A method for rapid determination of 11 synthetic pigments in pastries based on composite desorption mode extraction

Abstract

The present invention relates to a method for detecting pigments, specifically a method for rapidly determining 11 synthetic pigments in pastries based on a composite desorption extraction method. After sample extraction with a Tween-water-sodium-chloride-methanol solution (pH 8.0) with composite desorption, the sample is separated on a C18 chromatographic column using 20 mmol / L ammonium acetate and methanol as the mobile phase. Detection is performed using a diode array detector, and quantification is performed using an external standard method. This method is capable of rapidly and simultaneously determining 11 synthetic pigments in pastries: tartrazine, new red, amaranth, carmine, sunset yellow, allura red, acid red, erythrosine, brilliant blue, quinoline yellow, and indigo. This method is simple, rapid, and low-cost, suitable for batch testing of 11 pigments in pastries. This method can also be applied to matrices such as fruit products, candies, and solid beverages, providing a reference for the rapid detection of pigments in foods and offering technical support for regulatory oversight.

Description

Technical Field

[0001] The invention relates to a method for detecting pigments, and in particular to a method for rapidly detecting 11 synthetic pigments in cakes based on composite desorption mode extraction. Background Art

[0002] Synthetic pigments are a type of food additive that can impart or improve the color of food, thereby improving its sensory quality and increasing consumer purchasing desire. Due to their strong coloring power, good stability, and low cost, they are widely used in the food industry. However, excessive use of synthetic pigments is not advisable. Therefore, my country's food additive standard "GB2760-2024" has established maximum usage limits for various pigments, including tartrazine. Pastries, as a common food, often incorporate one or more pigments with different coloring effects for aesthetic purposes and vibrant fillings. To prevent companies from using synthetic pigments beyond the scope or limit, the various pigment indicators in pastries remain a key regulatory focus for these products.

[0003] GB 5009.35-2023 is the main standard method for detecting pigments in food. The compounds involved are tartrazine, new red, amaranth, carmine, sunset yellow, allura red, acid red, erythrosine, brilliant blue, quinoline yellow and indigo, a total of 11 pigments. However, its pretreatment process requires solid phase extraction column purification, nitrogen blowing and other steps. The process is complicated and time-consuming. In the pastry determination process, the recovery rates of indigo and erythrosine are low.

[0004] Currently, the methods reported in the literature for detecting synthetic pigments include spectrophotometry, liquid chromatography, and liquid chromatography-tandem mass spectrometry. Liquid chromatography, with its advantages of high accuracy, high sensitivity, and relatively low instrument cost, has become the primary method for detecting synthetic pigments. Pretreatment methods vary depending on the sample matrix, primarily solid-phase extraction (SPE) and direct extraction. For samples with complex matrices, SPE methods such as C18 and WAX are often used to remove impurities such as oil and sugar. For example, Ran Dan et al. developed a high-performance liquid chromatography (HPLC) method for the determination of 20 synthetic pigments in preserved fruits using post-extraction SPE column cleanup. Yang Rui et al. used SPE-HPLC to determine 11 sulfonic acid colorants in braised meats. Gao Jie et al. determined six synthetic pigments in soy sauce by liquid chromatography. While SPE-HPLC can achieve good purification results, the pretreatment process is relatively time-consuming, making it difficult to rapidly scale up for mass testing. To improve pretreatment efficiency, Xie Sai used a direct dilution method to determine erythrosine in beverages. Liu Ying et al. extracted erythrosine from cocoa butter substitute chocolate using an acidic aqueous solution containing 40% ethanol and directly measured it on a liquid chromatograph. Although the method is simple and fast, the matrices involved are mostly foods with relatively simple formulas such as beverages, and it can only meet the determination of erythrosine. Due to the high proportion of organic phase in the extract, there may be solvent effects when measuring on the machine, making it difficult to meet the requirements of simultaneous determination of multiple pigments. Xie Manli et al. obtained a method for extracting 10 pigments from beverages and candies using low eutectic solvent liquid-liquid microextraction technology. Although this method is simple and fast, the spiked recovery rate of erythrosine is only 40%, which is difficult to meet the determination requirements. Summary of the Invention

[0005] In response to the problems of low recovery rate and long pre-treatment process in the existing methods for synthetic pigment determination, the present invention explores a method that can directly analyze 11 pigments in pastries after extraction by high performance liquid chromatography. By adding a surfactant-Tween to the pigment extract using an extracting solution with a composite desorption mode, the non-specific adsorption of pigments during the extraction process is avoided, and an appropriate amount of sodium chloride is added to the extract to assist in desorption, thereby effectively improving the recovery rate of various pigments. At the same time, the proportion of organic phase in the extract is reduced, avoiding the influence of solvent effect on subsequent liquid chromatography determination. The present invention realizes the rapid and accurate determination of 11 pigments in pastries, greatly improves the detection efficiency and saves costs. At the same time, it has been verified by experiments that the present method can also be applied to matrices such as fruit products, candies, solid beverages and beverages. The present invention is of great significance for realizing batch inspection of pigments in food and formulating and revising relevant standards.

[0006] The present invention is achieved through the following technical solutions:

[0007] The method for rapid determination of 11 synthetic pigments in pastries based on composite desorption mode extraction includes the following steps:

[0008] (1) Sample pretreatment: Add Tween aqueous solution to the sample, adjust the pH value, add methanol and sodium chloride, sonicate and centrifuge to obtain the supernatant, and filter with a Tween water constant volume filter membrane;

[0009] (2) Prepare standard solution;

[0010] (3) High performance liquid chromatography (HPLC)

[0011] Furthermore, the volume percentage concentration of the Tween aqueous solution is 4%, and the pH value is 8.0.

[0012] Furthermore, the specific steps of sample pretreatment are as follows: accurately weigh 2 g of sample into a 50 mL centrifuge tube, add 25 mL of 4% Tween aqueous solution, adjust the pH to 8.0 with ammonia water, add 10.0 mL of methanol and 1.0 g of NaCl, vortex mix, ultrasonicate for 15 min, centrifuge at 8000 r / min for 2 min, take the supernatant into a 50 mL volumetric flask, dilute to the mark with 4% Tween aqueous solution, and filter through a 0.45 um filter membrane.

[0013] Furthermore, the 11 pigments are: tartrazine, quinoline yellow, new red, amaranth, carmine, sunset yellow, allura red, acid red and erythrosine, indigo, and brilliant blue, with concentrations of 0.04 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, and 10.0 μg / mL standard series working solutions.

[0014] Furthermore, the chromatographic column: XBridge C 18 Column (4.6 mm × 150 mm, 5 μm); column temperature: 40 °C; injection volume: 20 μL; detection wavelengths: 415 nm (tartrazine and quinoline yellow) / 520 nm (new red, amaranth, carmine, sunset yellow, allura red, acid red, and erythrosine) / 610 nm (indigo and brilliant blue); flow rate: 1.2 mL / min; mobile phase A was 20 mmol / L ammonium acetate solution, mobile phase B was methanol, and the gradient elution program was:

[0015] .

[0016] Beneficial effects

[0017] The present invention solves the problem of low recovery of pigments such as erythrosine when measuring matrices such as cakes from the perspective of desorption, and can directly perform liquid chromatography analysis after sample extraction, and simultaneously measure 11 kinds of pigments. By adding a surfactant, Tween, to the pigment extract, the nonspecific adsorption effect present in the extraction process is avoided, and an extracting solution with a composite desorption mode is obtained through the auxiliary desorption effect of sodium chloride, thereby solving the problem of poor extraction effect and low recovery of red pigments such as erythrosine, and obtaining a high performance liquid chromatography method that can quickly and simultaneously measure 11 kinds of synthetic pigments in cakes. After direct extraction, the sample can be entered into the liquid chromatograph for analysis, which shortens the analysis time, improves the detection efficiency, and saves the detection cost. The method has good recovery and precision and a good linear relationship, and can meet the measurement requirements of 11 kinds of synthetic pigments in cakes. At the same time, it has been verified that the method can also be applied to matrices such as fruit products, candies, and solid beverages. The method can meet the demand for batch detection of 11 kinds of synthetic pigments in food, and has practical application value for the supervision of synthetic pigments in food and the guarantee of food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of Tween addition in the extract on the recovery rate of 11 synthetic pigments in cakes;

[0019] Figure 2 The effect of precipitants on the recovery of 11 synthetic pigments in pastries;

[0020] Figure 3 The effect of the amount of NaCl added in the extract on the recovery rate of 11 synthetic pigments in cakes;

[0021] Figure 4 The effect of pH value of the extract on the recovery rate of 11 synthetic pigments in cakes;

[0022] Figure 5 Chromatogram of the standard solution at 415 nm and 520 nm;

[0023] Figure 6 The chromatogram of the standard solution is shown at 610 nm. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to specific examples. The examples are provided to better illustrate the present invention, but are not intended to limit the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified. Therefore, non-essential improvements and adjustments made by those skilled in the art based on the above invention are still within the scope of protection of the present invention.

[0025] Example

[0026] 1. Materials and Methods

[0027] 1.1 Materials and Reagents

[0028] Standard substances: Tartrazine (CAS 1934-21-0, 1000 μg / mL); New Red (CAS 220658-76-4, 1001.8 μg / mL); Amaranth (CAS 915-67-3, 1000.3 μg / mL); Carmine (CAS 2611-82-7, 1000 μg / mL); Sunset Yellow (CAS 2783-94-0, 1000 μg / mL); Allura Red (CAS 25956-17-6, 1000 μg / mL); Acid Red (CAS 3567-69-9, 1000.1 μg / mL); Erythrosine (CAS 16423-68-0, 1000.1 μg / mL); Brilliant Blue (CAS 3844-45-9, 1000.5 μg / mL); Quinoline Yellow (CAS The above standard substances (Bepure, CAS 8004-92-0, purity: 95%) were purchased from Beijing Manhag Biotechnology Co., Ltd. Indigo (CAS 860-22-0, 92.2%) was purchased from Tianjin Alta Technology Co., Ltd.

[0029] Tween 20 (analytical grade) was purchased from Shanghai Yien Chemical Technology Co., Ltd.; ammonia, potassium ferrocyanide, zinc acetate, sodium chloride, and ammonium acetate (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.; and methanol (chromatographic grade) was purchased from Merck, Germany. Bread and other foods used in the experiment were purchased from a local supermarket.

[0030] 1.2 Instruments and Equipment

[0031] Shimadzu LC-2030C 3DPlus HPLC all-in-one instrument; MS303S electronic balance, METTLERTOLEDO, Switzerland; UMV-2 multi-channel vortex mixer, Shandong Qingyun Laboratory Consumables Co., Ltd.; KQ-800DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; 3-18KS refrigerated centrifuge, Sigma, Germany; Milli-Q ultrapure water preparation device, Millipore, USA.

[0032] 1.3 Methods

[0033] 1.3.1 Preparation of standard solution

[0034] Accurately weigh 10.0 mg (after purity conversion) of quinoline yellow and indigo blue standard substances and dilute to a 10 mL volumetric flask with water to obtain a 1000 μg / mL standard stock solution. Pipette 2.5 mL of each pigment standard stock solution (1000 μg / mL) into a 25 mL volumetric flask to obtain a 100 μg / mL mixed standard intermediate solution. Dilute this mixed standard intermediate solution with water to prepare a series of standard working solutions with concentrations of 0.04 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, and 10.0 μg / mL.

[0035] 1.3.2 Sample pretreatment

[0036] Accurately weigh 2 g (accurate to 0.001 g) of sample into a 50 mL centrifuge tube, add 25 mL of 4% by volume Tween aqueous solution, adjust the pH to 8.0 with ammonia water, add 10.0 mL of methanol and 1.0 g of NaCl, vortex mix, ultrasonicate for 15 min, centrifuge at 8000 rpm for 2 min, collect the supernatant into a 50 mL volumetric flask, dilute to the mark with 4% by volume Tween aqueous solution, and filter through a 0.45 um filter membrane for testing.

[0037] 1.3.3 Chromatographic conditions

[0038] Column: XBridge C 18 Column (4.6 mm × 150 mm, 5 μm); column temperature: 40 °C; injection volume: 20 μL; detection wavelengths: 415 nm (tartrazine and quinoline yellow) / 520 nm (new red, amaranth, carmine, sunset yellow, allura red, acid red, and erythrosine) / 610 nm (indigo and brilliant blue); flow rate: 1.2 mL / min; mobile phase A was 20 mmol / L ammonium acetate solution, mobile phase B was methanol, and the gradient elution program was shown in Table 1.

[0039] Table 1 Gradient elution program

[0040] .

[0041] 1.3.4 Data Processing

[0042] LabSolutions chromatography workstation was used for data processing and OriginPro 9.0 was used for plotting.

[0043] 2. Results and Analysis

[0044] 2.1 Optimization of pretreatment conditions

[0045] 2.1.1 Determination of the methanol-ammonia system

[0046] The methanol-ammonia solution system can achieve effective extraction of synthetic pigments. However, the methanol content in the extract usually used is high, and liquid chromatography analysis can only be performed after solvent replacement. Otherwise, the solvent effect is easily caused by the mismatch between the extract and the mobile phase, affecting the peak shape of targets such as lemon yellow, and the solvent replacement process has the disadvantage of being time-consuming. The present invention explores a pretreatment method that can meet the simultaneous determination of 11 synthetic pigments in pastries, including lemon yellow, new red, amaranth, carmine, sunset yellow, allura red, acid red, erythrosine, brilliant blue, quinoline yellow and indigo, and can be directly subjected to liquid chromatography analysis after extraction. Therefore, the present invention investigates the effects of 0%, 10%, 20%, 30% and 40% methanol content in the extract on the extraction efficiency and peak shape of different targets. When the methanol content increased from 0% to 20%, the recovery rates of red pigments such as allura red and indigo increased. When the methanol content was 30%, the peak shape of lemon yellow began to show a shoulder peak due to the solvent effect. When the methanol content was 40%, the peak shapes of lemon yellow, new red and amaranth were all affected. Ultimately, the present invention selected a methanol content of 20% in the extract. Because the 11 pigments described in the present invention are all acidic group pigments that are easily extracted by alkaline extracts, the pH value of the extract was initially selected to be 8.0. The present invention preliminarily set the analysis process as follows: after the sample is extracted, impurities such as protein are precipitated with potassium ferrocyanide and zinc acetate, and then measured by liquid chromatography.

[0047] In summary, the present invention ultimately chose to extract samples using a methanol-ammonia solution with a 20% methanol content and a pH of 8.0, followed by protein precipitation using 1 mL of potassium ferrocyanide and 1 mL of zinc acetate as a pretreatment step for further optimization. The following experiments were conducted using spiked pastry samples as the analysis targets for the optimization process.

[0048] 2.1.2 Tween content in the extract

[0049] When the pigments in the pastry samples were extracted during the pre-treatment process, the recovery rate of erythrosine was 0%, which is easy to cause false negative results. The recovery rates of red pigments such as allura red and amaranth were relatively low at about 60%, and the recovery rate of brilliant blue was only 75%. It is speculated that the low recovery rate of the above-mentioned pigments may be related to the fact that red pigments such as erythrosine and brilliant blue are easily adsorbed by solid phase surfaces such as sample matrices and are not easy to be extracted. The present invention uses a methanol-ammonia water extract containing 2% Tween to extract pastry samples, and the recovery rates of pigments such as erythrosine are significantly improved. On the one hand, this is because Tween, as a surfactant, occupies the hydrophobic sites on the surface of the solid phase carrier, preventing the non-specific adsorption of the target substance and the solid surface. On the other hand, because Tween is an oil-in-water emulsifier, it can disperse the matrix that is not easily soluble in the extract, which has a solubilizing effect on the sample. In order to further obtain the optimal content of Tween in the extract, the influence of Tween content of 0%, 1%, 2%, 3%, 4% and 6% on the recovery rates of 11 pigments was investigated, and the results are as follows. Figure 1 shown.

[0050] Depend on Figure 1 It can be seen that the recovery rates of erythrosine, amaranth and carmine in the sample always show an upward trend as the Tween content increases. When the Tween content increases from 0% to 4%, the three pigment recovery rates show a significant upward trend. Among them, the recovery rate of erythrosine changes most significantly, increasing from 0% to about 70%, and the recovery rate of amaranth also increases significantly, increasing from 66% to 90%. When the amount of Tween increases from 4% to 6%, the recovery rate rises slowly, and the difference between the two is not significant. For the above three pigments, the optimal content of Tween in the extract is 4% or 6%. The recovery rates of allure red, acid red and brilliant blue in the sample show an upward trend when the Tween content increases from 0% to 1%. As the Tween content in the extract continues to rise, the recovery rates of the three pigments remain at a stable level and are all above 80%. Therefore, when the Tween content is 1%, it can meet the determination requirements of allure red, acid red and brilliant blue in the sample. When the Tween content in the extract ranged from 0% to 4%, the recovery of indigo remained around 90%. However, when the Tween content increased to 6%, the recovery dropped to 80%. This may be due to the poor stability of indigo, which is affected by a certain Tween concentration. Meanwhile, the three yellow pigments were not affected by changes in Tween content and remained above 90%. In summary, a 4% Tween content in the extract maintained an excellent recovery rate for all 11 pigments in the sample.

[0051] 2.1.3 Effect of precipitant

[0052] The addition of precipitants during the pretreatment process can precipitate impurities such as proteins for purification purposes, but considering that the precipitation process of the precipitant may affect the extraction of pigments, the present invention investigates the effects of adding and not adding precipitants on the recovery rates of 11 pigments, such as Figure 2 shown.

[0053] pass Figure 2 As can be known, compared with the pre-treatment process adding precipitation agent, not adding precipitation agent can effectively improve the recovery of other 5 kinds of red pigments except new red and quinoline yellow, and the recovery of sunset yellow and indigo is also slightly increased.Wherein, the recovery of temptation red and erythrosine changes the most obviously and increases to 100% and 86% by 83% and 68% respectively.This phenomenon may be relevant with the precipitation agent precipitation process that target object produces adsorption and embedding.Because cake class sample protein content is few, without precipitation agent processing, 8000r / min centrifugal rear extract is comparatively clear, and does not affect subsequent membrane filtration process.Therefore, the present invention selects pre-treatment process not to add precipitation agent processing, can make 11 kinds of pigment recovery rates all maintain more than 85% in the sample.

[0054] 2.1.4 Amount of NaCl added to the extract

[0055] The addition of NaCl to the pigment extract facilitates the extraction of the pigment. The present invention investigated the effect of adding 0 g, 1 g, 2 g, 3 g and 4 g of NaCl to the extract on the recovery rate of 11 pigments in the sample. Figure 3 shown.

[0056] Depend on Figure 3 As can be known, along with the increase of NaCl add-on in extracting solution, the recovery of amaranth, indigo is in a downward trend, and wherein the indigo recovery declines faster, and when the add-on of sodium chloride is 2g, the recovery drops to 77% from 91%.Contrary to above-mentioned two pigments, the red recovery of erythrosine is in an upward trend with the increase of NaCl add-on, and this may have certain electrostatic interaction with solid surface with NaCl, has occupied adsorption site, and the strongest erythrosine of adsorptivity is redly had to certain auxiliary desorption effect relevant.For further improving the red recovery of erythrosine, simultaneously, ensuring that the indigo recovery maintains more than 80%, it is 1 g that the present invention selects the add-on of NaCl.

[0057] 2.1.5 pH value of the extract

[0058] The structures of 11 pigments were analyzed. Erythrosine is a sodium benzoate structure, and the other 10 pigments are sodium benzenesulfonate structures. The pH value of the extract has an effect on the ionization state of the target, which may have a significant impact on the extraction efficiency of the pigment. The present invention compares the effects of pH values ​​of 6.0, 7.0, 8.0 and 9.0 on the recovery rates of 11 pigments in the sample. Figure 4 shown.

[0059] exist Figure 4 In the experiment, the change of pH value of the extract had a certain influence on the recovery rate of allura red, acid red, erythrosine, quinoline yellow and indigo. When the pH value of the solution changed from 6.0 to 8.0, the recovery rate of the above five pigments showed a significant upward trend. This is related to the fact that erythrosine has a sodium benzoate structure and other pigments have a sodium benzenesulfonate structure, and pigments with acidic groups are more easily extracted by alkaline extracts. When the pH value of the solution rose from 8.0 to 9.0, the recovery rate of indigo showed a significant downward trend. This is related to the unstable nature of indigo itself and its easy oxidation in an overly alkaline environment. Therefore, the pH value of the extract was finally determined to be 8.0.

[0060] 2.1.6 Ultrasound time

[0061] The experiment selected ultrasonic times of 15 min, 20 min, 25 min and 30 min to study the effects on the recovery rates of 11 pigments in the sample. As the ultrasonic time increased, the recovery rate of indigo in the sample showed a downward trend, but had little effect on the recovery rates of the other 10 pigments. When the ultrasonic time was longer than 20 min, the recovery rate of indigo dropped below 80%. On the one hand, this is related to the fact that as the ultrasonic time increased, the temperature increased, affecting the stability of indigo. On the other hand, this is related to the fact that the ultrasonic process may produce free radicals, making indigo susceptible to oxidation. In order to ensure the recovery rate of indigo and improve the analysis efficiency, the present invention selected an ultrasonic time of 15 min. Under this time length, the recovery rate of indigo in the sample was about 90%, and the recovery rates of other pigments could reach more than 90%.

[0062] 2.2 Selection of chromatographic conditions

[0063] 2.2.1 Selection of mobile phase flow rate

[0064] In order to realize the rapid and effective separation of 11 kinds of synthetic pigments, the present invention selects mobile phase flow rate to be 1.0 mL / min, 1.1 mL / min, 1.2 mL / min and 1.3 mL / min to carry out the test. The result shows that when the flow rate is 1.0 mL / min and 1.1 mL / min, the analysis time is extended. When the flow rate is 1.3 mL / min, it cannot meet the separation requirements. Therefore, selecting the mobile phase flow rate is 1.2 mL / min, at this time, both the separation requirements can be met and the analysis time can be shortened.

[0065] 2.2.2 Column selection

[0066] The experiment selected three types of chromatographic columns: Waters Symmetry C18 column (4.6 mm×250 mm, 5 μm), Waters XBridge C18 column (4.6 mm×150 mm, 5 μm) and Waters XBridge C18 column (4.6 mm×150 mm, 3.5 μm). The results showed that all three chromatographic columns can achieve effective separation of the target components. The Waters Symmetry C18 column is a long column, which prolongs the analysis time. Because the mobile phase flow rate of the present invention is 1.2 mL / min, the column pressure is higher when the Waters XBridge C18 column (3.5 μm) with a smaller particle size is used for analysis. The column pressure of the Waters XBridge C18 column (5 μm) is about 10 Mpa during the determination process, the pressure is low, and the analysis time is long and the column is short. Therefore, the Waters XBridge C18 column (4.6 mm×150 mm, 5 um) was selected as the analytical column, and the final analysis time was 20 min. The chromatogram of the obtained standard solution is shown in the figure below. Figure 5 and Figure 6 As shown, Brilliant Blue has two isomers, Brilliant Blue 1 and Brilliant Blue 2, and Quinoline Yellow has four isomers, quinoline yellow QYNa2I, quinoline yellow QYNa2II, quinoline yellow QYNaI and quinoline yellow QYNaII.

[0067] 2.3 Methodological Investigation

[0068] 2.3.1 Linear range, detection limit, and quantification limit

[0069] Standard working solutions prepared according to the procedure in 1.3.1 were injected into the HPLC instrument sequentially from low to high concentration. Linear fitting was performed using the standard solution concentration as the abscissa and the corresponding peak area as the ordinate. Good linear relationships were observed for all 11 synthetic pigments within the range of 0.04 μg / mL to 10 μg / mL. Specific results are shown in Table 2. Brilliant Blue has two isomers, Brilliant Blue 1 and Brilliant Blue 2, and quinoline yellow has four isomers, quinoline yellow QYNa2I, quinoline yellow QYNa2II, quinoline yellow QYNaI, and quinoline yellow QYNaII. Standard solutions of known concentration levels were spiked into the blank matrix. A signal-to-noise ratio of 3 was used as the limit of detection, and a signal-to-noise ratio of 10 was used as the limit of quantification. The limits of detection for all 11 pigments in this method were 0.35 mg / kg, and the limits of quantification were 1 mg / kg.

[0070] Table 2 Detection wavelengths and linear equations of 11 synthetic pigments

[0071]

[0072] 2.3.2 Recovery and precision

[0073] A bread sample without pigment was selected as a blank sample. The sample was spiked at three levels, with the spiked amounts being 1.0 mg / kg, 5.0 mg / kg, and 20.0 mg / kg, respectively. The measurement was repeated 6 times for each level, and the mean value and relative standard deviation (RSD) of the recoveries were calculated. The results are shown in Table 3. Brilliant Blue has two isomers, namely Brilliant Blue 1 and Brilliant Blue 2, and Quinoline Yellow has four isomers, namely Quinoline Yellow QYNa2I, Quinoline Yellow QYNa2II, Quinoline Yellow QYNaI, and Quinoline Yellow QYNaII. When the spiked concentration was 1.0 mg / kg, the recoveries ranged from 85.5% to 107.1% with RSDs of 1.1% to 4.7%. When the spiked concentration was 5.0 mg / kg, the recoveries ranged from 86.8% to 104.3% with RSDs of 1.8% to 4.7%. When the spiked concentration was 20.0 mg / kg, the recoveries ranged from 91.3% to 102.0% with RSDs of 1.1% to 5.1%. For the determination of 11 pigments in bread and other pastry samples, this method achieved recoveries exceeding 85% with excellent precision, meeting the requirements for pigment determination in related products.

[0074] Table 3 Recovery and precision of 11 synthetic pigments spiked at three levels

[0075]

[0076] 2.3.2 Comparison with national standard method

[0077] A bread sample and a mung bean cake sample were selected to compare the determination values ​​or recoveries of the method of the present invention and the national standard method. The results are shown in Table 4.

[0078] Table 4 Comparison of measured values ​​in samples with national standard methods

[0079]

[0080] Note: The spiked dosage in the figures is 5 mg / kg.

[0081] As shown in Table 4, the bread sample contains lemon yellow, and the mung bean cake sample contains lemon yellow and brilliant blue, and the measured values ​​of two kinds of assay methods obtained lemon yellow and brilliant blue are basically identical. The red and indigo recovery rates obtained by national standard method are lower, all do not reach 70%, and the two kinds of pigment recovery rates obtained by the inventive method are relatively high, more than 85.7%. Other pigment two method recovery differences are all less than 10%, and are all more than 85%.

[0082] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for rapid determination of 11 synthetic pigments in cakes based on composite desorption mode extraction, characterized in that: The following steps are involved: (1) Sample pretreatment: Accurately weigh 2 g of sample into a 50 mL centrifuge tube, add 25 mL of 4% Tween aqueous solution, adjust the pH to 8.0 with ammonia water, add 10.0 mL of methanol and 1.0 g of NaCl, vortex mix, ultrasonicate for 15 min, centrifuge at 8000 rpm for 2 min, take the supernatant into a 50 mL volumetric flask, dilute to the mark with 4% Tween aqueous solution, and filter through a 0.45 μm filter membrane; (2) Prepare standard solutions of 11 pigments with concentrations of 0.04 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, and 10.0 μg / mL standard series working solutions; the 11 pigments are: tartrazine, quinoline yellow, new red, amaranth, carmine, sunset yellow, allura red, acid red, erythrosine, indigo, and brilliant blue; (3) High performance liquid chromatography detection: Chromatographic column: XBridge C 18 Column, dimensions 4.6 mm × 150 mm, 5 μm; Column temperature: 40°C; injection volume: 20 μL; detection wavelength: 415 nm for tartrazine and quinoline yellow, 520 nm for new red, amaranth, carmine, sunset yellow, allura red, acid red, and erythrosine, and 610 nm for indigo and brilliant blue; flow rate: 1.2 mL / min; mobile phase A was 20 mmol / L ammonium acetate solution, mobile phase B was methanol, and the gradient elution program was:

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