CN116297920A - Rapid detection method for carotenoid in food - Google Patents

Abstract

The invention discloses a rapid detection method of 23 carotenoid in food, which comprises the steps of extracting by using a mixed solvent with the volume ratio of chloroform to methanol to water of 1:2:0.8, then adding water and chloroform, under the condition that no salt compound is added, automatically layering the mixed solvent into a water phase layer to an organic phase layer under the condition that the volume ratio of chloroform to methanol to water is 2:2:1.8, loading the water phase on an HLB column, eluting by using an organic phase and chloroform in sequence, and finally detecting and quantitatively analyzing by using high performance liquid chromatography-mass spectrometry. The invention simplifies the extraction steps, has low quantitative detection limit, is applied to the rapid detection and analysis of common 23 carotenoid in food, has recovery rate as high as 80.1-98.7 percent, and has higher sensitivity and reliability.

Description

Rapid detection method for carotenoid in food

Technical Field

The invention relates to the field of detection of natural pigment components in foods, in particular to a rapid detection method of carotenoid in foods.

Background

Carotenoids, which are important components of natural pigments, are composed of a series of conjugated isoprene units, and are extremely susceptible to oxidation and other chemical modifications, and the most common modification groups are hydroxyl, methoxy, carbonyl, carboxyl, and the like. Up to now, it has been reported that 750 or more carotenoids are classified into two main classes according to chemical structure, the first class being free carotenoids including oxygen-free alpha-carotene and beta-carotene, and oxygen-containing lutein, astaxanthin and the like, and the second class being esterified carotenoids, and being classified into carotenoid monoesters and carotenoid diesters according to the number of fatty acid chain linkages. In addition to their use as natural colorants, these photosensitive carotenoids have been reported to have increased value in maintaining human health, including improving immunity, improving vision, and the like, in addition to improving the organoleptic qualities of foods. The natural pigment is a necessary trend for healthy development of the food industry instead of the artificial synthetic pigment. For regulatory needs, it is necessary to detect and analyze carotenoids added to foods, however, the extreme hydrophobicity and high complexity of carotenoids in foods make rapid extraction, purification and analytical studies of this class of compounds a great challenge.

Traditional pretreatment technology for detecting carotenoid is mainly direct solvent extraction. The technology mainly adopts a single organic reagent as an extraction solvent, such as acetone, methanol or ethyl acetate, and the like, has the selectivity defect, especially can not be compatible with small molecular free carotenoid and low-polarity carotenoid ester at the same time, does not carry out purification treatment on the matrix property of the extracting solution, is only suitable for an analysis method for detecting certain carotenoid in a fixed food matrix, and has low sample treatment flux.

In the early stage, the subject group establishes a high-flux detection method of carotenoid in aquatic products aiming at the substrate attribute of aquatic organisms, realizes the detection and analysis of the carotenoid in the aquatic products, adopts the experimental thought of step-by-step extraction and graded purification in pretreatment, combines two purification technologies of solid phase extraction and substrate dispersion, but the pretreatment method is suitable for the aquatic organisms with simple and clean substrate, but is not suitable for the high-protein and high-fat food substrate, and the method is used for the food substrate, especially the high-fat food substrate, and finds that the food substrate contains abundant additive components when the carotenoid is extracted and purified, and has poor purification effect of free carotenoid due to the lack of leaching process when HLB is adopted for purification; in addition, neutral alumina powder is adopted to adsorb and remove grease components in the extracted esterified carotenoid, the applicability to food matrixes with higher grease content is poor, the overall recovery rate result is low, and the whole experimental process is complex and takes longer time due to step-by-step extraction and fractional purification. Therefore, a need exists for a more comprehensive and efficient pretreatment method for rapid detection and analysis of carotenoid species in food.

Disclosure of Invention

The invention aims to overcome the defects of the existing carotenoid detection method in food, such as selectivity, few detected types, low detection efficiency, low recovery rate and the like, and provides a rapid detection method suitable for carotenoids in complex food matrixes.

Considering that carotenoids cover a large number of species, the polarity difference between the different species is large; and the food has high protein and high fat matrix properties, so that a strong matrix effect is generated, and the detection result is seriously deviated. Thus, the present invention employs a modified Bligh-Dyer process to extract carotenoids from food substrates.

The Bligh-Dyer method is a traditional method for lipid analysis, and adopts chloroform-methanol-water as an extracting solution, wherein the polarity range of the extracting solution is wide, and the extracting solution can be actively layered into an aqueous phase layer and an organic phase layer at a certain ratio (2:2:1.8, v/v/v). Esterified carotenoids are similar in structure to lipids, and contain one or two fatty acids, and carotenoids are also known as lipid pigments. Therefore, according to the pKa value of the extraction solvent and the polar range of the target compound, chloroform-methanol-water is adopted as the extraction solvent, free carotenoid is mainly distributed in the water phase layer, part of low-grade free carotenoid and esterified carotenoid are mainly distributed in the organic phase layer, and the free carotenoid and the esterified carotenoid can be simultaneously reserved by synchronously collecting the water phase and the organic phase, so that the purification step is further optimized, and the rapid detection and analysis of the carotenoid in food can be realized.

The invention adopts the technical scheme that:

a method of detecting carotenoids in a food, the carotenoids comprising 23 carotenoids, being one or more of lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxyzeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, canthaxanthin, astaxanthin monoester of linoleic acid, astaxanthin monoester of docosahexaenoic acid, astaxanthin ester of distearate, astaxanthin ester of dipalmitate, the method comprising the steps of:

A. pretreatment:

(A1) Weighing 2g of food sample to be detected, repeatedly extracting with 7.5mL of chloroform-methanol-water solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol for 3 times, centrifuging, and combining the upper layer extract;

in the chloroform-methanol-water solution, the volume ratio of chloroform to methanol to water is 1:2:0.8;

vibrating for 5-10 min and ultrasonic extracting for 5-10 min;

centrifuging for 5-10 min at 12000 r/min;

2, 6-di-t-butyl-p-cresol (BHT) is an antioxidant useful for preventing oxidation of carotenoids;

(A2) Taking the upper layer extract obtained in the step (A1), sequentially adding 6.0mL of water and 6.0mL of chloroform into the upper layer extract, and automatically layering after centrifugation to obtain an upper layer water phase and a lower layer organic phase;

the centrifugation is generally carried out for 5-10 min at the rotation speed of 12000 r/min;

(A3) Taking the water phase obtained in the step (A2), loading the water phase to an HLB column, and then leaching the water phase by using 6.0ml of methanol-water solution;

in the methanol-water solution, the volume ratio of methanol to water is 3:2;

(A4) Eluting the organic phase in the step (A2) and 6.0mL of chloroform sequentially serving as an eluent of the HLB purification column, and collecting the eluting solution;

(A5) Concentrating and drying the eluent obtained in the step (A4), and then using a methanol solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol to fix the volume to 2.0ml, and filtering the eluent by a filter membrane to obtain a sample solution;

the concentration and drying are generally carried out by nitrogen blowing until the volume is no longer changed;

B. high performance liquid chromatography-mass spectrometry detection

Detecting the sample solution obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high-resolution mass spectrometer to obtain a high performance liquid chromatography-mass spectrogram of the sample solution, wherein the instrument conditions are as follows:

high performance liquid chromatography conditions:

chromatographic column Syncroniss C18 column (2.1 mm. Times.150 mm, particle size 1.7 μm);

mobile phase a: acetonitrile containing 10mM ammonium formate and a water volume ratio of 9:1;

mobile phase B: acetonitrile and isopropanol in a volume ratio of 7:3, a flow rate of 0.3mL/min, a sample injection amount of 5.0 mu L, a split ratio of 1:4, and gradient elution, wherein the elution procedure is preferably as shown in the following table 1:

table 1: gradient elution procedure

Mass spectrometry parameters: mass spectrum is subjected to full scan measurement in a positive ion conversion mode, and mass range is as follows: m/z 100-1200, resolution 70000, automatic gain control target value 5*e ⁵ The method comprises the steps of carrying out a first treatment on the surface of the Positive ion mode 3800V, ion transport tube temperature 300 ℃, desolvation gas nitrogen flow rate: 35L/h, auxiliary gas nitrogen flow rate: 15L/h, and the temperature of the gasification chamber is 350 ℃; performing positive ion correction on the instrument before the sample runs; the second stage adopts an automatic triggering mode, the resolution is 35000, and the automatic gain control target value 2*e ⁵ The normalized collision energy is 30%, 40% and 60% in sequence, and the retention time is +/-1.0 min according to the retention time of the target object;

quantitative analysis of C

And quantitatively detecting the carotenoid content in the sample solution by adopting an external standard method, and obtaining the 23 carotenoid content in the food sample to be detected by conversion.

Further, the quantitative analysis in the step C comprises the following steps:

(C1) Standard curves of standard products are prepared:

preparing standard products of lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, diatoxanthin, rhodoxanthin, canthaxanthin, astaxanthin monoester linoleate, astaxanthin monoester docosahexaenoic acid, astaxanthin monoester distearate and astaxanthin dipalmitate into mixed standard working solutions with different concentrations, detecting by high performance liquid chromatography-mass spectrometry according to the same conditions of the step B to obtain high performance liquid chromatography-mass spectrograms of the standard working solutions, and respectively drawing standard curves of the standard products according to the concentration and peak area of the standard products to obtain standard curves of the standard products;

(C2) Calculating the concentration of each component to be detected in the sample solution according to the high performance liquid chromatography-mass spectrogram of the sample solution obtained in the step B and the standard curve of the corresponding standard substance, and calculating the carotenoid content in the food sample to be detected according to the formula (1):

X＝C*V/m*1000

wherein:

the content of the to-be-detected substance in the X-sample is mg/kg;

c-concentration of the analyte in the sample solution, calculated according to a standard curve, in μg/L

V-constant volume in mL;

m-mass of food sample to be detected, the unit is g.

Further, the food sample to be detected in the step (A1) may be various solid or liquid foods, generally foods which can be directly eaten after processing, and contains various food additives including starch processed foods, oil processed foods, candy and chocolate processed foods, wines, beverages, seasonings and the like. Ham sausage, jam and cookies are selected as representative food substrates for the methodological verification of the invention. These foods cover different matrix attributes, including solid, liquid and high-fat foods, with the addition of pigment-rich, as a validated matrix for pretreatment techniques in commonly used foods.

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

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 an extraction solvent, and the three solvents have wide polarity coverage range, so that the extraction requirements of carotenoid compounds are completely met. Wherein, the methanol and the aqueous solution well ensure the extraction efficiency of the free carotene, and the chloroform extracted part mainly comprises the esterified carotene with low polarity and part of the free carotenoid. In addition, the existence of the aqueous solution can well prevent the emulsification phenomenon from happening, so that the layering interface is clearer, and the BHT can well avoid the carotenoid from undergoing oxidation reaction. The extracting solution obtained by extraction is a mixed solution of an organic phase and a water phase, water and chloroform are added after three times of extraction, and under the condition that no salt compound is added, the mixed solvent is prepared by the following steps: the chloroform-methanol-water ratio (2:2:1.8, v/v/v) automatically delaminates into an aqueous-organic phase layer (Bligh and dyr, 1959). This avoids contamination of the instrument by delamination with the salt solution. The medium polarity free carotenoids are predominantly distributed in the aqueous phase and the low polarity free and esterified carotenoids are predominantly distributed in the organic phase.

The principle of the purification step of the invention is that firstly, after the upper water phase obtained in the step (A2) is loaded on an HLB column, free carotenoid is reserved on the HLB column, after the optimized methanol-water leaching, the interference of hydrophilic compounds can be effectively removed, then during the elution, the eluent is divided into two parts and sequentially used as the eluent of the HLB purification column for eluting, the first part of eluent is the lower organic phase used as an auxiliary extractant in the step (A2), chloroform and part of methanol are separated from the extracting solution by adjusting the ratio (2:2:1.8, v/v/v) of chloroform-methanol-water, and the obtained organic phase layer is positioned at the lower layer and is used as the auxiliary extractant, so that the recovery rate of nonpolar carotenoid ester is well ensured. After the water phase layer is sampled, the organic phase at the lower layer is used as an eluent to be infiltrated step by step, so that the free carotenoid with the polarity and the nonpolar esterified carotenoid are eluted from the HLB purifying column in sequence. The second part of the eluent is chloroform solvent, and by optimizing the elution volume of chloroform, the interference of the grease compound is eliminated as much as possible while ensuring the complete elution of the carotenoid diester with strong hydrophobicity. Finally, carrying out data analysis by adopting high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS), and realizing rapid analysis and detection of carotenoid in food.

The high performance liquid chromatography-quadrupole electrostatic field orbit trap high resolution mass spectrum (HPLC-Q-Orbitrap-HRMS) technology adopted by the invention is different from the conventional triple quadrupole mass spectrum in that the method does not need characteristic ion fragments of known compounds to be detected, but directly adopts high-precision mass numbers (resolution is more than or equal to 70000 and m/z 200) to perform directional and non-directional full-scan detection on the samples to be detected, and when the target compounds are required to be added, the method does not need repeated sample injection, and can re-analyze the existing full-scan data, thereby being particularly suitable for multi-component simultaneous qualitative and quantitative analysis.

The invention has the beneficial effects that:

1. the carotenoid measurement lower limit is 0.02-0.05 mg/kg, and the standard curve is good in the linear range of 10 ng/mL-200 ng/mL (regression coefficient R2> 0.99);

2. the pretreatment uses chloroform-methanol-water as an extraction solvent, synchronously collects a water phase and an organic phase layer, and purifies by using a hydrophilic lipophilic balance column, so that the extraction and purification requirements of 2 large carotenoids with very different physicochemical properties can be met while the interference of hydrophilic compounds and neutral triglycerides is effectively eliminated, compared with the prior art, the extraction steps are simplified, the detection efficiency is improved, and the expensive time and the reagent cost consumption are reduced;

3. the method is applied to rapid detection and analysis of 23 common carotenoids in food, has recovery rate as high as 80.1-98.7%, has higher sensitivity and reliability, and the recovery rate of the detected carotenoids is superior to that of the existing detection method.

Drawings

FIG. 1 is a graph of the effect of chloroform elution volumes on elution of 4 esterified carotenoids and palmitic acid triglycerides.

The specific embodiment is as follows:

the technical scheme of the invention is further described below, but the protection scope of the invention is not limited to the scheme.

Instrument and reagent (Instruments and materials)

Q-exact quadrupole electrostatic field orbitrap high-resolution mass spectrometer (Semer Fielder technology ThermoFisher Scientific) was equipped with an H-ESI II source. The liquid chromatography system is an UltiMate3000 high pressure liquid chromatography band autosampler. Chromatographic column Syncronis C18 column (2.1 mm. Times.150 mm, particle size 1.7 μm). Milli-Q high purity water generator (Millipore Co., U.S.A.). Centrifuge (SIGMA, germany). Vortex shaker (Heldolph, germany). Filter (dimma, PTFE 0.22 μm). Waters Oasis HLB column (6 cc/200mg, activated with methanol, water equilibrated prior to loading).

Methanol, acetonitrile and ammonium acetate (chromatographic purity, sigma-Aldrich company, usa). Other reagents (chromatographic purity, merck, germany). The water used for the experiment was Milli-Q ultra pure water (18.2. OMEGA.M). Carotenoid standards were purchased from Sigma and Dr. Ehrensterfer, with purity > 95%. Including, free carotenoids: lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxyzeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, diatoxanthin, diasporin, canthaxanthin, astaxanthin; esterified carotenoids: astaxanthin monoesters of linoleic acid, astaxanthin monoesters of docosahexaenoic acid, astaxanthin distearate and astaxanthin dipalmitate.

Standard stock solution: and (3) weighing a proper amount of standard substances respectively, dissolving astaxanthin dipalmitate with dichloromethane, and dissolving the other standard substances except for the astaxanthin dipalmitate with methanol to a certain volume to prepare a standard stock solution of 0.1mg/mL, and preserving the standard stock solution at-20 ℃ in a dark place.

Standard working solution: 100. Mu.L of each component of the standard stock solution was taken up in a 10mL volumetric flask and the volume was fixed to the scale with methanol at a concentration of 1.0. Mu.g/mL.

Example 1

A. Pretreatment:

(A1) Weighing 2g of food to be detected, adding 7.5mL of chloroform-methanol-water solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol, vibrating for 5min, ultrasonically extracting for 5min, centrifuging for 5-10 min at a speed of 12000r/min to obtain an upper extract and residues, extracting for 2 times again, and combining the upper extract;

in the chloroform-methanol-water solution, the volume ratio of chloroform to methanol to water is 1:2:0.8;

(A2) Taking the upper layer extract obtained in the step (A1), sequentially adding 6.0mL of water and 6.0mL of chloroform into the upper layer extract, centrifuging at a rotating speed of 12000r/min for 5-10 min, automatically layering after completion to obtain an upper layer water phase and a lower layer organic phase,

(A3) Taking the water phase obtained in the step (A2), loading the water phase to an HLB column, and then leaching the water phase by adopting 6.0ml of methanol-water solution;

in the methanol-water solution, the volume ratio of methanol to water is 3:2;

(A4) Eluting the organic phase in the step (A2) and 6.0mL of chloroform sequentially serving as an eluent of the HLB purification column, and collecting the eluent to obtain an eluent;

(A5) Concentrating the eluent obtained in the step (A4) by nitrogen blowing until the volume is unchanged, then fixing the volume to 2.0ml by a methanol solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol, and obtaining a sample solution by a filter membrane with the pore diameter of 0.22 mu m;

B. detecting the sample solution obtained in the step (A5) by a high-performance liquid chromatography-quadrupole electrostatic field orbit trap high-resolution mass spectrometer to obtain a high-performance liquid chromatography-mass spectrogram of the sample solution, wherein the instrument conditions are as follows:

high performance liquid phase parameters:

chromatographic column Syncroniss C18 column (2.1 mm. Times.150 mm, particle size 1.7 μm);

mobile phase a: acetonitrile containing 10mM ammonium formate and a water volume ratio of 9:1;

mobile phase B: acetonitrile and isopropanol in a volume ratio of 7:3, the flow rate is 0.3mL/min, the sample injection amount is 5.0 mu L, the split ratio is 1:4, and the gradient elution program is shown in the following table 1:

TABLE 1 Positive ion mode gradient elution procedure

Mass spectrometry parameters: mass spectrum is subjected to full scan measurement in a positive ion conversion mode, and mass range is as follows: m/z 100-1200, resolution 70000, automatic gain control target value 5*e ⁵ The method comprises the steps of carrying out a first treatment on the surface of the Positive ion mode 3800V, ion transport tube temperature 300 ℃, desolvation gas nitrogen flow rate: 35L/h, auxiliary gas nitrogen flow rate: 15L/h, and the temperature of the gasification chamber is 350 ℃; performing positive ion correction on the instrument before the sample runs; the second stage adopts an automatic triggering mode, the resolution is 35000, and the automatic gain control target value 2*e ⁵ The normalized collision energy was 30% in sequence. 40% and 60% of retention time, according to target retention time + -1.0 min;

quantitative analysis of C

(C1) Drawing a standard curve of a standard substance:

preparing mixed standard working solutions with different concentrations from lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, diatoxanthin, canthaxanthin, astaxanthin monoester linoleate, astaxanthin monoester docosahexaenoic acid, astaxanthin bisstearates and astaxanthin bispalmate, testing according to the same conditions in the step B to obtain high performance liquid chromatography-mass spectrograms of the standard working solutions, and drawing standard curves according to the concentration and peak area of each standard product to obtain standard curves of the standard products; the results are shown in Table 3.

(C2) Calculating the concentration of each component to be detected in the sample solution according to the high performance liquid chromatography mass spectrogram of the sample solution obtained in the step B and the standard curve of the corresponding standard substance, and calculating the content of carotenoid in the food to be detected according to a formula (1):

X＝C*V/m*1000

wherein:

the content of the to-be-detected substance in the X-sample is mg/kg;

c-concentration of the analyte in the sample solution, calculated according to a standard curve, in μg/L

V-constant volume in mL;

m-mass of sample in g.

Comparative example 1 pretreatment a. Pretreatment was performed according to the method disclosed in CN111307976 a:

(A1) Weighing 1g of food to be detected, adding the food to be detected into 10mL of methanol solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol, vibrating for 5min, ultrasonically extracting for 20min, and centrifuging for 5-10 min at the rotation speed of 12000r/min at the temperature of 4-10 ℃ to obtain an organic phase and residues after completion;

(A2) Taking the organic phase obtained in the step (A1), adding at least 4 times of water to the organic phase to obtain a loading liquid, purifying the loading liquid through an HLB column to obtain a purified loading liquid, concentrating the purified loading liquid to be less than 2.0ml through nitrogen blowing, and fixing the volume to be 2.0ml through a methanol solution containing 0.1wt% of HF to obtain the fixed-volume purified loading liquid, wherein:

the HLB column is purified by using a methanol solution containing 0.1wt percent of 2, 6-di-tert-butyl-p-cresol as eluent;

(A3) Adding the residue obtained in the step (A1) into 10mL of methanol-dichloromethane solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol, vibrating for 5min, ultrasonically extracting for 20min, centrifuging for 5-10 min at the rotation speed of 12000r/min at the temperature of 4-10 ℃ to obtain an organic phase and residue, wherein:

in the methanol-dichloromethane solution, the volume ratio of methanol to dichloromethane is 1:1;

(A4) Taking the organic phase obtained in the step (A3), concentrating by nitrogen blowing until the volume is unchanged, then fixing the volume to 2.0ml by using a methanol solution containing 0.1wt% of HF, adding 1g of neutral alumina powder into the solution, fully oscillating the solution, and centrifuging the solution at the speed of 12000r/min at the temperature of 4-10 ℃ for 5-10 min to obtain supernatant and precipitate;

(A5) Combining the purified sample solution obtained in the step (A2) after volume fixing and the supernatant obtained in the step (A4), and then passing through a filter membrane with the pore diameter of 0.22 mu m to obtain a sample solution;

step B and step C were performed under the same conditions as in example 1.

The pretreatment methods of example 1 and comparative example 1 were used to detect and analyze carotenoid types in common foods (ham sausage, jam, cookies) respectively, and the standard addition amounts of the carotenoid types in the foods were 0.02 mg/kg, 0.05mg/kg and 0.1mg/kg, respectively, and then the carotenoid types were quantified according to a standard curve, and the recovery rate was calculated. The results are shown in Table 2. The results show that:

in the embodiment 1, chloroform-methanol-water is adopted as an extracting solution, and the extracting and purifying requirements of 23 carotenoid in total can be met while the interference of hydrophilic compounds and neutral triglyceride is effectively eliminated by synchronously collecting an aqueous phase layer and an organic phase layer and purifying by utilizing a hydrophilic lipophilic balance column. The carotenoid standard curve is good in the linear range of 10 ng/mL-200 ng/mL, the quantitative limit is 0.02-0.05 mg/kg, the recovery rate is as high as 80.1-98.7%, and the sensitivity and reliability are high.

Comparative example 1: the method realizes the high-flux screening of the carotenoid in the aquatic products, adopts the experimental thought of step-by-step extraction and graded purification in the pretreatment, combines two purification technologies of solid phase extraction and matrix dispersion, but can not well give consideration to the extraction and purification of the carotenoid in the food with more complex matrix components, the quantitative limit is 0.05-0.1 mg/kg, the recovery rate is 60.9-93.1%, the recovery rate is poor, and the quantitative accuracy of the carotenoid in the food matrix is poor. And the whole experimental procedure is time-consuming compared to example 1.

Table 2 experimental data (n=6) for recovery of 23 carotenoids in ham sausages, jams and cookies under both treatments.

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Table 323 Linear equation and regression coefficients for carotenoid standards

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Example 2 selection of elution conditions

A. Pretreatment:

(A1) Weigh 2g of blank ham sausage substrate sample and add 7.5mL of the mixture containing 0.1wt%

Vibrating chloroform-methanol-water solution of 2, 6-di-tert-butyl-p-cresol for 5min, ultrasonically extracting for 5min, centrifuging at 12000r/min for 5-10 min to obtain upper extract and residue, repeatedly extracting for 2 times, and mixing the upper extract;

in the chloroform-methanol-water solution, the volume ratio of chloroform to methanol to water is 1:2:0.8;

(A2) Taking the upper layer extract obtained in the step (A1), sequentially adding 6.0mL of water and 6.0mL of chloroform into the upper layer extract, centrifuging at a rotating speed of 12000r/min for 5-10 min, automatically layering after completion to obtain an upper layer water phase and a lower layer organic phase,

(A3) Taking the water phase obtained in the step (A2), adding the 23 carotenoid (C=1.0 mug/mL), loading the mixture into an HLB column, and then leaching the mixture by using 6.0mL of methanol-water solution;

in the methanol eluent, the volume ratio of methanol to water was 50%, 60%, 70%, 80 and 100%, respectively, and the influence of the methanol ratio on the recovery rate of the above-mentioned sample addition was examined.

(A4) And (3) drying the leaching solution, fixing the volume, and then performing high performance liquid chromatography-mass spectrometry detection in the step (B), wherein the concentration of the detected components is converted to obtain leaching loss rate, and the leaching loss rate is 100% -loss rate, namely the recovery rate, and the obtained results are shown in the following table 4.

The final detection result shows that the free carotenoid is prepared as the free carotenoid with the polarity, namely, fucoxanthin, lutein, astaxanthin and the like are greatly influenced by the proportion of methanol, and when the proportion of the methanol is more than or equal to 70%, the recovery rate starts to be reduced, and the recovery rate value is reduced from 94.9% to 12.3%; the recovery rate value of partial low-grade free carotenoid, namely alpha-carotene, beta-carotene and the like is not maintained at 100% when the methanol accounts for 50% -80%, and the recovery rate is reduced when the methanol accounts for 100%; the esterified carotenoid is not affected by the methanol ratio, and when the methanol ratio is 100%, the recovery rate tends to be stable and is maintained at 100%. Considering this together, the final rinse conditions were determined to be 60% methanol-water.

Table 4 effect of methanol ratio in leacheate on recovery of 23 carotenoids (n=3)

Example 3 selection of elution conditions

The eluent mainly comes from two parts, the first part is an organic phase used as an auxiliary extractant, and is positioned at the lower layer of the centrifuge tube after layering, and the eluent is used as the auxiliary extractant, so that the recovery rate of nonpolar carotenoid ester is well ensured. After the water phase layer is sampled, the organic phase at the lower layer is used as an eluent to be infiltrated step by step, so that free carotenoid is eluted from the HLB purifying column first. The second part of the eluent is chloroform solvent for ensuring the elution of carotenoid mono-and di-esters, but unavoidable grease compounds can generate co-elution phenomenon, thereby generating strong matrix inhibition effect.

In this example, the effect of chloroform as the eluting solvent volume on the results was examined.

The blank ham matrix sample was extracted according to step (A1) of example 1, and 4 kinds of esterified carotenoids, namely, astaxanthin monoester linoleate, astaxanthin monoester docosahexaenoic acid, astaxanthin distearate, astaxanthin dipalmitate, and triglyceride palmitate ([ m+na ] +, M/z 805.7) (c=1.0 μg/mL), which are common oily compounds in foods, were added to the resulting upper extract, respectively,

(A2) (A3) was the same as in example 1.

(A4) Eluting the organic phase and chloroform in the step (A2) sequentially as an eluent of an HLB purifying column, and collecting the eluent;

the volumes of chloroform as the eluting solvent were 0, 1, 2, 3, 4, 5, 6, 7 and 8mL, respectively, and the eluent was subjected to volume metering according to step (A5) of example 1, and then was detected according to step B, and graphs of the ion-extracting flow chromatographic peak areas of the corresponding compounds obtained from the different eluting volumes are shown in fig. 1. As can be seen from FIG. 1, when the chloroform volume was 5.0mL, the astaxanthin monoester linoleate and the astaxanthin monoester docosahexaenoic acid were eluted first, and the astaxanthin ester distearate and the astaxanthin ester dipalmitate were eluted at a chloroform volume of 7.0mL, and at the same time, the triglyceride palmitate began to elute simultaneously at a chloroform volume of 6.0mL. From the viewpoint of reducing the contamination with oil, the elution volume of chloroform as the eluent was determined to be 6.0mL.

Example 4 optimization of conditions for liquid phase Mass Spectrometry

The buffer salt is added into the mobile phase, so that the ionization is promoted, and meanwhile, the acid-base stability of the mobile phase can be well maintained, therefore, the carotenoid is detected in a positive ion mode, ammonium formate is added, meanwhile, the influence of the ammonium formate buffer solution on the ion intensity is further examined, and the concentration of the ammonium formate solution is changed from 2mmol/L to 20mmol/L. The results show that at an ammonium acetate concentration value of 10mmol/L, the optimal chromatographic peak shape, separation effect and mass spectrum signal response are obtained. The gradient elution mode is selected in the whole process, and the effective separation of the target mixture is realized by optimizing the gradient elution condition of the mobile phase. In addition, the SyncronisiC 18 column (2.1 mm. Times.150 mm, particle size 1.7 μm) and the HypersiloGoldC 18 column were compared, and the former was found to be superior in terms of isomer separation, successfully achieving effective separation of 5 isomers, namely lycopene, alpha-carotene, epsilon-carotene, beta-carotene and delta-carotene. Thus, the final column was a Syncronisc18 column (2.1 mm. Times.150 mm, particle size 1.7 μm).

In conclusion, the method has strong compatibility to different food matrixes and compounds with different physicochemical properties, the carotenoid detected by the method is more abundant in variety and more accurate in content value, and the reliability of the detection method is fully verified by comparing with the existing carotenoid detection method.

The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various modifications may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims (6)

1. A method for rapid detection of carotenoids in a food, characterized in that said carotenoids comprise 23 carotenoids, which are one or more of lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxyzeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, diadinoxanthin, canthaxanthin, astaxanthin monoester linoleate, astaxanthin monoester of docosahexaenoic acid, astaxanthin ester of distearate, astaxanthin ester of dipalmitin, said method comprising the steps of:

A. pretreatment:

(A1) Weighing 2g of food sample to be detected, repeatedly extracting with 7.5mL of chloroform-methanol-water solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol for 3 times, centrifuging, and combining the upper layer extract;

in the chloroform-methanol-water solution, the volume ratio of chloroform to methanol to water is 1:2:0.8;

(A2) Taking the upper layer extract obtained in the step (A1), sequentially adding 6.0mL of water and 6.0mL of chloroform into the upper layer extract, and automatically layering after centrifugation to obtain an upper layer water phase and a lower layer organic phase;

(A3) Taking the water phase obtained in the step (A2), loading the water phase to an HLB column, and then leaching the water phase by using 6.0ml of methanol-water solution;

in the methanol-water solution, the volume ratio of methanol to water is 3:2;

(A4) Eluting the organic phase in the step (A2) and 6.0mL of chloroform sequentially serving as an eluent of the HLB purification column, and collecting the eluting solution;

(A5) Concentrating and drying the eluent obtained in the step (A4), and then using a methanol solution containing 0.1wt% of 2, 6-di-tert-butyl-p-cresol to fix the volume to 2.0ml, and filtering the eluent by a filter membrane to obtain a sample solution;

B. high performance liquid chromatography-mass spectrometry detection

Detecting the sample solution obtained in the step (A5) by using a high performance liquid chromatography-quadrupole electrostatic field orbit trap high-resolution mass spectrometer to obtain a high performance liquid chromatography-mass spectrogram of the sample solution;

quantitative analysis of C

And quantitatively detecting the carotenoid content in the sample solution by adopting an external standard method, and obtaining the 23 carotenoid content in the food sample to be detected by conversion.

2. The method of claim 1, wherein in step (B), the conditions of high performance liquid chromatography are:

a chromatographic column Syncronis C18 column;

mobile phase a: acetonitrile containing 10mM ammonium formate and a water volume ratio of 9:1;

mobile phase B: acetonitrile and isopropanol in a volume ratio of 7:3, the flow rate is 0.3mL/min, the sample injection amount is 5.0 mu L, the split ratio is 1:4, and the gradient elution is carried out.

3. The method of claim 2, wherein the gradient elution procedure is as follows:

4. the method of claim 1, wherein in step B, the mass spectral parameters are: mass spectrum is subjected to full scan measurement in a positive ion conversion mode, and mass range is as follows: m/z 100-1200, resolution 70000, automatic gain control target value 5*e ⁵ The method comprises the steps of carrying out a first treatment on the surface of the Positive ion mode 3800V, ion transport tube temperature 300 ℃, desolvation gas nitrogen flow rate: 35L/h, auxiliary gas nitrogen flow rate: 15L/h, and the temperature of the gasification chamber is 350 ℃; performing positive ion correction on the instrument before the sample runs; the second stage adopts an automatic triggering mode, the resolution is 35000, and the automatic gain control target value 2*e ⁵ Normalized collision energy is 30%, 40% and 60% in order, and retention time is + -1.0 min according to target retention time.

5. The method of claim 1, wherein said step C quantitative analysis comprises the steps of:

(C1) Standard curves of standard products are prepared:

preparing standard products of lycopene, alpha-carotene, epsilon-carotene, beta-carotene, gamma-carotene, delta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein, zeaxanthin, epoxy zeaxanthin, rhodoxanthin, neoxanthin, fucoxanthin, diatoxanthin, rhodoxanthin, canthaxanthin, astaxanthin monoester linoleate, astaxanthin monoester docosahexaenoic acid, astaxanthin monoester distearate and astaxanthin dipalmitate into mixed standard working solutions with different concentrations, detecting by high performance liquid chromatography-mass spectrometry according to the same conditions of the step B to obtain high performance liquid chromatography-mass spectrograms of the standard working solutions, and respectively drawing standard curves of the standard products according to the concentration and peak area of the standard products to obtain standard curves of the standard products;

(C2) Calculating the concentration of each component to be detected in the sample solution according to the high performance liquid chromatography-mass spectrogram of the sample solution obtained in the step B and the standard curve of the corresponding standard substance, and calculating the carotenoid content in the food sample to be detected according to the formula (1):

X＝C*V/m*1000

wherein:

the content of the to-be-detected substance in the X-sample is mg/kg;

c-concentration of the analyte in the sample solution, calculated according to a standard curve, in μg/L

V-constant volume in mL;

m-mass of food sample to be detected, the unit is g.

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

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