CN115575527A - Method for identifying acetohydroxamic acid - Google Patents

Abstract

The invention discloses an identification method of acetohydroxamic acid. Relates to the technical field of detection. The method comprises the following steps: sample pretreatment: weighing a sample, adding a solvent, extracting, centrifuging, taking supernate, diluting and filtering to obtain a treated sample; and (4) detecting and identifying by liquid chromatography-mass spectrometry. The invention establishes a liquid chromatography-mass spectrometry identification process of acetohydroxamic acid in flour and flour treating agents. The target compound is extracted by methanol, diluted by water and directly tested on a machine, and the pretreatment operation is simple. The optimized liquid chromatography-mass spectrometry method is low in solvent effect, quantitative determination is carried out by adopting a matrix standard curve external standard method, and experimental steps are simplified. The method is simple to operate, high in sensitivity, good in accuracy, stable and reliable, is suitable for measuring the acetohydroxamic acid in the flour and flour treating agent, and provides technical support for establishing relevant standards and monitoring risks of supervision departments.

Description

Method for identifying acetohydroxamic acid

Technical Field

The invention relates to the technical field of detection, in particular to an identification method of acetohydroxamic acid.

Background

The acetohydroxamic acid is widely used in the medicine, feed additive and metal beneficiation agent in the medicine, animal husbandry and metallurgy industries, but is not a food raw material, and the country has no regulation to use the non-food raw material in flour or flour treatment agent, but the identification and detection research of the acetohydroxamic acid in the food field is lacked at present.

The existing literature reports about the acetohydroxamic acid are less, and the quantitative determination of the acetohydroxamic acid in the feed is carried out by utilizing the ultraviolet spectrophotometry under the wavelength of 420nm by utilizing the acetohydroxamic acid and ferric ions to form a red complex in the acidic condition of the Tengjiao; huang Shuling adopts acetohydroxamic acid and ferric chloride hydrochloric acid solution for color development, and measures the content of the acetohydroxamic acid in the acetohydroxamic acid capsule by ultraviolet spectrophotometry under the wavelength of 502 nm; the meaningful way adopts that under the acidic condition, the acetohydroxamic acid and ferric ions form a red compound, and the acetohydroxamic acid in the soil is measured at 420nm by a spectrophotometry; the documents refer to the research of methods of the acetohydroxamic acid in the non-food field, the pretreatment is complex, other reagents are introduced, the interference is large, the sensitivity is low, the specificity is poor, the false positive rate is high, and the method cannot be applied to food identification and trace detection.

Therefore, how to provide a method for identifying acetohydroxamic acid is a problem that needs to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of this, the invention provides an identification method of acetohydroxamic acid.

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

the method for identifying the acetohydroxamic acid is characterized by comprising the following steps of:

(1) Sample pretreatment: weighing a sample, adding a solvent, extracting, centrifuging, taking supernatant, diluting and filtering to obtain a treated sample;

(2) Carrying out liquid chromatography-mass spectrometry detection and identification on the sample treated in the step (1);

liquid chromatography conditions:

a chromatographic column: a T3 chromatographic column of 3.0X 100mm and 2.7 μm; a mobile phase A:0.1% aqueous formic acid; mobile phase B: methanol; flow rate 0.25mL/min, sample volume: 5 mu L of the solution; the column temperature is 35 ℃; gradient elution procedure is as in table 1:

TABLE 1 gradient elution procedure

Mass spectrum conditions:

an ion source: atmospheric pressure chemical ionization source APCI +; the scanning mode comprises the following steps: monitoring multiple reactions; ion discharge current: 4 muA; temperature of sheath gas: 350 ℃; ion transfer tube temperature: 325 ℃; flow rate of atomizing gas: 45Arb; flow rate of auxiliary gas: 5Arb; air flow speed of the air curtain: 1Arb;

mass spectral parameters and retention time table 2:

TABLE 2 Mass Spectrometry parameters and Retention time

Preferably: the solvent in the step (1) is methanol; the mass-to-volume ratio of sample to solvent was 2.5g:10ml.

Preferably: step (1) extraction: vortex for 10min, and ultrasonically extract for 10min.

Preferably, the following components: centrifuging: centrifuging at 10000r/min for 5min.

Preferably: diluting: centrifuging, mixing uniformly and taking supernatant, wherein the volume ratio of the supernatant to the diluted solution is 2:5.

preferably, the following components: filtering in step (1): filtration through a 0.22 μm filter.

The invention provides application of any one of the methods in food detection.

Preferably: the detection object is flour or a flour treating agent.

According to the technical scheme, compared with the prior art, the method for identifying the acetohydroxamic acid disclosed by the invention has the advantages of simple pretreatment, small introduced interference, high sensitivity, strong specificity and high accuracy, is suitable for identifying and detecting the acetohydroxamic acid doping in flour and flour processing agents, and has good practical application value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a general ion flow diagram of acetohydroxamic acid provided by the present invention.

FIG. 2 is a graph showing the comparison of the extraction efficiency of the same solvent repeatedly extracted according to the present invention.

FIG. 3 is a graph illustrating the effect of different weighing masses on the extraction efficiency provided by the present invention.

FIG. 4 is a diagram illustrating the effect of different extraction modes and extraction time on the extraction efficiency.

FIG. 5 is a molecular structure diagram of acetohydroxamic acid provided by the present invention.

FIG. 6 is a scanning diagram of the photon ion of acetohydroxamic acid provided by the present invention.

FIG. 7 is a total ion flow diagram of acetohydroxamic acid in the flour treatment agent provided by the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses an identification method of acetohydroxamic acid.

In the examples, the required raw materials and equipment are all commercial raw materials, and the method which is not mentioned is a conventional experimental method, such as:

Thermo Scientific ^TM TSQ Altis ^TM triple quadrupole mass spectrometer (with atmospheric pressure chemical ionization source APCI), FA2204N electronic balance (Shanghai precision instruments, inc.); a Multi-Tube vortex oscillator of Multi-Tube vortex VX-III type (Beijing Pedal science and technology Co., ltd.); KQ-500TDB ultrasonic cleaning instrument (Shanghai Huyue scientific instruments Co., ltd.); mili-Q ultra pure water machine (Millipore, USA).

Acetoxyhydroxamic acid (available from china altar ink quality testing technologies, ltd); methanol (chromatographically pure); formic acid (chromatographically pure), flour and flour treatment were purchased from local markets.

And will not be described in detail herein.

Example 1

Standard working solution preparation

The acetohydroxamic acid standard substance is accurately weighed, dissolved by methanol and subjected to constant volume to prepare a standard stock solution with the concentration of 1mg/mL, and the standard stock solution is stored at minus 4 ℃ in the dark. The standard stock solution of the acetohydroxamic acid is accurately transferred and diluted by methanol to prepare a mixed standard intermediate solution with the concentration of 10 mug/mL. Then diluted with methanol to prepare a series of standard working solutions with the concentrations of 0.025 mu g/mL, 0.05 mu g/mL, 0.10 mu g/mL, 0.20 mu g/mL, 0.50 mu g/mL, 1.00 mu g/mL, 2.00 mu g/mL and 5.00 mu g/mL.

Sample pretreatment

Pretreatment of flour: accurately weighing 2.5g (accurate to 0.001 g) of a sample, placing the sample in a 50mL centrifuge tube with a plug, accurately adding 10mL of methanol (final extraction solvent), carrying out vortex for 10min, carrying out ultrasonic extraction for 10min, centrifuging for 5min at 10000r/min, mixing uniformly, taking 2mL of supernate, placing the supernate in a 5mL volumetric flask, adding water to dilute to a scale, shaking uniformly, filtering through a 0.22 mu m filter membrane, and determining by using a liquid chromatography-tandem mass spectrometer.

Pretreatment of a flour treatment agent: accurately weighing 2.5g (accurate to 0.001 g) of a sample, placing the sample in a 50mL centrifuge tube with a plug, accurately adding 10mL of methanol (final extraction solvent), carrying out vortex for 10min, carrying out ultrasonic extraction for 10min, centrifuging for 5min at 10000r/min, taking 2mL of supernate, placing the supernate in a 5mL volumetric flask, adding water to dilute the supernate to a scale, shaking the supernate uniformly, filtering the supernate with a 0.22 mu m filter membrane, and determining the supernate by using a liquid chromatography-tandem mass spectrometer.

Conditions of instrumental analysis

Liquid chromatography

The chromatographic conditions are as follows: a chromatographic column: a T3 chromatographic column of 3.0X 100mm and 2.7 μm; mobile phase A:0.1% aqueous formic acid; mobile phase B: methanol; flow rate 0.25mL/min, sample volume: 5 mu L of the solution; the column temperature is 35 ℃; the gradient elution procedure is detailed in table 1:

TABLE 1 gradient elution procedure

Conditions of Mass Spectrometry

An ion source: atmospheric pressure chemical ionization source APCI +; the scanning mode comprises the following steps: monitoring multiple reactions; ion discharge current: 4 muA; temperature of sheath gas: 350 ℃; ion transfer tube temperature: 325 ℃; flow rate of atomizing gas: 45Arb; flow rate of auxiliary gas: 5Arb; air flow speed of the air curtain: 1Arb;

mass spectral parameters and retention time table 2:

TABLE 2 Mass Spectrometry parameters and Retention time

* : and (4) quantifying ions.

Comparative experiment 1

Optimization of processing conditions

Selection of solvents

The acetohydroxamic acid is a strong polar weak acid compound and is easy to dissolve in water, methanol and ethanol. According to the principle of similarity and compatibility, the extraction efficiency of five extraction solvents of water, methanol, ethanol, methanol/water (5, v), acetonitrile/water (8, v); the methanol is used as an extraction solvent, the sample extracting solution is clear, and the extraction efficiency is optimal when the methanol is used as the extraction solvent.

Methanol was therefore chosen as the final extraction solvent.

Comparative experiment 2

When a sample solution obtained after methanol extraction is directly injected into a liquid chromatogram-mass spectrometer for testing, serious solvent effect, poor chromatographic peak shape and serious tailing exist, and the ratio of methanol extract to water is considered to be 9: 1. 8: 2. 7: 3. 6: 4. 5: 5. 4: 6. 3: 7. 2: 8. 1:9, shows that the solvent effect can be completely eliminated to improve the peak shape when the proportion of water is more than 60%. Thus, considering the extraction with methanol, 2ml of the solution after extraction was taken, diluted to 5ml with water, shaken well filtered and then measured on the machine, see FIG. 1.

Comparative experiment 3

Selection of extraction volume

Weighing 2.5g of sample, adding standard solution, extracting with 5mL of methanol under shaking for 10min, ultrasonically extracting for 10min, centrifuging at 8000r/min at 4 ℃ for 5min, taking out all supernatant, weighing 2mL, adding water to dilute to 5mL, shaking, filtering, and measuring on the machine.

5mL of methanol was further added to the remaining residue, and the solutions obtained by repeating the above operations were all subjected to the on-machine measurement. The examination of the volume of the extraction solvent was carried out by repeating the extraction with 3 additions of 5mL of the same solvent, the results of which are shown in FIG. 2.

The result shows that the recovery rate reaches 68.24% -73.52% in the first 5mL solvent extraction, the recovery rate is 20.86% -27.21% in the second 5mL solvent extraction, and the recovery rate is 3.06% -4.90% in the third 5mL solvent extraction. The sum of the recovery rates of the first extraction and the second extraction reaches 90.16-96.80%, and the recovery rate of the third extraction is less than 5%, so that the previous two extractions are basically completely extracted, and the final extraction volume is 10mL in order to save the solvent.

Comparative experiment 4

Determination of the weighing mass

Respectively weighing 0.5g, 1.0g, 2.0g, 2.5g and 5.0g of samples, respectively adding standard solutions to simulate positive samples, performing shaking extraction for 10min by using 10mL of methanol, performing ultrasonic extraction for 10min, centrifuging for 5min at 8000r/min at 4 ℃, taking 2mL of supernate, adding water to dilute the supernate to 5mL, shaking up, filtering and then performing on-machine determination, wherein the result is shown in figure 3. The result shows that the influence of the sample weighing on the recovery rate is not large, the standard recovery rate of the two samples is between 81.18 and 108.17 percent, and the two samples can meet the experimental requirements. In view of detection limit and saving of sample amount, the finally used sample amount was 2.5g.

Comparative experiment 5

Optimization of extraction mode and extraction time

Weighing 2.5g of sample, adding a certain amount of acetohydroxamic acid standard substance into the blank matrix sample, using 10mL of methanol, inspecting the optimal extraction mode and extraction time according to the difference between vortex oscillation time and ultrasonic extraction time, centrifuging the extraction solution at 4 ℃ for 5min at 8000r/min, measuring 2mL, adding water to dilute to 5mL, shaking up, filtering, and then measuring on the machine, wherein the result is shown in FIG. 4. The result shows that for the standard sample, the extraction mode and the extraction time have no obvious influence on the recovery rate. In the positive sample, on the basis of vortex oscillation for 10min, the influence of continuous ultrasonic for 10min, 20min and 30min on the extraction effect is not obvious (81.6-90.0%), but the recovery rate of the target object in the positive sample of the noodles is obviously reduced (26.3-28.2%) without ultrasonic extraction, and the recovery rate of the target object in the positive sample of the wheat flour treating agent is slightly reduced (77.1-79.5%). The fixed ultrasonic time is 10min, the oscillation time is 10min, 20min and 30min respectively, the influence on the extraction effect is not obvious (82.9-91.9%), and the recovery rate of two matrix positive samples is reduced (59.4-69.2%) when the oscillation is not carried out. And (3) comprehensively considering the extraction efficiency and the time cost, and finally selecting vortex oscillation for 10min and ultrasonic extraction for 10min as the optimal extraction mode and time.

Example 2

A peristaltic pump was used to directly inject the standard solution of acetoxyhydroxamic acid (1. Mu.g/mL) to optimize each parameter. The molecular structure of the acetohydroxamic acid is shown in figure 5, the molecular weight is 75.07, an atmospheric pressure chemical ionization source is adopted for ionization, and hydrogen generation parent ions [ M + H ] +, are obtained in a Q1 full-scan mode; then, a Production mode is adopted, the daughter ions are scanned by changing collision energy, fig. 6 is a scanning diagram of the daughter ions of the acetohydroxamic acid under the condition that the collision energy is 10ev, and the information of acetohydroxamic acid fragments is rich, namely the fragment ions of 34.14, 57.97 and 43.07 can be seen from the scanning diagram. Then, all fragment ions found in the parent ion [ M + H ] + mode and the Production mode found in the Q1MS mode are introduced into the MRM scan mode list, and systematic optimization is performed on parameters such as collision energy, and ions having the highest abundance of 34.14 and 43.07 and stable and interference-free signals in the measurement of actual samples are found out of all fragment ions, so that the fragments of 34.14 and 43.07 are preferred as monitor ions.

Linear range, detection limit, quantitation limit, recovery and precision

Injecting the prepared matrix standard working curve solution (a blank matrix solution prepared standard working curve solution for eliminating matrix effect) into a liquid chromatogram-atmospheric pressure chemical ionization source-tandem mass spectrometer for detection, and drawing a standard curve by taking the peak area (Y) of the standard working solution as a vertical coordinate and the concentration (X) of the standard working solution as a horizontal coordinate, wherein the result shows that the correlation coefficient r is more than 0.9990 and the linearity is good when the concentration is in the range of 0.025-5.0 mu g/mL.

Comparative experiment 6

The method of adding the target compound into a blank sample matrix is adopted, and the detection limit and the quantification limit of the method are determined. And taking the mass concentration corresponding to the 3-time signal-to-noise ratio of the chromatographic peak response value as a detection limit of the method, and taking the lowest point of the curve concentration capable of being accurately quantified as a quantification limit of the method.

The acetohydroxamic acid with the quantitative limit, the 2-time quantitative limit and the 10-time quantitative limit is added into the blank flour and the flour treating agent to carry out a standard addition recovery test, so that the accuracy and precision of the method are verified. Each concentration level was measured in duplicate 6 times and averaged to calculate recovery and precision, see table 3.

TABLE 3 detection limits, quantitation limits, linear range, spiking recovery and precision of acetohydroxamic acid

Example 3

Sample assay

The commercial flour and the inspected 20 batches of flour and 20 batches of flour treatment agent samples are subjected to acetohydroxamic acid determination according to the invention, the method has good applicability, and 1 flour treatment agent positive sample is found, and the detected content is 26.1mg/kg, which is shown in figure 7.

In conclusion, the invention establishes a liquid chromatography-mass spectrometry identification process of acetohydroxamic acid in the flour and flour treating agent. The target compound is extracted by methanol, diluted by water and directly tested on a machine, and the pretreatment operation is simple. The optimized liquid chromatography-mass spectrometry method is low in solvent effect, quantitative determination is carried out by adopting a matrix standard curve external standard method, and experimental steps are simplified. The method is simple to operate, high in sensitivity, good in accuracy, stable and reliable, is suitable for measuring the acetohydroxamic acid in the flour and flour treating agent, and provides technical support for establishing relevant standards and monitoring risks of supervision departments.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. The method for identifying the acetohydroxamic acid is characterized by comprising the following steps of:

(1) Sample pretreatment: weighing a sample, adding a solvent, extracting, centrifuging, taking supernate, diluting and filtering to obtain a treated sample;

(2) Carrying out liquid chromatography-mass spectrometry detection and identification on the sample treated in the step (1);

the liquid chromatography conditions are as follows:

a chromatographic column: a T3 chromatographic column of 3.0X 100mm and 2.7 μm; mobile phase A:0.1% aqueous formic acid; and (3) mobile phase B: methanol; flow rate 0.25mL/min, sample volume: 5 mu L of the solution; the column temperature is 35 ℃; gradient elution procedure is as in table 1:

TABLE 1 gradient elution procedure

Mass spectrum conditions:

an ion source: atmospheric pressure chemical ionization source APCI +; the scanning mode is as follows: monitoring multiple reactions; ion discharge current: 4 muA; temperature of sheath gas: 350 ℃; ion transfer tube temperature: 325 ℃; flow rate of atomizing gas: 45Arb; flow rate of auxiliary gas: 5Arb; air flow speed of air curtain: 1Arb;

mass spectral parameters and retention time table 2:

TABLE 2 Mass Spectrometry parameters and Retention time

2. The method of claim 1, wherein the solvent in step (1) is methanol; the mass-to-volume ratio of the sample to the solvent is 2.5g:10ml.

3. The authentication method according to claim 2, wherein the extracting of step (1): vortex for 10min, and ultrasonically extract for 10min.

4. The method of claim 3, wherein said centrifugation of step (1): centrifuging at 10000r/min for 5min.

5. The method of claim 4, wherein said diluting of step (1): and (3) centrifuging, uniformly mixing, and taking supernatant, wherein the volume ratio of the supernatant to the diluted solution is 2:5.

6. the authentication method of claim 5, wherein said filtering of step (1): filtration through a 0.22 μm filter.

7. Use of the method of any one of claims 1 to 6 for food testing.

8. The use of claim 7, wherein the test object is flour or a flour treatment agent.

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