Method for detecting pigment in condiment and application of method

Abstract

The invention relates to the technical field of analytical chemistry and biological detection, in particular to a method for detecting pigments in condiments and application of the method. The detection method comprises the following steps: pretreating a sample to obtain a to-be-detected sample solution; and determining and analyzing the to-be-detected sample solution by adopting ultra-high performance liquid chromatography-mass spectrometry. According to the present invention, the 115 pigments in the seasoning are detected by combining the liquid chromatography with the mass spectrometry, the positive and negative ion switching mode is adopted, the anions and the cations of the pigments are simultaneously detected, the versatility is strong, the detection object coverage range is wide, the method is simple, and the performance and the technical advantages of high speed, high efficiency, high resolution, trace detection and automatic analysis are provided; the leak detection risk is reduced; the method has the advantages of short analysis time, high sensitivity, low detection limit and wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting pigments in condiments and application thereof. Background Art

[0002] At present, consumers' requirements for food safety are constantly increasing, and achieving a virtuous interaction between high-quality development and high-level safety has become the constant pursuit of more and more food companies. For the seasoning industry, due to the wide source of raw materials and large differences in varieties, the quality control pressure of products is huge, and there is an urgent need to establish rapid and high-throughput detection technology to ensure the quality level of raw materials. This project is mainly aimed at industrial pigments that may be illegally added to seasoning raw materials, taking the possible illegal addition of pepper raw materials as the scenario, to develop and apply related high-throughput screening technologies.

[0003] Currently, there are many types of methods to determine colorants in food, including capillary electrophoresis (CE), thin layer chromatography, ion chromatography, high performance liquid chromatography (HPLC) tandem ultraviolet / visible detector (UV / Vis) or diode array detector (DAD), liquid chromatography-mass spectrometry detection. Among these methods, HPLC-UV / Vis or HPLC-DAD are the most commonly used detection methods because colorants have strong absorbance at visible wavelengths. However, since there are many isomers and structural analogs that are difficult to separate in colorants, these methods cannot simultaneously screen a large number of prohibited colorants. In addition, this detection method is also prone to false positives or false negatives. In order to solve this problem, tandem quadrupole mass spectrometry can be used to detect colorants in food because it can provide detailed structural information, especially in the selected reaction monitoring (SRM) mode, specific SRM channels (precursor ions and product ions) can exclude the appearance of interfering substances, so the accuracy of quantification can be greatly improved. Although tandem triple quadrupole mass spectrometry has such potential, the simultaneous detection of hundreds of industrial pigments is still a huge challenge for triple quadrupole mass spectrometry. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for detecting pigments in condiments.

[0005] The invention also proposes the application of the above detection method.

[0006] In one aspect of the present invention, a method for detecting pigments in condiments is provided, the method comprising the following steps:

[0007] S1, pre-treating the sample to obtain a sample solution to be tested;

[0008] S2, measuring and analyzing the sample solution by ultra-high performance liquid chromatography-mass spectrometry;

[0009] The ultra-high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium acetate solution with a concentration of 5 to 15 mmol / L; and the mobile phase B is acetonitrile;

[0010] The mobile phase was eluted according to the following gradient:

[0011] 0.00-1.00min, the volume percentage of mobile phase B is 2%-6%, and the balance is mobile phase A;

[0012] 1.00-4.00min, the volume percentage of mobile phase B is 5%-50%, and the balance is mobile phase A;

[0013] 4.00-8.00 min, the volume percentage of mobile phase B is 50%-95%, and the balance is mobile phase A;

[0014] 8.00-12.00 min, the volume percentage of mobile phase B is 92%-98%, and the balance is mobile phase A;

[0015] 12.00-12.10min, the volume percentage of mobile phase B is 92%-98% to 1%-5%, and the balance is mobile phase A;

[0016] 12.10 -15.00min, the volume percentage of mobile phase B is 2%-6%, and the balance is mobile phase A.

[0017] In some embodiments of the present invention, the pigment includes carminic acid, lemon yellow, new red, amaranth, acid blue 74 / indigo carmine, carmine, acid red 18 / carmine / ponceau 4R, quinoline yellow, sunset yellow, orange yellow G, allura red, acid yellow 17, red 2G / acid yellow 1, mordant blue 13, acid red, carmine SX, acid red 26, acid blue 9, brilliant blue, acid red 87 / eosin, fluorescein sodium, reactive brilliant blue KN-R, acid orange 20, amido black 10B / amido black, acid blue 1, acid red 73 / acid scarlet GR , Acid Green 16, Acid Green 25, Acid Red 52, Patent Blue V, Congo Red / Direct Red 28, Acid Orange 7 / Acid Orange II, Acid Blue 3, Crocus Orange G, Methyl Orange, Acid Blue 41, Soap Yellow / Acridine Yellow / Acid Golden Yellow, Acid Violet 17, Acid Violet 43, Acid Blue 7, Acid Blue 113, Acid Orange 3, Acid Blue 90, Disperse Yellow 1, Sudan Orange G, Disperse Yellow 49, Para Red, Disperse Yellow 23, Indigo, Rhodamine 110, Erythrosine, Azure B, Basic Blue 17, Methylene Blue, Neutral Red, Thioflavin T / Basic Yellow 1, Basic Red 9, Basic Red 2, basic red 14 / cationic brilliant red 5GN, basic tender yellow O, rhodamine B, basic violet 14, basic orange 21, disperse red 19, basic violet 2 / new fuchsin, disperse red 11, basic orange 2, basic blue 1, disperse yellow 9, disperse red 17 / disperse red GG, basic brilliant green, vat red 6B / basic violet 7, disperse yellow 39, basic orange 22, basic blue 12 / nile blue, solvent yellow 1, curcumin, basic red 1 / rhodamine 6G, disperse yellow 3, disperse orange 11, disperse red 1, basic violet 5BN, disperse orange 3, disperse blue 14, basic green 1, One or more of Disperse Blue 124, Disperse Orange 25, Solvent Yellow 3, Basic Blue 11, Methyl Yellow / Solvent Yellow 2, Sudan Red 197, Victoria Blue, Disperse Orange 37 / 76, Crystal Violet Lactone, Solvent Yellow 114, Solvent Red 80, Solvent Red 1 / Sudan Red G, Sudan Yellow, Pigment Red 3, Sudan Yellow 56, Sudan 1, Basic Blue 7 Leuco Malachite Green, Crystal Violet, Disperse Orange 1, Disperse Orange 149, Basic Violet, Sudan 2, Sudan Blue II, Sudan 3, Disperse Orange 13, Sudan Red 7B, Sudan Red B / Solvent Red 25, Sudan 4 and Sudan Black B.

[0018] In some embodiments of the present invention, the pretreatment includes the following steps: adding an extractant to the sample to be tested, mixing, and collecting supernatant A after centrifugation; repeating the extraction once and combining the extracts; adding a C18 adsorbent to the extract, collecting supernatant B after centrifugation, and filtering the supernatant B to obtain a sample solution to be tested.

[0019] In some embodiments of the present invention, the extractant is a mixture of acetonitrile and methanol, and the volume percentage of acetonitrile in the extractant is 20% to 80%.

[0020] In some embodiments of the present invention, the extractant is a mixture of acetonitrile and methanol, and the volume percentage of acetonitrile in the extractant is 50%.

[0021] In some embodiments of the present invention, the added mass volume ratio of the sample to be tested to the extractant is 1:(4-6) g / mL.

[0022] In some embodiments of the present invention, the added mass volume ratio of the sample to be tested to the extractant is 1:5 g / mL.

[0023] In some embodiments of the present invention, the centrifugation condition is 6000-10000 r / min for 4-6 min.

[0024] In some embodiments of the present invention, the centrifugation condition is 8000r / min for 5min.

[0025] In some embodiments of the present invention, the added amount of the C18 adsorbent is 20-30 mg.

[0026] In some embodiments of the present invention, the mobile phase is subjected to gradient elution according to the following gradient:

[0027] 0.00-1.00min, the volume percentage of mobile phase B is 5%, and the balance is mobile phase A;

[0028] 1.00-4.00min, the volume percentage of mobile phase B is 5%-50%, and the balance is mobile phase A;

[0029] 4.00-8.00 min, the volume percentage of mobile phase B is 50%-95%, and the balance is mobile phase A;

[0030] 8.00-12.00 min, the volume percentage of mobile phase B is 95%, and the balance is mobile phase A;

[0031] 12.00-12.10min, the volume percentage of mobile phase B is 95% to 5%, and the balance is mobile phase A;

[0032] 12.10 -15.00min, the volume percentage of mobile phase B is 5%, and the balance is mobile phase A.

[0033] According to some embodiments of the present invention, the ultra-high performance liquid chromatography conditions include: the chromatographic column is Waters Acquity UPLC BEH Shield RP18, 2.1 mm×100 mm, and the particle size is 1.7 μm.

[0034] In some embodiments of the present invention, the ultra-high performance liquid chromatography conditions include: a flow rate of 0.8 mL / min-1.2 mL / min.

[0035] In some embodiments of the present invention, the ultra-high performance liquid chromatography conditions include: an injection volume of 3-7 μL and a column temperature of 30° C.-40° C.

[0036] In some embodiments of the present invention, the mass spectrometer is a quadrupole electrostatic field orbital trap high-resolution mass spectrometer.

[0037] In some embodiments of the present invention, the mass spectrometry detection adopts a primary parent ion full scan plus a data-dependent secondary daughter ion scan mode to collect data, and a primary full scan is set to cover the mass number range of 150 to 900 m / z of the target compound.

[0038] In some embodiments of the present invention, the mass spectrometry conditions include: heated electrospray ion source, positive and negative ion modes; spray voltage: positive ion mode 3.8-4.2 kV, negative ion mode 2.8-3.5 kV; capillary temperature 300-340 ° C; sheath gas: 32-37 arb; auxiliary gas: 8-12 arb; scanning mode: Full MS / dd-MS 2 ; Level 1 full scan resolution: 68000-77000FWHM, AGC maximum capacity: 2×10 6 -4×10 6 , AGC maximum injection time: 80-120ms; data dependent secondary product ion scanning resolution: 17000-18000FWHM, trigger threshold maximum capacity: 0.8×10 5 -1.2×10 5 , maximum injection time: 40-60ms.

[0039] In some embodiments of the present invention, the mass spectrometry qualitative and quantitative ions and retention times of the pigment are shown in the following table:

[0040]

[0041]

[0042] In some embodiments of the present invention, the mass spectrometry conditions include: heated electrospray ion source, positive and negative ion modes; spray voltage: 4.0 kV in positive ion mode, 3.2 kV in negative ion mode; capillary temperature 320°C; sheath gas: 35 arb; auxiliary gas: 10 arb; scan mode: Full MS / dd-MS 2 ; Level 1 full scan resolution: 70000FWHM, AGC maximum capacity: 3×10 6, AGC maximum injection time: 100ms; data dependent secondary product ion scanning resolution: 17500FWHM, trigger threshold maximum capacity: 1×10 5 , maximum injection time: 50ms.

[0043] In some embodiments of the present invention, the temperature of the heated electrospray ion source is 450-550°C.

[0044] In some embodiments of the present invention, the temperature of the heated electrospray ion source is 500°C.

[0045] In some embodiments of the present invention, the mass spectrometry conditions further include: the drying gas is nitrogen with a flow rate of 14-19 L / min.

[0046] In some embodiments of the present invention, the mass spectrometry conditions further include: capillary spray voltage: 5000-6000V; nebulizing gas pressure: 30-50psi.

[0047] In some embodiments of the present invention, the method also includes the step of processing the data of the sample to be tested obtained by the measurement and analysis, and the data processing includes importing the data of the sample to be tested into the Tracefinder 4.1 data workstation software for processing, and setting the precise mass number deviation, retention time deviation, and compound minimum response value parameters as search and filtering conditions, comparing the data of the sample to be tested with the standard data to determine the target pigment.

[0048] In some embodiments of the present invention, the accurate mass deviation is 4-6 ppm.

[0049] In some embodiments of the present invention, the accurate mass deviation is 5 ppm.

[0050] In some embodiments of the present invention, the retention time deviation is 0.1-0.3 min.

[0051] In some embodiments of the present invention, the retention time deviation is 0.2 min.

[0052] In some embodiments of the present invention, the minimum response value of the compound is 8000-12000 counts.

[0053] In some embodiments of the present invention, the minimum response value of the compound is 10,000 counts.

[0054] In some embodiments of the present invention, the identification criteria for determining the target pigment are:

[0055] 1) When the parent ion (MS) and retention time (RT) match the standard mass library, and the secondary fragment (MS2) matches the standard data by more than 55% (mzVault score>55), the compound can be confirmed;

[0056] 2) When the retention time was inconsistent, but the matching percentage of the parent ion (MS) and secondary fragments with the constructed standard data was greater than 55%, the compound was classified as a suspected compound.

[0057] According to a second aspect of the present invention, application of the above method in detecting pigments in condiments is proposed.

[0058] In some embodiments of the present invention, the condiment includes one of chili oil, chili powder and chili flakes.

[0059] According to some embodiments of the present invention, the following beneficial effects are achieved: the present invention uses liquid chromatography combined with mass spectrometry to detect 115 pigments in condiments, adopts a positive and negative ion switching mode, and simultaneously detects the anions and cations of the pigments. It has strong versatility, a wide range of detection objects, a simple method, and has the performance and technical advantages of high speed, high efficiency, high resolution, trace detection and automated analysis, which reduces the risk of missed detection; the analysis time is short, the sensitivity is high, the detection limit is low, and it has broad application prospects.

[0060] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0062] Figure 1 These are three kinds of pepper samples in the embodiments of the present invention;

[0063] Figure 2 This is the total ion current diagram of the oil chili sample without adding pigment in the positive ion mode in the embodiment of the present invention;

[0064] Figure 3 This is the total ion current diagram of the oil chili sample without adding pigment in the negative ion mode in the embodiment of the present invention;

[0065] Figure 4 This is the total ion current diagram of a chili powder sample without pigment added in the positive ion mode in an embodiment of the present invention;

[0066] Figure 5 This is the total ion current diagram of a chili powder sample without pigment added in the negative ion mode in an embodiment of the present invention;

[0067] Figure 6 This is the total ion current diagram of the chili powder sample without adding pigment in the positive ion mode in the embodiment of the present invention;

[0068] Figure 7 This is the total ion current diagram of a chili powder sample without pigment added in the negative ion mode in an embodiment of the present invention;

[0069] Figure 8 This is the total ion current diagram of the chili oil sample with 115 kinds of pigment standards added in the positive ion mode in the embodiment of the present invention;

[0070] Fig. 9 This is the total ion current diagram of the chili oil sample with 115 pigments added in the negative ion mode in the embodiment of the present invention;

[0071] Fig.10 This is the total ion current diagram of a chili powder sample with 115 pigments added in the positive ion mode in an embodiment of the present invention;

[0072] Fig.11 This is the total ion current diagram of a chili powder sample with 115 pigments added in the negative ion mode in an embodiment of the present invention;

[0073] Fig.12 This is the total ion current diagram of the chili powder sample with 115 pigments added in the positive ion mode in the embodiment of the present invention;

[0074] Fig.13 This is the total ion current diagram of the chili powder sample with 115 pigments added in the negative ion mode in the embodiment of the present invention;

[0075] Fig.14 This is a test result diagram of Sudan 1 spiked with 0.1 mg / kg in an embodiment of the present invention;

[0076] Fig.15 This is a test result diagram of Sudan 1 spiked with 0.2 mg / kg in an embodiment of the present invention;

[0077] Fig.16 This is a test result diagram of Sudan 1 spiked with 1 mg / kg in an embodiment of the present invention;

[0078] Fig.17 This is the test result of Sudan 4 spiked with 0.1 mg / kg in the embodiment of the present invention.

[0079] Fig.18 This is the test result of Sudan 4 spiked with 0.2 mg / kg in the embodiment of the present invention.

[0080] Fig.19 This is the test result of Sudan 4 spiked with 1 mg / kg in the embodiment of the present invention.

[0081] Fig. 20 This is a test result diagram of Rhodamine B spiked with 0.1 mg / kg in an embodiment of the present invention;

[0082] Fig.21 This is a test result diagram of the Rhodamine B spiked with 0.2 mg / kg in the embodiment of the present invention;

[0083] Fig. 22 This is a test result diagram of the Rhodamine B spiked with 1 mg / kg in an embodiment of the present invention;

[0084] Fig.23 This is a test result diagram of Basic Orange 2 spiked with 0.1 mg / kg in an embodiment of the present invention;

[0085] Fig.24 This is a test result diagram of Basic Orange 2 spiked with 0.2 mg / kg in an embodiment of the present invention;

[0086] Fig.25 This is a test result diagram of Basic Orange 2 spiked with 1 mg / kg in an embodiment of the present invention;

[0087] Fig.26 This is a test result diagram of basic tender yellow O spiked with 0.1 mg / kg in an embodiment of the present invention;

[0088] Fig. 27 This is a test result diagram of basic tender yellow O spiked with 0.2 mg / kg in an embodiment of the present invention;

[0089] Fig.28 This is a test result diagram of basic tender yellow O spiked with 1 mg / kg in an embodiment of the present invention;

[0090] Fig.29 This is a test result diagram of the disperse red 1 spiked with 0.1 mg / kg in the embodiment of the present invention;

[0091] Fig.30 This is a test result diagram of the disperse red 1 spiked with 0.2 mg / kg in the embodiment of the present invention;

[0092] Fig.31 This is a test result diagram of the disperse red 1 spiked with 1 mg / kg in an embodiment of the present invention;

[0093] Fig.32 This is a test result graph of sunset yellow spiked with 0.1 mg / kg in an embodiment of the present invention;

[0094] Fig.33 This is a test result graph of sunset yellow spiked with 0.2 mg / kg in an embodiment of the present invention;

[0095] Fig.34 This is a test result diagram of sunset yellow spiked with 1 mg / kg in an embodiment of the present invention;

[0096] Fig.35 It is the test result diagram of the red erythrosine spiked with 0.1 mg / kg in the embodiment of the present invention;

[0097] Fig.36 It is the test result diagram of the red erythrosine spiked with 0.2 mg / kg in the embodiment of the present invention;

[0098] Fig.37 It is the test result diagram of the red erythrosine spiked with 1 mg / kg in the embodiment of the present invention;

[0099] Fig.38 This is a test result diagram of Acid Orange 7 / Acid Orange II spiked with 0.1 mg / kg in an embodiment of the present invention;

[0100] Fig.39 This is a test result diagram of Acid Orange 7 / Acid Orange II spiked with 0.2 mg / kg in an embodiment of the present invention;

[0101] Fig.40 This is a test result diagram of Acid Orange 7 / Acid Orange II spiked with 1 mg / kg in an embodiment of the present invention;

[0102] Fig.41 This is a test result diagram of Acid Red 52 spiked with 0.1 mg / kg in an embodiment of the present invention;

[0103] Fig.42 This is a test result diagram of Acid Red 52 spiked with 0.2 mg / kg in an embodiment of the present invention;

[0104] Fig.43 This is a test result diagram of Acid Red 52 spiked with 1 mg / kg in an embodiment of the present invention;

[0105] Fig.44 This is a test result graph of the new red spiked with 0.1 mg / kg in the embodiment of the present invention;

[0106] Fig.45 This is a test result graph of the new red spiked with 0.2 mg / kg in the embodiment of the present invention;

[0107] Fig.46 This is a test result graph of the new red spiked with 1 mg / kg in an embodiment of the present invention. DETAILED DESCRIPTION

[0108] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0109] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for other reagents or instruments used, they are all conventional products that can be purchased commercially.

[0110] Testing instruments: UltiMate 3000-Q / Exactive high performance liquid chromatography tandem high resolution mass spectrometry (Thermo Fisher Scientific, USA), Milli-R04 ultrapure water generator (Millipore, Germany); Allegra X-22R refrigerated centrifuge (Beckman Coulter, USA); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Company); VORTEXKB-3 vortex oscillator (Scientific Industries, USA); Mettler Toledo analytical balance (precision: 0.0001g, Mettler Toledo, USA), MR9501 grinder (Beijing Morphy Electric Co., Ltd.); 1mL syringe (Jiangsu Huada Medical Instrument Co., Ltd., Jiangsu, China); 0.2μm filter membrane (organic filter membrane) (Tianjin Jinteng Technology Co., Ltd., Tianjin, China).

[0111] Colorant standards: 115 colorant standards (standard information is shown in Table 1) were purchased from Tianjin Alta Company. Information on each compound is shown in Table 1. HPLC-grade methanol and acetonitrile were purchased from Fisher (Pittsburgh, PA, USA). Ultrapure water was obtained from a Milli-Q water purifier (Millipore, Bedford, MA, USA). Analytical-grade formate and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0112] Mixed standard solution: The 115 kinds of colorant standards purchased above were prepared into a mixed standard solution for analysis within 1 month after purchase. During the analysis, the concentration of each colorant standard in the mixed standard solution was 0.1 mg / L.

[0113] Table 1

[0114]

[0115]

[0116]

[0117] Example 1 A method for detecting pigments in condiments

[0118] This embodiment provides a method for detecting pigments in condiments, the method comprising the following steps:

[0119] 1. Sample pretreatment

[0120] The specific steps are as follows:

[0121] Accurately weigh 1.00g of the sample to be tested into a 50mL centrifuge tube, add 5mL of acetonitrile-methanol (50:50, V:V), vortex on a vortex mixer for 1min, centrifuge at 8000r / min for 5min, take 1mL of supernatant into a 10mL colorimetric tube, add an appropriate amount of acetonitrile-methanol (50:50, V:V) solution to 10mL, mix well, take out 1mL into a 2mL centrifuge tube, add 25mg C18 purifier, vortex for 1min, centrifuge at 8000r / min for 5min. Take the supernatant and filter it through a 0.22μm PTFE filter membrane (discard the first 2 drops of filtrate) and wait for the determination on the machine.

[0122] 2. Ultra-high performance liquid chromatography-mass spectrometry analysis

[0123] Chromatographic column: Waters Acquity UPLC BEH Shield RP18 (2.1mm×100mm, 1.7μm); column temperature: 35°C; injection volume: 5μL; mobile phase A is 10mmol / L ammonium acetate aqueous solution, and B is acetonitrile; flow rate is 0.3mL / min; in terms of volume percentage, the gradient elution conditions are: 0-1.0min, 5%B; 1.0-4.0min, 5%-50%B; 4.0-8.0min, 50-95%B; 8.0-12.0min, 95%B; 12.0-12.1min, 95-5%B; 12.1-15.0min, 5%B, and the mobile phase A is used to make up the remaining volume percentage in each elution time period.

[0124] The mass spectrometer used a quadrupole electrostatic field orbital trap high-resolution mass spectrometer, and the mass spectrometer conditions were as follows: heated electrospray ion source (HESI), ion source temperature of 500°C, simultaneous monitoring of positive and negative ion modes; spray voltage: 4.0 kV in positive ion mode, 3.2 kV in negative ion mode; capillary temperature: 320°C; capillary spray voltage: 5500 V, nebulizer gas pressure: 40 psi; sheath gas: nitrogen, flow rate of 35 arb; auxiliary gas: nitrogen, flow rate of 10 arb; dry gas: nitrogen, flow rate of 17 L / min; scan mode: full scan of primary parent ion and data-dependent secondary daughter ion scan (Full MS / dd-MS 2 ) mode. Primary parent ion full scan resolution: 7×10 4 Half peak width (FWHM) (7×10 4 ~7.5×10 4 The mass spectrometer scanning range is: m / z 150~900, the automatic gain control (AGC) and the maximum injection time (IT) are 3×10 6 , 100ms. Data dependent secondary product ion scanning resolution: 1.75×10 4 FWHM; trigger threshold and maximum injection time (IT) are: 1×10 5 The collision energy (NCE) values ​​of each compound were uniformly set to 25, 35, and 45 eV. The specific mass spectrometry parameter information of each pigment is shown in Table 2.

[0125] 3. Data processing

[0126] Thermo Fisher Scientific TraceFinder4.1 software (Thermo Fisher Scientific, USA) was used for data collection, database screening, and quantitative analysis. During the screening, the key information of the 115 pigment compounds, such as the name, molecular formula, primary accurate mass number, retention time, and characteristic fragment ions, were entered into the TraceFinder4.1 software, and then the secondary mass spectra collected under different collision energy conditions were imported into the above software to form a screening mass spectrometry database for 115 industrial pigment compounds in condiment raw materials.

[0127] Table 2

[0128]

[0129]

[0130]

[0131] Example 2

[0132] This embodiment optimizes the mass spectrometry scanning acquisition mode, primary scanning resolution, and secondary scanning resolution in the method of Embodiment 1.

[0133] 1. Optimization of mass spectrometry scanning acquisition method

[0134] This study investigated three mass spectrometry acquisition modes (Full MS, Targeted-SIM / dd-MS 2 and Full MS / dd-MS 2 ) effect, and the mass spectrometry conditions were the same as in Example 1.

[0135] The results showed that although Full MS can achieve quantitative detection of the target, its qualitative screening is based only on the accurate mass number and retention time of the parent ion, resulting in false positives for some compounds; Targeted-SIM / dd-MS 2 The qualitative identification ability is enhanced, but the quantitative analysis level is relatively low; Full MS / dd-MS 2 Qualitative screening and quantitative detection can be achieved simultaneously. This mode can be combined with the HCD fragment ion mass spectrum for accurate qualitative analysis to meet detection needs.

[0136] 2. Optimization of primary scanning resolution and secondary scanning resolution

[0137] The experimental method is the same as that of Example 1, except that the primary scanning resolution (140000, 75000, 50000) and the secondary scanning resolution (40000, 25000, 17500) are combined to examine the selectivity and sensitivity.

[0138] The results show that when the primary scanning resolution is 140000 and the secondary scanning resolution is 40000, 25000, and 17500, the scanning selectivity is very strong but the sensitivity is low, and the number of points collected is small or even no peak; when the primary scanning resolution is 75000 and the secondary scanning resolution is 25000 and 17500, the matrix effect is relatively reduced and the response value is improved. Among them, when the primary scanning resolution is 75000 and the secondary scanning resolution is 17500, most of the pigments to be tested can obtain excellent accuracy and sensitivity. When the primary scanning resolution is 50000 and the secondary scanning resolution is 25000 and 17500, the scanning speed is very fast, but it is difficult to identify substances with similar molecular weights. Considering the selectivity and sensitivity settings of multiple components, the resolution combination of 75000 for the primary scanning resolution and 17500 for the secondary scanning resolution is better.

[0139] Example 3 Methodological Evaluation

[0140] This example verifies the use of the method for detecting pigments in condiments in Example 1 to detect pigments in specific condiments (chili oil, chili powder and chili shreds samples).

[0141] Samples and sample sources: The three chili samples were purchased from the market, namely chili oil (LJ01), chili powder (LJ02) and chili flakes (LJ03). Figure 1 shown.

[0142] 1. Detection method of pigments in chili oil

[0143] (1) Pigment extraction

[0144] Weigh about 20.00g of sample into a grinder and grind for 3min. Then weigh 1.00g of the powdered sample accurately into a 50mL centrifuge tube. Add different concentrations of pigment standard solution, add 5mL of acetonitrile-methanol mixed solution (50:50, V:V), vortex for 1min on a vortex mixer, centrifuge at 8000r / min for 5min, take 1mL of supernatant into a 10mL colorimetric tube, add appropriate amount of acetonitrile-methanol (50:50, V:V) solution and make it up to 10mL. After mixing, take out 1mL into a 2mL centrifuge tube, add 25mg C18 purifier, vortex for 1min, centrifuge at 8000r / min for 5min. Take the supernatant and filter it through a 0.22μm PTFE filter (discard the first 2 drops of filtrate) and then measure it on the machine.

[0145] (2) UPLC-MS / MS analysis

[0146] The extract obtained by the above pigment extraction was subjected to UPLC-MS / MS analysis. The chromatographic conditions and mass spectrometry conditions were consistent with those in Example 1. During the sample analysis, a complete molecular ion peak could be extracted from the sample, the signal-to-noise ratio was greater than 3, and the absolute signal stress was greater than 1×10 5 The data processing method is the same as that in Example 1.

[0147] (3) Data processing

[0148] The HRMS data collected from the seasoning raw material extract was imported into the Tracefinder 4.1 data workstation software for data preprocessing and compound identification. By setting the parameters such as the accurate mass deviation (5ppm), retention time deviation (0.2min), and the minimum response value of the compound (10000counts) as the search and filtering conditions, the detection data was searched and identified with the industrial chromatographic mass spectrometry database constructed in the laboratory in Example 1.

[0149] The identification criteria are: 1) When the parent ion (MS) and retention time (RT) match the standard mass library, and the secondary fragment (MS2) matches the standard mass library at a percentage greater than 55% (mzVault score>55), the compound can be confirmed; 2) When the retention time is inconsistent, but the parent ion (MS) and secondary fragment match the constructed standard mass library at a percentage greater than 55%, the compound is classified as a suspected compound. Some compounds cannot obtain secondary fragment information due to low primary peak response, but the parent ion (MS) and retention time match the standard mass library, and are also classified as suspected compounds. Suspected compounds need to be confirmed using standards.

[0150] 2. Detection method of pigments in chili powder and chili powder samples

[0151] (1) Pigment extraction

[0152] Weigh 1.00g chili powder or chili powder sample into a 50mL centrifuge tube, add different concentrations of pigment standard solution, add 5mL acetonitrile-methanol mixed solution (50:50, V:V), vortex on a vortex mixer for 1min, centrifuge at 8000r / min for 5min, take 1mL supernatant into a 10mL colorimetric tube, add appropriate amount of acetonitrile-methanol (50:50, V:V) solution and adjust the volume to 10mL, mix well and take out 1mL into a 2mL centrifuge tube, add 25mg C18 purifier, vortex for 1min, centrifuge at 8000r / min for 5min. Take the supernatant and filter it through a 0.22μm PTFE filter (discard the first 2 drops of filtrate) and then measure it on the machine.

[0153] (2) UPLC-MS / MS analysis

[0154] The extract obtained by the above pigment extraction was subjected to UPLC-MS / MS analysis. The chromatographic conditions and mass spectrometry conditions were consistent with those in Example 1. During the sample analysis, a complete molecular ion peak could be extracted from the sample, the signal-to-noise ratio was greater than 3, and the absolute signal stress was greater than 1×10 5 as a standard for detection limit.

[0155] (3) Data processing

[0156] The HRMS data collected from the seasoning raw material extract was imported into the Tracefinder 4.1 data workstation software for data preprocessing and compound identification. By setting the parameters such as the accurate mass deviation (5ppm), retention time deviation (0.2min), and the minimum response value of the compound (10000counts) as the search and filtering conditions, the detection data was searched and identified with the industrial chromatographic mass spectrometry database constructed in the laboratory in Example 1.

[0157] The identification criteria are: 1) When the parent ion (MS) and retention time (RT) match the standard mass library, and the secondary fragment (MS2) matches the standard mass library at a percentage greater than 55% (mzVault score>55), the compound can be confirmed; 2) When the retention time is inconsistent, but the parent ion (MS) and secondary fragment match the constructed standard mass library at a percentage greater than 55%, the compound is classified as a suspected compound. Some compounds cannot obtain secondary fragment information due to low primary peak response, but the parent ion (MS) and retention time match the standard mass library, and are also classified as suspected compounds. Suspected compounds need to be confirmed using standards.

[0158] The detection limit analysis results of pigments in chili oil, chili powder and chili pieces are listed in Table 3.

[0159] Table 3 Detection limits of 115 pigments in chili oil, chili flakes and chili powder (unit: mg / Kg)

[0160]

[0161]

[0162]

[0163] As can be seen from Table 3, the scheme of the present invention can accurately detect 115 pigments in chili oil, chili powder and chili pieces with a low detection limit.

[0164] Full MS / dd-MS of blank and spiked samples 2 Analytical chromatograms such as Figure 2-Figure 13 As shown in the figure, it can be seen that no industrial pigments were detected in the actual three pepper samples. For these pepper samples, when 0.2 mg / kg of pigment was added to the pepper samples, 92 pigments could be detected; when 0.5 mg / kg of pigment was added, 98 pigments could be detected; when 1.0 mg / kg of pigment was added, 101 pigments could be detected; when 10.0 mg / kg of pigment was added, 110 pigments could be detected; the remaining five pigments, including new red, quinoline yellow, mordant blue 13, disperse yellow 9, and acid red 73, need to be added at a concentration of 50.0 mg / kg before they can be detected.

[0165] Related studies have shown that pigments added to food will only make the food color after the concentration of 1.0 mg / kg. Using this method, more than 100 pigments can be detected at the same time after 1.0 mg / kg of pigment is added, which shows that this method can be used to screen banned colorants in condiments.

[0166] 3. Recovery rate determination

[0167] Using the method for detecting pigments in condiments provided in Example 1, taking 11 pigments as examples, the recovery rates of the 11 pigments in condiments were measured.

[0168] The sample pretreatment method, ultra-high performance liquid chromatography and mass spectrometry conditions, and mass spectrometry analysis parameters are the same as those in Example 1.

[0169] (1) Linear range and correlation coefficient of the method

[0170] The linear range of the 11 pigments was determined by external standard method. The results showed that the 11 pigments had good linear relationship within their respective linear ranges, and the correlation coefficient R 2 The specific results are shown in Table 4.

[0171] Table 4 Linear range and correlation coefficient of the method

[0172] Serial number Compound Linear regression equation <![CDATA[Coefficient of correlation R 2 > Linear range (ng / mL) 1 Sudan 1 y=2943.78063x+1300947 0.99977 50～2500 2 Sudan 4 y=993.98235x-31460.8 0.99956 50～2500 3 Rhodamine B y=62387.6x+6347580 0.99798 50～2500 4 Basic Orange 2 y=10117.5x+69640.2 0.99971 50～2500 5 Alkaline Yellow O y=40797x+4131260 0.99732 50～2500 6 Disperse Red 1 y=4516.134x-139969 0.99939 50～2500 7 Sunset Yellow y=59.97094x+9696.94138 0.99856 50～2500 8 Erythrosin y=485.75291x-61716.7 0.99296 50～2500 9 Acid Orange 7 / Acid Orange Ⅱ y=1038.83074x-70848.1 0.99915 50～2500 10 Acid Red 52 y=40.37729x-959.83857 0.99983 50～2500 11 New Red y=0.08009x-391.94448 0.99413 5000～500000

[0173] (2) Recovery rates of 11 pigments

[0174] Blank samples were selected for recovery and precision tests. The three spiked concentrations of 10 industrial pigments, including Sudan 1, Sudan 4, Rhodamine B, Basic Orange 2, Basic Tender Yellow O, Disperse Red 1, Sunset Yellow, Erythrosine, Acid Orange 7 / Acid Orange II, and Acid Red 52, were 0.1 mg / kg, 0.2 mg / kg, and 1.0 mg / kg, respectively; the spiked concentrations of New Red were 50.0 mg / kg, 100.0 mg / kg, and 500.0 mg / kg, respectively. After preparation according to the pretreatment method, the sample was tested. Figure 14 to Figure 46 As can be seen from Table 5, the average spiked recoveries of the 11 industrial pigments were 70.3-114.4% (n=3).

[0175] Table 5 Recovery of chili powder matrix calculated by solution standard curve

[0176]

[0177]

[0178]

[0179] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for detecting pigments in condiments, characterized in that: The detection method comprises the following steps: S1, pre-treating the sample to obtain a sample solution to be tested; S2, measuring and analyzing the sample solution by ultra-high performance liquid chromatography-mass spectrometry; The ultra-high performance liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an ammonium acetate solution with a concentration of 5 to 15 mmol / L; and the mobile phase B is acetonitrile; The mobile phase was eluted according to the following gradient: 0.00-1.00min, the volume percentage of mobile phase B is 2%-6%, and the balance is mobile phase A; 1.00-4.00min, the volume percentage of mobile phase B is 5%-50%, and the balance is mobile phase A; 4.00-8.00 min, the volume percentage of mobile phase B is 50%-95%, and the balance is mobile phase A; 8.00-12.00 min, the volume percentage of mobile phase B is 92%-98%, and the balance is mobile phase A; 12.00-12.10min, the volume percentage of mobile phase B is 92%-98% to 1%-5%, and the balance is mobile phase A; 12.10-15.00min, the volume percentage of mobile phase B is 2%-6%, and the balance is mobile phase A.

2. The detection method according to claim 1, characterized in that: The pigments include carminic acid, lemon yellow, new red, amaranth, acid blue 74 / indigo carmine, carmine, acid red 18 / carmine / ponceau 4R, quinoline yellow, sunset yellow, orange yellow G, allura red, acid yellow 17, red 2G / acid yellow 1, mordant blue 13, acid red, carmine SX, acid red 26, acid blue 9, brilliant blue, acid red 87 / eosin, sodium fluorescein, reactive brilliant blue KN-R, acid orange 20, amido black 10B / amido black, acid blue 1, acid red 73 / acid bright red GR, acid green 16, acid Green 25, Acid Red 52, Patent Blue V, Congo Red / Direct Red 28, Acid Orange 7 / Acid Orange II, Acid Blue 3, Crocus Orange G, Methyl Orange, Acid Blue 41, Soap Yellow / Acridine Yellow / Acid Golden Yellow, Acid Violet 17, Acid Violet 43, Acid Blue 7, Acid Blue 113, Acid Orange 3, Acid Blue 90, Disperse Yellow 1, Sudan Orange G, Disperse Yellow 49, Para Red, Disperse Yellow 23, Indigo, Rhodamine 110, Erythrosine, Azure B, Basic Blue 17, Methylene Blue, Neutral Red, Thioflavin T / Basic Yellow 1, Basic Red 9, Basic Red 2, Basic Red 14 / Cationic Brilliant Red 5GN, Basic Tender Yellow O, Rhodamine B, Basic Violet 14, Basic Orange 21, Disperse Red 19, Basic Violet 2 / New Fuchsin, Disperse Red 11, Basic Orange 2, Basic Blue 1, Disperse Yellow 9, Disperse Red 17 / Disperse Red GG, Basic Brilliant Green, Vat Red 6B / Basic Violet 7, Disperse Yellow 39, Basic Orange 22, Basic Blue 12 / Nile Blue, Solvent Yellow 1, Curcumin, Basic Red 1 / Rhodamine 6G, Disperse Yellow 3, Disperse Orange 11, Disperse Red 1, Basic Violet 5BN, Disperse Orange 3, Disperse Blue 14, Basic Green 1, Disperse Blue One or more of 124, Disperse Orange 25, Solvent Yellow 3, Basic Blue 11, Methyl Yellow / Solvent Yellow 2, Sudan Red 197, Victoria Blue, Disperse Orange 37 / 76, Crystal Violet Lactone, Solvent Yellow 114, Solvent Red 80, Solvent Red 1 / Sudan Red G, Sudan Yellow, Pigment Red 3, Sudan Yellow 56, Sudan 1, Basic Blue 7 Leucomalachite Green, Crystal Violet, Disperse Orange 1, Disperse Orange 149, Basic Violet, Sudan 2, Sudan Blue II, Sudan 3, Disperse Orange 13, Sudan Red 7B, Sudan Red B / Solvent Red 25, Sudan 4 and Sudan Black B.

3. The detection method according to claim 1, characterized in that: The pretreatment comprises the following steps: adding an extractant to the sample to be tested, mixing, and collecting supernatant A after centrifugation; repeating the extraction once and combining the extracts; adding a C18 adsorbent to the extract, collecting supernatant B after centrifugation, and filtering the supernatant B to obtain a sample solution to be tested; Preferably, the extractant is a mixture of acetonitrile and methanol, and the volume percentage of acetonitrile in the extractant is 20%-80%.

4. The detection method according to claim 1, characterized in that: The ultra-high performance liquid chromatography conditions include: the chromatographic column is Waters Acquity UPLC BEH Shield RP18, 2.1 mm×100 mm, and the particle size is about 1.7 μm; And / or, the ultra-high performance liquid chromatography conditions include: a flow rate of 0.8 mL / min-1.2 mL / min; And / or, the ultra-high performance liquid chromatography conditions include: an injection volume of 3-7 μL and a column temperature of 30° C.-40° C.

5. The detection method according to claim 1, characterized in that: The mass spectrometry detection adopts a primary parent ion full scan plus a data-dependent secondary daughter ion scan mode to collect data, and a primary full scan is set to cover the mass number range of 150 to 900 m / z of the target compound.

6. The detection method according to claim 1, characterized in that: The mass spectrometry conditions include: heated electrospray ion source, positive and negative ion modes; spray voltage: 3.8-4.2 kV in positive ion mode, 2.8-3.5 kV in negative ion mode; capillary temperature 300-340° C.; sheath gas: 32-37 arb; auxiliary gas: 8-12 arb; scanning mode: Full MS / dd-MS 2 ; Level 1 full scan resolution: 68000-77000FWHM, AGC maximum capacity: 2×10 6 -4×10 6 , AGC maximum injection time: 80-120ms; data dependent secondary product ion scanning resolution: 17000-18000FWHM, trigger threshold maximum capacity: 0.8×10 5 -1.2×10 5 , Maximum injection time: 40-60ms; And / or, the mass spectrometry qualitative and quantitative ions of the pigment and the retention time of each component are shown in the following table:

7. The detection method according to claim 1, characterized in that: The method also includes the step of processing the data of the sample to be tested obtained by the determination and analysis, wherein the data processing includes importing the data of the sample to be tested into Tracefinder4.1 data workstation software for processing, setting the accurate mass number deviation, retention time deviation, and compound minimum response value parameters as search and filtering conditions, comparing the data of the sample to be tested with the standard data, and determining the target pigment.

8. The detection method according to claim 7, characterized in that: The accurate mass deviation is 4-6ppm; And / or, the retention time deviation is 0.2 min; And / or, the minimum response value of the compound is 8000-12000 counts.

9. Use of the detection method according to any one of claims 1 to 8 in detecting pigments in condiments.

10. The use according to claim 9, characterized in that: The condiment comprises one of chili oil, chili powder and chili flakes.

Images