A method for detecting preservatives in food

Abstract

This invention belongs to the field of food testing technology, specifically relating to a method for detecting preservatives in food. The method involves acidifying the sample, extracting with acetonitrile, removing grease and impurities with alkane and saturated sodium chloride solution, reconstituted with nitrogen, and then determining the preservatives using gas chromatography-mass spectrometry (GC-MS). This method can simultaneously determine 12 preservatives (acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben). The procedure is simple and rapid, requires small sample volumes, and has high sensitivity. The method detection limit ranges from 0.04 to 2.00 mg / kg, and the quantitation limit ranges from 0.12 to 6.67 mg / kg.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting preservatives in food. Background Technology

[0002] Food preservatives, also known as antimicrobial agents, can directly or indirectly act on the proteins, genetic material, and enzyme systems of microorganisms to interfere with their growth, reproduction, and metabolism. This effectively slows down food spoilage, extends shelf life, enhances economic benefits, and reduces food poisoning caused by microbial growth. Food preservatives can be classified according to their function into bactericides and bacteriostatic agents, and according to their composition and source into chemical preservatives and natural preservatives (also known as biological preservatives). Chemical preservatives can be further subdivided into three main types: acidic preservatives, ester-based preservatives, and inorganic salt preservatives. Currently, the chemical preservatives permitted for use in my country include benzoic acid and its salts, sorbic acid and its salts, dehydroacetic acid and its sodium salt, and parabens (para-hydroxybenzoic acid esters).

[0003] With the increasing variety of food preservatives, the misuse of food preservatives is common in the market. my country has formulated GB 2760-2014, the National Food Safety Standard for the Use of Food Additives, to regulate the use of various preservatives. Current national and industry standards also introduce a series of testing methods for different preservatives in different food matrix components. However, as food safety monitoring requirements become increasingly stringent, the scope of coverage expands, and the testing workload grows, there is an urgent need to develop a high-throughput detection method capable of simultaneously detecting multiple preservatives. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting additives in food. The detection method of this invention can simultaneously detect acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben, achieving efficient and rapid detection of 12 preservatives.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting preservatives in food, comprising the following steps:

[0007] The sample to be tested, inorganic acid, and acetonitrile were mixed and extracted to obtain the extract;

[0008] The extract, alkane, and saturated sodium chloride solution were mixed and purified. The purified solution was then subjected to nitrogen blowing and redissolution to obtain the test solution. The alkane included n-hexane and / or n-heptane.

[0009] The test solution was analyzed by gas chromatography-mass spectrometry, and the content of preservatives was obtained according to a predetermined standard curve. The preservatives included acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben.

[0010] The gas chromatography-mass spectrometry (GC-MS) conditions included: DB-FFAP capillary column; temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; injection port temperature: 300℃; carrier gas: helium; carrier gas flow rate: 1.0 mL / min; split ratio: 5–10:1; injection volume: 1.0 μL.

[0011] The mass spectrometry conditions for the gas chromatography-mass spectrometry analysis include: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; and selection of ion monitoring mode.

[0012] Preferably, the ratio of the sample to be tested to acetonitrile is 1-2 g: 10-15 mL.

[0013] Preferably, the inorganic acid includes hydrochloric acid.

[0014] Preferably, the volume ratio of the extract to the alkane is 5:1.5 to 2.5.

[0015] Preferably, the volume ratio of the extract to the saturated sodium chloride solution is 5:1.5 to 2.5.

[0016] Preferably, the solvent for redissolution is methanol.

[0017] Preferably, the volume ratio of the purified liquid to the reconstituted solvent is 5 to 10:1.

[0018] Preferably, the predetermined standard curve uses the concentration ratio of preservative to internal standard as the abscissa and the peak area ratio of preservative to internal standard as the ordinate. The concentrations of the preservatives are: acetic acid, propionic acid, sorbic acid, dehydroacetic acid, and benzoic acid, each independently ranging from 2.5 to 50 μg / mL; and dimethyl fumarate, methylparaben, ethylparaben, propylparaben, butylparaben, isopropylparaben, and isobutylparaben, each independently ranging from 1 to 20 μg / mL.

[0019] Preferably, the internal standard includes ethyl D4-p-hydroxybenzoate.

[0020] Preferably, the concentration of D4-p-hydroxybenzoate ethyl ester is 40-60 μg / mL.

[0021] This invention provides a method for detecting preservatives in food, comprising the following steps:

[0022] The sample to be tested, inorganic acid, and acetonitrile were mixed and extracted to obtain the extract;

[0023] The extract, alkane, and saturated sodium chloride solution were mixed and purified. The purified solution was then subjected to nitrogen blowing and redissolution to obtain the test solution. The alkane included n-hexane and / or n-heptane.

[0024] The test solution was analyzed by gas chromatography-mass spectrometry, and the content of preservatives was obtained according to a predetermined standard curve. The preservatives included acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben.

[0025] The gas chromatography-mass spectrometry (GC-MS) conditions included: DB-FFAP capillary column; temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; injection port temperature: 300℃; carrier gas: helium; carrier gas flow rate: 1.0 mL / min; split ratio: 5–10:1; injection volume: 1.0 μL.

[0026] The mass spectrometry conditions for the gas chromatography-mass spectrometry analysis include: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; and selection of ion monitoring mode.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention involves acidifying the sample, extracting it with acetonitrile, removing grease and impurities with alkane and saturated sodium chloride solution, reconstituted by nitrogen blowing, and then determining it using gas chromatography-mass spectrometry (GC-MS). It can simultaneously determine 12 preservatives (acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben), with high sensitivity; the method detection limit ranges from 0.04 to 2.00 mg / kg, and the quantitation limit ranges from 0.12 to 6.67 mg / kg.

[0029] Furthermore, the internal standard method used in this invention reduces errors caused by pretreatment, and the retention time and characteristic ion qualitative analysis reduce the probability of false positives. This method is suitable for rapid and effective detection of preservatives in food, facilitating the regulation of preservatives. The detection method of this invention requires small sample volumes, has simple and rapid operation steps, and uses minimal reagents, thus reducing laboratory contamination. The detection method of this invention provides accurate and reliable results with good precision. The 12 preservatives exhibit good linearity within the concentration range of 1.0–50 μg / mL, with correlation coefficients all greater than 0.99. The average recoveries of samples at low, medium, and high spiked concentrations ranged from 70.18% to 109.22%, with relative standard deviations ranging from 1.82% to 9.79% (n=6). Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Chromatograms of 12 food preservatives (all at a concentration of 0.1 mg / mL) and their internal standard solutions;

[0032] Figure 2 Chromatograms using a VF-WAXms column;

[0033] Figure 3 The chromatogram of the scallion-flavored soda cracker sample;

[0034] Figure 4 Chromatogram of a sample of steamed bread from Hong Kong Rong;

[0035] Figure 5 This is the chromatogram of the condensed milk toast sample. Detailed Implementation

[0036] This invention provides a method for detecting preservatives in food, comprising the following steps:

[0037] The sample to be tested, inorganic acid, and acetonitrile were mixed and extracted to obtain the extract;

[0038] The extract, alkane, and saturated sodium chloride solution were mixed and purified. The purified solution was then subjected to nitrogen blowing and redissolution to obtain the test solution. The alkane included n-hexane and / or n-heptane.

[0039] The test solution was analyzed by gas chromatography-mass spectrometry, and the content of preservatives was obtained according to a predetermined standard curve. The preservatives included acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben.

[0040] The gas chromatography-mass spectrometry (GC-MS) conditions included: DB-FFAP capillary column; temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; injection port temperature: 300℃; carrier gas: helium; carrier gas flow rate: 1.0 mL / min; split ratio: 5–10; injection volume: 1.0 μL.

[0041] The mass spectrometry conditions for the gas chromatography-mass spectrometry analysis include: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; and selection of ion monitoring mode.

[0042] Unless otherwise specified, all reagents, materials and equipment used in this invention are commercially available products in the field.

[0043] This invention involves mixing the sample to be tested, inorganic acid, and acetonitrile for extraction to obtain an extract.

[0044] In this invention, the sample to be tested is preferably a pulverized pastry. This invention does not have special requirements for the pulverization method, and any method commonly used by those skilled in the art is acceptable. The pastry preferably includes shortbread, soda crackers, cakes, or toast. When the sample to be tested is a liquid sample, this invention does not have special requirements for the preparation method of the liquid sample, and it is acceptable to mix it evenly.

[0045] In this invention, the inorganic acid preferably includes hydrochloric acid, which is preferably obtained by diluting commercially available concentrated hydrochloric acid with water. The mass percentage concentration of the commercially available concentrated hydrochloric acid is preferably 36-38%, the volume ratio of the commercially available concentrated hydrochloric acid to water is preferably 1:1, and the volume ratio of hydrochloric acid to acetonitrile is preferably 0.4-0.6:10-15, more preferably 0.5:10. The addition of hydrochloric acid can eliminate the interference of weak acid anions and create an acidic extraction environment.

[0046] In this invention, the preferred ratio of the sample to acetonitrile is 1-2 g: 10-15 mL, more preferably 2 g: 10 mL.

[0047] In this invention, the mixing method is preferably a vortex. This invention does not have special requirements for the parameters of the vortex, and methods commonly used by those skilled in the art can be adopted.

[0048] In this invention, the mixing process preferably includes centrifugation, during which the preservative is extracted into acetonitrile, and the supernatant obtained by centrifugation is the extract.

[0049] After obtaining the extract, the present invention purifies the extract, alkanes and saturated sodium chloride solution by mixing them, and then subjecting the purified solution to nitrogen blowing and redissolution in sequence to obtain the test solution; the alkanes include n-hexane and / or n-heptane.

[0050] In this invention, the extract and alkane are first mixed, the alkane layer is discarded after separation, and then a saturated sodium chloride solution is added for a second mixing. After further separation, a purified liquid is obtained, wherein the purified liquid is the upper acetonitrile layer. The first and second mixing methods are both preferably vortexed, and the mixing time for both is preferably 1 min. The vortex speed is preferably 3000-5000 r / min. The separation and re-separation methods are both preferably centrifuged, and the centrifugation speed is preferably 5000 r / min, and the centrifugation time is preferably 3 min. The vortexing and centrifugation conditions described in this invention can reduce the occurrence of emulsification.

[0051] In this invention, the volume ratio of the extract to the alkane is preferably 5:1.5 to 2.5, more preferably 5:2; the alkane is preferably n-hexane, and the function of the alkane is to remove fat. The number of times the fat is removed is preferably 1 to 3 times. When the number of times the fat is removed is multiple times, this invention preferably removes fat multiple times consecutively and then adds saturated sodium chloride solution for impurity removal.

[0052] In this invention, the volume ratio of the extract to the saturated sodium chloride solution is preferably 5:1.5 to 2.5, more preferably 5:2; the function of the saturated sodium chloride solution is to remove impurities (including removing water, sugar, and hydrochloric acid); the number of impurity removal operations is preferably 1 to 3 times. The saturated sodium chloride solution also serves to: increase surface tension to prevent emulsification; salt out, reduce the solubility of the organic phase (acetonitrile) in the aqueous phase, and facilitate layering and extraction of the target preservative.

[0053] The present invention does not have special requirements for the parameters of the nitrogen blowing; methods commonly used by those skilled in the art can be adopted.

[0054] In this invention, the solvent for resolution is preferably methanol, and the volume ratio of the purified liquid to the solvent for resolution is preferably 5 to 10:1, more preferably 5:1.

[0055] After obtaining the test solution, the present invention performs gas chromatography-mass spectrometry analysis on the test solution and obtains the content of preservatives according to a predetermined standard curve; the preservatives include acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben;

[0056] The gas chromatography-mass spectrometry (GC-MS) conditions included: DB-FFAP capillary column; temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; injection port temperature: 300℃; carrier gas: helium; carrier gas flow rate: 1.0 mL / min; split ratio: 5–10:1; injection volume: 1.0 μL.

[0057] The mass spectrometry conditions for the gas chromatography-mass spectrometry analysis include: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; and selection of ion monitoring mode.

[0058] In this invention, the quantitative method for obtaining the content of preservatives according to a predetermined standard curve is preferably the internal standard method; preferably, before mixing the sample to be tested, inorganic acid and acetonitrile, an internal standard is added to the sample to be tested, and the internal standard preferably includes D4-ethyl p-hydroxybenzoate.

[0059] In this invention, the retention time, quantitative and qualitative ions of the preservatives (12 kinds) and internal standards are preferably as shown in Table 1:

[0060] Table 1. Retention time, quantitative and qualitative ions of 12 preservatives and internal standards

[0061]

[0062] In this invention, the predetermined standard curve preferably uses the concentration ratio of preservative to internal standard as the abscissa and the peak area ratio of preservative to internal standard as the ordinate. The concentration of the preservative is preferably 2.5-50 μg / mL for acetic acid, propionic acid, sorbic acid, dehydroacetic acid, and benzoic acid, more preferably 2.5, 5, 12.5, 25, and 50 μg / mL; the concentrations of dimethyl fumarate, methylparaben, ethylparaben, propylparaben, butylparaben, isopropylparaben, and isobutylparaben are preferably 1-20 μg / mL, more preferably 1, 2, 4, 10, and 20 μg / mL; the internal standard preferably includes ethylparaben, and the concentration of ethylparaben is preferably 40-60 μg / mL, more preferably 50 μg / mL.

[0063] In this invention, the helium gas is preferably high-purity helium gas, and the purity of the high-purity helium gas is preferably ≥99.999%.

[0064] In this invention, the split ratio is preferably 6 to 8:1, and more preferably 7:1.

[0065] In this invention, the mass spectrometry conditions preferably include a solvent delay, and the solvent delay time is preferably 4 min.

[0066] To further illustrate the present invention, the detection method for additives in food according to the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0067] In specific embodiments of the present invention, the following instruments and reagents are included:

[0068] Gas chromatography-triple quadrupole mass spectrometry system (Agilent Technologies, USA); vortex mixer (Shanghai Jingke); constant temperature water bath (Shanghai Beilun Instrument Equipment Co., Ltd.); ultrasonic generator (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu Province); analytical balance: sensitivity 0.0001g; grinder; nitrogen blower; centrifuge.

[0069] Acetonitrile (CH3CN): chromatographic grade; Ethanol (C2H5OH): chromatographic grade; n-hexane (C6H 14 Chromatographic grade; Hydrochloric acid (HCl): Analytical grade; Methanol (CH3OH): Chromatographic grade.

[0070] The purity of D4-ethylparaben, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, dimethyl fumarate, acetic acid, propionic acid, dehydroacetic acid, benzoic acid, and sorbic acid standards were all ≥99.0% and were purchased from the National Institute of Metrology, China or Tanmo Technology.

[0071] Example 1

[0072] 1. Preparation of standard solutions

[0073] Accurately weigh 0.1000 g each of methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, isobutylparaben, dimethyl fumarate, acetic acid, propionic acid, dehydroacetic acid, benzoic acid, and sorbic acid standards into 10 mL volumetric flasks. Dilute with methanol and bring to the mark to prepare standard stock solutions with a concentration of 10 mg / mL. Store below 4°C.

[0074] Accurately pipette 1 mL of each 10 mg / mL standard stock solution into a 10 mL volumetric flask, dilute with methanol to volume, and prepare a 1 mg / mL standard intermediate solution.

[0075] Accurately weigh 10 mg of D4-p-hydroxybenzoate into a 10 mL volumetric flask, dilute with methanol to the mark, and prepare a 1 mg / mL internal standard solution.

[0076] Before use, the solutions were diluted with methanol to prepare a series of standard solutions of different concentrations. The concentrations of each target compound are shown in Table 2. The concentration of the internal standard D4-ethyl p-hydroxybenzoate was 50 μg / mL.

[0077] Table 2 Concentrations of each target compound in the standard series solutions

[0078]

[0079] 2. Sample pretreatment

[0080] Weigh 2g (accurate to 0.0001g) of the sample to be tested into a 50mL plastic centrifuge tube, add 100μL of D4-ethyl p-hydroxybenzoate internal standard solution (1mg / mL), add 0.5mL of hydrochloric acid solution (commercially available concentrated hydrochloric acid to water volume ratio of 1:1) for acidification, extract by vortexing with 10mL of acetonitrile (extraction solvent) and centrifuge, take 5mL of the upper acetonitrile extract into a 15mL plastic centrifuge tube, add 2mL of n-hexane to remove fat, discard the upper n-hexane, add 2mL of saturated sodium chloride solution to remove impurities, vortex centrifuge, aspirate the acetonitrile extract and concentrate it to near dryness by nitrogen blowing, take 1mL of methanol to redissolve by vortexing and then perform analysis.

[0081] 3. Instrument conditions

[0082] Chromatographic conditions: DB-FFAP capillary column (30m×250μm×0.25μm); Temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; Injector temperature: 300℃; Carrier gas: high-purity helium; Carrier gas flow rate: 1.0 mL / min; Split ratio: 7:1; Injection volume: 1.0 μL.

[0083] Mass spectrometry conditions: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; solvent delay: 4min; selected ion monitoring mode (SIM).

[0084] Selection of characteristic ions

[0085] Twelve target compounds at a concentration of 1 mg / mL were determined using full-scan mode. Characteristic ions with high peak intensity, low interference, and good matching were selected from each ion fragment as qualitative and quantitative ions for the target compounds, and their retention times were determined. Selected ion monitoring (SIM) mode was then used to improve the method's selectivity and sensitivity. Chromatograms of the 12 food preservatives (all at 0.1 mg / mL) and their internal standard solutions are shown below. Figure 1 As shown in Table 1, the retention times and characteristic ions are specific to each other.

[0086] Example 2

[0087] An optimization experiment was conducted according to the pretreatment and detection conditions in Example 1. The pretreatment condition optimization experiment selected pastry samples, while other processing steps remained unchanged.

[0088] 1. Selection of internal standard

[0089] Because food matrix compositions are complex and the content of target compounds is relatively low, they are easily masked by interference, requiring pretreatment methods such as purification and concentration. However, some preservatives, such as acetic acid, propionic acid, and dimethyl fumarate, have small molecular weights, low boiling points, and are prone to sublimation, which can easily lead to the loss of target compounds during pretreatment and affect the recovery rate. Therefore, selecting a suitable internal standard is essential.

[0090] Ethyl D4-p-hydroxybenzoate has similar physicochemical properties to the target analyte, is completely soluble in the sample, and is not present in the sample. It does not react with the sample and can be separated from the chromatographic peaks of other components in the sample. The peak positions are close and do not co-elute, effectively avoiding sensitivity differences caused by instrument instability. Therefore, this invention selects ethyl D4-p-hydroxybenzoate as an internal standard for the determination of 12 preservatives, including acetic acid, propionic acid, benzoic acid, sorbic acid, and parabens.

[0091] 2. Selection of chromatographic column

[0092] According to the detection conditions in Example 1, the present invention uses a DB-FFAP column (specification: 30m×250μm×0.25μm) and a VF-WAXms column (specification: 30m×250μm×0.25μm) to determine the 0.1 mg / mL standard solution.

[0093] Figure 1 The chromatogram is obtained using a DB-FFAP column. Figure 2The chromatograms obtained using the VF-WAXms column are shown. Results indicate that on the VF-WAXms column, the chromatogram baseline is uneven, the peak shapes of the target analytes are relatively poor, and tailing is observed; while on the DB-FFAP column, the separation of each target analyte is good, the peak shapes are good, and the response values ​​are high. Therefore, the DB-FFAP column is selected in this invention for the determination of preservatives.

[0094] 3. Extraction solvent

[0095] This invention compared the extraction effects of acetonitrile, acetone, methanol, water, ethanol, chloroform, ethyl acetate, petroleum ether-diethyl ether (volume ratio 3:1), and n-hexane-ethyl acetate (volume ratio 1:1) as extraction solvents. The results showed that the extraction efficiencies of acetic acid, propionic acid, and dimethyl fumarate were similar across the solvents. Dehydroacetic acid showed relatively high extraction efficiency with ethyl acetate and acetonitrile, with less peak tailing, while extraction with methanol and ethanol showed lower efficiency, and extraction with chloroform and acetone caused interference. Diethyl ether, petroleum ether, and n-hexane were highly volatile and unstable. Water had a significant impact on acetic acid and propionic acid, resulting in poor extraction efficiency. Considering both safety and extraction efficiency, this invention ultimately selected acetonitrile as the extraction solvent.

[0096] Because acetic acid, propionic acid, and dehydroacetic acid are volatile, unstable, and have low extraction efficiency, this experiment used acetonitrile-water solutions with different volume ratios of acetonitrile and water (3:1, 4:1, 5:1, 8:3, 10:3, 10:0) for extraction. The extract was dehydrated with 2g of anhydrous sodium sulfate and then defatted with n-hexane. The results showed that the target compounds (acetic acid, propionic acid, and dehydroacetic acid) were significantly lost during the dehydration process, and the extraction efficiency did not increase significantly. Finally, acetonitrile was chosen as the extraction solvent.

[0097] Meanwhile, this invention compared the effects of direct injection of acetonitrile extract with that of nitrogen blowing concentration of acetonitrile extract followed by methanol reconstitution before injection and determination. It was found that the peak shapes of acetic acid, propionic acid, and dehydroacetic acid were more stable and the precision was better after reconstitution with methanol, while the results of direct injection were unstable. Therefore, this invention uses methanol reconstitution before instrumental determination.

[0098] 4. Purification conditions

[0099] This invention uses 2 mL of n-hexane to conduct continuous degreasing experiments at different frequencies (1 time, 2 times, 3 times). The results show that vortex degreasing with 2 mL of n-hexane once can meet the experimental requirements.

[0100] This experiment used 2 mL of saturated sodium chloride solution to conduct continuous impurity removal experiments at different frequencies (1 time, 2 times, 3 times). The results showed that vortexing with 2 mL of saturated sodium chloride solution once was sufficient to meet the experimental requirements.

[0101] High-speed, long-duration vortexing and centrifugation can easily cause emulsification and interfere with the experiment. After multiple experiments and adjustments, it was found that a vortex speed of 3000-5000 r / min for 1 min and a centrifugation speed of 5000 r / min for 3 min can achieve better vortexing and centrifugation effects without causing sample emulsification.

[0102] Example 3 Methodological Validation

[0103] 1. Linear range, limit of detection, limit of quantitation

[0104] The linear range, limit of detection (LOD), and limit of quantitation (LOQ) of the method were validated under the instrument conditions described in Example 1, and the results are shown in Table 3. The results indicate that the 12 preservatives exhibit good linearity in the ranges of 1.0–20 μg / mL and 2.5–50 μg / mL, with correlation coefficients greater than 0.99 for all values. Based on a signal-to-noise ratio of 3, the LOD range was calculated to be 0.04–2.00 mg / kg, and based on a signal-to-noise ratio of 10, the LOQ range was calculated to be 0.12–6.67 mg / kg.

[0105] Table 312 shows the linear range, linear equation, correlation coefficient, limit of detection, and limit of quantitation for 12 preservatives.

[0106]

[0107]

[0108] 2. Accuracy and precision

[0109] Following the detection method described in Example 1, spiking experiments were conducted at three different concentration levels (low, medium, and high) on blank samples containing none of the 12 preservatives. Each spiking level was repeated six times in parallel to examine the accuracy and precision of the method. The results are shown in Tables 4 and 5. The results indicate that the average recoveries of the 12 preservatives ranged from 70.18% to 109.22%, and the relative standard deviations ranged from 1.82% to 9.79%. The method is accurate and reliable, and suitable for the detection of 12 preservatives, including benzoic acid, sorbic acid, and parabens, in food.

[0110] Table 4. Accuracy and precision of 12 food preservatives (n=6)

[0111]

[0112] Table 5. Accuracy and precision of 12 food preservatives (n=6)

[0113]

[0114] Example 4: Analysis of Actual Samples

[0115] Following the detection method in Example 1, preservatives were tested on pastries such as shortbread, soda crackers, cakes, and toast. The results are shown in Tables 6 and 7. Figure 3 The chromatogram of the scallion-flavored soda cracker sample shows the detection of acetic acid, propionic acid, dehydroacetic acid, and benzoic acid. Figure 4 The chromatogram of the steamed bread sample from Gangrong (detection of acetic acid, propionic acid, sorbic acid, dehydroacetic acid, and benzoic acid) is shown. Figure 5 The chromatogram of the condensed milk toast sample shows the detection of acetic acid, propionic acid, sorbic acid, dehydroacetic acid, and benzoic acid.

[0116] The results showed that preservatives were detected in all the samples. Acetic acid, dehydroacetic acid, sorbic acid, and butyl paraben had the highest detection rates. The detection range for acetic acid was ND ~ 73.82 mg / kg, for propionic acid it was ND ~ 150.53 mg / kg, for dimethyl fumarate it was ND ~ 1.85 mg / kg, for sorbic acid it was ND ~ 540.56 mg / kg, for dehydroacetic acid it was ND ~ 272.68 mg / kg, and for benzoic acid it was ND ~ 25.69 mg / kg. The detection ranges for isopropylparaben (ND) and methylparaben (ND) were 2.42 mg / kg, methylparaben (ND) and 2.85 mg / kg, ethylparaben (ND) and 2.94 mg / kg, and propylparaben (ND) and 2.71 mg / kg, respectively. Isobutylparaben was not detected, and butylparaben (ND) was detected in the range of 2.47 mg / kg. All levels were within the maximum permissible limits stipulated in the National Food Safety Standard (GB2760). Most samples tested positive for more than one preservative, and the total detected values ​​complied with the provisions of GB2760-2014, "National Food Safety Standard for the Use of Food Additives," regarding the mixed use of the same functional food additives. Some samples showed discrepancies between the actual detected preservatives and the information on the sample label. Furthermore, the preservative content was determined using the national standard method, and the results were consistent with those of this invention, demonstrating the accuracy and reliability of the detection method.

[0117] Table 6. Detection results of 12 food preservatives in pastry samples (unit: mg / kg)

[0118]

[0119]

[0120] Table 7. Detection results of 12 food preservatives in pastry samples (unit: mg / kg)

[0121]

[0122]

[0123] The method for determining 12 preservatives in pastry foods using gas chromatography-mass spectrometry with internal standard in this embodiment of the invention is simple to operate, rapid in detection, accurate and reliable in results, highly sensitive and precise. The internal standard method can reduce the error caused by pretreatment, and the qualitative analysis by retention time and characteristic ions can reduce the probability of false positives. It is suitable for the rapid and effective detection of preservatives in pastry foods.

[0124] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting preservatives in food, characterized in that, Includes the following steps: The sample to be tested, an inorganic acid, and acetonitrile were mixed and extracted to obtain an extract; the inorganic acid was hydrochloric acid. The extract, alkane, and saturated sodium chloride solution were mixed and purified. The purified solution was then subjected to nitrogen blowing and redissolution to obtain the test solution. The alkane included n-hexane and / or n-heptane. The test solution was analyzed by gas chromatography-mass spectrometry, and the content of preservatives was obtained according to a predetermined standard curve. The preservatives included acetic acid, propionic acid, dehydroacetic acid, benzoic acid, sorbic acid, dimethyl fumarate, methylparaben, ethylparaben, propylparaben, isopropylparaben, butylparaben, and isobutylparaben. The gas chromatography-mass spectrometry (GC-MS) conditions included: DB-FFAP capillary column; temperature program: initial temperature 60℃, hold for 1 min, increase to 220℃ at 15℃ / min, then increase to 245℃ at 5℃ / min, hold for 10 min; injection port temperature: 300℃; carrier gas: helium; carrier gas flow rate: 1.0 mL / min; split ratio: 5~10:1; injection volume: 1.0 μL. The mass spectrometry conditions for the gas chromatography-mass spectrometry analysis include: EI ion source; ion source temperature: 230℃; quadrupole temperature: 150℃; transfer line temperature: 280℃; ionization energy: 70eV; and selection of ion monitoring mode.

2. The detection method according to claim 1, characterized in that, The ratio of the sample to acetonitrile used is 1~2g:10~15mL.

3. The detection method according to claim 1, characterized in that, The volume ratio of the extract to the alkane is 5:1.5~2.

5.

4. The detection method according to claim 1 or 3, characterized in that, The volume ratio of the extract to the saturated sodium chloride solution is 5:1.5~2.

5.

5. The detection method according to claim 1, characterized in that, The solvent for the resolution is methanol.

6. The detection method according to claim 1 or 5, characterized in that, The volume ratio of the purified liquid to the reconstituted solvent is 5~10:

1.

7. The detection method according to claim 1, characterized in that, The predetermined standard curve uses the concentration ratio of preservative to internal standard as the x-axis and the peak area ratio of preservative to internal standard as the y-axis. The concentrations of the preservatives are: acetic acid, propionic acid, sorbic acid, dehydroacetic acid, and benzoic acid, each independently ranging from 2.5 to 50 μg / mL; and dimethyl fumarate, methylparaben, ethylparaben, propylparaben, butylparaben, isopropylparaben, and isobutylparaben, each independently ranging from 1 to 20 μg / mL.

8. The detection method according to claim 7, characterized in that, The internal standard includes ethyl D4-p-hydroxybenzoate.

9. The detection method according to claim 8, characterized in that, The concentration of D4-p-hydroxybenzoate ethyl ester is 40~60 μg / mL.

Images