A rapid method for detecting carotenoids in food

Abstract

The application discloses a kind of rapid detection methods of 23 kinds of carotenoids in food, using chloroform-methanol-water volume ratio 1:2:0.8 mixed solvent extraction, then add water and chloroform, without adding any salt compound, mixed solvent in chloroform-methanol-water volume ratio 2:2:1.8, automatically layered into water phase layer-organic phase layer, water phase HLB column, sequentially eluted with organic phase and chloroform, finally detected by high performance liquid chromatography-mass spectrometry, quantitative analysis.The application simplifies the extraction step, the quantitative detection limit is low, applied to the rapid detection and analysis of 23 kinds of carotenoids commonly seen in food, the recovery rate is as high as 80.1-98.7%, with high sensitivity and reliability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detecting natural pigment ingredients in food, in particular to a rapid detection method of carotenoids in food. BACKGROUND

[0002] Carotenoids are an important part of natural pigments, composed of a series of conjugated isoprene units, and are extremely susceptible to oxidation and other chemical modifications. The most common modification groups are hydroxyl, methoxyl, carbonyl, carboxyl, etc. So far, more than 750 kinds of carotenoids have been found and reported. According to the chemical structure, they can be divided into two categories. The first category is free carotenoids, including α-carotene and β-carotene without oxygen, and lutein, astaxanthin and other oxygen-containing carotenoids. The second category is esterified carotenoids, which can be divided into carotenoid monoesters and carotenoid diesters according to the number of fatty acid chain bonds. These photosensitive carotenoids, in addition to being used as natural colorants to improve the sensory quality of food, have gradually been reported to have value in maintaining human health, including improving human immunity and improving vision. The substitution of natural pigments for synthetic pigments will be an inevitable trend for the healthy development of the food industry. Due to regulatory needs, it is necessary to detect and analyze the carotenoids added to food. However, the extremely hydrophobic and highly complex nature of carotenoids in food poses a huge challenge to the rapid extraction, purification and analysis of these compounds.

[0003] The traditional pretreatment technology for detecting carotenoids is direct solvent extraction. This technology mainly uses a single organic reagent as the extraction solvent, such as acetone, methanol or ethyl acetate, etc. However, this technology has the disadvantage of selectivity, especially for small molecule free carotenoids and low polarity carotenoid esters, which cannot be simultaneously compatible. Moreover, there is no purification treatment for the matrix properties of the extraction solution, and it is only suitable for analysis methods for detecting a certain type of carotenoid in a fixed food matrix, with low sample processing throughput.

[0004] In the early stage, the research group established a high-throughput detection method for carotenoids in aquatic organisms according to the matrix properties of aquatic organisms, realized the detection and analysis of carotenoids in aquatic products, and adopted step-by-step extraction, grading purification experimental ideas, combined with solid phase extraction and matrix dispersion two purification technologies. However, this pretreatment method is suitable for aquatic organisms with simple and clean matrix, but not suitable for food matrix with high protein and high fat. When the method is used for food matrix, especially high-fat food matrix, it is found that due to the rich additive components in the food matrix, the purification effect of free carotenoids is poor due to the lack of elution process during HLB purification. In addition, the neutral aluminum oxide powder in the method is used to adsorb and remove the oil components in the extracted esterified carotenoids, which has poor applicability for food matrix with high fat content, resulting in low overall recovery rate. In addition, the step-by-step extraction and grading purification lead to a complex and time-consuming experimental process. Therefore, a more comprehensive and efficient pretreatment method is needed to rapidly detect and analyze the types of carotenoids in food. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the existing carotenoid detection method in food, such as poor selectivity, low detection efficiency, low recovery rate, etc., and to provide a rapid detection method for carotenoids in complex food matrix.

[0006] Considering that carotenoids cover a wide range of types and have large polarity differences between different categories, and that food has high protein and high fat matrix properties, which further produces strong matrix effect, resulting in serious deviation of the detection results. Therefore, the improved Bligh-Dyer method is used to extract carotenoids in food matrix.

[0007] Bligh-Dyer method is a traditional method for lipid analysis, which uses chloroform-methanol-water as the extraction solvent. The extraction solvent has a wide polarity range and can actively separate into water phase and organic phase under a certain proportion (2:2:1.8, v / v / v). Esterified carotenoids are similar in structure to lipids, both containing one or two fatty acids, and carotenoids are also called lipid pigments. Therefore, according to the pKa value of the extraction solvent and the polarity range of the target compound, chloroform-methanol-water is used as the extraction solvent, free carotenoids are mainly distributed in the water phase, and part of the low-level free carotenoids and esterified carotenoids are mainly distributed in the organic phase. By synchronously collecting water phase and organic phase, free carotenoids and esterified carotenoids can be reserved at the same time, and the purification step is further optimized, so that the rapid detection and analysis of carotenoids in food can be realized.

[0008] The technical scheme adopted by the present application is:

[0009] A method for detecting carotenoids in food, the carotenoids including 23 kinds of carotenoids, one or more of lycopene, α-carotene, ε-carotene, β-carotene, γ-carotene, δ-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein, zeaxanthin, epoxide zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatomeaxanthin, canthaxanthin, astaxanthin, astaxanthin monolaurate, astaxanthin monolinoleate, astaxanthin distearate, astaxanthin dipalmitate, the method comprising the following steps:

[0010] A. Pretreatment:

[0011] (A1) Take 2 g of food sample to be detected, repeatedly extract 3 times with 7.5 mL of chloroform-methanol-water solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, centrifuge, and combine the upper extract;

[0012] In the chloroform-methanol-water solution, the volume ratio of chloroform, methanol and water is 1:2:0.8;

[0013] Generally, the extraction time is 5-10 min, and the ultrasonic extraction time is 5-10 min;

[0014] The centrifugation is generally carried out at a speed of 12000 r / min for 5-10 min;

[0015] 2,6-di-tert-butyl-p-cresol (BHT) is an antioxidant that can be used to prevent oxidation of carotenoids;

[0016] (A2) Take the upper extract obtained in step (A1), and sequentially add 6.0 mL of water and 6.0 mL of chloroform to it, and after centrifugation, automatically separate the layers to obtain the upper aqueous phase and the lower organic phase;

[0017] The centrifugation is generally carried out at a speed of 12000 r / min for 5-10 min;

[0018] (A3) Take the aqueous phase obtained in step (A2), and load it onto an HLB column, and then elute it with 6.0 mL of methanol-water solution;

[0019] In the methanol-water solution, the volume ratio of methanol to water is 3:2;

[0020] (A4) Elute the organic phase of step (A2) and 6.0 mL of chloroform with 6.0 mL of chloroform as the eluent of the HLB purification column, and collect the eluate;

[0021] (A5) The eluate obtained from step (A4) is concentrated and dried, then dissolved with methanol containing 0.1 wt% 2,6-di-tert-butyl-p-cresol to 2.0 ml, and filtered through a membrane to obtain a test sample solution;

[0022] The concentration and drying is generally performed by nitrogen blowing to a constant volume;

[0023] B. High performance liquid chromatography-mass spectrometry detection

[0024] The test sample solution obtained from step (A5) is detected by high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometer to obtain a high performance liquid chromatography-mass spectrum of the test sample solution, wherein the instrument conditions are as follows:

[0025] High performance liquid chromatography conditions:

[0026] Chromatographic column: Syncronis C18 column (2.1 mm x 150 mm, particle size 1.7 μm);

[0027] Mobile phase A: a solution containing 10 mM ammonium formate in acetonitrile, water in a volume ratio of 9:1;

[0028] Mobile phase B: a solution of acetonitrile, isopropanol in a volume ratio of 7:3, flow rate: 0.3 mL / min, injection volume: 5.0 μL, split ratio 1:4, gradient elution, and the elution program is preferably as shown in Table 1:

[0029] Table 1: Gradient elution program

[0030]

[0031] Mass spectrometry parameters: mass spectrometry is performed in full scan mode under positive ion conversion mode, mass range: m / z 100-1200, resolution 70000, automatic gain control target value 5*e 5 ; positive ion mode 3800 V, ion transmission tube temperature 300°C, desolvation gas nitrogen flow rate: 35 L / h, auxiliary gas nitrogen flow rate: 15 L / h, gasification chamber temperature 350°C; the instrument is calibrated in positive ion mode before sample operation; secondary stage is in automatic trigger mode, resolution 35000, automatic gain control target value 2*e 5 , normalized collision energy in turn 30%, 40% and 60%, and retention time according to target retention time ± 1.0 min;

[0032] C Quantitative analysis

[0033] The carotenoid content in the test sample solution is detected by external standard method, and the content of 23 kinds of carotenoids in the food sample to be detected is converted and obtained.

[0034] Further, the step C quantitative analysis comprises the following steps:

[0035] (C1) Preparation of standard curve of standard sample:

[0036] Take the standard samples of lycopene, a-carotene, e-carotene, b-carotene, g-carotene, d-carotene, a-cryptoxanthin, b-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatomeaxanthin, canthaxanthin, astaxanthin, astaxanthin monolaurate, astaxanthin monolinoleate, astaxanthin distearate, and astaxanthin dipalmitate, and prepare mixed standard working solutions with different concentrations. Perform high performance liquid chromatography-mass spectrometry detection under the same conditions as step B to obtain the high performance liquid chromatography-mass spectrometry chromatogram of the standard working solution. Draw the standard curve for each standard sample according to its concentration and peak area to obtain the standard curve of the above-mentioned standard samples.

[0037] (C2) According to the high performance liquid chromatography-mass spectrometry chromatogram of the test sample solution obtained in step B and the standard curve of the corresponding standard sample, calculate the concentration of each component to be measured in the test sample solution. Calculate the content of carotenoids in the food sample to be tested according to formula (1):

[0038] X = C * V / m * 1000

[0039] In the formula:

[0040] X - the content of the measured substance in the sample, in mg / kg;

[0041] C - the concentration of the measured substance in the test sample solution, calculated according to the standard curve, in pg / L

[0042] V - the constant volume, in mL;

[0043] m - the mass of the food sample to be tested, in g.

[0044] Further, the food sample to be tested in step (A1) can be various solid and liquid foods, generally processed foods that can be directly consumed, containing various food additives, including starch processed foods, oil processed foods, candy and chocolate foods, wine, beverages, seasonings, etc. The present application selects ham sausages, jams and cookies as representative food matrices for the validation of the method. These foods cover different matrix properties, including solids, liquids and high-fat foods, and are rich in added pigments, which are commonly used food validation matrices for pretreatment techniques.

[0045] Further, the concentrations of the mixed standard working solutions in step (C1) are 0 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL and 200 ng / mL, respectively.

[0046] In the extraction step, according to the Bligh-Dyer method, a chloroform-methanol-water (1:2:0.8, v / v / v, 0.1% BHT) mixture is selected as the extraction solvent, the polarity of the three solvents covers a wide range, which fully meets the extraction requirements of carotenoids. Among them, the methanol and water solution can well ensure the extraction efficiency of free carotenoids, and the chloroform-extracted part mainly includes low-polarity esterified carotenoids and part of free carotenoids. In addition, the presence of the water solution can well prevent the occurrence of emulsification, making the layering interface clearer, and BHT can well avoid the oxidation reaction of carotenoids. The extracted extract is a mixture of organic and aqueous phases, after three extractions, water and chloroform are added, without adding any salt compounds, the mixed solvent is automatically layered into the water phase layer-organic phase layer at a fixed ratio, i.e. the ratio of chloroform-methanol-water (2:2:1.8, v / v / v) (Bligh and Dyer, 1959). This can avoid the pollution of the instrument caused by the salt solution layering. The medium-polarity free carotenoids are mainly distributed in the aqueous phase, and the low-polarity free and esterified carotenoids are mainly distributed in the organic phase.

[0047] The principle of the purification step of the present application is that, first, the upper aqueous phase obtained in step (A2) is loaded onto the HLB column, the free carotenoids are retained on the HLB column, and after methanol-water elution, the interference of hydrophilic compounds can be effectively removed, then during elution, the eluent is divided into two parts, which are sequentially eluted as the eluent of the HLB purification column, the first part of the eluent is the lower organic phase in step (A2) as the auxiliary extractant, by adjusting the ratio of chloroform-methanol-water (2:2:1.8, v / v / v), chloroform and part of methanol are separated from the extract, and the obtained organic phase is located in the lower layer, which is used as an auxiliary extractant, which can well ensure the recovery rate of non-polar carotenoid esters. After loading the aqueous phase, the organic phase in the lower layer is used as the eluent, which makes the polar free carotenoids and non-polar esterified carotenoids eluted from the HLB purification column in turn. The second part of the eluent is chloroform solvent, by optimizing the elution volume of chloroform, the interference of oil compounds can be eliminated as much as possible while ensuring the complete elution of strong hydrophobic carotenoid diesters. Finally, high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) is used for data analysis, which can realize the rapid analysis and detection of carotenoids in food.

[0048] The difference between the high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS) technology adopted by the present application and the conventional triple quadrupole mass spectrometry is that the HPLC-Q-Orbitrap-HRMS does not need the characteristic ion fragments of the known to-be-detected compound, but directly uses the high-precision mass number (the resolution is greater than or equal to 70000, m / z 200) to perform the directional and non-directional full-scan detection on the to-be-detected sample, and when the target compound needs to be increased, the existing full-scan data can be reanalyzed without repeating the sampling, which is especially suitable for the simultaneous qualitative and quantitative analysis of multiple components.

[0049] The present application has the following beneficial effects:

[0050] 1. The determination limit of the carotenoid in the present application is 0.02-0.05 mg / kg, and the standard curve is good in the linear range of 10 ng / mL-200 ng / mL (the regression coefficient R2 is greater than 0.99).

[0051] 2. The pretreatment uses chloroform-methanol-water as the extraction solvent, synchronously collects the water phase and the organic phase layer, and purifies by using a hydrophilic-lipophilic balance column, so that the interference of hydrophilic compounds and neutral glycerin triesters is effectively eliminated, the extraction and purification requirements of the two categories of carotenoids with very different physicochemical properties are met, the extraction steps are simplified compared with the prior art, the detection efficiency is improved, and the consumption of expensive time and reagent costs is reduced.

[0052] 3. The method is applied to the rapid detection and analysis of 23 common carotenoids in food, and the recovery rate is as high as 80.1-98.7%, has high sensitivity and reliability, and the recovery rate of the detected carotenoids is better than that of the existing detection methods. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 Figure of the effect of chloroform elution volume on the elution of four esterified carotenoids and glyceryl tri-palmitate. DETAILED DESCRIPTION

[0054] The technical solutions of the present application are further described below, but the protection scope of the present application is not limited thereto.

[0055] Instruments and materials

[0056] Q-Exactive quadrupole electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific), equipped with H-ESI II source. The liquid chromatography system is UltiMate 3000 high-pressure liquid chromatography with automatic sampler. The chromatographic column is Syncronis C18 column (2.1 mm x 150 mm, particle size 1.7 μm). Milli-Q high-purity water generator (Millipore, USA). Centrifuge (SIGMA, Germany). Vortex (Heldolph, Germany). Filter membrane (DIKMA, PTFE 0.22 μm). Waters Oasis HLB small column (6 cc / 200 mg, activated with methanol before loading, water balance).

[0057] Methanol, acetonitrile and ammonium acetate (chromatographically pure, Sigma-Aldrich, USA). Other reagents (chromatographically pure, Merck, Germany). Experimental water is Milli-Q ultra-pure water (18.2 ΩM). Carotenoid standards are purchased from Sigma and Dr. Ehrenstorfer, purity ≥95%. Including, free carotenoids: lycopene, α-carotene, ε-carotene, β-carotene, γ-carotene, δ-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein, zeaxanthin, epoxide zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatoxanthin, canthaxanthin, astaxanthin; esterified carotenoids: astaxanthin monolinoieate, astaxanthin monolinoieate, astaxanthin distearate and astaxanthin dipalmitate.

[0058] Standard stock solution: weigh an appropriate amount of standard, except for astaxanthin dipalmitate which is dissolved in dichloromethane, and methanol is used for constant volume, and the others are dissolved in methanol to prepare a standard stock solution of 0.1 mg / mL, stored at -20°C in the dark.

[0059] Standard working solution: 100 μL of each component of the standard stock solution is taken into a 10 mL volumetric flask, respectively, and methanol is used for constant volume to the mark, with a concentration of 1.0 μg / mL.

[0060] Example 1

[0061] A. Pretreatment:

[0062] (A1) Weigh 2 g of food to be tested, add 7.5 mL of chloroform-methanol-water solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, ultrasonic extraction for 5 min, and then centrifuge at 12000 r / min for 5-10 min. After completion, the upper extract and residue are obtained, and the extraction is repeated twice, and the upper extract is combined;

[0063] chloroform-methanol-water solution, volume ratio of chloroform, methanol, water is 1:2:0.8;

[0064] (A2) taking the upper extraction liquid obtained from step (A1), and sequentially adding 6.0 mL of water and 6.0 mL of chloroform thereto, centrifuging at a speed of 12000 r / min for 5-10 min, and after completion, automatically separating into upper and lower layers to obtain an upper aqueous phase and a lower organic phase,

[0065] (A3) taking the aqueous phase obtained from step (A2), and loading it into an HLB column, and then eluting it with 6.0 mL of a methanol-water solution;

[0066] methanol-water solution, volume ratio of methanol to water is 3:2;

[0067] (A4) sequentially eluting the organic phase of step (A2) and 6.0 mL of chloroform as eluent of an HLB purification column, and collecting the eluate;

[0068] (A5) taking the eluate obtained from step (A4), and concentrating it by nitrogen blowing until the volume does not change, then adding methanol containing 0.1wt% 2,6-di-tert-butyl-p-cresol to 2.0 mL, and passing through a filter membrane with a pore size of 0.22μm to obtain a test sample solution;

[0069] B. detecting the test sample solution obtained from step (A5) by high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometer to obtain the high performance liquid chromatography-mass spectrum of the test sample solution, wherein the instrument conditions are as follows:

[0070] High performance liquid chromatography parameters:

[0071] Chromatographic column: Syncronis C18 column (2.1mm x 150mm, particle size 1.7μm);

[0072] Mobile phase A: solution containing 10mM ammonium formate, volume ratio of acetonitrile to water is 9:1;

[0073] Mobile phase B: solution of acetonitrile and isopropanol, volume ratio is 7:3, flow rate: 0.3mL / min, injection amount: 5.0μL, split ratio 1:4 injection, gradient elution program is shown in Table 1 below:

[0074] Table 1 Gradient elution program in positive ion mode

[0075]

[0076] Mass spectrometry parameters: mass spectrometry is determined in positive ion conversion mode, mass range: m / z 100-1200, resolution 70000, automatic gain control target value 5*e 5; positive ion mode 3800V, ion transfer tube temperature 300℃, desolvation gas nitrogen flow rate: 35 L / h, auxiliary gas nitrogen flow rate: 15 L / h, vaporization chamber temperature 350℃; the instrument was calibrated in positive ion mode before sample running; the secondary stage was in automatic trigger mode, resolution 35000, automatic gain control target value 2*e 5 , the normalized collision energy was 30%, 40% and 60% in turn, and the retention time was according to the target retention time ± 1.0 min;

[0077] C quantitative analysis

[0078] (C1) Draw the standard curve of the standard sample:

[0079] Take lycopene, α-carotene, ε-carotene, β-carotene, γ-carotene, δ-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatomeaxanthin, canthaxanthin, astaxanthin, astaxanthin monolaurate, astaxanthin monomyristate, astaxanthin distearate, astaxanthin dipalmitate, and prepare mixed standard working solutions of different concentrations. Test them according to the same conditions of step B to obtain the high performance liquid chromatography-mass spectrometry (HPLC-MS) chromatogram of the standard working solution. Draw the standard curve of each standard sample according to its concentration and peak area. The standard curves of the above standard samples are obtained. The results are shown in Table 3.

[0080] (C2) Calculate the concentration of each component to be tested in the test sample solution according to the HPLC-MS chromatogram of the test sample solution obtained in step B and the standard curve of the corresponding standard sample. Calculate the content of carotenoids in the food to be tested according to formula (1):

[0081] X = C*V / m*1000

[0082] In the formula:

[0083] X - the content of the test substance in the sample, in mg / kg;

[0084] C - the concentration of the test substance in the test sample solution, calculated according to the standard curve, in μg / L

[0085] V - the constant volume, in mL;

[0086] m - the mass of the sample, in g.

[0087] Comparative Example 1. Pretreatment according to the method disclosed in CN111307976A.

[0088] (A1) Take 1 g of the food to be tested and add 10 mL of a methanol solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, ultrasonic extraction for 20 min, and centrifuge at 12000 r / min at 4-10 degrees Celsius for 5-10 min. After completion, the organic phase and residue are obtained;

[0089] (A2) Take the organic phase obtained in step (A1) and add at least 4 times the volume of water to obtain a sample solution. Then purify the sample solution through an HLB column to obtain a purified sample solution. Then concentrate the purified sample solution to <2.0 mL by nitrogen blowing, and then dilute to 2.0 mL with a methanol solution containing 0.1 wt% HF to obtain a diluted and purified sample solution, wherein:

[0090] The HLB column purification is eluted with a methanol solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol as the eluent;

[0091] (A3) Add the remaining residue of step (A1) to 10 mL of a methanol-dichloromethane solution containing 0.1 wt% 2,6-di-tert-butyl-p-cresol, shake for 5 min, ultrasonic extraction for 20 min, and centrifuge at 12000 r / min at 4-10 degrees Celsius for 5-10 min. After completion, the organic phase and residue are obtained, wherein:

[0092] The volume ratio of methanol to dichloromethane in the methanol-dichloromethane solution is 1:1;

[0093] (A4) Take the organic phase obtained in step (A3), concentrate it to a constant volume by nitrogen blowing, then dilute it to 2.0 mL with a methanol solution containing 0.1 wt% HF, then add 1 g of neutral alumina powder and shake well, and centrifuge at 12000 r / min at 4-10 degrees Celsius for 5-10 min. After completion, the supernatant and precipitate are obtained;

[0094] (A5) Combine the diluted and purified sample solution obtained in step (A2) and the supernatant obtained in step (A4), and then pass through a filter membrane with a pore size of 0.22 μm to obtain a test sample solution;

[0095] Steps B and C are carried out under the same conditions as in Example 1.

[0096] Using the pretreatment methods of Example 1 and Comparative Example 1, the types of carotenoids in common foods (ham sausage, jam, and cookie) were detected and analyzed. The addition amount of the standard in the food was 0.02, 0.05, and 0.1 mg / kg, respectively. Then the standard curve was quantified and the recovery rate was calculated. The results are shown in Table 2. The results show that:

[0097] Example 1: The chloroform-methanol-water was used as the extraction solution, the water phase and organic phase were collected synchronously, and the hydrophilic-lipophilic balance column was used for purification. The method can meet the extraction and purification requirements of 23 carotenoids with large differences in physicochemical properties, while effectively eliminating the interference of hydrophilic compounds and neutral triglycerides. The standard curve of carotenoids was linear in the range of 10 ng / mL-200 ng / mL, the limit of quantification was 0.02-0.05 mg / kg, the recovery rate was as high as 80.1-98.7%, and the method had high sensitivity and reliability.

[0098] Comparative Example 1: This method was established by the research group for the matrix properties of aquatic organisms. The method realized high-throughput screening of carotenoids in aquatic products. The pretreatment used step-by-step extraction and fractional purification, combined with solid-phase extraction and matrix dispersion purification technology. However, the method cannot well accommodate the extraction and purification of carotenoids in food matrices with more complex components. The limit of quantification was 0.05-0.1 mg / kg, and the recovery rate was 60.9-93.1%, which was poor. The quantitative accuracy of carotenoids in food matrices was poor. The entire experimental process was time-consuming compared to Example 1.

[0099] Table 2: Recovery rate data of 23 carotenoids in ham sausages, jams, and cookies under two treatment methods (n=6).

[0100]

[0101]

[0102]

[0103]

[0104] Table 3: Linear equation and regression coefficient of 23 carotenoid standards

[0105]

[0106]

[0107]

[0108] Example 2: Selection of elution conditions

[0109] A. Pretreatment:

[0110] (A1) Weigh 2 g of blank ham sausage matrix sample, add 7.5 mL of 0.1 wt% BHT methanol solution, 7.5 mL of 0.1 wt% BHT chloroform solution, and 7.5 mL of 0.1 wt% BHT methanol solution, and mix well.

[0111] 2,6-di-tert-butyl-p-cresol in chloroform-methanol-water solution, oscillation for 5 min, ultrasonic extraction for 5 min, and centrifugation at 12000 r / min for 5-10 min, to obtain the supernatant and residue, and the supernatant was combined and extracted twice more;

[0112] The volume ratio of chloroform, methanol and water in the chloroform-methanol-water solution was 1:2:0.8;

[0113] (A2) The supernatant obtained in step (A1) was taken and 6.0 mL of water and 6.0 mL of chloroform were sequentially added thereto, and centrifugation was performed at 12000 r / min for 5-10 min, and the supernatant was obtained after automatic layering, to obtain the supernatant and the lower organic phase,

[0114] (A3) The supernatant obtained in step (A2) was taken, 23 kinds of carotenoids (C = 1.0 μg / mL) were added, and the supernatant was loaded onto an HLB column, and then eluted with 6.0 mL of a methanol-water solution;

[0115] The volume ratio of methanol and water in the methanol eluent was 50%, 60%, 70%, 80% and 100%, respectively, and the influence of the methanol ratio on the recovery rate of the above test substance was investigated.

[0116] (A4) After the eluent was blown dry and made up to volume, high performance liquid chromatography-mass spectrometry detection was performed in step B, the concentration of the detected components was converted to obtain the elution loss rate, and the recovery rate was 100%-loss rate. The results are shown in Table 4 below.

[0117] The final test results show that free carotenoids, especially free carotenoids with special polarity, i.e., fucoxanthin, lutein and astaxanthin, are greatly affected by the methanol ratio. When the methanol ratio is ≥70%, the recovery rate begins to decrease, and the recovery rate value decreases from 94.9% to 12.3%. Some low-polarity free carotenoids, i.e., α-carotene and β-carotene, maintain a recovery rate value of 100% when the methanol ratio is 50%-80%, and the recovery rate begins to decrease when the methanol ratio is 100%. Esterified carotenoids are not affected by the methanol ratio, and the recovery rate tends to be stable at 100% when the methanol ratio is 100%. In summary, the elution condition is determined to be 60% methanol-water.

[0118] Table 4 Influence of methanol ratio in eluent on recovery rate of 23 kinds of carotenoids (n = 3)

[0119]

[0120]

[0121] Example 3 Selection of elution conditions

[0122] The eluent is mainly from two parts, the first part is the organic phase as an auxiliary extractant, which is located in the lower layer of the centrifuge tube after layering, and it serves as an auxiliary extractant, which well ensures the recovery rate of non-polar carotenoid esters. After the water phase layer is loaded, the organic phase located in the lower layer as an eluent gradually penetrates, so that the free carotenoids are first eluted from the HLB purification column. The second part of the eluent is chloroform solvent, which is used to ensure the elution of carotenoid mono- and di-esters, but the inevitable oil compounds will produce co-elution phenomenon, and then produce strong matrix inhibition effect.

[0123] In this example, the influence of the volume of chloroform as an elution solvent on the results is investigated.

[0124] According to the procedure (A1) of Example 1, a blank ham sausage matrix sample is extracted, and 4 esterified carotenoids, i.e. astaxanthin linoleate ester, astaxanthin docosahexaenoate ester, astaxanthin distearate ester, astaxanthin dipalmitate ester, and a common oil compound in food-palmitic acid triglyceride ([M+Na]+, m / z 805.7) (C = 1.0 μg / mL) are added to the obtained upper extraction liquid, respectively,

[0125] (A2), (A3) are the same as Example 1.

[0126] (A4) The organic phase and chloroform of step (A2) are sequentially used as eluents of the HLB purification column for elution, and the eluent is collected;

[0127] The volume of chloroform as an elution solvent is 0, 1, 2, 3, 4, 5, 6, 7, 8 mL, respectively, the eluent is diluted according to the procedure (A5) of Example 1, then the detection is carried out according to procedure B, and the corresponding compound extraction ion chromatogram peak area curves obtained by different elution volumes are shown in Figure 1 As can be seen from Figure 1 when the volume of chloroform is 5.0 mL, astaxanthin linoleate ester and astaxanthin docosahexaenoate ester are first eluted, astaxanthin distearate ester and astaxanthin dipalmitate ester are eluted when the volume of chloroform is 7.0 mL, and at the same time, palmitic acid triglyceride starts to be eluted when the volume of chloroform is 6.0 mL. From the perspective of reducing oil pollution, the elution volume of chloroform as an eluent is determined to be 6.0 mL.

[0128] Optimization of liquid chromatography-mass spectrometry conditions in Example 4

[0129] The addition of buffer salt in the mobile phase can promote ionization and maintain the acid-base stability of the mobile phase well. Therefore, for the detection of carotenoids in positive ion mode, ammonium formate is added, and the effect of ammonium formate buffer on the ion strength is further investigated. The concentration of ammonium formate solution is changed from 2 mmol / L to 20 mmol / L. The results show that when the concentration of ammonium acetate is 10 mmol / L, the optimal chromatographic peak shape, separation effect and mass spectrum signal response are obtained. The whole process selects gradient elution mode, and the effective separation of the target mixture is realized by optimizing the gradient elution conditions of the mobile phase. In addition, the Syncronis C18 column (2.1 mm x 150 mm, particle size 1.7 μm) chromatographic column and the Hypersile Gold C18 chromatographic column are compared, and it is found that the former is superior in isomer separation, and the effective separation of five isomers, i.e. lycopene, alpha-carotene, epsilon-carotene, beta-carotene and delta-carotene, is realized. Therefore, the chromatographic column is finally selected as the Syncronis C18 column (2.1 mm x 150 mm, particle size 1.7 μm).

[0130] In summary, the method of the present application has strong compatibility for different food matrices and compounds with different physicochemical properties. The method can detect more types of carotenoids and obtain more accurate content values. The reliability of the detection method is fully verified by comparing with existing carotenoid detection methods.

[0131] The above describes the embodiments of the present application in detail. However, the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application.

Claims

1. A method for rapid detection of carotenoids in food products, characterized in that The carotenoids include the following 23 kinds: lycopene, a-carotene, ε-carotene, β-carotene, γ-carotene, δ-carotene, a-cryptoxanthin, β-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatomeeaxanthin, canthaxanthin, astaxanthin, astaxanthin monolaurate, astaxanthin monolinoleate, astaxanthin distearate, astaxanthin dipalmitate, the method comprising the following steps: A. Pretreatment: (A1) Take 2 g of the food sample to be detected, and repeatedly extract 3 times with 7.5 mL of a chloroform-methanol-water solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol, centrifuge, and combine the upper extraction liquid; In the chloroform-methanol-water solution, the volume ratio of chloroform, methanol and water is 1:2:0.8; (A2) Take the upper extraction liquid obtained in step (A1), and sequentially add 6.0 mL of water and 6.0 mL of chloroform thereto, and after centrifugation, automatically separate the layers to obtain the upper aqueous phase and the lower organic phase; (A3) Take the aqueous phase obtained in step (A2), and load it onto an HLB column, and then perform elution with 6.0 ml of a methanol-water solution; In the methanol-water solution, the volume ratio of methanol to water is 3:2; (A4) Elute the organic phase of step (A2) and 6.0 mL of chloroform sequentially as eluent of the HLB purification column, and collect the eluent flowing out; (A5) Take the eluent obtained in step (A4), concentrate and dry, then use a methanol solution containing 0.1wt% 2,6-di-tert-butyl-p-cresol to make up to 2.0 ml, and filter membrane filter as the test sample solution; B. High performance liquid chromatography-mass spectrometry detection The test sample solution obtained in step (A5) is detected by high performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometer to obtain the high performance liquid chromatography-mass spectrum of the test sample solution; C Quantitative analysis The external standard method is used for quantitative analysis, and the content of carotenoids in the test sample solution is detected, and the content of carotenoids in the food sample to be detected is converted.

2. The method of claim 1, wherein In step (B), The high performance liquid chromatography conditions are as follows: The chromatographic column is Syncronis C18 column; The mobile phase A is a solution containing 10mM ammonium formate in acetonitrile and water with a volume ratio of 9:1; The mobile phase B is a solution of acetonitrile and isopropanol with a volume ratio of 7:3, the flow rate is 0.3 mL / min, the injection amount is 5.0 μL, the split ratio is 1:4, and the gradient elution is used.

3. The method of claim 2, wherein The program of the gradient elution is as follows: 。 4. The method of claim 1, wherein The mass spectrum parameters in step B are as follows: the mass spectrum is determined by full scan in positive ion conversion mode, the mass range is m / z 100-1200, the resolution is 70000, and the automatic gain control target value is 5*e 5 ; the positive ion mode is 3800 V, the ion transmission tube temperature is 300 DEG C, the desolvation gas nitrogen flow rate is 35 L / h, the auxiliary gas nitrogen flow rate is 15 L / h, the gasification chamber temperature is 350 DEG C; the instrument is corrected by positive ion before sample operation; the secondary stage is in automatic trigger mode, the resolution is 35000, the automatic gain control target value is 2*e 5 , the normalized collision energy is 30%, 40% and 60% in sequence, and the retention time is according to the target retention time ±1.0 min.

5. The method of claim 1, wherein The step C quantitative analysis includes the following steps: (C1) Prepare a standard curve of the standard product: The standard samples of lycopene, α-carotene, ε-carotene, β-carotene, γ-carotene, δ-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, violaxanthin, neoxanthin, fucoxanthol, fucoxanthin, diadinoxanthin, diatomeaxanthin, canthaxanthin, astaxanthin, astaxanthin monoeicosadienoate, astaxanthin monoeicosapentaenoate, astaxanthin distearate, astaxanthin dipalmitate are prepared into mixed standard working solutions with different concentrations, and the high performance liquid chromatography-mass spectrometry detection is carried out according to the same conditions in step B to obtain the high performance liquid chromatography-mass spectrometry diagram of the standard working solution; the standard curve of each standard sample is drawn according to the concentration and peak area, and the standard curve of the above standard sample is obtained. (C2) According to the high performance liquid chromatography-mass spectrometry diagram of the test sample solution obtained in step B, the standard curve of the corresponding standard sample, the concentration of each component to be measured in the test sample solution is calculated, and the content of carotenoid in the food sample to be detected is calculated according to formula (1): X=C*V / m*1000 In the formula: X-the content of the measured substance in the sample, unit: mg / kg; C-the concentration of the measured substance in the test sample solution, which is calculated according to the standard curve, unit: μg / L V-the constant volume, unit: mL; m-the mass of the food sample to be detected, unit: g.

6. The method of claim 1, wherein In the step (A1), the extraction process is oscillation for 5-10 min and ultrasonic extraction for 5-10 min.

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