阅读说明：本技术 一种检测食品中稻曲菌素a或稻曲菌素b的方法 (Method for detecting ustilagin A or ustilagin B in food ) 是由 刘春生 孙倩 孔任 于 2020-12-01 设计创作，主要内容包括：本发明属于分析化学领域,公开了一种检测食品中稻曲菌素A或稻曲菌素B的方法。该方法的步骤包括：待测样品冷冻干燥并粉碎过筛,加入超纯水溶液超声提取,提取液加入二氯甲烷涡旋离心,收集的上清液采用WAX固相萃取柱净化。净化后的待测样品借助超高效液相色谱-电喷雾质谱联用仪分析稻曲菌素A和稻曲菌素B的含量。本方法可同时实现对大米、谷糠、米粉、青贮饲料等多种稻谷制品中UA和UB的定性定量检测,回收率高可达到87%-96%,检出限可低至40 ng/kg,在食品及饲料中毒素残留分析方面具有较大的应用前景。(The invention belongs to the field of analytical chemistry, and discloses a method for detecting ustilaginoidin A or ustilaginoidin B in food. The method comprises the following steps: freeze-drying, pulverizing, sieving, adding ultrapure water solution, ultrasonic extracting, adding dichloromethane into the extractive solution, centrifuging, and purifying the collected supernatant with WAX solid phase extraction column. And analyzing the contents of the ustilaginoidin A and the ustilaginoidin B in the purified sample to be detected by means of an ultra-high performance liquid chromatography-electrospray mass spectrometer. The method can simultaneously realize qualitative and quantitative detection of UA and UB in various rice products such as rice, bran coat, rice flour, silage and the like, has high recovery rate of 87-96 percent and detection limit as low as 40ng/kg, and has wide application prospect in the aspect of toxin residue analysis in food and feed.)

1. A method for detecting ustiloxin A and/or ustiloxin B in food, comprising the following steps:

(1) sample extraction:

freeze-drying and pulverizing the product to be tested, adding water, oscillating, performing ultrasonic treatment in ice bath, centrifuging, collecting the supernatant, continuously extracting the residue according to the steps, and mixing the supernatants; adding dichloromethane into the supernatant, shaking, centrifuging and collecting the upper-layer water phase to obtain a solution to be purified;

(2) solid phase micro-extraction column purification

Adjusting the pH range of the solution to be purified to 4.0-6.0 by adopting 0.1-1% formic acid solution, and passing through a WAX column which is activated in advance at the flow rate of 0.1-1mL/min to discard effluent; leaching the solid phase extraction small column with 1-4mL of formic acid solution with pH of 4-6 and 1-4mL of methanol in sequence, finally eluting with 1-5% ammoniated methanol, and collecting eluent; blowing the elution liquid nitrogen to be clean and dry, re-dissolving the elution liquid nitrogen with 0-10% methanol-water, filtering the elution liquid nitrogen by a filter membrane, and performing detection on the elution liquid nitrogen on a machine;

the WAX activation conditions were: sequentially measuring 2-4mL of methanol and 2-4mL of aqueous formic acid solution with pH of 4-6, and allowing the aqueous formic acid solution to pass through a WAX column at a flow rate of 0.1-1 mL/min;

(3) detection of ultra-high performance liquid chromatography-triple quadrupole mass spectrometer

Detecting the liquid to be detected by using a ultra-high liquid chromatography-triple quadrupole tandem mass spectrometer (LC-MS/MS), and quantifying by adopting an external standard method to detect UA and UB in the sample to be detected; wherein:

(a) ultra-high performance liquid chromatography conditions: the chromatographic column is an ACQUITY UPLC HSS T3 column (2.1 mm. times.100 mm,1.8 μm); the column temperature is 30-45 ℃; the sample injection volume is 1-5 mu L; the flow rate is 0.20-0.40 mL/min; the mobile phase adopts LC-MS grade methanol (A) and 0.01-0.1% formic acid-water (B), the operation gradient is 0-2min, 2-5% A; 2-6min, 40-95% A; 6-8min, 95-100% A; 8-8.5min, 2-5% A; 8.5-10min, 2-5% of A.

(b) Mass spectrum conditions: the ion source is ESI (+), the capillary voltage is 0.5-1.0KV, the desolvation temperature is 300-400 ℃, the taper hole voltage is 20-60V, and the desolvation flow rate and the collision air flow rate are respectively 600-800 and 100-200L/h; and (5) monitoring the multistage reaction.

2. The method of claim 1, wherein: the qualitative ion pairs of UA and UB are m/z 674.30>209.00 and m/z646.30>187.00 respectively; the quantitative ion pairs of UA and UB are m/z 674.30>187.00 and m/z646.30> 181.00 respectively.

3. The method of claim 1, wherein: the WAX solid phase extraction column is loaded with formic acid water solution with pH value of 5.0, and the maximum loading volume is 20 mL.

4. The method of claim 1, wherein: the elution condition of the WAX solid phase extraction column is 1% ammoniated methanol solution.

5. The method of claim 1, wherein: the liquid phase mobile phase in the step (3) (a) is a methanol and 0.01% formic acid-water system.

6. The method of claim 1, wherein: the activation conditions for the WAX column were: 3mL of methanol and 3mL of aqueous formic acid solution at pH 5.0 were sequentially measured and passed through a WAX column at a flow rate of 1 mL/min.

7. The method of claim 1, wherein: specific mass spectral parameters of UA and UB:

mass spectrometry conditions for UA and UB detection

8. Use of the method of claim 1 for the detection of UA and/or UB in rice, rice bran, rice hulls, rice, silage.

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting ustilagin A or ustilagin B in food.

Background

Rice is one of three most important grain crops in the world, and provides a food source for nearly 21% of people in the world. In recent years, along with large-area popularization of high-yield and high-spike-density hybrid rice, change of rice cultivation modes and the like, false smut becomes one of three fungal diseases in rice production in China, and a main secondary metabolite, namely ustilagin, of the false smut is widely concerned. The ustilaginoidea virens is a water-soluble cyclopeptide mycotoxin, and 6 structurally similar compounds are isolated and identified currently, wherein ustilaginoidea virens A (UA) and ustilaginoidea virens B (UB) are toxins with the highest content and the highest toxicity. The ustilaginoidin is a potential eukaryotic antimitotic agent and has wide biological activity on animal and plant cells. It has been reported that excessive ingestion of ustiloxin can cause multiple organ lesions and even death of the individual. In view of the prevalence and high frequency of false smut, selective monitoring of UA and UB residual levels in rice and its products is of great importance in assessing food safety.

At present, the conventional detection method of mycotoxin mainly adopts a chromatographic analysis method, a liquid chromatography-mass spectrometry technology and an immune enzyme-linked adsorption method. Among them, the immune enzyme-linked method is liable to cause false positive, and when a plurality of ustilaginoids are present simultaneously, it is impossible to accurately provide the precise content of each toxin simultaneously. The liquid chromatography-mass spectrometry (LC-MS/MS) is a method of detection which has been developed most rapidly in recent years, and is favored by researchers because of its advantages such as good selectivity and high sensitivity. In recent years, some groups of subjects have achieved good results in the detection of ustilaginoidea virens by LC-MS/MS technology. For example, Shiburong et al uses deionized water containing PEG4000 as extraction solvent, uses C18 column solid phase extraction column as purification column, and realizes the detection of oryzanol in rice by LC-MS/MS, wherein the detection limit of UA is 5.0 μ g/L. Chen and the like combine the molecular structure characteristics of UA and UB, utilize the characteristic that the two can be positively charged under the acidic condition, and construct a quantitative analysis method for UA and UB in polished rice by taking a PCX mixed cation exchange column as a purifying column, wherein the quantitative limit of UA and UB is 1 mug/kg. Although the methods show excellent performance when detecting the ustilagin in the rice, the methods have the defects of large sample requirement (generally 5-20g), low sensitivity and the like, and are not suitable for detecting the trace amount of the ustilagin in other rice products. The main reason is that the content of the ustilaginoidin in the rice products such as silage, rice husks and the like is relatively low, and the rice products contain various pigments with large polarity and organic acid interferents, so that the pigments are difficult to remove in the sample pre-purification and enrichment steps, and the serious matrix inhibition effect is easily caused in the mass spectrometry.

When the medium trace substance of a complex matrix is analyzed, an effective pre-purification and enrichment means is one of the methods for improving the detection limit of a sample. It is to be noted that the kind of the mobile phase not only affects the separation effect of the target compound, but also determines the ionization efficiency of the component to be measured. For example, the ionization efficiency of estrogen in NH4F system is obviously higher than that of ammonia water system; the ionization efficiency of various mycotoxins such as aflatoxin, fumonisin, and fumagillin in an ammonium acetate system is obviously higher than that of a formic acid system. Therefore, optimizing chromatographic conditions is also one of the most effective methods to increase the sensitivity of the method.

Aiming at the technical problems in the prior art, the invention aims to provide a pretreatment method for realizing the rapid, accurate and high-sensitivity detection of UA and UB in food.

Disclosure of Invention

The invention aims to provide a method for detecting ustilagin A or ustilagin B in food, which can quickly detect trace amounts of ustilagin A and ustilagin B in rice, rice bran, rice husk, rice, silage and the like, and realize quick, accurate and high-sensitivity detection.

In order to achieve the purpose, the invention adopts the following technical measures:

a method for detecting ustiloxin A and/or ustiloxin B in food, comprising the following steps:

(1) sample extraction:

freeze-drying and pulverizing the product to be tested, adding water, oscillating, performing ultrasonic treatment in ice bath, centrifuging, collecting the supernatant, continuously extracting the residue according to the steps, and mixing the supernatants; adding dichloromethane into the supernatant, shaking, centrifuging and collecting the upper-layer water phase to obtain a solution to be purified;

(2) solid phase micro-extraction column purification

Adjusting the pH range of the solution to be purified to 4.0-6.0 by adopting 0.1-1% formic acid solution, and passing through a WAX column which is activated in advance at the flow rate of 0.1-1mL/min to discard effluent; leaching the solid phase extraction small column with 1-4mL of formic acid solution with pH of 4-6 and 1-4mL of methanol in sequence, finally eluting with 1-5% ammoniated methanol, and collecting eluent; blowing the elution liquid nitrogen to be clean and dry, re-dissolving the elution liquid nitrogen with 0-10% methanol-water, filtering the elution liquid nitrogen by a filter membrane, and performing detection on the elution liquid nitrogen on a machine;

the WAX activation conditions were: sequentially measuring 2-4mL of methanol and 2-4mL of aqueous formic acid solution with pH of 4.0-6.0, and allowing the aqueous formic acid solution to pass through a WAX column at a flow rate of 0.1-1 mL/min;

(3) detection of ultra-high performance liquid chromatography-triple quadrupole mass spectrometer

Detecting the liquid to be detected by using a ultra-high liquid chromatography-triple quadrupole tandem mass spectrometer (LC-MS/MS), and quantifying by adopting an external standard method to detect UA and UB in the sample to be detected; wherein:

(a) ultra-high performance liquid chromatography conditions: the chromatographic column is an ACQUITY UPLC HSS T3 column (2.1 mm. times.100 mm,1.8 μm); the column temperature is 30-45 ℃; the sample injection volume is 1-5 mu L; the flow rate is 0.20-0.40 mL/min; the mobile phase adopts LC-MS grade methanol (A) and 0.01-0.1% formic acid-water (B), the operation gradient is 0-2min, 2-5% A; 2-6min, 40-95% A; 6-8min, 95-100% A; 8-8.5min, 2-5% A; 8.5-10min, 2-5% of A.

(b) Mass spectrum conditions: the ion source is ESI (+), the capillary voltage is 0.5-1.0KV, the desolvation temperature is 300-400 ℃, the taper hole voltage is 20-60V, and the desolvation flow rate and the collision air flow rate are respectively 600-800 and 100-200L/h; and (5) monitoring the multistage reaction.

In the above-described method, preferably, the pair of qualitative ions of UA and UB is m/z 674.30>209.00 and m/z646.30>187.00, respectively; the quantitative ion pairs of UA and UB are m/z 674.30>187.00 and m/z646.30> 181.00 respectively.

In the above method, preferably, the loading conditions of the WAX solid phase extraction column are a pH 5.0 aqueous formic acid solution, and the maximum loading volume is 20 mL.

In the above method, preferably, the elution condition of the WAX solid phase extraction column is 1% ammoniated methanol solution.

In the above-mentioned method, preferably, the mobile phase of the liquid phase in step (3) (a) is methanol and 0.01% formic acid-water system.

In the above method, preferably, the activation conditions of the WAX column are: 3mL of methanol and 3mL of aqueous formic acid solution at pH 5.0 were sequentially measured and passed through a WAX column at a flow rate of 1 mL/min.

In the above method, preferably, the specific mass spectrum parameters of UA and UB are:

mass spectrometry conditions for UA and UB detection

The method is suitable for detecting UA and UB in rice, rice bran, rice hulls, rice and silage. Compared with the prior art, the invention has the following advantages:

the invention establishes a liquid quality detection method for the ustilagin, can effectively detect the ustilagin in rice products such as rice, bran coat, rice paste, silage and the like, and has important significance for developing the environmental distribution and environmental hazard evaluation of the ustilagin.

The method has the advantages of high sensitivity, good repeatability and the like, the standard addition recovery rate of UA and UB is 87-96%, the limit of quantitative detection can be as low as 40ng/kg, and the sample matrix basically has no inhibition effect on target molecules, so that the method can accurately analyze the residue condition of the ustilaginoidin A or the ustilaginoidin B in food or feed.

3 the invention adopts weak anion solid phase exchange column to extract and enrich the trace amount of ustilaginoidea virens A and ustilaginoidea virens B in the paddy and the related products. In the system, interferents such as sugar, organic alkali, organic weak acid and the like are basically not reserved or weakly reserved on the WAX column, so that the first purification of the sample to be detected is realized. And then, the non-specifically adsorbed large polar pigment molecules on the chromatogram can be effectively removed by eluting with a pure methanol solution, so that the UA in the sample to be detected is further purified and enriched, and the matrix effect and the baseline noise of the sample are reduced to the maximum extent.

Drawings

FIG. 1 shows the recovery rate of UA and UB from rice by various types of solid phase extraction columns.

Fig. 2 shows the distribution of UA and UB in 44 commercial rice portions.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available. The WAX column described in the examples of the invention was purchased from Anan Spectrum (CNW, 200mg,3 CC).

Example 1:

a method for detecting ustilagin A or ustilagin B in food, comprising the steps of:

the invention

1.1 sample extraction:

freeze drying the product, crushing with high speed crusher and 100 mesh sieving. Weighing 0.5g of crushed sample to be tested, adding a mixed standard solution of ustiloxin (containing 100 mug/L UA and 100 mug/L UB) to ensure that the final concentrations of the UA and the UB in the sample are 0 and 0 mug/kg respectively; 2 and 2. mu.g/kg; 10 and 10. mu.g/kg (with UA and UB concentrations of 0 and 0. mu.g/kg added as blank matrix samples, i.e., blank controls, both 2 and 10. mu.g/kg of labeled blank matrix samples), were equilibrated at room temperature for 30min after mixing. Adding 6mL of ultrapure water, vortexing for 1min, then oscillating for 15min, performing ice bath ultrasound for 15min, centrifuging at 7000rpm for 10min, and collecting supernatant. Extracting the residue twice with 6mL of 2 water, and mixing the supernatants to obtain extractive solution;

the sample to be detected is as follows: rice, rice bran, rice hull, rice, silage without UA and UB.

1.2 liquid-liquid extraction:

adding 15mL of dichloromethane into the extract, carrying out vortex oscillation for 1min, centrifuging at 7000rpm for 5min, and collecting the upper aqueous phase to be purified.

1.3 solid phase extraction:

the pH of the solution to be purified was adjusted to 5.0 with 0.1% formic acid solution, and the solution was passed through a WAX column activated in advance at a flow rate of 1mL/min (in the activation manner: 3mL of methanol and 3mL of aqueous formic acid solution having a pH of 5.0 were used in this order, and passed through the WAX column at a flow rate of 1mL/min, CNW 200mg,3CC), and the effluent was discarded. And (3) sequentially eluting the solid-phase extraction column with 2mL of aqueous formic acid solution with the pH value of 5.0 and 2mL of methanol at the flow rate of 1mL/min, discarding the eluent, and pumping by a vacuum pump for 20 min. Finally, the eluent is eluted twice (the flow rate is controlled within 1 mL/min) by using 2mL of 1% ammoniated methanol solution, and the eluates are combined. And (4) blowing the elution liquid nitrogen to be clean and dry, fixing the volume to 200 mu L by adopting 5% methanol-water, filtering the solution by using a 0.22 mu M filter membrane, transferring the solution to an interpolation machine for detection.

1.4 LC-MS/MS quantitative determination of ustilagin A and ustilagin B

And (3) diluting the solution in the step 1.3 by a 5% methanol-water gradient to enable the final concentration of UA and UB to be 0.05-100 mu g/L, and detecting the total ion flow graph of the solution in the presence of UA and UB with the aid of LC-MS/MS. And then respectively establishing standard curves of UA and UB by taking the concentration as an abscissa and taking the peak area of a selective detection graph of the quantitative ion pair as an ordinate.

The LC-MS/MS detection equipment comprises: waters ACQUITYH-plus (UHPLC) ultra performance liquid chromatography tandemTQ-XS triple quadrupole mass spectrometry (Milford, MA, USA).

The liquid chromatography adopts ACQUITY UPLC HSS T3 column (2.1mm × 100mm,1.8 μm) and mobile phase of methanol (A) and 0.01% formic acid water (B); the column temperature was 40 ℃; the sample injection volume is 2 mu L; the flow rate was 0.3 mL/min. The mobile phase used LC-MS grade methanol (a) and 0.01% formic acid-water (B) and the running gradient is as shown in the table below.

Liquid phase operating gradient

Time (min)	A(％)
		0	5
0.5	5
		6	95
8	100
		8.2	5
10	5

An electrospray ionization (ESI +) ion source is adopted in a mass spectrum, the capillary voltage is 1.0KV, the desolvation temperature is 300 ℃, the taper hole voltage is 30V, the desolvation flow rate and the collision air flow rate are respectively 600L/h and 150L/h, a multi-reaction monitoring (MRM) mode is used for detecting and scanning a target analyte, and the qualitative ion pairs of UA and UB are respectively m/z 674.30>209.00 and m/z646.30> 187.00; the quantitative ion pairs of UA and UB are m/z 674.30>187.00 and m/z646.30> 181.00 respectively.

Mass spectrometry conditions for UA and UB detection

In the range of 0.05-100 mug/L, the concentrations of UA and UB and the corresponding chromatographic peak areas show good linear relation, and R²>0.999. The recovery rate of the method and the matrix effect of the sample are respectively evaluated by adopting a standard addition recovery method and a standard addition method (refer to Waters sample pretreatment and chromatographic column handbook)Angle.) results are shown in the table below.

In the rice products of rice, rice bran, rice and silage, the recovery rate of UA is 87.3-92.1%, the recovery rate of UB is 87.6-95.7%, the matrix effect of UA is 91.2-96.2%, and the matrix effect of UB is 93.4-96.2%. And (4) calculating the minimum quantitative limit according to the S/N-10 method, wherein the lower limit of the quantitative detection of UA and UB is 40ng/kg and 25ng/kg respectively (remarks: the detection limit is converted into the mass concentration of the sample to be detected). Therefore, the method has the advantages of good accuracy and high sensitivity.

Spiked recovery of UA and UB in different matrix samples and matrix effects

Example 2:

influence of different extracts of to-be-detected products on final detection sensitivity

0.5g of a blank rice sample is weighed and a volume of a mixed solution of UA and UB is added so that the mass concentration of UA and UB in the rice is 2 mug/kg.

UA and UB are both water-soluble cyclic peptides, which have better water solubility in water and methanol, and in step 1.1 of example 1, the applicant selects methanol-water (10: 0-0: 10) with different ratios as extraction solvents. The rest of the experimental conditions were the same as in example 1.

Experiments show that the concentration of methanol does not influence the extraction efficiency of UA and UB, but high concentration of methanol is easy to extract organic acid, pigment, fat and the like in a sample, so that the baseline noise of the sample is increased.

Example 3:

adsorption effect of ion exchange columns of different brands and functions on UA and/or UB:

0.5g of a blank rice sample is weighed and a volume of a mixed solution of UA and UB is added so that the mass concentration of UA and UB in the rice is 2 mug/kg.

UA and UB are both amphipathic molecules with pKa1 and pKa2 of 1.05 and 8.80, respectively, and their charging properties in free solution depend on the pH of the solution.

The applicants have replaced the ion exchange column of the process described in example 1, step 1.3, with the following respective ion exchange columns: agilent PCX mixed cation exchange column (200mg,3CC), Agilent PAX mixed anion exchange column (200mg,3CC), Waters Oasis MCX mixed cation exchange column (200mg,3CC), Waters Oasis MAX mixed anion exchange column (200mg,3CC), Waters Sep-Pak aminopropyl (NH2) solid phase extraction column (200mg,3CC), Waters Oasis WAX mixed weak anion exchange column (200mg,3CC), Anam CNW WAX weak anion exchange column (200mg,3 CC).

The remaining parameters were identical to step 1.3 to examine the effect of different ion exchange columns on the adsorption capacity of UA and UB:

the results are shown in FIG. 1. Besides the ion exchange effect, PCX, PAX, MCX and MAX have stronger non-specific adsorption effect on UA and UB, and the recovery rate of UA and UB under the conventional elution condition is lower than 70%. Oasis WAX and Sep-PakNH₂And the pH value is 0-6, and the UA and the UB are not retained. The CNW WAX weak anion exchange column has the retention efficiency of up to 95% for UA and UB in the pH range of 4-6, and the recovery rate of UA and UB is more than 85%.

Example 4:

effect of mobile phase formic acid concentration on sensitivity in liquid chromatography:

0.5g of a blank rice sample not containing UA and UA was weighed and added with a volume of a mixed solution of UA and UB such that the mass concentrations of UA and UB in the rice were 2. mu.g/kg.

The procedure and parameters were the same as in example 1 except that the concentration of formic acid (0 to 0.1%) in the mobile phase in the liquid chromatography in step 1.4 of example 1 was changed.

The experimental result shows that 0.1% formic acid is generally adopted for elution and separation in the prior art, but in the invention, 0.01% formic acid can obviously reduce the half-peak width of UA and UB and enhance the mass spectrum response, but high concentration formic acid (0.1%) can inhibit the ionization efficiency of UA and UB and reduce the sensitivity of the method. Subsequently, the separation of UA and UB was examined using methanol-water and acetonitrile-water as mobile phases, respectively. The results show that the ionization efficiency of UA and UB in a methanol-water system is significantly higher than that of an acetonitrile-water system.

Example 5:

effect of different chromatography columns on sensitivity:

0.5g of a blank silage sample is weighed, and a certain volume of mixed solution of UA and UB is added, so that the mass concentration of UA and UB in the sample is 2 mug/kg.

The column type and mobile phase gradient of the liquid chromatography in step 1.4 of example 1 were changed, and the rest of the steps and parameters were the same as those of example 1.

The specific types of the chromatographic column are as follows: ACQUITY UPLC BEH C18 column (2.1 mm. times.100 mm,1.7 μm), ACQUITY UPLC HSS T3 column (2.1 mm. times.100 mm,1.8 μm)

The experimental results show that the C18 column has poor retention capacity for UA and UB under most mobile phase conditions and essentially no separation capacity for UA and UB. In addition, when methanol and 0.01% formic acid-water are used as mobile phases, the UA and UB in the sample to be tested have serious matrix inhibition effect, and the sensitivity of the method is reduced. When T3 is used as a chromatographic separation column, UA and UB have better separation degree, can realize better baseline separation with impurities in a sample to be detected, and have negligible matrix effect, thereby greatly increasing the sensitivity of the method.

Example 6:

influence of different loading conditions and elution conditions in the WAX solid phase extraction column on the sensitivity:

0.5g of a blank silage sample is weighed, and a certain volume of mixed solution of UA and UB is added, so that the mass concentration of UA and UB in the sample is 2 mug/kg.

The loading conditions, the washing conditions and the elution conditions of the WAX column in step 1.3 of example 1 were changed, and the rest of the steps and parameters were the same as those of example 1.

UA and UB are amphoteric molecules and are electronegative under weak acidic conditions. First, the effect of the pH of the loading solution on the adsorption effect of UA and UB on the weak anion exchange column WAX was compared. The experimental results show that UA and UB have good retention capacity on WAX column when pH of the loading solution is between 4-6; the retention capacity is weak when the pH of the solution is too high or too low. Subsequently, the applicant found that the concentration of ammonium acetate in the loading solution significantly affected the retention of UA and UB, and that the adsorption of UA and UB tended to be inversely related to the salt concentration in the loading solution. On the basis, the elution and elution conditions of the WAX column are further optimized, and finally, when the small column is sequentially eluted by 2mL of formic acid aqueous solution with the pH value of 5 and 2mL of methanol solution and eluted by 0.5% -2% of ammoniated methanol, the high recovery rate can be obtained, impurities such as pigment in a sample to be detected can be effectively removed, and the baseline noise is reduced. Considering individual differences of the WAX columns, complexity of actual samples and experimental stability comprehensively, formic acid solution with pH of 5.0 is selected as loading solution, 2mL of water and 2mL of methanol are selected as eluting solvent, and 1% ammonia water methanol solution is selected as sample eluent in the embodiment 1 of the invention.

Example 7:

the method disclosed by the invention is applied to the detection of residues of UA and UB in commercial rice:

the residue of UA and UB in 44 rice samples collected in en shi city, hubei province in 2020 was analyzed and conducted as described in example 1.

1) Sample extraction:

the rice samples were freeze dried and crushed using a high speed crusher and sieved through a 100 mesh sieve. 0.5g of the pulverized sample was weighed, 6mL of ultrapure water was added, vortexed for 1min, shaken for 15min and sonicated for 15min (4 ℃), centrifuged at 7000rpm for 10min, and the supernatant was collected. The residue was extracted twice more with 6mL x 2 water and the supernatants were combined.

2) Liquid-liquid extraction:

adding 15mL of dichloromethane into the extract, carrying out vortex oscillation for 1min, centrifuging at 7000rpm for 5min, and collecting the upper aqueous phase to be purified.

3) Solid phase extraction:

the pH of the above-mentioned solution to be purified was adjusted to 5.0 with 0.1% formic acid solution, and the effluent was discarded by passing through a previously activated WAX column (3mL of methanol, 3mL of aqueous 5.0 formic acid solution) at a flow rate of 1 mL/min. And (3) sequentially leaching the solid-phase extraction column by using 2mL of aqueous formic acid solution with the pH value of 5.0 and 2mL of methanol, discarding the leaching solution, and pumping out for 20min by using a vacuum pump. Finally, the eluent was eluted twice with 2mL of 1% ammoniated methanol solution and combined. And (4) blowing the elution liquid nitrogen to be clean and dry, fixing the volume to 200 mu L by adopting 5% methanol-water, filtering the solution by using a 0.22 mu M filter membrane, transferring the solution to an interpolation machine for detection.

4) LC-MS/MS quantitative detection of UA and UB

Preparing a mixed solution containing UA and UB, diluting the solution in a gradient manner to enable the final concentration of UA and UB to be 0.05-100 g/L, and detecting a total ion flow diagram of the solution in the presence of UA and UB with the aid of LC-MS/MS. And then respectively establishing standard curves of UA and UB by taking the concentration as an abscissa and taking the peak area of a selective detection graph of the quantitative ion pair as an ordinate. Wherein the LC-MS/MS detection conditions are as follows: waters ACQUITYH-plus (UHPLC) ultra performance liquid chromatography tandemTQ-XS triple quadrupole mass spectrometry (Milford, MA, USA). Wherein the mass spectrum adopts an electrospray ionization (ESI +) ion source and a multi-reaction monitoring (MRM) mode to detect and scan target analytes, and the qualitative ion pairs of UA and UB are respectively m/z 674.30>209.00 and m/z646.30>187.00, respectively; quantitative ion pairs of UA and UB are m/z 674.30 respectively>187.00 and m/z646.30>181.00. The liquid chromatography adopts ACQUITY UPLC HSS T3 column (2.1mm × 100mm,1.8 μm) and mobile phase of methanol (A) and 0.01% formic acid water (B); the column temperature was 40 ℃; the sample injection volume is 2 mu L; the flow rate was 0.3 mL/min. The mobile phase adopts LC-MS grade methanol (A) and 0.01% formic acid-water (B), and the operation gradient is 0min and 5% A; 0.5min, 5% A; 6min, 95% A; 8min, 100% A; 8.2min, 5% A; 10min, 5% A.

The detection result is shown in figure 2, the detection rate of UA is up to 100%, and the average value is 1.69 mug/kg; the detection rate of UB was 86.4%, and the average value was 0.35. mu.g/kg.