WO2022121942A1

WO2022121942A1 - Method for extracting canthaxanthin from egg yolk and purifying same, and solid phase extraction high performance liquid chromatography method for determining canthaxanthin in egg yolk

Info

Publication number: WO2022121942A1
Application number: PCT/CN2021/136420
Authority: WO
Inventors: 王凤芹; 曹进平; 汪以真; 路则庆; 程远之
Original assignee: 浙江大学
Priority date: 2020-12-08
Filing date: 2021-12-08
Publication date: 2022-06-16
Also published as: CN112379024A; CN112379024B

Abstract

Disclosed is a solid phase extraction high performance liquid chromatography method for determining canthaxanthin in egg yolk. The method involves extracting 5 g of egg yolk with acetonitrile using ultrasonic assistance, diluting an extract with water, then directly loading the diluted extract into a PRiME HLB solid phase extraction column, firstly rinsing the column with 5 mL of an acetonitrile aqueous solution (a volume ratio of 1:1), then eluting the column with 3 mL of acetonitrile, drying the eluent in a blowing manner with nitrogen at 50°C, and finally, separating same using a Welch C18 chromatographic column with acetonitrile-water (95:5, V/V) as a mobile phase and a flow rate of 1.2 mL/min by means of isocratic elution; and detecting same at an ultraviolet-visible absorption wavelength of 474 nm, and then using an external standard method to quantitatively analyze canthaxanthin and isomers thereof. The method can meet the requirements for rapid and accurate determination of canthaxanthin and isomers thereof in egg yolk.

Description

Method for extracting and purifying cantharidin in egg yolk, solid-phase extraction-high performance liquid chromatography for determination of cantharidin in egg yolk

This application claims the priority of the Chinese patent application filed with the China Patent Office on December 08, 2020, the application number is 202011421913.2, and the invention name is "a solid-phase extraction-high performance liquid chromatography method for the determination of cantharidin in egg yolk" , the entire contents of which are incorporated herein by reference.

technical field

The invention relates to a solid phase extraction-high performance liquid chromatography method for the determination of cantharidin in egg yolk.

Background technique

Eggs are one of the important foods in many countries, and they contain, in addition to nutrients, carotenoids, which are deposited in the yolk to give it a golden or orange color (Brulc, 2013). The chemical name of cantharidin is β,β-carotene-4,4-dione, and there are mainly all-trans (Fig. 1) and cis (Fig. 2) isomers (Wenzel, 2010), which is an important carotenoid Vegetarian red pigments are used as poultry colorants to enhance the skin, muscle and egg yolk color of poultry (Choi, 2005). Besides that, like other carotenoid pigments, cantharidin is a potent antioxidant (Weber, 2013). However, excessive intake of cantharidin may deposit on the retina of humans and animals, causing visual problems (Surai, 2012). In addition, the practice of using caged chickens and eggs to pretend to be high-priced native chickens and native eggs harms the interests of consumers. To this end, in 2019, 315 exposed the "makeup" egg. In 2003, the United Kingdom stipulated the addition amount of cantharidin in meat and egg poultry feeds, which should not exceed 25 mg/kg and 8 mg/kg in meat and egg poultry compound feeds respectively (Weber, 2013). In 2017, the Ministry of Agriculture and Rural Affairs of my country also made the same restrictions (Ministry of Agriculture and Rural Affairs Announcement No. 2625). The European Food Safety Authority and the Japanese Ministry of Health, Labour and Welfare have respectively stipulated that the maximum residue limits of cantharidin in egg yolks are 30 μg/g and 25 μg/g (Furusawa, 2011).

At present, literature reports on the detection of cantharidin in egg yolk mainly include high performance liquid chromatography (HPLC) (Furusawa, 2014; Bononi, 2002; Schlatterer, 2006), high performance liquid chromatography-mass spectrometry (HPLC-MS) ( Wenzel, 2010; Nimalaratne, 2012; Tsiaka, 2018). The instruments and methods for the analysis of cantharidin in egg yolk based on high performance liquid chromatography and high performance liquid chromatography-mass spectrometry are relatively mature, and the simultaneous detection of multiple pigments has also been realized, but the cis-isomer of cantharidin is often ignored. Quantitative (Li, 2005; Furusawa, 2011; Brucl, 2013; Chen, 2019). According to literature reports, the cis isomer of cantharidin has stronger antioxidant properties than the all-trans isomer (Venugopalan, 2013). In addition, it is a serious challenge to reduce the interference of lipid substances in egg yolk to obtain a high recovery, simple extraction and purification method, and thus to establish an accurate and rapid quantitative analysis method for cis-trans isomers of cantharidin in egg yolk.

There are three main ways to remove lipids in egg yolk. Saponification method: Larsen et al. (Larsen, 2005) used potassium hydroxide methanol solution to extract lutein, but the saponification process will lead to pigment degradation (Wenzel, 2010), so this method is rarely reported in the follow-up literature. Solid-phase extraction method: First, extract cantharidin from the sample matrix with organic solvents. Common solvents include acetonitrile (He, 2012; GB/T 22958-2008), methanol (Wenzel, 2011), acetone (Li, 2005), petroleum Ether-methanol-ethyl acetate mixed solution (Schlatterer, 2006), acetonitrile-ethyl acetate mixed solution (Chen, 2019) and absolute ethanol-dichloromethane mixed solution (GB/T 18970-2003) etc. 18-SPE, silica gel-SPE, cyanopropyl-SPE, neutral alumina-SPE cleanup (Brulc, 2013; Furusawa, 2010). The above extraction systems such as ethyl acetate and dichloromethane can completely extract cantharidin from egg yolk, but the extract contains a large amount of lipids, which causes blockage of the solid-phase extraction column, which obviously causes the recovery rate of cantharidin to be too low. In the process, the cantharidin extract is purified by solid phase extraction columns such as C18-SPE, silica gel-SPE, cyanopropyl-SPE and neutral alumina-SPE. Most of them adopt complex activation and equilibration steps and operate under vacuum pump, which is time-consuming and labor-intensive. . Liquid-liquid extraction method: Use n-hexane and other non-polar solvents to extract and remove fat from egg yolk extract (GB/T 22958-2008). Acetonitrile has good extraction efficiency for cantharidin in egg yolk. Although the cost of secondary extraction and degreasing is low, the consumption of organic solvent is large, and n-hexane causes part of the loss of cantharidin in the process of extraction and degreasing, which also leads to a low recovery rate of cantharidin.

SUMMARY OF THE INVENTION

Aiming at the problems that the pretreatment method of the existing method for detecting cantharidin in egg yolk is too complicated, the recovery rate is low, and the cis-trans isomers of cantharidin cannot be simultaneously quantified, the present invention provides a solid method for the determination of cantharidin in egg yolk. Phase Extraction - High Performance Liquid Chromatography. In this method, acetonitrile was used to extract cantharidin from egg yolk under the assistance of ultrasonic waves. The extract was diluted with water and then directly loaded on a PRiME solid-phase extraction column for purification. Finally, it was separated by Welch C18 chromatographic column. The external standard method was used to quantify cantharidin and its isomers. body. Through the implementation case, it is found that the method is also suitable for the quantitative detection of cantharidin in market commodity egg yolk.

A solid-phase extraction-high performance liquid chromatography method for the determination of cantharidin in egg yolk, 5 g of egg yolk is extracted with acetonitrile under the assistance of ultrasonic waves, the extract is diluted with water and directly loaded on a PRiME HLB solid-phase extraction column, and 5 mL of acetonitrile is used first. Aqueous solution, eluted with a volume ratio of 1:1, and then eluted with 3 mL of acetonitrile. The eluent was dried with nitrogen at 50 °C, and finally separated by a Welch C18 chromatographic column. The mobile phase was acetonitrile-water, 95:5, V/V, The flow rate was 1.2mL/min, isocratic elution, detection at UV-visible absorption wavelength 474nm, external standard method for quantitative analysis of cantharidin and its isomers.

The solid-phase extraction-high performance liquid chromatography method for the determination of cantharidin in egg yolks is to take fresh eggs, separate the yolks, then mix the yolks evenly, and then weigh 5g, accurate to 0.001g, and place them in a 50mL centrifuge tube , and add 10g anhydrous sodium sulfate and 20mL acetonitrile, stir well with a glass rod, mix well, ultrasonically extract for 10min, then centrifuge at 8000rpm for 10min, and collect the extract into a 50mL volumetric flask; repeat the extraction with 20mL acetonitrile once, Combine the extracts in a volumetric flask, make up to the mark with acetonitrile, shake well, and set aside; accurately pipette 5mL of the extracts into a 15mL centrifuge tube, add 5mL of water, mix by ultrasonic, and pass through Oasis

PRiME HLB solid phase extraction column, add 1 mL of acetonitrile and 1 mL of water to the centrifuge tube successively, wash the centrifuge tube, and pass the washing liquid through the column together; rinse the solid phase extraction column with 5 mL of acetonitrile aqueous solution, volume ratio, 1:1, and finally use Elute with 3 mL of acetonitrile, collect the eluent and dry it with nitrogen at 50°C, add 0.5 mL of acetonitrile accurately to dissolve the residue, centrifuge at 8000 rpm for 5 min, take the supernatant and separate it by a Welch C18 chromatographic column, the mobile phase is acetonitrile-water, 95:5 , V/V, flow rate of 1.2mL/min, isocratic elution, detection at UV-visible absorption wavelength of 474nm, external standard method for quantitative determination of cantharidin and its isomers, the detection limit of cantharidin in egg yolk is 0.05mg/kg, the limit of quantification is 0.1mg/kg.

Beneficial effects of the present invention:

In this experiment, acetonitrile was used for ultrasonic extraction of cantharidin from egg yolk, and part of the extract was mixed with water and directly loaded onto a PRiMEHLB solid-phase extraction column for purification. The eluate was concentrated and detected by RP-HPLC. Compared with the method reported in the literature, this method was simpler, easier to operate and had higher recovery rate, and could realize the rapid and accurate quantification of cis-trans isomers of Cantharidinium cantharidinum in egg yolk.

Description of drawings

Fig. 1 is the molecular structure of all-trans cantharidin;

Fig. 2 is the 9-cis isomer structure of cantharidin;

Figure 3 is a chromatogram of cantharidin standard solution (1.0 μg/mL);

Fig. 4 is the influence figure of different pretreatments to Cantharidin recovery rate;

Figure 5 is the chromatogram of six pigments (Rt 6.418: astaxanthin, Rt 9.653: lutein, Rt 9.844: zeaxanthin, Rt 11.764: all-trans cantharidin, Rt 13.519: β-apo-8' - carotene aldehyde, Rt19.929: β-apo-8'-carotene acid ethyl ester);

Figure 6 is a chromatogram of blank egg yolk added with cantharidin.

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments.

Method principle

The cantharidin in egg yolk was extracted with acetonitrile. After the extract was purified by solid-phase extraction column, the cantharidin extract was separated by reversed-phase C18 chromatographic column and detected at the ultraviolet-visible absorption wavelength of 474nm. Construct.

Steps

Take a fresh egg, separate the yolk, then mix the egg yolk evenly, weigh 5g (accurate to 0.001g) into a 50mL centrifuge tube, add 10g anhydrous sodium sulfate and 20mL acetonitrile, stir well with a glass rod, and mix well , ultrasonic extraction for 10min, centrifugation at 8000rpm for 10min, and the extract was collected into a 50mL volumetric flask. The precipitate was repeatedly extracted and centrifuged with 20 mL of acetonitrile, and the extracts were combined in a volumetric flask, and the volume was adjusted to the mark with acetonitrile, shaken, and set aside. Accurately pipette 5mL of the extract into a 15mL centrifuge tube, add 5mL of water, mix by ultrasonic, and load the sample into Oasis

PRiME HLB solid phase extraction column, add 1 mL of acetonitrile and 1 mL of water to the centrifuge tube successively, wash the centrifuge tube, and pass the washing liquid through the column together. The solid phase extraction column was rinsed with 5 mL of acetonitrile aqueous solution (volume ratio, 1:1), and finally eluted with 3 mL of acetonitrile. The supernatant was separated by Welch C18 chromatographic column, the mobile phase was acetonitrile-water (95:5, V/V), the flow rate was 1.2mL/min, isocratic elution was performed, and the detection was performed at the ultraviolet-visible absorption wavelength of 474nm. Standard method for quantification of cantharidin and its isomers. The linearity, minimum detection limit, minimum quantification limit, repeatability, accuracy and precision of the analytical method were investigated.

Instruments and reagents

Cantharidin (purity ≥95.2%) was purchased from LGC Labor GmbH, Germany. Chromatographic grade acetonitrile was purchased from SigmaAldrich Company, USA. Oasis PRiME HLB solid phase extraction cartridge (60 mg, 3 mL) was purchased from Waters Company, USA. Analytical grade acetonitrile, n-hexane, ethyl acetate, anhydrous ethanol, dichloromethane and anhydrous sodium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.

(e2695, American Waters Company), ultrasonic cleaner (KQ-500E, Kunshan Ultrasonic Instrument Co., Ltd.), water purifier (Millipore, Germany EMD Millipore Company), electronic analytical balance (ME204E, Switzerland MettlerToledo Company), centrifuge ( ST 40R, American Thermo Scientific Company), nitrogen blowing instrument (N-EVAP 112, American Organization Company).

Fresh white eggs, red eggs, whole grain eggs and local eggs are purchased from general supermarkets.

The drawing of embodiment 1 standard curve

Weigh 62.5 mg of cantharidin (accurate to 0.0001 g), dissolve with dichloromethane and dilute to a 50 mL brown volumetric flask, mix well, and prepare a standard stock solution of cantharidin with a concentration of 1250 μg/mL. Store at -20°C after aliquoting. Accurately pipette 1250 μg/mL cantharidin standard stock solution and prepare standard working solutions of 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 10.0 μg/mL with acetonitrile. Available now.

Example 2 Extraction and purification of samples

Do 3 parallel experiments. Take a fresh egg, separate the yolk, then mix the yolk evenly, weigh 5g (accurate to 0.001g) and place it in a 50mL centrifuge tube, add 10g anhydrous sodium sulfate and 20mL acetonitrile, stir well with a glass rod, and mix well , ultrasonic extraction for 10min, centrifugation at 8000rpm for 10min, and the extract was collected into a 50mL volumetric flask. The precipitate was repeatedly extracted and centrifuged with 20 mL of acetonitrile, and the extracts were combined in a volumetric flask, and the volume was adjusted to the mark with acetonitrile, shaken, and set aside. Accurately pipette 5mL of the extract into a 15mL centrifuge tube, add 5mL of water, mix with ultrasound, and pass through the HLB solid-phase extraction column. Add 1mL of acetonitrile and 1mL of water to the centrifuge tube successively, wash the centrifuge tube, and pass the washing solution through the column. . The solid phase extraction column was rinsed with 5 mL of acetonitrile aqueous solution (volume ratio, 1:1), and finally eluted with 3 mL of acetonitrile. Take the supernatant and measure it on the machine.

The recovery rate experiment of cantharidin in the sample of embodiment 3

Screen eggs without cantharidin yolk-blank eggs, separate the yolks, then mix the yolks evenly, weigh 5g (accurate to 0.001g) and place them in a 50mL centrifuge tube, add 10g anhydrous sodium sulfate, and then add them separately. Add 0.2 mL of cantharidin standard solution with concentrations of 1.25 μg/mL, 25.0 μg/mL and 100.0 μg/mL respectively, so that the addition levels of cantharidin in egg yolk are 0.1 μg/g, 1.0 μg/g and 4.0 μg/g, press 1.2 Steps of extraction and purification, calculation of recovery and coefficient of variation. The results are shown in Table 1. The recovery of cantharidin was 85.9%-98.6% and the relative standard deviation (RSD) was 1.7%-8.0% in the range of 0.1-4.0 μg/g addition level.

Other pigment interference experiments in the sample of Example 4

Prepare mixed standard solutions of astaxanthin, zeaxanthin, lutein, cantharidin, β-apo-8'-carotene aldehyde, β-apo-8'-carotene acid ethyl ester, and measure on the machine. The chromatographic separation of the above pigments was investigated.

The application of embodiment 5 detection method

The purchased white eggs, red eggs, whole-grain eggs and this egg were pre-treated by the steps of Example 2, and the cantharidin content was analyzed by HPLC. The results are shown in Table 2.

The results and discussions obtained according to the above Examples 1-5:

Drawing of the standard curve

According to the steps of Example 1, the results show that the cantharidin is in the concentration range of 0.1-10.0 μg/mL, the standard curve equation is y=173321x-9521, the correlation coefficient (R ² )=0.9999, and the chromatogram is shown in Figure 3 .

Optimization of sample pretreatment methods

Considering that egg yolk contains a lot of lipids, this experiment firstly carried out the acetonitrile extract, using n-hexane fractional extraction and degreasing, as a control for the acetonitrile extract to be further purified with a solid phase extraction column:

Do 3 parallel experiments. Take a fresh egg, separate the yolk, then mix the yolk evenly, weigh 5g (accurate to 0.001g) and place it in a 50mL centrifuge tube, add 10g anhydrous sodium sulfate and 20mL acetonitrile, stir well with a glass rod, and mix well , ultrasonically extracted for 10 min, and then centrifuged at 8000 rpm for 10 min, the extract was extracted with 20 mL of acetonitrile-saturated n-hexane, and the acetonitrile phase was collected into a 50 mL volumetric flask. The precipitate was repeatedly extracted with 20 mL of acetonitrile, and then extracted with 20 mL of acetonitrile-saturated n-hexane. The acetonitrile phases were combined in a volumetric flask, and the volume was adjusted to the mark with acetonitrile, shaken, and set aside. Accurately pipet 5mL of the extract into a 10mL centrifuge tube, concentrate to dryness by blowing nitrogen in a water bath at 50°C, add 0.5mL of acetonitrile to dissolve the residue, centrifuge at 8000rpm for 5min, and take the supernatant to measure on the machine.

At the same time, this experiment investigated the extraction efficiency of Cantharidin from egg yolk by two different mixed solvent extraction systems of acetonitrile-ethyl acetate and anhydrous ethanol-dichloromethane, as the control of acetonitrile extract:

Do 3 parallel experiments. Take a fresh egg, separate the yolk, then mix the egg yolk evenly, weigh 5g (accurate to 0.001g) and place it in a 50mL centrifuge tube, and add 10g anhydrous sodium sulfate and 20mL acetonitrile-ethyl acetate (volume ratio, 1 : 1)/absolute ethanol-dichloromethane (volume ratio, 1:1), fully stirred with a glass rod, mixed evenly, ultrasonically extracted for 10 min, and then centrifuged at 8000 rpm for 10 min, and the extract was collected into a 50 mL volumetric flask. Repeat the extraction of the precipitate with 20 mL of the corresponding extracting solution once, combine the extracting solutions in a volumetric flask, use the corresponding extraction solvent to make up to the mark, shake well, and set aside. Accurately pipet 5mL of the extract into a 10mL centrifuge tube, concentrate it to dryness by blowing nitrogen in a water bath at 50°C, add 3mL of acetonitrile, sonicate for 15 seconds, then add 3mL of water, ultrasonically mix it through a PRiME HLB solid phase extraction column, and pour it into the centrifuge tube successively. Add 1 mL of acetonitrile and 1 mL of water, wash the centrifuge tube, and pass the washing solution through the column. Rinse the solid phase extraction column with 5 mL of acetonitrile aqueous solution (volume ratio, 1:1), drain the eluent, and elute with 3 mL of acetonitrile. Centrifuge at 8000 rpm for 5 min, and take the supernatant to measure on the machine.

Acetonitrile was degreased with n-hexane and extracted with three extraction solvents, acetonitrile-ethyl acetate, acetonitrile and anhydrous ethanol-dichloromethane, and then purified by solid-phase extraction column. The recovery results are shown in Figure 4.

By comparing two degreasing methods for acetonitrile extracts-n-hexane fractional extraction degreasing and solid phase extraction column cleanup, and three different solvent extraction systems of acetonitrile, acetonitrile-ethyl acetate, absolute ethanol-dichloromethane, egg yolk The extraction efficiency of Chinese cantharidin found that acetonitrile has a good extraction efficiency for cantharidin in egg yolk, and n-hexane is used for fractional extraction and degreasing. Although the cost is low, the consumption of organic solvents is large, and n-hexane is used in the process of extraction and degreasing. This resulted in the loss of part of cantharidin in egg yolk, resulting in low recovery of cantharidin; from the obtained lighter residue color, it was inferred that the acetonitrile-ethyl acetate and absolute ethanol-dichloromethane extraction system could completely extract cantharidin from egg yolk, but the extraction of cantharidin was complete. The liquid contained a large amount of lipids, which caused blockage of the solid-phase extraction column, which could not be further purified on the solid-phase extraction column, and the matrix significantly affected the recovery rate of cantharidin. Secondly, during the extraction of cantharidin, the unpurified matrix impurities were adsorbed on the chromatographic column and could not be eluted, resulting in a decrease in the column efficiency of the chromatographic column and an increase in the system pressure at the same time.

Cantharidin is lipid-soluble and highly hydrophobic, while PRiME HLB, as a reversed-phase solid-phase extraction adsorbent, has good retention of cantharidin. The entire purification process does not require activation and equilibration, and the sample is purified under normal pressure. It is reported in the literature that the extract of cantharidin is purified by C18-SPE, silica gel-SPE, cyanopropyl-SPE and neutral alumina-SPE and other solid phase extraction columns. Most of them adopt complex activation and equilibration steps and operate under vacuum pump.

Acetonitrile is used as the extraction solvent of cantharidin. After mixing with water, it is directly passed through the solid-phase extraction column without concentration to dryness, which avoids the decomposition of cantharidin that may be caused by heating.

Therefore, in this example, acetonitrile was used for ultrasonic extraction of cantharidin from egg yolk, and a part of the extract was mixed with water and directly loaded onto a PRiME HLB solid-phase extraction column for purification.

Other pigment interference experiments in the sample of Example 4

Commonly used livestock and poultry colorants such as lutein, β-apo-8'-carotene aldehyde, and β-apo-8'-carotene acid ethyl ester are often added to feed by feed manufacturers and farms. The chromatographic separation of the mixed standard solutions of astaxanthin, zeaxanthin, lutein, cantharidin, β-apo-8'-carotene aldehyde, and β-apo-8'-carotene acid ethyl ester found that in The retention times were around 11.7 and 12.7 minutes, and other common pigment peaks did not interfere with the cis-trans isomer peaks of cantharidin, indicating that the detection method had good specificity. (see Figure 5).

methodological findings

Cantharidin has a good linear relationship in the concentration range of 0.1-10μg/mL, and the correlation coefficient (R ² ) is greater than 0.999; the detection limit of cantharidin in egg yolk is 0.05mg/kg, and the limit of quantification is 0.1mg/kg (see Figure 6), the method has good sensitivity; at low, medium and high concentration levels, the average recovery of cantharidin is greater than 85%, and the relative standard deviation is less than 10%, the method has high accuracy and precision ; The application of this method is found to be suitable for rapid and accurate detection of cantharidin yellow in fresh commercial egg yolks in the market.

Quantitative Calculation of Cantharidin

The main components of cantharidin standard product are all-trans isomers, with a content of more than 95%, followed by some cis isomers. During the analysis of cantharidin in egg yolk, it was found that the cis-cantharidin isomer accounted for about 15%. The total amount of the two isomers was counted in the quantification of cantharidin.

Determination of cantharidin in commercially available eggs

The above-mentioned acetonitrile extraction, solid phase extraction-high performance liquid chromatography method was used to detect the cantharidin yolk in fresh commercial egg yolks on the market. The content varies widely, ranging from less than 0.1 μg/g to as high as 8 μg/g.

Table 1 Experimental structure of cantharidin recovery (n=3)

Table 2 The experimental results of the determination of cantharidin yellow in different types of commercially available eggs (n=3)

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims

The invention discloses a method for extracting cantharidin from egg yolk, which comprises ultrasonically extracting cantharidin from egg yolk with acetonitrile.
Extraction method according to claim 1, is characterized in that, the consumption ratio of described egg yolk and acetonitrile is 5g: 20mL.
The extraction method according to claim 1, wherein the ultrasonic extraction also adds anhydrous sodium sulfate; the mass ratio of the egg yolk to the anhydrous sodium sulfate is 5g:10g.
The method for purifying the extract obtained by the extraction method according to any one of claims 1 to 3, wherein the method comprises the following steps:

The extract was mixed with water and directly loaded onto a PRiMEHLB solid phase extraction column for purification, eluted at normal pressure, and the obtained eluate was concentrated.
Purification method according to claim 4, is characterized in that, the model of described PRiME HLB solid phase extraction column is 60mg, 3mL.
The purification method according to claim 4, wherein the elution is 3 mL of acetonitrile elution.
The purification method according to claim 6, characterized in that, before the eluting, further comprising rinsing the PRiMEHLB solid phase extraction column; the rinsing is 5 mL of acetonitrile aqueous solution with a volume ratio of 1:1.
purifying method according to claim 4, is characterized in that, described concentrating is 50 ℃ of nitrogen blow-drying.
A solid-phase extraction-high performance liquid chromatography method for measuring cantharidin in egg yolk, characterized in that it comprises the following steps:

The residue obtained by the purification method according to any one of claims 4 to 8 is detected by RP-HPLC, and the RP-HPLC detection is Welch C18 chromatographic column separation, and external standard method is used to quantify cantharidin and its isomers.
A solid-phase extraction-high performance liquid chromatography method for the determination of cantharidin in egg yolk, characterized in that: 5 g of egg yolk is extracted with acetonitrile under the assistance of ultrasonic waves, and the extract is diluted with water and directly loaded onto a PRiME HLB solid-phase extraction column, First, rinse with 5 mL of acetonitrile aqueous solution with a volume ratio of 1:1, then with 3 mL of acetonitrile, dry the eluent with nitrogen at 50 °C, and finally separate it by a Welch C18 chromatographic column. The mobile phase is acetonitrile with a volume ratio of 95:5. -Water, flow rate is 1.2mL/min, isocratic elution, detection at UV-visible absorption wavelength 474nm, external standard method for quantitative analysis of cantharidin and its isomers.
The solid-phase extraction-high performance liquid chromatography method according to claim 10, wherein the 5g egg yolk is extracted with acetonitrile under the assistance of ultrasonic waves, and the 5g egg yolk is extracted with 20mL of acetonitrile under the assistance of ultrasonic waves.
The solid phase extraction-high performance liquid chromatography method according to claim 10, wherein the extraction of the 5g egg yolk with acetonitrile under ultrasonic assistance also comprises adding anhydrous sodium sulfate; the quality of the egg yolk and the anhydrous sodium sulfate The ratio is 5g:10g.
The solid phase extraction-high performance liquid chromatography method according to any one of claims 9 to 12, characterized in that: take a fresh egg, separate out the egg yolk, then mix the egg yolk evenly, then weigh 5 g, accurate to 0.001 g, Put it in a 50mL centrifuge tube, add 10g anhydrous sodium sulfate and 20mL acetonitrile, fully stir with a glass rod, mix well, ultrasonically extract for 10min, then centrifuge at 8000rpm for 10min, and collect the extract into a 50mL volumetric flask; precipitation use 20mL Repeat the extraction with acetonitrile once, combine the extracts in a volumetric flask, make up to the mark with acetonitrile, shake well, and set aside; accurately pipette 5mL of the extract into a 15mL centrifuge tube, add 5mL of water, and ultrasonically mix evenly and pass through Oasis
HLB solid phase extraction column, add 1 mL of acetonitrile and 1 mL of water to the centrifuge tube successively, wash the centrifuge tube, and pass the washing liquid through the column together; rinse the solid phase extraction column with 5 mL of acetonitrile aqueous solution with a volume ratio of 1:1, and finally use 3 mL of Elute with acetonitrile, collect the eluent and blow dry with nitrogen at 50 °C, add 0.5 mL of acetonitrile to dissolve the residue, centrifuge at 8000 rpm for 5 min, take the supernatant and separate it by Welch C18 chromatographic column, the mobile phase is acetonitrile with a volume ratio of 95:5 -Water, flow rate is 1.2mL/min, isocratic elution, detection at UV-visible absorption wavelength 474nm, external standard method for quantitative determination of cantharidin and its isomers, the detection limit of cantharidin in egg yolk is 0.05mg /kg, the limit of quantification is 0.1 mg/kg.