CN114720598A - Method for determining residual quantity of validamycin A in infant rice flour by hydrophilic interaction chromatography-tandem mass spectrometry - Google Patents

Abstract

The invention discloses a method for determining residual quantity of validamycin A in infant rice flour by a hydrophilic interaction chromatography-tandem mass spectrometry method, which comprises the following steps: extracting a sample, and redissolving an ammonium acetate solution; purifying; the measurement is carried out by adopting liquid chromatography-tandem mass spectrometry. The method has good linear relation (R) within the range of 0.1-5.0 ng/mL²> 0.99), a limit of detection (LOD) of 2.0. mu.g/kg and a limit of quantitation (LOQ) of 5.0. mu.g/kg. The method is characterized in that 3 horizontal standard adding recovery experiments of 5.0, 50 and 200 mug/kg are carried out in blank infant rice flour samples, the average recovery rate of the method is 88.5% -100.5%, and the Relative Standard Deviation (RSD) is 4.84% -10.08%. The method is simple, convenient, rapid, sensitive and good in repeatability, and can be used for qualitative and quantitative analysis of validamycin A in infant rice flour.

Description

Method for determining residual quantity of validamycin A in infant rice flour by hydrophilic interaction chromatography-tandem mass spectrometry

Technical Field

The invention belongs to the field of food detection, and particularly relates to a method for determining residual quantity of validamycin A in infant rice flour by a hydrophilic interaction chromatography-tandem mass spectrometry method.

Background

The validamycin is an agricultural antibiotic produced by streptomyces hygroscopicus validamycin variants, and comprises six components A-F, wherein the main active substance is validamycin A. Validamycin has broad-spectrum antifungal activity, has the strongest antibacterial activity on rhizoctonia solani, and has good effect of preventing and treating plant diseases. Because of the characteristics of strong therapeutic action, low toxicity, low price and the like, validamycin A has become one of the most important agricultural antibiotics with the largest yield in China. Due to potential food safety risks, countries around the world have relevant regulations on the limited value of validamycin. The european union banned the use of validamycin (validamycin); the japanese positive list specifies the residual limits of validamycin in brown rice, soybeans, vegetables and fruits; GB 2763-. The infant cereal complementary food is commonly called infant rice flour and is the most ideal infant complementary food recommended by the World Health Organization (WHO). The infant supplement food is prepared by taking grains such as rice, millet, wheat and the like as main raw materials, selectively adding white granulated sugar, vegetables, fruits, eggs, meat and the like, and adding minerals such as calcium, phosphorus, iron and the like, vitamins and the like. The infant has single dietary type, the intestinal function is weak, and the dietary safety of the infant is strictly controlled. At present, the existing literature has few reports about validamycin residue detection, research substrates mainly comprise rice, cane shoots, bighead atractylodes rhizome and the like, and only a few literatures develop researches on various food substrates. The detection method of validamycin in infant rice flour is not reported, so that the research establishes a detection method of validamycin residue in infant rice flour so as to provide technical support for the safety of infant food.

Currently, the analysis methods of validamycin a mainly include capillary electrophoresis, ion exchange chromatography, gas chromatography, liquid chromatography and liquid chromatography-tandem mass spectrometry. Validamycin A belongs to aminoglycoside compounds, has strong polarity and is difficult to gasify, and the gas chromatography requires derivatization and has complicated steps. Meanwhile, the ultraviolet absorption wavelength of the fluorescent powder is 210 nm, and the fluorescent powder is easily interfered by a matrix when analyzed by a liquid chromatography or a capillary electrophoresis method, so that the accuracy of a qualitative and quantitative result is influenced. The liquid chromatography-tandem mass spectrometry has the advantages of strong anti-interference capability, high sensitivity and the like, and is more and more widely applied to detection of pesticide and veterinary drug residues.

Disclosure of Invention

In the study, validamycin A in infant rice flour is extracted by methanol-water, and is analyzed and determined after being purified by an MCX column. The method has simple pretreatment process and short detection period, and the methodological indexes of sensitivity, recovery rate and the like of the method also meet the qualitative and quantitative requirements.

The invention aims to establish an analysis method for determining validamycin A by using a hydrophilic interaction chromatography-tandem mass spectrometry, wherein validamycin A in a sample is extracted by methanol-water (70 + 30), 1mL of extracted liquid nitrogen is dried and then redissolved by using 20 mmol/L ammonium acetate (pH = 3.0), purified by an MCX solid phase extraction column, separated by a BEH Amide chromatographic column and scanned by adopting an electrospray mass spectrometry Multiple Reaction Monitoring (MRM) mode, and quantified by an external standard method. The method has good linear relation (R) within the range of 0.1-5.0 ng/mL²> 0.99), a limit of detection (LOD) of 2.0. mu.g/kg and a limit of quantitation (LOQ) of 5.0. mu.g/kg. The method is characterized in that the standard adding recovery experiments of 5.0, 50 and 200 mug/kg 3 levels are carried out in blank infant rice flour samples, and the average recovery rate of the method is 88.5% -10%0.5% and the Relative Standard Deviation (RSD) is between 4.84 and 10.08%. The method is simple, convenient, rapid, sensitive and good in repeatability, and can be used for qualitative and quantitative analysis of validamycin A in infant rice flour.

The invention is realized by the following technical scheme:

a method for determining residual quantity of validamycin A in infant rice flour by using a hydrophilic interaction chromatography-tandem mass spectrometry method comprises the following steps:

(1) sample extraction: adding the sample into the extracting solution, carrying out secondary extraction, centrifuging, combining the two supernatants, drying by nitrogen, re-dissolving by using an ammonium acetate solution, and purifying;

(2) purifying: purifying the MCX solid phase extraction column, leaching, eluting the elution solution, blowing nitrogen, redissolving the initial mobile phase, filtering by a microporous filter membrane, and performing analysis and determination by a liquid chromatography-tandem mass spectrometer;

(3) and (4) measuring by using liquid chromatography-mass spectrometry.

Further, the conditions of the liquid chromatography are: and (3) chromatographic column: waters ACQUITY UPLC BEH Amide (100X 2.1mm, 1.7 μm); mobile phase: the phase A is 5 mmol/L ammonium acetate water solution, and the phase B is acetonitrile; a gradient elution procedure; column temperature: at 40 ℃; sample introduction amount: 5 μ L.

Further, the mass spectrometry conditions were: an ion source: an electrospray ion source; the detection mode is as follows: monitoring multiple reactions; the scanning mode is as follows: scanning in a positive ion mode; electrospray voltage: 5500V; atomizer pressure: 50L/h; air curtain air pressure: 35L/h; auxiliary gas pressure: 65L/h; collision gas pressure: 9L/h; ion source temperature: at 600 ℃. The atomized gas and the auxiliary gas are compressed air, and the air curtain gas and the collision gas are high-purity nitrogen. The qualitative ion pair, the quantitative ion pair, the collection time, the declustering voltage and the collision energy are shown in the table.

Further, the specific steps of the step (1) are as follows: accurately weighing 2.5 g of sample in a 50 mL centrifuge tube, adding 15 mL of extracting solution, uniformly mixing by vortex, ultrasonically extracting for 15 min, centrifuging for 5 min at 8000 r/min, and taking out supernatant in a 25 mL colorimetric tube; adding 8 mL of extraction solution for second extraction, centrifuging, mixing the two supernatants, and finally fixing the volume to the scale with the extraction solution and shaking up; 1mL of the suspension was removed and dried at 40 ℃ with nitrogen, and then reconstituted with 1mL of 20 mmol/L ammonium acetate solution (pH = 3.0).

Further, the specific steps of the step (2) are as follows: activating the MCX solid phase extraction column by using 3 mL of methanol and 3 mL of water in sequence; the composite solution is completely passed through a solid phase extraction column at a flow rate of not more than 1.0 mL/min; leaching with 3 mL of ultrapure water and 3 mL of methanol, discarding leacheate, and draining the small column; eluting with 5.0 mL of elution solution, blowing dry with nitrogen at 40 ℃, redissolving with 5.0 mL of initial mobile phase, and filtering with a microporous membrane.

Advantageous effects

An analysis method for determining validamycin A in infant rice flour by using a hydrophilic interaction chromatography-tandem mass spectrometry method is established. Extracting with methanol-water, re-dissolving ammonium acetate solution after blowing and concentrating with appropriate amount of extracted liquid nitrogen, purifying with MCX solid phase extraction column, and quantifying with matrix matching with standard solution; the recovery rate, precision and sensitivity of the method can meet various detection requirements. The method has simple and rapid pretreatment process, and is suitable for qualitative and quantitative analysis of validamycin A residue in infant rice flour.

Drawings

FIG. 1 is a structural formula of validamycin A;

FIG. 2 influence of methanol content on validamycin A recovery rate;

FIG. 3 shows the effect of different solid phase extraction columns on the recovery rate of validamycin A;

FIG. 4 influence of pH of the loading solution on validamycin A recovery rate;

FIG. 5 is a mass spectrum of quantitative limit-plus-standard multiple reaction monitoring in infant rice flour.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

1 experimental part

1.1 instruments, reagents and materials

AB SCIEX Triple Quad 6500+ ultra high performance liquid chromatography-tandem mass spectrometer (equipped with electrospray ionization source) (AB SCIEX corporation, usa); 3-18K bench high speed centrifuge (Sigma, Germany); BT 125D electronic balance (Sartorius, germany); vortex mixer model MS3 (IKA, germany); SB-800DTD ultrasonic cleaning apparatus (Ningbo Xinzhi Biotech Co., Ltd.); Milli-Q ultrapure water system (Millipore, USA).

Methanol, acetonitrile (chromatographically pure, Merck, Germany), ammonium acetate (chromatographically pure, Sigma-aldrich, USA); acetic acid, ammonia (analytical pure, chemical reagents of national drug group, ltd.); ammonium acetate (analytically pure, west longa science ltd); validamycin A standard (CAS number: 37248-47-8, purity 86%) was purchased from Dr.Ehrenstontorfer, Germany; MCX solid phase extraction column (60 mg, 3 mL, Waters, USA); BEH Amide column (100X 2.1mm, 1.7 μm, Waters Corp.); the experimental water was prepared from an ultrapure water meter (Millipore corporation, usa); the infant rice flour for experiments is purchased from various supermarkets in Jinan city.

Preparation of the solution

20 mmol/L ammonium acetate buffer solution (pH = 3.0): 1.54 g of ammonium Acetate (AR) was weighed out and dissolved in 950 water, the pH was adjusted to 3.0 with acetic acid and the volume was adjusted to 1000 mL with water.

Extracting solution: 300 mL of water was removed and mixed well with 700 mL of methanol.

Eluent (0.5% ammonia methanol solution): measuring 0.5 mL of ammonia water, diluting to 100 mL with methanol, and mixing.

Preparation of samples

Taking about 200 g of a representative sample, grinding the sample by a grinder, sieving the ground sample by a round-hole sieve of 2.0 mm, mixing the ground sample and the round-hole sieve uniformly, putting the mixture into a clean container, sealing the container, marking the container, and refrigerating the container at 0-4 ℃.

Sample pretreatment

1.4.1 sample extraction

Accurately weighing 2.5 g of sample (accurate to 0.01 g) in a 50 mL centrifuge tube, adding 15 mL of extracting solution, uniformly mixing by vortex, carrying out ultrasonic extraction for 15 min, centrifuging for 5 min at 8000 r/min, and taking out supernatant in a 25 mL volumetric flask; adding 8 mL of extraction solution for second extraction, centrifuging, mixing the two supernatants, adding the extraction solution to constant volume to scale, and shaking. 1mL of the suspension was removed, dried at 40 ℃ under nitrogen, and then reconstituted with 1mL of 20 mmol/L ammonium acetate (pH = 3.0) for purification.

Purification

The MCX solid phase extraction column was activated sequentially with 3 mL of methanol, 3 mL of water. The complex solution passes through a solid phase extraction column at a flow rate not higher than 1.0 mL/min; leaching with 3 mL of ultrapure water and 3 mL of methanol, discarding leacheate, and draining the small column; eluting with 5.0 mL of elution solution, drying with nitrogen at 40 deg.C, re-dissolving with 5.0 mL of initial mobile phase, filtering with microporous membrane, and analyzing and determining with HPLC-tandem mass spectrometer.

Conditions of instrumental analysis

1.5.1 chromatographic conditions

A chromatographic column: waters ACQUITY UPLC BEH Amide (100X 2.1mm, 1.7 μm); mobile phase: the phase A is 5 mmol/L ammonium acetate water solution, and the phase B is acetonitrile; the gradient elution procedure is shown in table 1; column temperature: 40 ℃; sample introduction amount: 5 μ L.

TABLE 1 HPLC gradient elution procedure

Table 1 Gradient elution program for HPLC separation

1.5.2 Mass Spectrometry conditions

An ion source: electrospray ion source (ESI source); the detection mode is as follows: multiple Reaction Monitoring (MRM); the scanning mode is as follows: scanning in a positive ion mode; electrospray voltage: 5500V; atomizer pressure: 50L/h; air curtain pressure: 35L/h; auxiliary gas pressure: 65L/h; collision gas pressure: 9L/h; ion source temperature: at 600 ℃. The atomized gas and the auxiliary gas are compressed air, and the air curtain gas and the collision gas are high-purity nitrogen. The qualitative ion pair, the quantitative ion pair, the acquisition time, the declustering voltage and the collision energy are shown in table 2.

TABLE 2 Mass Spectrometry parameters for validamycin A

Table 2 MS/MS parameters of validamycin A

* Quantitative ion

2 results and discussion

2.1 optimization of the pretreatment method

2.1.1 selection of extraction solvent

Validamycin a is readily soluble in water and methanol, and thus methanol or methanol-water may be selected for extraction. The experiment examines the influence of the proportion of methanol in the extraction solvent on the extraction efficiency. Four extraction solvents with methanol contents of 10%, 50%, 70% and 90% were compared, respectively, and the results are shown in FIG. 2. When the content of the methanol is lower than 70 percent, the recovery rate is less than 80 percent; when the methanol content is 70-90%, the recovery rate is higher than 85%, and the extract is clear. The higher the methanol content, the more volatile the solvent, and for environmental and laboratory operator safety reasons we chose a methanol-water (70 + 30) solution as the extraction solvent.

Selection of purification mode

The infant rice flour has complex matrix, and besides main raw materials such as rice, wheat and millet, vegetables, fruits, meat, eggs and milk powder are often added, and a plurality of mineral substances and vitamins are also added, so that a proper purification mode needs to be selected for purifying a target object. The solid phase extraction column has the characteristics of simple operation, solvent saving, good reproducibility and the like, and is widely applied to the purification of food matrixes. This experiment examined the effect of three solid phase extraction columns, Waters Oasis HLB (500 mg, 6 mL), SCX (500 mg, 6 mL) and MCX (60 mg, 3 mL) on validamycin A recovery (see FIG. 3). The HLB column retains relatively weakly polar compounds such as proteins and phospholipids, and thus has been used as a flow-through solid-phase extraction column in this study. Experiments show that the validamycin A can be partially retained on an HLB column, and the recovery rate is low; the recovery rate of SCX is higher than 80%, and can meet the detection requirement of pesticide residue, but SCX is a strong cation exchange column bonded on a silica gel matrix, is not resistant to drying and has high requirement on the operation process. The MCX column belongs to a mixed type strong cation exchange column of a polymer matrix, is resistant to drying and better in reproducibility, the validamycin A is well reserved by adjusting the pH value of a redissolving solvent, and the recovery rate is higher than 90%. Therefore, MCX was chosen as the purification column.

pH selection of the Loading solution

When a solid phase extraction column is used for purifying a target substance, particularly ion exchange SPE is used, the pH value of a sample loading solution has a remarkable influence on the recovery rate. The pKa of validamycin A is 6.0, the influence of the pH value of the loading solution in the range of 2.0-8.0 on the recovery rate of validamycin A is intensively examined in the research, and the experimental result is shown in FIG. 4. The results show that the recovery rate is higher than 85% between pH 3.0 and 4.0, and the recovery rate is optimal when the pH is 3.0.

The addition of vegetables, fruits and vitamins in the infant rice flour can cause the pH value of the extracting solution to be different among different products, and the subsequent purification effect is influenced. Methanol can generate certain influence on the pH value of the solution, when the concentration of the methanol solution is different, the generated influence is different, and the influence is more obvious when the concentration is higher^[18]. Experimental verification shows that if the pH of the methanol-water (70 + 30) extraction solution is directly adjusted to 3.0 and purification is continued, the recovery rate is low and unstable. This may be due to the presence of methanol, which affects the accuracy of the solution pH. Therefore, 1mL of methanol-water extraction solution is accurately transferred in the experiment, and after nitrogen blowing concentration, the solution is redissolved by using 20 mmol/L ammonium acetate (pH = 3.0), and the solution has better buffer capacity, so that the pH value of the redissolution of rice noodles with different tastes is stable.

Selection of elution solvent

The experiment also examined the effect of the concentration of ammonia in the eluent and the volume of eluent on recovery. The elution capability of 0.5%, 1.0, 2.0 and 5.0% ammonia water methanol solutions is investigated in experiments, and the result shows that the 0.5% ammonia water methanol solution can ensure that the target object is completely eluted. Validamycin A has the best stability in a solution with pH of 3-9, and the stability is influenced by too high concentration of ammonia water, so 0.5% ammonia water-methanol (v/v) solution is selected as the eluent. The recovery rate of validamycin A is also affected by the using amount of the eluent, the target substance can be completely eluted only when the volume of the eluent is not less than 5 mL, the subsequent concentration time is prolonged when the using amount of the eluent is too much, and finally 5.0 mL of eluent is selected for elution.

TABLE 3 influence of eluent dosage on validamycin A recovery

Table 3 Effect of eluent dosages on the recoveries of validamycin A

2.2 optimization of chromatographic conditions

2.2.1 selection of chromatography columns

Validamycin A belongs to aminoglycoside compounds, has strong polarity and is weakly retained on a common C18 chromatographic column. Hydrophilic interaction chromatography is one of the common ways to improve retention of polar compounds, and the method can effectively avoid the use of ions for reagents, and has better compatibility with mass spectrometry, so that a hydrophilic interaction chromatographic column is selected for analyzing a target substance in the experiment.

The retention behavior of validamycin A on Waters BEH HILIC (100 mm. times.2.1 mm, particle size 1.7 μm) and Waters BEH Amide (100 mm. times.2.1 mm, particle size 1.7 μm) 2-column chromatography was compared. The result shows that the retention time of the target object on the HILIC chromatographic column is 2.56 min, the response value is low, and the peak shape is asymmetric; on the Amide chromatographic column, the retention time is 2.83 min, the response value is improved by nearly 10 times, and the peak shape is better. Finally BEH Amide was selected for sample analysis.

2.2.2 selection of the Mobile phase

The composition of the mobile phase affects the chromatographic peak shape, retention time and response of the target. In the experiment, the retention behaviors of the target substance on a chromatographic column are firstly examined when ammonium acetate solutions with the concentrations of 2 mmol/L, 5 mmol/L and 10 mmol/L, namely acetonitrile, are respectively used as mobile phases. Experiments show that the retention time of validamycin A is gradually shortened along with the increase of the concentration of ammonium acetate in the mobile phase, and the response value is the highest in a 5 mmol/L ammonium acetate solution, so that the 5 mmol/L ammonium acetate solution is selected as the mobile phase. This experiment was compared with the addition of 0.1% and 0.2% formic acid to 5 mmol/L ammonium acetate. As a result, it was found that as the concentration of formic acid added to the mobile phase increases, the target retention time decreases and the response value also decreases. Therefore 5 mmol/L ammonium acetate solution-acetonitrile was chosen as the mobile phase.

Methodology validation

2.3.1 Linear relationship with detection Limit

A series of standard working solutions with mass concentrations of 0.1, 0.2, 0.5, 2 and 5 ng/mL are measured under the condition of '1.5', and a standard curve is drawn by taking the mass concentration (X) as an abscissa and the quantitative ion peak area (Y) as an ordinate. The result shows that the linear relation of validamycin A is good in the range of 0.1-5 ng/mL, and the linear equations are as follows: y = 6.799 × 10⁵X + 5955.3, correlation coefficient 0.9996. The concentration corresponding to 3 times the signal-to-noise ratio of the peak was taken as the method detection Limit (LOD), and the concentration corresponding to 10 times the signal-to-noise ratio of the peak was taken as the method quantitation lower Limit (LOQ). The detection limit of validamycin A is 2.0 mug/kg, the quantification limit is 5.0 mug/kg, and the sensitivity of the method is higher than GB 23200.74. The MRM of the quantitative limit plus standard in infant rice flour is shown in figure 5.

Recovery and precision

Original nutritious rice flour (sample 1), coarse cereal and millet rice flour (sample 2) and mixed cereal nutritious rice flour (sample 3) are selected as blank samples, and the recovery rate and precision of the method are examined. And (4) adding marks of a low level (5.0 mug/kg), a medium level (50.0 mug/kg) and a high level (200 mug/kg) to the blank sample respectively, and performing parallel determination for 6 times. The average recovery rate of the method is 88.5-100.5%, the precision is 4.84-10.08%, and the specific data are shown in Table 5.

Table 5 recovery and precision of validamycin a (n = 6)

Table 5 Recovery and relative standard deviation of validamycin A (n=6).

2.4 determination of the quantitative mode

In the liquid chromatography-tandem mass spectrometry analysis, the matrix effect becomes one of the key factors influencing the accuracy and the sensitivity, so the matrix effect needs to be evaluated. In the experiment, the purification effects of the three infant rice flour are examined in a mode of ME (%) = (response value of blank matrix solution diluted standard solution/response value of pure solvent diluted standard solution-1) × 100. In the experiment, compared with the concentration of 4 ng/mL, the ME values of validamycin A in the samples 1-3 are 72.5%, 58.1% and 63.4% respectively. The absolute values of ME are all more than 50%, indicating that the matrix effect is obvious. Thus, the method uses matrix matching standard curves for quantification.

Analysis of actual samples

The method established in the text is adopted to analyze 15 batches of infant rice flour purchased in each supermarket in denna, and validamycin A is not detected in the result.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (5)

1. A method for determining residual quantity of validamycin A in infant rice flour by using a hydrophilic interaction chromatography-tandem mass spectrometry method is characterized by comprising the following steps:

(1) sample extraction: adding the sample into the extracting solution, carrying out secondary extraction, centrifuging, combining the two supernatants, drying by nitrogen, re-dissolving by using an ammonium acetate solution, and purifying;

(2) purification: activating an MCX solid-phase extraction column, loading a sample, leaching, eluting an elution solution, blowing nitrogen, re-dissolving an initial mobile phase, filtering by a microporous filter membrane, and performing analysis and determination by a liquid chromatography-tandem mass spectrometer;

(3) and (4) measuring by adopting liquid chromatography-mass spectrometry.

2. The method for determining the residual quantity of validamycin A in infant rice flour by using the hydrophilic interaction chromatography-tandem mass spectrometry as claimed in claim 1, wherein the conditions of the liquid chromatography are as follows: and (3) chromatographic column: waters ACQUITY UPLC BEH Amide (100X 2.1mm, 1.7 μm); mobile phase: the phase A is 5 mmol/L ammonium acetate water solution, and the phase B is acetonitrile; a gradient elution procedure; column temperature: 40 ℃; sample introduction amount: 5 mu L of the solution;

。

3. the method for determining the residual quantity of validamycin A in infant rice flour by using the hydrophilic interaction chromatography-tandem mass spectrometry as claimed in claim 1, wherein the mass spectrometry conditions are as follows: an ion source: an electrospray ion source; the detection mode is as follows: monitoring multiple reactions; the scanning mode is as follows: scanning in a positive ion mode; electrospray voltage: 5500V; atomizer pressure: 50L/h; air curtain pressure: 35L/h; auxiliary gas pressure: 65L/h; collision gas pressure: 9L/h; ion source temperature: 600 ℃; the atomized gas and the auxiliary gas are compressed air, and the air curtain gas and the collision gas are high-purity nitrogen; the qualitative ion pair, the quantitative ion pair, the acquisition time, the cluster removing voltage and the collision energy are shown in a table;

。

4. the method for determining the residual quantity of validamycin A in infant rice flour by using the hydrophilic interaction chromatography-tandem mass spectrometry as claimed in claim 1, wherein the specific steps in the step (1) are as follows:

accurately weighing 2.5 g of sample in a 50 mL centrifuge tube, adding 15 mL of extracting solution, uniformly mixing by vortex, ultrasonically extracting for 15 min, centrifuging for 5 min at 8000 r/min, and taking out supernatant in a 25 mL volumetric flask; adding 8 mL of extraction solution for second extraction, centrifuging, mixing the two supernatants, and finally fixing the volume to the scale with the extraction solution and shaking up; 1mL of the suspension was removed and dried at 40 ℃ with nitrogen, and then reconstituted with 1mL of 20 mmol/L ammonium acetate solution (pH = 3.0).

5. The method for determining the residual quantity of validamycin A in infant rice flour by using the hydrophilic interaction chromatography-tandem mass spectrometry as claimed in claim 1, wherein the step (2) comprises the following specific steps: activating the MCX solid phase extraction column by using 3 mL of methanol and 3 mL of water in sequence; the composite solution is completely passed through a solid phase extraction column at a flow rate of not more than 1.0 mL/min; leaching with 3 mL of ultrapure water and 3 mL of methanol, discarding leacheate, and pumping the small column to be dry; eluting with 5.0 mL of elution solution, blowing dry with nitrogen at 40 ℃, redissolving with 5.0 mL of initial mobile phase, and filtering with a microporous membrane.

Images