A high-throughput detection method for fat-soluble synthetic dyes in food

Abstract

The present invention proposes a high-throughput detection method for fat-soluble synthetic dyes in food, which comprises the following steps: 1) extracting and centrifuging the sample to obtain a sample extract, which is then diluted and purified by dispersed solid phase extraction to obtain a sample test solution; 2) using ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry to measure the sample test solution, establishing a calibration curve with a matrix standard solution, and quantifying by an external standard method. The present invention establishes a screening and quantitative detection method for 104 fat-soluble synthetic dyes in food, wherein the sample is extracted, diluted, and purified, and then measured using the Fullms-ddMS2 mode of ultra-high performance liquid chromatography-high resolution mass spectrometry, quantifying the primary parent ion, distinguishing non-isomers by the primary parent ion, and distinguishing isomers by retention time, and a secondary mass spectrometry full scan is used for auxiliary qualitative determination, thereby achieving qualitative and quantitative determination of 104 kinds of dyes, including 6 groups of isomers. The method has good linearity and is characterized by high recovery and precision.

Description

Technical Field

[0001] The invention relates to a method for quickly screening synthetic pigments, in particular to a high-throughput detection method for fat-soluble synthetic dyes in food. Background Art

[0002] Food color is a significant factor influencing consumer preferences, choices, and appetite. Compared to natural dyes, synthetic dyes are favored by food manufacturers due to their low cost, strong coloring power, high effectiveness, and long-lasting color. However, over time, a growing number of studies have revealed that many synthetic dyes, such as azo dyes, disperse dyes, and other fat-soluble synthetic dyes, pose potential hazards to human health and may even be teratogenic or carcinogenic. For example, Sudan dyes and rhodamine B are classified as Group 3 carcinogens by the International Agency for Research on Cancer, while basic yellow O and dimethyl yellow are classified as Group 2B carcinogens.

[0003] A variety of methods have been studied for the detection of fat-soluble synthetic dyes both domestically and internationally. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is currently the most commonly used technique, but it suffers from drawbacks such as a limited range of detection targets, a single target, complex pretreatment, significant matrix effects, low MS resolution, weak interference immunity, and a high false-negative rate. Furthermore, this technique relies on qualitative standards for identification, making it difficult to identify unknown compounds without control standards. This can lead to missed detection of some unknown synthetic dyes with similar structural properties, posing a food safety risk. Compared to LC-MS / MS, ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) offers the advantages of highly accurate mass measurement and rich fragmentation information, enabling more accurate compound identification while avoiding interference from complex matrices. In particular, the Q-Orbitrap analyzer, utilizing an Orbitrap mass analyzer and a quadrupole mass filter, performs a primary full scan and a secondary full scan for specific ions in FullMS-ddMS2 mode. This allows for qualitative screening of suspected substances by analyzing the secondary mass spectral information. Consequently, Q-Orbitrap HRMS has been applied to the quantitative detection and screening of substances.

[0004] However, there are few research reports on the use of Q-Orbitrap HRMS to analyze banned synthetic dyes in food, and no research has been conducted on the separation and detection of isomeric synthetic dyes. Therefore, establishing a method for the simultaneous screening, identification, and quantitative determination of multiple banned dyes in food is of great significance for improving regulatory efficiency and ensuring food safety. Summary of the Invention

[0005] The purpose of the present invention is to propose a high-throughput detection method for fat-soluble synthetic dyes in food. The method can simultaneously screen and quantify 104 fat-soluble synthetic dyes in food, thus overcoming the shortcomings of existing methods.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A high-throughput detection method for fat-soluble synthetic dyes in food, characterized by comprising the following steps:

[0008] Step 1) The sample is extracted and centrifuged to obtain a sample extract, which is then diluted and purified by dispersed solid phase extraction to obtain a sample test solution;

[0009] Step 2) The sample test solution of step 1) is measured by ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry, a calibration curve is established with a matrix standard solution, and quantification is performed by external standard method.

[0010] Furthermore, in the high-throughput detection method for fat-soluble synthetic dyes in food, the names and quantitative ions of the fat-soluble synthetic dyes are: basic green 5, 329.1067; basic red 9, 288.1495; basic blue 9, 284.1216; basic blue 17, 270.1059; riboflavin, 377.1456; basic violet 14, 302.1652; thioflavin T, 283.1264; rhodamine 123, 345.1234; basic red 14, 344.2121; basic red 5, 253.1448; basic orange 2, 213.1135; basic red 46, 321.1822; corynebacterium iodide O, 252.1495; basic red 2, 315.1604; basic thiophene. Basic Orange 14, 266.1652; Basic Violet 2, 330.1965; Basic Blue 41, 371.1536; Basic Yellow 51, 292.1808; Basic Yellow O, 268.1808; Basic Yellow 24, 322.1910; Basic Orange 21, 315.1856; New Methylene Blue 24, 312.1529; Basic Red 13 , 353.1779; hexahydrocurcumin, 373.1657; basic blue 1, 363.1623; basic green 4, 329.2012; basic orange 22, 391.2169; basic blue 12, 318.1601; basic violet 7, 381.2092; basic violet 1, 358.2278; rhodamine 6G, 443.2329 ; Crystal violet, 372.2434; Fluorescein, 333.0758; Disperse violet 1, 239.0815; Basic green 1, 385.2638; Basic blue 11, 422.2591; Rhodamine B, 443.2329; Disperse yellow 9, 275.0775; Disperse red 11, 269.0921; Disperse blue 7, 359.1238; Solvent red 72, 490.8958; Basic blue 26, 470.2591; N,N-dimethyl-4,4-azodiphenylamine, 241.1448; Disperse yellow 1, 274.0469; Solvent yellow 1, 198.1026; Desdimethoxycurcumin, 309.1121; Demethoxycurcumin, 339.1227; Basic blue 7,478.3217; curcumin, 369.1333; Disperse Red 58, 373.1329; Disperse Red 19, 331.1401; Basic Violet 4, 456.3373; Disperse Orange 3, 243.0877; 4-Hydroxy-4-dimethylaminoazobenzene, 242.1288; Disperse Blue 102, 366.1231; Methyl Red, 270.1237; 4-Hydroxyazobenzene, 199.0866; Solvent Yellow 33, 274.0863; Disperse Orange 11, 238.0863; Disperse Red 15, 240.0655; Disperse Red 17, 345.1557; Sudan Orange G, 215.0815; Solvent Red 197, 382.1662; Disperse Red 7, 365.1011; Disperse Blue 14, 267.1128; Disperse Yellow 3, 270.1237; Solvent Yellow 3, 226.1339; Disperse Orange 25, 324.1455; Disperse Red 9, 238.0863; Disperse Red 1, 315.1452; Disperse Blue 124, 378.1231; Disperse Brown, 433.0232; Sudan Yellow, 226.1339; Disperse Violet 28, 307.0036 ; Disperse Orange 37, 392.0676; Disperse Red 13, 349.1062; Para Red, 294.0873; Disperse Orange 61, 481.9645; Solvent Red 3, 293.1285; Solvent Red 169, 266.1176; 2-phenylazo-4-methylphenol, 213.1022; Vat Red 41, 297.0039; Toluidine Red, 308.103; Tangerine Red, 309. 1234; Diethyl Yellow, 254.1652; Solvent Yellow 16, 279.124; Sudan Red G, 279.1128; Disperse Orange 1, 319.1190; Sudan Red I, 249.1022; Disperse Yellow 23, 303.1240; Disperse Yellow 7, 317.1397; Oil Orange SS, 263.1179; Sudan Blue, 343.1441; 808 Scarlet, 368.1394; Solvent Solvent Blue 35, 351.2053; Disperse Orange 13, 353.1397; Sudan Red II, 277.1335; Solvent Violet 13, 330.1125; Oil Blue N, 379.2380; Sudan Red III, 353.1397; Sudan Red 7B, 380.1870; Solvent Green 3, 419.1754; Sudan Red IV, 381.1710; Solvent Red 25, 381.1710.

[0011] Furthermore, in the high-throughput detection method for fat-soluble synthetic dyes in food, the specific steps of step 1) are as follows:

[0012] Step 1.1) Extraction: Weigh 1-3 g of sample into a centrifuge tube, add 5-15 mL of Extraction Solution A, vortex, sonicate, and centrifuge. Remove the supernatant and add 5-15 mL of Extraction Solution A to the centrifuge tube again. Repeat the extraction once, remove the supernatant, combine the supernatants, and dilute to 10-30 mL to obtain the sample extract.

[0013] Step 1.2) Dilution: Pipette 1 mL of sample extract and dilute with diluent A to obtain sample diluent;

[0014] Step 1.3) Purification: Pipette 1 mL of the sample dilution solution into a dispersive solid phase extraction tube for purification, vortex, centrifuge, and pass the supernatant through a PTFE membrane to obtain the sample test solution.

[0015] Furthermore, the extracting solution A in step 1.1) is a mixture of formic acid and acetonitrile, and the volume ratio of formic acid to acetonitrile in the extracting solution A is (0.1-2): (88-99.9).

[0016] Furthermore, the diluent A in step 1.2) is methanol, and the dilution ratio of the sample extract to methanol is 1:(5-10).

[0017] Furthermore, in the high-throughput detection method for fat-soluble synthetic dyes in food, the mass spectrometry conditions of the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 2) are:

[0018] Ion source parameters: positive and negative ion modes, with full acquisition in positive ion mode and acquisition times of 3.0–3.5 min, 4.1–5.3 min, and 8.7–9.5 min in negative ion mode, respectively; Spray Voltage (±) 3.0 kV, Capillary Temperature 320°C, Sheath Gas 40 arb, Aux Gas 10 arb, Probe Heater Temperature 350°C, S-Lens RF level 55;

[0019] Full MS (full scan) mode, with the following conditions: mass spectrometry resolution of 70,000, AGC target of 3e6, Maximum IT of 200 ms, and scan range of 100–1000 m / z;

[0020] Furthermore, in the step 2), the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry was also performed in a data-dependent secondary mass spectrometry full scan mode under the mass spectrometry conditions.

[0021] ddMS2 (data-dependent secondary mass spectrometry full scan) mode, with the following conditions: mass spectral resolution 17500, AGC target (automatic gain control) 2e5, Maximum IT (maximum dwell time) 50 ms, Loop count 5, CE (collision energies) 15, 30, and 45, Isolation window 1.0 m / z, scan range 50 - parent ion * charge number + 20, Intensity threshold 8.0e4, Apex trigger time 2–6 s, Dynamic exclusion time 8.0 s;

[0022] Instrument acquisition and data processing were performed by Xcalibur 4.1 and TraceFinder 4.3 software (ThermoScientific, Waltham, MA, USA), respectively.

[0023] Furthermore, the specific liquid chromatography conditions of the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 1.1) are:

[0024] Chromatographic conditions were as follows: chromatographic column: Waters Acquity UPLC BEH (150 mm × 2.1 mm, 1.7 μm); mobile phase A: 0.1% formic acid in water (containing 10% methanol), mobile phase B: 0.1% formic acid in methanol; gradient elution; column temperature: 40°C; injection volume: 5 μL; flow rate: 0.3 mL / min; injection volume: 5.0 μL;

[0025] In terms of volume percentage, the elution gradient is: from 0 to 0.5 min, phase A is 100% to 60%; from 0.5 to 3.0 min, phase A is 60% to 35%; from 3.0 to 5.5 min, phase A is 35% to 30%; from 5.5 to 8.0 min, phase A is 30% to 2%; from 8.0 to 14.0 min, phase A is 2%; from 14.0 to 14.5 min, phase A is 2% to 100%; from 14.5 to 18.0 min, phase A is 100%.

[0026] The present invention has the following outstanding substantive features and significant advancements:

[0027] 1. The present invention establishes a method for screening and quantitative detection of 104 fat-soluble synthetic dyes in food. The samples are extracted, diluted, and purified, and then measured using the FullMS-ddMS2 mode of ultra-high performance liquid chromatography-high-resolution mass spectrometry and quantified by the external standard method. The method is quantitatively analyzed by primary parent ion, distinguishing non-isomer dyes by primary parent ion, and distinguishing isomer dyes by retention time, achieving qualitative and quantitative determination of 104 types, including 6 groups of isomer dyes. The method has good linearity (correlation coefficient R>0.99), a recovery rate of 61.3%-118.8%, a precision of <20%, a limit of quantification of 0.05-0.5 mg / kg, and a screening limit of 2.5-25 μg / kg. It has high recovery and precision and is suitable for rapid, efficient, and accurate detection of daily foods.

[0028] 2. The present invention further uses a full scan of the secondary mass spectrometry to assist in qualitative analysis during screening. When the "primary parent ion meets the requirements" and the "retention time meets the requirements", the "secondary fragment ions of the secondary mass spectrometry" also meet the requirements. These three conditions are met as triple qualitative conditions, avoiding false positives caused by matrix interference, thereby effectively ensuring the reliability and scientificity of the detection.

[0029] 3. The present invention utilizes the high resolution, high precision and high mass number of Q-Orbitrap HRMS to establish a mass spectrum database of 104 fat-soluble synthetic dyes, including Basic Red 46, Rhodamine 123, Rhodamine 6G, Basic Fuchsin and other newly emerged illegal synthetic dyes. In order to obtain reliable results, the liquid chromatography conditions and mass spectrometry parameters were investigated and optimized. At the same time, the linear range, matrix effect, sensitivity, recovery rate and precision of the method were verified. Studies have shown that this method can achieve efficient, accurate and reliable screening and quantitative determination of 104 lipophilic synthetic dyes, i.e. fat-soluble dyes, in food, and can meet the regulatory needs for illegal synthetic dyes in food, especially new dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the recovery and precision distribution of the four samples of tomato sauce, cooked meat, eggs and chili powder in Example 1. DETAILED DESCRIPTION

[0031] In order to more clearly, comprehensively and completely illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments:

[0032] The present invention provides a high-throughput detection method for fat-soluble synthetic dyes in food. The fat-soluble synthetic dyes include the following 104 synthetic dyes, as shown in Table 1.

[0033]

[0034]

[0035]

[0036]

[0037] The above 104 synthetic dyes include 6 groups of 12 isomers of pigments, as shown in Table 2.

[0038]

[0039]

[0040]

[0041]

[0042] The above 104 synthetic pigments were prepared into standard stock solutions using organic solvents such as methanol and acetonitrile according to their chemical properties, and then the matrix standard working solutions were prepared using the corresponding blank sample extracts.

[0043] Ultra performance liquid chromatography-quadrupole electron orbital ion trap mass spectrometry was used for determination, and a calibration curve was established based on peak area and concentration.

[0044] Ultra-high performance liquid chromatography quadrupole electron orbital ion trap mass spectrometry detection conditions are as follows:

[0045] Mass spectrometry conditions are:

[0046] Ion source parameters: positive and negative ion modes, where the acquisition time for positive ion mode ESI+ is 3.0-3.5 min, the acquisition time for negative ion mode ESI- is 4.1-5.3 min, and the acquisition time for negative ion mode ESI- is 8.7-9.5 min, respectively; Spray Voltage (±) 3.0 kV, Capillary Temperature 320°C, Sheath Gas 40 arb, AuxGas 10 arb, Probe Heater Temperature 350°C, S-Lens RF level 55;

[0047] Full MS (full scan) mode: mass spectrum resolution is 70000, AGC target (automatic gain control target) is 3e6, Maximum IT (maximum dwell time) is 200 ms, and the scan range is 100-1000 m / z.

[0048] The ddMS2 (data-dependent secondary mass spectrometry full scan) mode was used with the following conditions: mass resolution of 17500, AGC target of 2e5, Maximum IT of 50 ms, Loop count of 5, CE of 15, 30, and 45, Isolation window of 1.0 m / z, scan range of 50-parent ion * charge number + 20, Intensity threshold of 8.0e4, Apex trigger time of 2–6 s, and Dynamic exclusion time of 8.0 s.

[0049] The 104 dyes contain 6 groups of isomeric dyes. As shown in Table 2, the isomeric synthetic pigments with identical molecular formulas have identical primary parent ion mass numbers (quantitative ions in the corresponding table 3 of the primary parent ion mass numbers) and cannot be distinguished and quantitatively determined by the primary parent ions. In order to address the deficiencies in the detection standards of the above-mentioned prior art, the present invention separates and accurately quantifies the 104 dyes including the 6 groups of isomeric pigments using the following specific liquid chromatography elution conditions.

[0050] The following are the specific liquid chromatography conditions of the present invention:

[0051] Chromatographic conditions: Column: Waters Acquity UPLC BEH (150 mm × 2.1 mm, 1.7 μm); Mobile phase A: 0.1% formic acid in water (containing 10% methanol); Mobile phase B: 0.1% formic acid in methanol; Gradient elution; Column temperature: 40°C; Injection volume: 5 μL; Flow rate: 0.3 mL / min. Injection volume: 5.0 μL.

[0052] In terms of volume percentage, the elution gradient is: from 0 to 0.5 min, phase A is 100% to 60%; from 0.5 to 3.0 min, phase A is 60% to 35%; from 3.0 to 5.5 min, phase A is 35% to 30%; from 5.5 to 8.0 min, phase A is 30% to 2%; from 8.0 to 14.0 min, phase A is 2%; from 14.0 to 14.5 min, phase A is 2% to 100%; from 14.5 to 18.0 min, phase A is 100%.

[0053] Under the specific liquid chromatography conditions described above, the six groups of isomers were chromatographically separated by retention time. Combined with the primary precursor ion mass (quantitative ion in Table 3) in Full MS (full scan mass spectrometry) mode, qualitative and quantitative detection of isomeric pigments was achieved. Specific quantitative parameters for the 104 synthetic dyes are shown in Table 3:

[0054]

[0055]

[0056]

[0057] The 104 dyes in the present invention are all fat-soluble substances, including non-isomer dyes and 6 groups of isomer dyes as shown in Table 2. During the detection, (1) 104 dyes are used as detection objects, and all are subjected to a full mass spectrometry scan in FullMS mode. The primary parent ions of the non-isomer dyes are different, and the primary mass spectra are collected in FullMS mode to perform qualitative and quantitative analysis of the non-isomer dyes in the present invention; (2) Since the 6 groups of isomer dyes have the same molecular weight, their primary parent ions are also exactly the same, and the FullMS mode cannot be used for qualitative analysis. Therefore, the present invention adopts the specific liquid chromatography separation to distinguish them, and then the 6 groups of isomer dyes are separated by the optimized elution conditions in the specific liquid chromatography. As described in Tables 2 and 3, although the primary parent ions of each pair of isomer dyes are exactly the same, the retention times are different. The 6 groups of isomer dyes are distinguished by the difference in retention time, and the 6 groups of isomer dyes are quantitatively analyzed by the FullMS mode. (3) The full scan of the secondary mass spectrometry in ddMS2 mode is used to assist qualitative analysis during screening. The principle is that the primary parent ion meets the requirements, the retention time meets the requirements, and the secondary fragment ions of the secondary mass spectrometry also meet the requirements. These three conditions are met in the triple qualitative conditions, avoiding false positives caused by matrix interference, thereby effectively ensuring the reliability and scientificity of the detection.

[0058] The methodological parameters such as linear range, calibration equation, correlation coefficient, and detection limit of 104 synthetic dyes are shown in Table 4.

[0059]

[0060]

[0061]

[0062]

[0063] The application of the present invention in food is described in detail below with reference to specific embodiments:

[0064] Example 1

[0065] Spike recovery test investigation: Tomato sauce, cooked meat, eggs, and chili powder were selected as samples for spike recovery test. Tomato sauce, cooked meat, eggs, and chili powder corresponded to Figure 1 Add tomato sauce, dried pork, egg, and chilli powder. The specific steps are as follows:

[0066] Step 1) Sample spike

[0067] Step 1.1) Sample extraction:

[0068] Weigh 2.00 g of sample into a 50 mL centrifuge tube, add an appropriate volume of synthetic pigment standard solution to the sample (the spiked amount is 1 times, 2 times, 5 times and 10 times the quantification limit, respectively), add 10 mL of extracting solution A, wherein the extracting solution A is 0.1% formic acid-acetonitrile, vortex, centrifuge at a speed of 8000 r / min for 3 min, transfer the supernatant to another 50 mL centrifuge tube, add 10 mL of 0.1% formic acid-acetonitrile (extracting solution A) and repeat the extraction once, combine the supernatants, and make up to volume with 0.1% formic acid-acetonitrile to obtain the sample extract.

[0069] Step 1.2) Dilution: Dilute the supernatant with methanol (diluent A) at a dilution ratio of 1:5 to obtain a sample dilution solution.

[0070] Step 1.3) Purification: Pipette 1 mL of the sample dilution solution into a dispersed solid phase C18 extraction tube for purification, vortex, and centrifuge. The supernatant is passed through a 0.22 μm PTFE membrane to obtain the sample test solution.

[0071] At the same time, prepare blank sample test solution according to the same method to prepare matrix standard working solution.

[0072] Step 2) The sample test solution of step 1) is measured by ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry, a calibration curve is established with matrix standard working solution, and external standard method is used for quantification.

[0073] (1) Preparation of standard solution and sample determination

[0074] A mixed standard solution of 104 pigments was drawn up, and a matrix standard working solution was prepared with a blank sample test solution. The matrix standard working solution and the sample test solution were determined by ultra-performance liquid chromatography-quadrupole static electron orbital ion trap mass spectrometry. A calibration curve was established based on the peak area and concentration, and the samples were quantitatively analyzed.

[0075] Mass spectrometry conditions:

[0076] Ion source parameters: positive and negative ion mode, spray voltage (±) spray voltage 3.0 kV, capillary temperature ion transfer tube temperature 320 ° C, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, probe heater temperature, spray needle temperature 350 ° C, S-Lens RF level ion lens voltage 55;

[0077] Full MS (full scan) mode: mass spectrum resolution is 70000, AGC target (automatic gain control target) is 3e6, Maximum IT (maximum dwell time) is 200 ms, and the scan range is 100-1000 m / z.

[0078] The ddMS2 (data-dependent secondary mass spectrometry full scan) mode was used with the following conditions: mass resolution of 17500, AGC target of 2e5, Maximum IT of 50 ms, Loop count of 5, CE of 15, 30, and 45, Isolation window of 1.0 m / z, scan range of 50-parent ion * charge number + 20, Intensity threshold of 8.0e4, Apex trigger time of 2–6 s, and Dynamic exclusion time of 8.0 s.

[0079] Specific HPLC conditions:

[0080] Chromatographic conditions were as follows: chromatographic column: Waters Acquity UPLC BEH (150 mm × 2.1 mm, 1.7 μm); mobile phase A: 0.1% formic acid in water (containing 10% methanol), mobile phase B: 0.1% formic acid in methanol; gradient elution; column temperature: 40°C; injection volume: 5 μL; flow rate: 0.3 mL / min;

[0081] The elution conditions are as follows: from 0 to 0.5 min, phase A changes uniformly from 100% to 60%; from 0.5 to 3.0 min, phase A changes uniformly from 60% to 35%; from 3.0 to 5.5 min, phase A changes uniformly from 35% to 30%; from 5.5 to 8.0 min, phase A changes uniformly from 30% to 2%; from 8.0 to 14.0 min, phase A is 2%; from 14.0 to 14.5 min, phase A changes uniformly from 2% to 100%; from 14.5 to 18.0 min, phase A is 100%.

[0082] (2) Result analysis

[0083] The sample spike recovery test results are shown in Figure 1 For the HRMS method, the limit of quantification is defined as the lowest spike level with a recovery of 60%-120% and a repeatability RSD < 20%. Figure 1As shown in the figure, the recoveries of tomato paste, eggs, cooked meat, and chili powder ranged from 61.8% to 111.2%, 61.3% to 118.8%, 61.8% to 117.7%, and 61.4% to 117.7%, respectively, with RSDs of <20%. These results indicate that the method has good recovery and precision.

[0084] Example 2

[0085] Screening of 104 fat-soluble synthetic dyes in commercial condiments, cooked meat, eggs, and chili powder samples

[0086] In this example, 104 fat-soluble synthetic dyes were screened in 389 samples of commercially available condiments, cooked meat, eggs, chili powder, etc. A total of 1 chili powder, 1 cooked meat, and 1 egg were detected to contain fat-soluble positive synthetic dyes. Among them, rhodamine B was detected in the chili powder and cooked meat samples, and rhodamine 6G was detected in the egg sample. The specific screening results of positive samples are shown in Table 5.

[0087]

[0088] Although the above examples do not cover the application of the present invention in various foods, the concept of the present invention can effectively eliminate the influence of matrix effects and detect samples of various types and matrices. In addition, since isomers can be distinguished, the accuracy of the detection conclusion is ensured, which has great promotional significance.

Claims

1. A high-throughput detection method for fat-soluble synthetic dyes in food, characterized in that: The following steps are involved: Step 1), the sample is extracted and centrifuged to obtain a sample extract, which is diluted and purified by dispersed solid phase extraction to obtain a sample test solution; the extract is a mixture of formic acid and acetonitrile; Step 2) using ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry to measure the sample test solution of step 1), establish a calibration curve with a matrix standard solution, and quantify by external standard method; The mass spectrometry conditions of the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 2) are as follows: ion source parameters: positive and negative ion modes, wherein the positive ion mode is fully acquired, and the negative ion mode acquisition time is 3.0-3.5 min, 4.1-5.3 min, and 8.7-9.5 min, respectively; the first-stage mass spectrometry is in full scan mode; In step 2), the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry was also performed in a data-dependent secondary mass spectrometry full scan mode under mass spectrometry conditions; the data-dependent secondary mass spectrometry full scan mode was performed under the following conditions: collision energies of 15, 30, and 45; The specific liquid chromatography conditions for ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 2) are: Chromatographic conditions were as follows: chromatographic column: Waters Acquity UPLC BEH, 150 mm × 2.1 mm, 1.7 μm; mobile phase A: 0.1% formic acid in water containing 10% methanol; mobile phase B: 0.1% formic acid in methanol; gradient elution; In terms of volume percentage, the elution gradient is: from 0 to 0.5 min, phase A is 100% to 60%; from 0.5 to 3.0 min, phase A is 60% to 35%; from 3.0 to 5.5 min, phase A is 35% to 30%; from 5.5 to 8.0 min, phase A is 30% to 2%; from 8.0 to 14.0 min, phase A is 2%; from 14.0 to 14.5 min, phase A is 2% to 100%; from 14.5 to 18.0 min, phase A is 100%; The names and quantitative ions of the fat-soluble synthetic dyes are: basic green 5, 329.1067; basic red 9, 288.1495; basic blue 9, 284.1216; basic blue 17, 270.1059; riboflavin, 377.1456; basic violet 14, 302.1652; thioflavin T, 283.1264; rhodamine 123, 345.1234; basic red 14, 344.2121; basic red 5, 253.1448; basic orange 2, 213.1135; basic red 46, 321.1822; coriophosphine O, 252.1495; basic red 2, 315.1604; basic orange 14, 266.1652; basic violet 2, 330.19 65; basic blue 41, 371.1536; basic yellow 51, 292.1808; basic yellow O, 268.1808; basic yellow 24, 322.1910; basic orange 21, 315.1856; new methylene blue 24, 312.1529; basic red 13, 353.1779; hexahydrocurcumin, 373.1657; basic blue 1, 363.1623; basic green 4, 329.2012; basic orange 22, 391.2169; basic blue 12, 318.1601; basic violet 7, 381.2092; basic violet 1, 358.2278; rhodamine 6G, 443.2329; crystal violet, 372.2434; fluorescein, 333. 0758; Disperse Violet 1, 239.0815; Basic Green 1, 385.2638; Basic Blue 11, 422.2591; Rhodamine B, 443.2329; Disperse Yellow 9, 275.0775; Disperse Red 11, 269.0921; Disperse Blue 7, 359.1238; Solvent Red 72, 490.8958; Basic Blue 26, 470.2591; N,N-dimethyl-4,4-azodiphenylamine, 241.1448; Disperse Yellow 1, 274.0469; Solvent Yellow 1, 198.1026; Desdimethoxycurcumin, 309.1121; Demethoxycurcumin, 339.1227; Basic Blue 7, 478.3217; Curcumin, 369 .1333; Disperse Red 58, 373.1329; Disperse Red 19, 331.1401; Basic Violet 4, 456.3373; Disperse Orange 3, 243.0877; 4-Hydroxy-4-dimethylaminoazobenzene, 242.1288; Disperse Blue 102, 366.1231; Methyl Red, 270.1237; 4-Hydroxyazobenzene, 199.0866; Solvent Yellow 33, 274.0863; Disperse Orange 11, 238.0863; Disperse Red 15, 240.0655; Disperse Red 17, 345.1557; Sudan Orange G, 215.0815; Solvent Red 197, 382.1662; Disperse Red 7, 365.1011; Disperse Blue 14, 267.1128; Disperse Yellow 3, 270.1237; Solvent Yellow 3, 226.1339; Disperse Orange 25, 324.1455; Disperse Red 9, 238.0863; Disperse Red 1, 315.1452; Disperse Blue 124, 378.1231; Disperse Brown, 433.0232; Sudan Yellow, 226.1339; Disperse Violet 28, 307.0036; Disperse Orange 37, 392.0676; Disperse Red 13, 349.1062; Para Red, 2 94.0873; Disperse Orange 61, 481.9645; Solvent Red 3, 293.1285; Solvent Red 169, 266.1176; 2-phenylazo-4-methylphenol, 213.1022; Vat Red 41, 297.0039; Toluidine Red, 308.103; Tangerine Red, 309.1234; Diethyl Yellow, 254.1652; Solvent Yellow 16 , 279.124; Sudan Red G, 279.1128; Disperse Orange 1, 319.1190; Sudan Red I, 249.1022; Disperse Yellow 23, 303.1240; Disperse Yellow 7, 317.1397; Oil Orange SS, 263.1179; Sudan Blue, 343.1441; 808 Scarlet, 368.1394; Solvent Blue 35, 351.205 3; Disperse Orange 13, 353.1397; Sudan Red II, 277.1335; Solvent Violet 13, 330.1125; Oil Blue N, 379.2380; Sudan Red III, 353.1397; Sudan Red 7B, 380.1870; Solvent Green 3, 419.1754; Sudan Red IV, 381.1710; Solvent Red 25, 381.1710.

2. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 1, wherein: The specific steps of step 1) are as follows: Step 1.1) Extraction: Weigh 1-3 g of sample into a centrifuge tube, add 5-15 mL of Extraction Solution A, vortex, sonicate, and centrifuge. Remove the supernatant and add 5-15 mL of Extraction Solution A to the centrifuge tube again. Repeat the extraction once, remove the supernatant, combine the supernatants, and dilute to 10-30 mL to obtain the sample extract. Step 1.2) Dilution: Pipette 1 mL of sample extract and dilute with diluent A to obtain sample diluent; Step 1.3) Cleanup: Pipette 1 mL of the sample dilution solution into a dispersive solid phase extraction tube for cleanup. Vortex and centrifuge. Pass the supernatant through a PTFE membrane to obtain the sample test solution.

3. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 2, wherein: The extracting solution A in step 1.1) is a mixture of formic acid and acetonitrile, and the volume ratio of formic acid to acetonitrile in the extracting solution A is (0.1-2): (88-99.9).

4. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 2, wherein: The diluent A in step 1.2) is methanol, and the dilution ratio of the sample extract to methanol is 1:(5-10).

5. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 1, wherein: The mass spectrometry conditions of the ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 2) are: The spray voltage was 3.0 kV, the ion transfer tube temperature was 320 °C, the sheath gas flow rate was 40 arb, the auxiliary gas flow rate was 10 arb, the spray needle temperature was 350 °C, and the ion lens voltage was 55; The conditions for the first-stage mass spectrometry full scan mode were as follows: mass spectrometry resolution of 70,000, automatic gain control target of 3e6, maximum dwell time of 200 ms, and scan range of 100-1000 m / z.

6. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 1, wherein: In step 2), the data relies on the full scan mode of the secondary mass spectrometer, and the conditions are: mass spectrometer resolution is 17500, automatic gain control is 2e5, maximum dwell time is 50ms, number of cycles is 5, isolation window is 1.0 m / z, scan range is 50-parent ion*charge number + 20, intensity threshold is 8.0e4, apex trigger time is 2–6 s, and dynamic exclusion time is 8.0 s.

7. The high-throughput detection method for fat-soluble synthetic dyes in food according to claim 1, wherein: The specific liquid chromatography conditions for ultra-high performance liquid chromatography quadrupole static electron orbital ion trap mass spectrometry in step 2) are: The chromatographic conditions were as follows: column temperature: 40°C; injection volume: 5 μL; flow rate: 0.3 mL / min; injection volume: 5.0 μl.

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