A method for identifying acetohydroxamic acid

Abstract

The invention discloses a method for identifying acetohydroxamic acid. It relates to the field of detection technology. It includes: sample pretreatment: weighing a sample, adding a solvent, centrifuging after extraction, taking the supernatant, diluting and filtering, and obtaining a treated sample; liquid chromatography-mass spectrometry detection and identification. The present invention establishes a liquid chromatography-mass spectrometry identification process for acetohydroxamic acid in flour and flour treatment agents. The target compound is extracted with methanol, diluted with water and directly measured on the machine, and the pretreatment operation is simple. The optimized liquid chromatography-mass spectrometry method has low solvent effect, and the matrix standard curve external standard method is used for quantitative determination, which simplifies the experimental steps. The method is simple to operate, highly sensitive, accurate, stable and reliable, and is suitable for the determination of acetohydroxamic acid in flour and flour treatment agents. At the same time, it provides technical support for the establishment of relevant standards and risk monitoring of regulatory departments.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and more particularly to an identification method for acetohydroxamic acid. Background Art

[0002] As a drug, feed additive and metal beneficiation agent, acetohydroxamic acid is widely used in the pharmaceutical, animal husbandry and metallurgical industries, but it is a non-food raw material. The state has expressly prohibited the use of non-food raw materials in flour or flour processing agents, but there is currently a lack of identification and detection research on acetohydroxamic acid in the food field.

[0003] There are relatively few literature reports on acetohydroxamic acid. Teng Jiaoqin used acetohydroxamic acid to form a red complex with trivalent iron ions under acidic conditions, and used ultraviolet spectrophotometry at a wavelength of 420nm to quantitatively determine acetohydroxamic acid in feed; Huang Shuling used acetohydroxamic acid and ferric chloride hydrochloric acid solution for color development, and determined the content of acetohydroxamic acid in acetohydroxyamine capsules by ultraviolet spectrophotometry at a wavelength of 502nm; Jun Yinghua used acetohydroxamic acid to form a red compound with trivalent iron ions under acidic conditions, and used spectrophotometry to determine acetohydroxamic acid in soil at 420nm; all of the above documents are method studies on acetohydroxamic acid in non-food fields, with complex pretreatment, introduction of many other reagents, large interference, low sensitivity, poor specificity, and high false positive rate, and are not suitable for food identification and trace detection.

[0004] Therefore, how to provide a method for identifying acetohydroxamic acid is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a method for identifying acetohydroxamic acid.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for identifying acetohydroxamic acid, characterized in that it comprises the following steps:

[0008] (1) Sample pretreatment: weigh the sample, add solvent, extract and centrifuge, take the supernatant and dilute and filter to obtain the treated sample;

[0009] (2) subjecting the sample treated in step (1) to liquid chromatography-mass spectrometry for identification;

[0010] Liquid chromatography conditions:

[0011] Chromatographic column: T3 chromatographic column 3.0×100 mm, 2.7 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; flow rate 0.25 mL / min, injection volume: 5 μL; column temperature 35°C; gradient elution program as shown in Table 1:

[0012] Table 1 Gradient elution program

[0013]

[0014] Mass spectrometry conditions:

[0015] Ion source: atmospheric pressure chemical ionization source APCI+; scanning mode: multiple reaction monitoring; ion discharge current: 4μA; sheath gas temperature: 350℃; ion transfer tube temperature: 325℃; nebulizer gas flow rate: 45Arb; auxiliary gas flow rate: 5Arb; curtain gas flow rate: 1Arb;

[0016] Mass spectrometry parameters and retention time table 2:

[0017] Table 2 Mass spectrometry parameters and retention time

[0018]

[0019] Preferably, the solvent in step (1) is methanol; and the mass volume ratio of the sample to the solvent is 2.5 g:10 ml.

[0020] Preferably: step (1) extraction: vortex for 10 min, ultrasonic extraction for 10 min.

[0021] Preferably: step (1) centrifugation: 10000r / min centrifugation for 5min.

[0022] Preferably: step (1) dilution: after centrifugation, mix and take the supernatant, the volume ratio of the supernatant to the diluted solution is 2:5.

[0023] Preferably: step (1) filtration: 0.22 μm filter membrane filtration.

[0024] The present invention provides application of any of the above methods in food testing.

[0025] Preferably, the detection object is flour or a flour treating agent.

[0026] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a method for identifying acetohydroxamic acid, which has the technical effects of simple pretreatment, small interference, high sensitivity, strong specificity, and high accuracy. It is suitable for the identification and detection of acetohydroxamic acid adulteration in flour and flour treatment agents, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The accompanying drawing is a total ion current diagram of acetohydroxamic acid provided by the present invention.

[0029] Figure 2 The accompanying drawing is a comparison diagram of extraction efficiency when the same solvent is repeatedly extracted provided by the present invention.

[0030] Figure 3 The accompanying drawing is a diagram showing the influence of different weighing masses on the extraction efficiency provided by the present invention.

[0031] Figure 4 The accompanying drawing is a diagram showing the effects of different extraction methods and extraction times on the extraction efficiency provided by the present invention.

[0032] Figure 5 The accompanying drawing is a molecular structure diagram of acetohydroxamic acid provided by the present invention.

[0033] Figure 6 The attached drawing is a daughter ion scanning diagram of acetohydroxamic acid provided by the present invention.

[0034] Figure 7 The accompanying drawing is a total ion flow diagram of acetohydroxamic acid in the flour treating agent provided by the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The embodiment of the invention discloses a method for identifying acetohydroxamic acid.

[0037] In the examples, the required raw materials and equipment are all commercially available raw materials, and the methods not mentioned are conventional experimental methods, such as:

[0038] Thermo Scientific TM TSQ Altis TMTriple quadrupole mass spectrometer (equipped with atmospheric pressure chemical ionization source APCI), FA2204N electronic balance (Shanghai Precision Instrument Co., Ltd.); Multi-Tube Vortexer VX-Ⅲ multi-tube vortex oscillator (Beijing Tajin Technology Co., Ltd.); KQ-500TDB ultrasonic cleaner (Shanghai Huyueming Scientific Instrument Co., Ltd.); Mili-Q ultrapure water machine (Millipore Corporation, USA).

[0039] Acetohydroxamic acid (purchased from China Tanmo Quality Inspection Technology Co., Ltd.); methanol (chromatographic grade); formic acid (chromatographic grade); flour and flour treatment agents were purchased from the local market.

[0040] I will not go into details here.

[0041] Example 1

[0042] Preparation of standard working solution

[0043] Accurately weigh the acetohydroxamic acid standard, dissolve it in methanol and make up to volume to prepare a standard stock solution with a concentration of 1 mg / mL, and store it at -4°C in the dark. Accurately pipette the acetohydroxamic acid standard stock solution and dilute it with methanol to prepare a mixed standard intermediate solution with a concentration of 10 μg / mL. Then dilute it with methanol to prepare a series of standard working solutions with concentrations of 0.025 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.20 μg / mL, 0.50 μg / mL, 1.00 μg / mL, 2.00 μg / mL, and 5.00 μg / mL.

[0044] Sample preparation

[0045] Flour pretreatment: Accurately weigh 2.5 g of the sample (accurate to 0.001 g), place it in a 50 mL stoppered centrifuge tube, accurately add 10 mL of methanol (final extraction solvent), vortex for 10 min, ultrasonically extract for 10 min, centrifuge at 10000 r / min for 5 min, mix well, take 2 mL of the supernatant, transfer it to a 5 mL volumetric flask, dilute to the scale with water, shake well, pass through a 0.22 μm filter membrane, and provide for liquid chromatography-tandem mass spectrometry determination.

[0046] Pretreatment of flour treatment agent: accurately weigh 2.5 g of the sample (accurate to 0.001 g), place it in a 50 mL stoppered centrifuge tube, accurately add 10 mL of methanol (final extraction solvent), vortex for 10 min, ultrasonically extract for 10 min, centrifuge at 10000 r / min for 5 min, take 2 mL of the supernatant, transfer it to a 5 mL volumetric flask, dilute it to the scale with water, shake well, pass it through a 0.22 μm filter membrane, and provide it for determination by liquid chromatography-tandem mass spectrometry.

[0047] Instrument analysis conditions

[0048] Liquid chromatography

[0049] Chromatographic conditions are as follows: Chromatographic column: T3 chromatographic column 3.0×100 mm, 2.7 μm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: methanol; Flow rate 0.25 mL / min, injection volume: 5 μL; Column temperature 35°C; Gradient elution program as shown in Table 1:

[0050] Table 1 Gradient elution program

[0051]

[0052] Mass spectrometry conditions

[0053] Ion source: atmospheric pressure chemical ionization source APCI+; scanning mode: multiple reaction monitoring; ion discharge current: 4μA; sheath gas temperature: 350℃; ion transfer tube temperature: 325℃; nebulizer gas flow rate: 45Arb; auxiliary gas flow rate: 5Arb; curtain gas flow rate: 1Arb;

[0054] Mass spectrometry parameters and retention time table 2:

[0055] Table 2 Mass spectrometry parameters and retention time

[0056]

[0057] *: Quantitative ion.

[0058] Comparative experiment 1

[0059] Optimization of treatment conditions

[0060] Choice of solvent

[0061] Since acetohydroxamic acid is a strong polar weak acid compound, it is easily soluble in water, methanol, and ethanol. According to the principle of like dissolves like, the experiment compared the extraction efficiency of five extraction solvents: water, methanol, ethanol, methanol / water (5:5, v:v), and acetonitrile / water (8:2, v:v). Since flour treatment agents contain a lot of starch, if water is added to the extraction solvent, the flour treatment agent will become viscous and gelatinized, and it is impossible to obtain a clear filtrate; when methanol is used as the extraction solvent, the sample extracts are all clear, and the extraction efficiency is the best when methanol is used as the extraction solvent.

[0062] Therefore, methanol was selected as the final extraction solvent.

[0063] Comparative experiment 2

[0064] When the sample solution after methanol extraction is directly injected into the liquid chromatography-mass spectrometer for testing, there is a serious solvent effect, poor chromatographic peak shape, and severe tailing. The experiment investigated the chromatographic peak shape when the ratio of methanol extract to water was 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9. It was shown that when the proportion of water was greater than 60%, the solvent effect could be completely eliminated and the peak shape could be improved. Therefore, methanol extraction was considered. After extraction, 2 ml of the solution was measured, diluted to 5 ml with water, shaken and filtered, and then measured on the machine. Figure 1 .

[0065] Comparative experiment 3

[0066] Selection of extraction volume

[0067] Weigh 2.5g sample, add standard solution, use 5mL methanol, shake and extract for 10min, ultrasonic extract for 10min, centrifuge at 4℃8000r / min for 5min, take out all the supernatant, measure 2mL, dilute to 5ml with water, shake and filter, and then measure on the machine.

[0068] 5 mL of methanol was added to the remaining residue, and the solution after repeating the above operation was measured on the machine. The volume of the extraction solvent was investigated by adding 5 mL of the same solvent for three times. The results are shown in Figure 2 .

[0069] The results showed that the recovery rate reached 68.24% to 73.52% when the first 5mL solvent was extracted, 20.86% to 27.21% when the second 5mL solvent was extracted, and 3.06% to 4.90% when the third 5mL solvent was extracted. The sum of the recovery rates of the first and second extractions reached 90.16% to 96.80%, and the recovery rate of the third extraction was less than 5%. Therefore, the first two extractions were basically complete. In order to save solvent, the final extraction volume was 10mL.

[0070] Comparative experiment 4

[0071] Determination of weighing mass

[0072] Weigh 0.5g, 1.0g, 2.0g, 2.5g, and 5.0g of the sample respectively, add the standard solution to simulate the positive sample, use 10mL of methanol, shake and extract for 10min, ultrasonic extract for 10min, centrifuge at 4℃8000r / min for 5min, take 2mL of the supernatant, dilute to 5ml with water, shake and filter, and then measure on the machine. The results are shown in the figure. Figure 3 The results showed that the sample weight had little effect on the recovery rate, and the recovery rates of the two samples were between 81.18% and 108.17%, which met the experimental requirements. Considering the detection limit and saving sample volume, the final sample weight used was 2.5g.

[0073] Comparative experiment 5

[0074] Optimization of extraction method and extraction time

[0075] Weigh 2.5g sample, add a certain amount of acetohydroxamic acid standard to the blank matrix sample, use 10mL methanol, and investigate the best extraction method and extraction time according to the different vortex oscillation time and ultrasonic extraction time. Centrifuge the extraction solution at 8000r / min at 4℃ for 5min, measure 2mL, dilute with water to 5ml, shake well, filter and then measure on the machine. The results are shown in Figure 4 . The results showed that for spiked samples, the extraction method and extraction time had no significant effect on the recovery rate. For positive samples, on the basis of vortex oscillation for 10 minutes, continuing ultrasonic oscillation for 10 minutes, 20 minutes, and 30 minutes had no significant effect on the extraction effect (81.6% to 90.0%), but without ultrasonic extraction, the recovery rate of the target in the noodle positive sample was significantly reduced (26.3% to 28.2%), and the recovery rate of the target in the wheat flour treatment agent positive sample was slightly reduced (77.1% to 79.5%). When the ultrasonic time was fixed at 10 minutes, and the oscillation time was 10 minutes, 20 minutes, and 30 minutes, respectively, the effect on the extraction effect was not obvious (82.9% to 91.9%), and the recovery rates of the positive samples of the two matrices were reduced when there was no oscillation (59.4% to 69.2%). Considering the extraction efficiency and time cost, vortex oscillation for 10 minutes and ultrasonic extraction for 10 minutes were finally selected as the optimal extraction method and time.

[0076] Example 2

[0077] A peristaltic pump was used to directly inject the acetohydroxamic acid standard solution (1 μg / mL) to optimize the parameters. The molecular structure of acetohydroxamic acid can be found in Figure 5 , molecular weight 75.07, ionized by atmospheric pressure chemical ionization source, and obtained hydrogen generation parent ion [M+H]+ in Q1 full scan mode; then the production mode was used to scan the daughter ions by changing the collision energy, Figure 6 This is the daughter ion scan of acetohydroxamic acid at a collision energy of 10ev. It can be seen from the figure that the fragment information of acetohydroxamic acid is very rich, mainly 34.14, 57.97, and 43.07 fragment ions. Then the parent ion [M+H]+ found in the Q1MS mode and all the fragment ions found in the Production mode are imported into the list of the MRM scanning mode, and the parameters such as the collision energy are systematically optimized. It is found that 34.14 and 43.07 have the highest abundance among all the fragment ions, and the signals are stable and non-interfering in the determination of actual samples. Therefore, the fragments of 34.14 and 43.07 are preferred as monitoring daughter ions.

[0078] Linear range, detection limit, quantitation limit, recovery and precision

[0079] The prepared matrix standard working curve solution (the standard working curve solution prepared with the blank matrix solution to eliminate the matrix effect) was injected into the liquid chromatography-atmospheric pressure chemical ionization source-tandem mass spectrometer for detection. The standard curve was drawn with the peak area (Y) of the standard working solution as the ordinate and the concentration (X) of the standard working solution as the abscissa. The results showed that the correlation coefficient r of this method was greater than 0.9990 within the concentration range of 0.025 to 5.0 μg / mL, and the linearity was good.

[0080] Comparative experiment 6

[0081] The detection limit and quantification limit of the method were determined by adding the target compound to the blank sample matrix. The mass concentration corresponding to 3 times the signal-to-noise ratio of the chromatographic peak response value was taken as the detection limit of the method, and the lowest point of the curve concentration that could be accurately quantified was taken as the quantification limit of the method.

[0082] The accuracy and precision of the method were verified by adding acetohydroxamic acid at the limit of quantification, 2 times the limit of quantification, and 10 times the limit of quantification to blank flour and flour treatment agents. Six parallel determinations were performed at each concentration level, and the average value was taken to calculate the recovery rate and precision, as shown in Table 3.

[0083] Table 3 Detection limit, quantification limit, linear range, spike recovery and precision of acetohydroxamic acid

[0084]

[0085] Example 3

[0086] Sample determination

[0087] The acetohydroxamic acid in the samples of 20 batches of flour and 20 batches of flour treatment agents obtained from the market and the audit was determined according to the present invention. The method has good applicability and one positive sample of flour treatment agent was found with a detected content of 26.1 mg / kg. Figure 7 .

[0088] In summary, the present invention establishes a liquid chromatography-mass spectrometry identification process for acetohydroxamic acid in flour and flour treatment agents. The target compound is extracted with methanol, diluted with water and directly measured on the machine, and the pre-treatment operation is simple. The optimized liquid chromatography-mass spectrometry method has low solvent effect, and the matrix standard curve external standard method is used for quantitative determination, which simplifies the experimental steps. The method is simple to operate, highly sensitive, accurate, stable and reliable, and is suitable for the determination of acetohydroxamic acid in flour and flour treatment agents. At the same time, it provides technical support for the establishment of relevant standards and risk monitoring by regulatory authorities.

[0089] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0090] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying acetohydroxamic acid, characterized in that: The following steps are involved: (1) Sample pretreatment: weigh the sample, add solvent, extract and centrifuge, take the supernatant and dilute and filter to obtain the treated sample; (2) subjecting the sample treated in step (1) to liquid chromatography-mass spectrometry for identification; The liquid chromatography conditions are: Chromatographic column: T3 chromatographic column 3.0×100 mm, 2.7 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; flow rate 0.25 mL / min, injection volume: 5 μL; column temperature 35°C; gradient elution program as shown in Table 1: Table 1 Gradient elution program Mass spectrometry conditions: Ion source: atmospheric pressure chemical ionization source APCI+; scanning mode: multiple reaction monitoring; ion discharge current: 4μA; sheath gas temperature: 350℃; ion transfer tube temperature: 325℃; nebulizer gas flow rate: 45Arb; auxiliary gas flow rate: 5Arb; curtain gas flow rate: 1Arb; Mass spectrometry parameters and retention time table 2: Table 2 Mass spectrometry parameters and retention time The solvent in step (1) is methanol; The sample is flour or a flour treating agent.

2. The identification method according to claim 1, characterized in that: The mass volume ratio of the sample and the solvent in step (1) is 2.5 g:10 ml.

3. The identification method according to claim 2, characterized in that: The extraction in step (1) was performed by vortexing for 10 min and ultrasonic extraction for 10 min.

4. The identification method according to claim 3, characterized in that: The centrifugation in step (1) was carried out at 10,000 rpm for 5 min.

5. The identification method according to claim 4, characterized in that: The dilution in step (1): after centrifugation, the mixture is mixed and the supernatant is obtained, wherein the volume ratio of the supernatant to the diluted solution is 2:

5.

6. The identification method according to claim 5, characterized in that: The filtration in step (1) is carried out through a 0.22 μm filter membrane.

7. Application of the method according to any one of claims 1 to 6 in food testing, characterized in that: The test objects are flour or flour treatment agents.

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