the corresponding injection volume; c0 is the detection limit of benzoic acid, sorbic acid, sodium saccharin, sweetener, acesulfame in the blank, which is 0 ng / mL; m is the mass of the sample taken, which is 2 g; V is the total volume of the sample solution, which is 25 mL; X is the determination limit.

[0052] Table 2 Signal-to-noise ratio, detection limit (ng / mL) and determination limit (mg / kg) of each food additive

[0053] Conclusion: The results in Table 2 show that the detection limit and determination limit of each food additive are 0.1-0.5 ng / mL and 0.002-0.005 mg / kg, respectively.

[0054] Effect Example 2 Precision and accuracy test

[0055] (1) Precision test: The same positive sample of cakes was pretreated according to the previous method, and 6 parallel determinations were performed on the machine. The precision test data are shown in Table 3.

[0056] Among them, the relative standard deviation is calculated according to the following formula:

[0057] In formula (5)-(7): x k The kth test result of the sample; - The average value of sample testing; S- the standard deviation of sample testing; RSD- the relative standard deviation of sample testing, n-6 parallel determinations.

[0058] The calculation formula of reproducibility R is:

[0059] Table 3 Precision test data of food additives

[0060] (2) Accuracy test

[0061] Accurately weigh 1.0 g of the cake sample (negative matrix) without the target compound into a 25 mL volumetric flask, accurately add a certain amount of each additive standard solution with a micro pipette, and dilute to the mark with the initial mobile phase. Mix well to prepare the quality control (QC) samples at low, medium, and high concentration levels. Each concentration is determined in 6 parallel tests.

[0062] The calculation formula of relative error is:

[0063] In formula (8): - the average value of the test of a certain concentration standard substance; μ - the concentration of the standard substance; RE - the relative error of the test of a certain concentration standard substance;

[0064] The calculation formula of the recovery rate of the spiked sample is:

[0065] In formula (9): - the average value of the test of a certain concentration sample; - - the average value of the test of the spiked sample; P - the recovery rate of the spiked sample;

[0066] The calculated values of the measurements of the spiked samples are shown in Table 4:

[0067] Table 4 Recovery rate and relative error (RE) of the spiked samples of food additives (n = 6)

[0068] Conclusion: The results in Table 4 show that each food additive at low, medium, and high concentration levels in the cake has good accuracy and precision. The recovery rate of each target compound in the cake is 95-110%, and the relative error (RE) is -3.5-9%;

[0069] Effect Example 3 Application of the detection method

[0070] According to the detection method provided in Example 1, the food additives in 15 commercially available breads, 15 biscuits, 8 shortcakes, and 8 cakes were determined.

[0071] Among them, sorbic acid was detected in bread and cake, with a content of 0.03-0.5 g / kg and 0.03-0.61 g / kg, respectively. Sorbic acid was detected in biscuits and shortcakes, with a content of 0.03-0.30 g / kg and 0.03-0.25 g / kg, respectively. Among them, the detection rate of sorbic acid as a food additive in bread and cake is about 90%; the detection rate of sorbic acid in biscuits and shortcakes is 79%; and the detection rate of benzoic acid, sodium saccharin, cyclamate, and acesulfame in bread, cake, biscuits, and shortcakes is almost 0.

[0072] The above merely provides the preferred embodiments of the present application, and the protection scope of the present application is not limited thereto, and any changes or substitutions within the technical scope disclosed by the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for determining food additives in cakes by high performance liquid chromatography / mass spectrometry, characterized by, Includes the following steps: (1) Pretreatment of the sample to be tested: The pastry sample was vortexed, ultrasonicated in water bath, impurities removed, vortexed, and centrifuged to obtain supernatant and residue; the residue was vortexed and centrifuged to obtain supernatant. Then combine the supernatants, make up to volume, filter, and obtain the sample solution to be tested; (2) Determine the parameter conditions for high performance liquid chromatography and mass spectrometry; (3) Determine the peak time and monitor the ion pairs for each food additive; (4) Test the sample solution to be tested.

2. The method for determining food additives in cakes according to claim 1, characterized in that, The food additives include one or more of acesulfame potassium, benzoic acid, sorbic acid, cyclamate, and sodium saccharin.

3. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 1, characterized in that, The impurity removal process described in step (1) is as follows: potassium ferrocyanide solution and zinc acetate solution are added for impurity removal.

4. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 3, characterized in that, The concentrations of the potassium ferrocyanide solution and the zinc acetate solution were 92 g / L and 183 g / L, respectively.

5. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 1, characterized in that, The parameter conditions for the high performance liquid chromatography method described in step (2) are as follows: Mobile phase: methanol and water, with a volume ratio of 40:60; column temperature: 40℃; elution mode: isocratic elution; flow rate: 0.4 mL / min; detection time: 2 min.

6. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 1, characterized in that, The parameter conditions for the mass spectrometry method in step (2) are as follows: The ionization mode was electrospray ionization; the scanning method was Negalive; the detection method was MRM; the capillary was -4000V; the nebulizer was 15Psi; the gas flow was 11.0L / min; the gas temperature was 300℃; the fragmentor was 50-130V; and the collision energy was 0-20eV.

7. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 1, characterized in that, The specific steps for determining the peak time and monitoring ion pairs for each food additive in step (3) are as follows: After the negative sample of pastry is pretreated as described in step (1), it is mixed with the standard substance solution and the volume is adjusted to obtain the standard working solution; The standard working solution was scanned for precursor ions using a mass spectrometer to determine the monitoring ion pairs for each food additive. The standard working solution was subjected to high performance liquid chromatography to determine the elution time of each food additive.

8. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 7, characterized in that, The standard substance solution is: A single food additive solution with a concentration of 1000 ng / mL.

9. The method for determining food additives in pastries by high performance liquid chromatography / mass spectrometry according to claim 1, characterized in that, The injection volume of the sample solution to be tested in step (4) is 20 μL.

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