Method for detecting residues of 35 forbidden colorants in preserved fruit and dried fruit products

Abstract

The invention relates to a method for detecting 35 forbidden colorant residues in preserved fruit and dried fruit products, which comprises the following steps: fully extracting weighed and homogenized samples with water and acetonitrile, classifying and researching water solubility, fat solubility, acidity and alkalinity, and performing multi-column series liquid-liquid extraction by using solid-phase extraction small columns with different mechanisms, thereby fully extracting a target object in one step, and determining the content of the forbidden colorant residues in the preserved fruit and dried fruit products. And determining by using an ultra-high performance liquid chromatography-tandem mass spectrometer, and quantifying by using an external standard method and an internal standard method. The content of the coloring agent in the preserved fruit and dried fruit products is rapidly screened and confirmed, the food safety risk of forbidden coloring agents is comprehensively checked, and the method has important practical significance in safety risk assessment work of forbidden coloring agents in the preserved fruit and dried fruit products.

Description

Technical Field

[0001] The present invention relates to a method for detecting colorants, and in particular to a method for detecting the residues of 35 banned colorants in candied fruits and dried fruit products. Background Art

[0002] Banned colorants are artificial or natural colorants that are expressly prohibited from use in food according to food safety regulations or standards. These substances may be banned due to potential health risks such as toxicity, carcinogenicity, and allergic reactions. Examples include Sudan Red, Malachite Green, and Rhodamine B. These banned colorants are often carcinogenic and mutagenic, can cause liver and kidney damage, are highly toxic to the human body, and may also harm the nervous and blood systems.

[0003] Preserved fruit is a traditional food made from fruit or other plants, processed by sugaring, salting, or adding other flavorings. Dried fruit, a fruit product made through natural or artificial dehydration techniques, retains the fruit's flavor and nutrients, is portable, and is often used as a snack or baking ingredient, representing a healthy snack. Preserved fruit and dried fruit, as essential components of the snack food market, are deeply loved by the Chinese public for their rich flavor, long shelf life, and healthy image. With rising consumption trends and the widespread adoption of healthy eating habits, preserved fruit and dried fruit will continue to hold a significant position in China's snack food market.

[0004] However, due to the production and manufacturing processes of air-drying, sun-drying, and oven-toasting, the food itself undergoes a browning process, making the finished product's color significantly different from that of fresh fruits and vegetables. Consequently, vendors often add colorants to improve the color. Some even illegally pass off inferior products as genuine ones, using damaged fruit as raw material and then using colorants to repair the color. Despite strict government regulations on the use of colorants in food, many vendors still take risks to save costs by using excessive amounts and beyond the specified limits.

[0005] As early as 2005, relevant EU agencies highlighted the issue of Sudan Red, Para Red, and similar banned colorants in seasonings and foods. They required that any food used in excess or beyond the specified range be recalled. In 2021, the International Organization for Standardization (ISO) released ISO 13496:2021(E) for the determination of colorants in meat and meat products. This standard uses high-performance liquid chromatography (HPLC) to determine ten colorants in meat and meat products. This method has drawbacks such as cumbersome pretreatment, long sample run times, and high detection limits.

[0006] Currently, the main methods used in China to determine banned colorants include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-mass spectrometry (LC-MS / MS). The nine common synthetic banned colorants listed in my country's Food Additive Hygiene Standard GB 2760-2014 are primarily detected using HPLC. This method suffers from drawbacks such as low efficiency, cumbersome pretreatment, and a high risk of false positives. LC-MS / MS offers advantages such as excellent sensitivity and precision, making it more suitable for trace analysis of various banned colorants. The main domestic method for high-throughput LC-MS determination of banned colorants in food is the import and export standard SN / T 3540-2013. However, this standard categorizes banned colorants into three major categories: water-soluble alkaline pigments, water-soluble acidic pigments, and fat-soluble pigments. The three separate pretreatment methods are required for detection and analysis, which is time-consuming and cumbersome, making it inadequate for high-throughput rapid screening. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for detecting the residues of 35 banned colorants in candied fruits and dried fruit products.

[0008] This is achieved specifically through the following technical solutions:

[0009] A method for detecting residues of 35 banned colorants in candied fruits and dried fruit products, the method comprising the following steps:

[0010] 1) Take the edible part of the candied fruit or dried fruit sample, grind it into a homogenate and set aside;

[0011] 2) Place 2 g of the sample treated in step 1) in a 50 mL centrifuge tube, add ultrapure water and acetonitrile in sequence, vortex, and perform ultrasound-assisted extraction. After extraction, add anhydrous ammonium sulfate salting-out agent and manually shake for 30 seconds. Centrifuge to separate the aqueous and organic phases, collect the aqueous and organic phases separately, and set aside.

[0012] 3) Assembling a composite column, wherein the composite column is composed of a polyamide column and a diatomaceous earth column connected in series, with the polyamide column located above the diatomaceous earth column, passing the aqueous phase solution through the series columns and reacting on the diatomaceous earth column for 15 minutes, and eluting the series columns with the organic phase solution as an eluent to receive the primary eluent;

[0013] 4) After dissociation of the tandem columns, the polyamide column was eluted with 5 mL of a 10% ammonia-methanol solution and the eluate was collected and combined with the primary eluate. The eluate was concentrated to dryness under nitrogen in a water bath, then reconstituted with supernatant water and filtered through a 0.22 μm aqueous microporous membrane to obtain a sample for analysis;

[0014] 5) Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used for determination, with both external and internal standard methods used for quantification. The peak areas of the molecular weights and retention times obtained from the candied fruit and dried fruit samples were substituted into the corresponding standard curves of mixed standards for 35 banned colorants. The content of each banned colorant in the candied fruit and dried fruit was then calculated.

[0015] Furthermore, in step 2), the amount of ultrapure water added was 5 mL, the amount of acetonitrile added was 10 mL, the mixture was vortexed for 1 min, the ultrasound-assisted extraction was performed for 10 min, the amount of anhydrous ammonium sulfate salting-out agent added was 1 g, and the centrifugation conditions were 4000 r / min for 3 min.

[0016] Furthermore, in step 4), the water bath temperature is 40°C.

[0017] Furthermore, the 35 prohibited colorants in step 5) are: malachite green, basic violet 5BN, basic rhodamine B, basic orange II, basic tender yellow O, basic fuchsin, brilliant green, solvent yellow 2, solvent yellow 1, solvent yellow 3, disperse red 1, disperse orange 37 / 76, disperse orange 3, disperse yellow 3, disperse blue 3, citrus red No. 2, Sudan red I, Sudan red II, Sudan red III, Sudan red IV, acid red No. 52, red 2G, quinoline yellow, patent blue, acid red 26, acid orange II, lemon yellow, sunset yellow, amaranth, carmine, erythrosine, indigo, brilliant blue, new red, and allura red.

[0018] Furthermore, the internal standards used in the internal standard method in step 5) are malachite green-D5, basic violet 5BN-D6, basic rhodamine B-D4, solvent yellow 2-D5, disperse red 1-D3, citrus red No. 2-D6, Sudan red I-D5, Sudan red II-D6, Sudan red III-D6, and Sudan red IV-D6.

[0019] Furthermore, the liquid chromatography conditions used in step 5) are: chromatographic column Poroshell 120SB-Aq, 2.1×100 mm, 1.9 μm, column temperature: 40° C., injection volume: 5 μL, mobile phase A is 0.1% formic acid-acetonitrile solution, B is 10 mmol / L ammonium acetate solution, and gradient elution.

[0020] Furthermore, the mass spectrometry conditions used in step 5) are: ESI source, positive and negative ion mode; spray voltage (IS): 5000 V (ESI+), 4500 V (ESI-); ion source temperature: 550°C; nebulizing gas pressure (GS1): 50.0 psi; auxiliary gas pressure (GS2): 55.0 psi; curtain gas pressure (CUR): 30.0 psi; scanning mode: multiple reaction monitoring (MRM).

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention establishes a method for analyzing the residual amounts of 35 banned colorants in candied fruits and dried fruits using ultra-high performance liquid chromatography-tandem mass spectrometry. This method has the advantages of one-step rapid extraction pretreatment, good accuracy, minimal matrix interference, high recovery, and good stability. It overcomes the barriers and difficulties in traditional analytical techniques for banned colorants and provides a basic experimental approach for the determination of banned colorants in candied fruits, dried fruits, and even snack foods.

[0023] 2) The present invention achieves separation of 35 banned colorants within 15 minutes, with good linearity within the detection ranges of 10.0, 20.0, 50.0, 80.0, and 100 μg / L, with a linear correlation coefficient (R²) greater than 0.990. For preserved fruit, the limits of detection for each substance were 0.05-2.50 μg / kg, the limits of quantification were 0.15-8.33 μg / kg, and the recoveries were 80.33-117.10%. For dried fruit, the limits of detection for each substance were 0.05-1.79 μg / kg, the limits of quantification were 0.15-5.95 μg / kg, and the recoveries were 80.45-111.86%.

[0024] 3) The reproducibility of 35 banned colorants was good. In the candied fruit matrix, the relative standard deviation (RSD) was 0.14-3.08%; in the dried fruit matrix, the relative standard deviation (RSD) was 0.21-7.84%.

[0025] 4) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is more susceptible to matrix interference than gas chromatography-tandem mass spectrometry (GC-MS / MS). Furthermore, electrospray ionization (ESI) produces ionization suppression that is more pronounced than atmospheric pressure chemical ionization (APCI). To effectively eliminate the influence of matrix effects, the present invention uses isotope-labeled internal standards for calibration. This method establishes a precise compensation mechanism through the simultaneous detection of isotope-labeled internal standards, significantly improving the accuracy of test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 and Figure 2 is the matrix standard curve;

[0027] Figure 3 The mass spectrum of the substance detected in the sample. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0029] Main instruments and reagents:

[0030] Agilent 1290 / 5500Qtrap liquid chromatography-tandem mass spectrometer (AB SCIEX, USA), Milli-Q ultrapure water instrument, high-speed refrigerated centrifuge ST16R (Thermos), vortex mixer VG3 S025 (IKA), high-speed homogenizer T25 (IKA), knife mill GM200 (Retsch).

[0031] Malachite green, basic violet 5BN, basic rhodamine B, basic orange II, basic tender yellow O, basic fuchsin, brilliant green, solvent yellow 2, solvent yellow 1, solvent yellow 3, disperse red 1, disperse orange 37 / 76, disperse orange 3, disperse yellow 3, disperse blue 3, citrus red No. 2, Sudan red I, Sudan red II, Sudan red III, Sudan red IV, acid red No. 52, red 2G, quinoline yellow, patent blue, acid red 26, acid orange II, lemon yellow, sunset yellow, amaranth, carmine, erythrosine, indigo, brilliant blue, new red, allura red, were purchased from Dr. Ehrenstorfer, and the concentration of each was 100 mg / L.

[0032] Solvent Yellow 2-D5, Disperse Red 1-D3, Sudan Red 1-D5, Sudan Red 2-D6, Sudan Red 3-D6, Sudan Red 4-D6, Tangerine Red No. 2-D6, Malachite Green-D5, Crystal Violet 5BN-D6, and Rhodamine B-D4 were all purchased from Dr. Ehrenstorfer at a concentration of 100 mg / L.

[0033] Acetonitrile, chromatographic grade, Merk, Germany; ammonium acetate, chromatographic grade, Aladdin Reagent Co., Ltd.; formic acid, chromatographic grade, Thermofisher (USA); anhydrous ammonium sulfate, analytical grade, Wuxi Zhanwang Chemical Reagent Co., Ltd.; ammonia, chromatographic grade, Sigma-Aldrich, USA; diatomaceous earth column, 500 mg / 6 mL; polyamide column, 500 mg / 6 mL, Shandong Qingyun Chemical Technology Co., Ltd.

[0034] Samples: 10 batches of candied fruits and dried fruits purchased online were randomly inspected.

[0035] Example

[0036] Sample Preparation: Take the edible portion of the sample and homogenize it using a grinder. Weigh 2.00 g (accurate to ±0.01 g) of the processed sample into a 50 mL centrifuge tube. Add 5.0 mL of ultrapure water and 10.0 mL of acetonitrile, sequentially. Vortex for 1 minute. Perform ultrasound-assisted extraction for 10 minutes. Add 1.0 g of anhydrous ammonium sulfate salting-out agent and manually vortex for 30 seconds. Centrifuge at 4000 rpm for 3 minutes to separate the aqueous and organic phases. Allow to pass through the column.

[0037] A composite column purification strategy was employed, combining a polyamide column (pre-conditioned with 3 mL of methanol and 3 mL of ultrapure water) and a diatomaceous earth column in series. The aqueous phase was passed through the columns and allowed to equilibrate within the diatomaceous earth column for 15 minutes. The acetonitrile layer then eluted the columns, collecting the primary eluate.

[0038] After dissociation of the column system, the polyamide column was eluted with 5 mL of 10% ammonia methanol solution and collected. The two eluates were combined and concentrated to dryness in a 40°C constant temperature water bath under nitrogen. The sample was reconstituted with 1.0 mL of ultrapure water and filtered through a 0.22 μm water-based microporous filter before being analyzed by the instrument.

[0039] Ultra-high performance liquid chromatography-tandem mass spectrometry detection:

[0040] Chromatographic conditions: Poroshell 120SB-Aq column, 2.1 × 100 mm, 1.9 μm. Column temperature: 40°C. Injection volume: 5 μL. Mobile phase A: 0.1% formic acid in acetonitrile, mobile phase B: 10 mmol / L ammonium acetate. Gradient elution details are shown in Table 1.

[0041] Table 1: Mobile phase gradient elution program

[0042]

[0043] Mass spectrometry conditions: ESI source, positive and negative ion modes; spray voltage (IS): 5000 V (ESI+), 4500 V (ESI-); ion source temperature: 550°C; nebulizer gas pressure (GS1): 50.0 psi; auxiliary gas pressure (GS2): 55.0 psi; curtain gas pressure (CUR): 30.0 psi; scan mode: multiple reaction monitoring (MRM). The mass spectrometry parameters for the 35 banned colorant standards and internal standard are shown in Table 2.

[0044] Table 2: Main mass spectrometry parameters of the compounds

[0045]

[0046]

[0047] Quantification of 35 banned colorants in the sample to be tested:

[0048] (1) Preparation of mixed standard stock solution: Measure the single standard of each of the 35 banned colorants and the internal standard, dilute to volume with chromatography-grade acetonitrile in a 10 mL volumetric flask, and prepare a mixed standard stock solution with a concentration of approximately 1 mg / L for each substance.

[0049] (2) Preparation of standard curve: The prepared mixed standard stock solution was measured and diluted step by step to obtain mixed standard samples of 35 banned colorants at 5 different concentrations of 10.0, 20.0, 50.0, 80.0, and 100 μg / L, with an internal standard concentration of 50.0 μg / L. The mixed standard samples of each concentration were tested in turn, and 35 standard curves were drawn with the ratio of the internal and external standard areas as the vertical axis Y and the mass concentration of the banned colorant substance as the horizontal axis X. Specific parameters are shown in Table 3, and the standard linear graphs of each substance are shown in Figure 1 、 2 .

[0050] Table 3

[0051]

[0052]

[0053]

[0054] As shown in the table above, the 35 banned colorants in the candied fruits and dried fruits were separated within 15 minutes, and had a good linear relationship in the detection range of 10.0-100 μg / L. The correlation coefficient R 2 The detection limits of various substances in candied fruit were 0.05-2.50 μg / kg, the limits of quantification were 0.15-8.33 μg / kg, and the recoveries were 80.33-117.10%. The detection limits of various substances in dried fruit were 0.05-1.79 μg / kg, the limits of quantification were 0.15-5.95 μg / kg, and the recoveries were 80.45-111.86%.

[0055] (3) Determination of candied fruit and dried fruit samples: Take the processed candied fruit and dried fruit samples for testing, substitute the peak area obtained by the test into the standard curve of the corresponding substance, and analyze and calculate the content of 35 banned colorants in candied fruit and dried fruit. The mass spectrum of the detected sample is shown in Figure 3 The results are shown in Table 4 below.

[0056] Table 4: Content of 35 banned colorants in samples (unit: μg / kg)

[0057]

[0058] (Note: ND means not detected)

[0059] As shown in the table above, tartrazine, indigo, carmine, and brilliant blue were detected in the eight batches of candied fruit and dried fruit, while the remaining 31 banned colorants were not detected. The highest detected residue reached 39,259.989 μg / kg.

Claims

1. A method for detecting the residues of 35 banned colorants in candied fruits and dried fruit products, characterized in that: The detection method comprises the following steps: 1) Take the edible part of the candied fruit or dried fruit sample, grind it into a homogenate and set aside; 2) Place 2 g of the sample treated in step 1) in a 50 mL centrifuge tube, add ultrapure water and acetonitrile in sequence, vortex, and perform ultrasound-assisted extraction. After extraction, add anhydrous ammonium sulfate salting-out agent and manually shake for 30 seconds. Centrifuge to separate the aqueous and organic phases, collect the aqueous and organic phases separately, and set aside. 3) Assembling a composite column, wherein the composite column is composed of a polyamide column and a diatomaceous earth column connected in series, with the polyamide column located above the diatomaceous earth column, passing the aqueous phase solution through the series columns and reacting on the diatomaceous earth column for 15 minutes, and eluting the series columns with the organic phase solution as an eluent to receive the primary eluent; 4) After dissociation of the tandem columns, the polyamide column was eluted with 5 mL of a 10% ammonia-methanol solution and the eluate was collected and combined with the primary eluate. The eluate was concentrated to dryness under nitrogen in a water bath, then reconstituted with supernatant water and filtered through a 0.22 μm aqueous microporous membrane to obtain a sample for analysis; 5) Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used for determination, with both external and internal standard methods used for quantification. The peak areas of the molecular weights and retention times obtained from the candied fruit and dried fruit samples were substituted into the corresponding standard curves of mixed standards for 35 banned colorants. The content of each banned colorant in the candied fruit and dried fruit was then calculated.

2. The method for detecting 35 kinds of prohibited colorants residues in candied fruit and dried fruit products according to claim 1, characterized in that: In step 2), the amount of ultrapure water added was 5 mL, the amount of acetonitrile added was 10 mL, the mixture was vortexed for 1 min, ultrasound-assisted extraction was performed for 10 min, the amount of anhydrous ammonium sulfate salting-out agent added was 1 g, and the centrifugation conditions were 4000 r / min for 3 min.

3. The method for detecting 35 kinds of prohibited colorants residues in candied fruit and dried fruit products according to claim 1, characterized in that: In step 4), the water bath temperature is 40°C.

4. The method for detecting 35 kinds of prohibited colorants residues in candied fruit and dried fruit products according to claim 1, characterized in that: The 35 prohibited colorants in step 5) are: malachite green, basic violet 5BN, basic rhodamine B, basic orange II, basic tender yellow O, basic fuchsin, brilliant green, solvent yellow 2, solvent yellow 1, solvent yellow 3, disperse red 1, disperse orange 37 / 76, disperse orange 3, disperse yellow 3, disperse blue 3, citrus red No. 2, Sudan red I, Sudan red II, Sudan red III, Sudan red IV, acid red No. 52, red 2G, quinoline yellow, patent blue, acid red 26, acid orange II, lemon yellow, sunset yellow, amaranth, carmine, erythrosine, indigo, brilliant blue, new red, and allura red.

5. The method for detecting 35 kinds of prohibited colorants residues in candied fruits and dried fruit products according to claim 1, characterized in that: The internal standards used in the internal standard method in step 5) are malachite green-D5, basic violet 5BN-D6, basic rhodamine B-D4, solvent yellow 2-D5, disperse red 1-D3, citrus red No. 2-D6, Sudan red I-D5, Sudan red II-D6, Sudan red III-D6, and Sudan red IV-D6.

6. The method for detecting 35 kinds of prohibited colorants residues in candied fruits and dried fruit products according to claim 1, characterized in that: The liquid chromatography conditions used in step 5) are: chromatographic column Poroshell 120SB-Aq, 2.1×100 mm, 1.9 μm, column temperature: 40° C., injection volume: 5 μL, mobile phase A: 0.1% formic acid-acetonitrile solution, mobile phase B: 10 mmol / L ammonium acetate solution, gradient elution.

7. The method for detecting 35 kinds of prohibited colorants residues in candied fruits and dried fruit products according to claim 1, characterized in that: The mass spectrometry conditions used in step 5) are: ESI source, positive and negative ion modes; spray voltage (IS): 5000 V (ESI+), 4500 V (ESI-); Ion source temperature: 550°C; nebulizer gas pressure (GS1): 50.0 psi; auxiliary gas pressure (GS2): 55.0 psi; curtain gas pressure (CUR): 30.0 psi; scan mode: multiple reaction monitoring (MRM).

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