CN114236007B - Method for measuring acetohydroxamic acid in wheat flour and flour treatment agent thereof - Google Patents
Method for measuring acetohydroxamic acid in wheat flour and flour treatment agent thereof Download PDFInfo
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- CN114236007B CN114236007B CN202111551639.5A CN202111551639A CN114236007B CN 114236007 B CN114236007 B CN 114236007B CN 202111551639 A CN202111551639 A CN 202111551639A CN 114236007 B CN114236007 B CN 114236007B
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- RRUDCFGSUDOHDG-UHFFFAOYSA-N acetohydroxamic acid Chemical compound CC(O)=NO RRUDCFGSUDOHDG-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 229960001171 acetohydroxamic acid Drugs 0.000 title claims abstract description 61
- 235000013312 flour Nutrition 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 28
- 241000209140 Triticum Species 0.000 title claims abstract description 26
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 26
- 235000010037 flour treatment agent Nutrition 0.000 title claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000004817 gas chromatography Methods 0.000 claims abstract description 10
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000004458 analytical method Methods 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 6
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000010926 purge Methods 0.000 claims description 6
- 125000006850 spacer group Chemical group 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 239000012159 carrier gas Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 238000004451 qualitative analysis Methods 0.000 claims 1
- 238000004445 quantitative analysis Methods 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 7
- 230000014759 maintenance of location Effects 0.000 abstract description 5
- 238000010812 external standard method Methods 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000011002 quantification Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 31
- 239000011159 matrix material Substances 0.000 description 13
- 239000012488 sample solution Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 1
- 239000004390 EU approved flour treatment agent Substances 0.000 description 1
- 229940090496 Urease inhibitor Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000013215 result calculation Methods 0.000 description 1
- 210000004767 rumen Anatomy 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000002601 urease inhibitor Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method for measuring acetohydroxamic acid in wheat flour and a flour treatment agent thereof. Belongs to the technical field of chemical analysis. The method comprises the following steps: extracting a sample; purifying and analyzing; drawing a standard curve; and (5) qualitatively and quantitatively identifying the sample. The invention detects the acetohydroxamic acid based on the gas chromatography technology, and C is extracted by ethanol 18 Purifying by solid phase extraction column, filtering, detecting by gas chromatograph equipped with nitrogen-phosphorus detector, and quantifying by gas chromatograph retention time qualitative and external standard method. The method is simple and convenient, has high sensitivity, and is very suitable for the measurement of the acetohydroxamic acid in the wheat flour and the flour treatment agent thereof. The detection limit is 10mg/kg; the limit of quantification was 30mg/kg.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for measuring acetohydroxamic acid in wheat flour and a flour treatment agent thereof.
Background
Acetohydroxamic acid (Acetohydroxamic acid, AHA), also known as acetohydroxamic acid, mycoston, and the like, is a hydroxamic acid-type compound, CAS is 546-88-3, and the molecular formula is C 2 H 5 NO 2 The molecular weight was 75.0666. the-CONHOH functional group in the acetohydroxamic acid structure is an excellent organic ligand and is an important beneficiation reagent; acetohydroxamic acid was widely used in animal husbandry in the nineties of the last century as a good rumen microbial urease inhibitor.
Flour is one of the staple foods most frequently eaten by the northern people in China, is also the main raw material of a plurality of baked and other processed foods, and can prevent the flour and products thereof from being colored and browned by adding the acetohydroxamic acid into the flour and the flour treating agent.
However, acetohydroxamic acid is a non-food material. Currently there is no standard or method of measurement for acetohydroxamic acid in flour or flour treatment.
Thus, establishing and monitoring the assay of acetohydroxamic acid in wheat flour and flour treatments thereof is a problem that needs to be addressed by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides a method for measuring acetohydroxamic acid in wheat flour and its flour treatment agent.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for measuring acetohydroxamic acid in wheat flour and a flour treatment agent thereof comprises the following steps:
(1) Sample extraction: adding polyethylene glycol into the sample, mixing uniformly, adding absolute ethyl alcohol, oscillating, performing ultrasonic extraction, and centrifuging to obtain a supernatant for later use;
(2) Purification analysis: transferring the supernatant obtained in step (1) to activated C 18 Collecting effluent liquid in the solid phase extraction column, filtering to obtain subsequent filtrate, and performing gas chromatographic analysis;
(3) Drawing a standard curve;
(4) And (5) qualitatively and quantitatively analyzing the sample.
Preferably: the mass volume ratio of the sample in the step (1), the polyethylene glycol and the absolute ethyl alcohol is 1g:0.4g:10mL; vortex oscillation is used for oscillation, and the time is 10min; the power of the ultrasonic wave is 100kHz, and the extraction time is 10min; the centrifugation conditions were: 8000r/min at 4℃and 5min.
Preferably: and (2) activating: activation with 6mL methanol, 6mL ethanol; and (3) collecting effluent liquid: discarding the initial 3/5 volume of the effluent; and (3) filtering: passing through 0.22 μm organic filter membrane; continuing filtrate: discarding 2-5 drops of primary filtrate to obtain subsequent filtrate.
Preferably: conditions of step (2) gas chromatography:
chromatographic column specification: 30 m.times.320 μm.times.0.25. Mu.m;
carrier gas: the purity of the high-purity nitrogen is more than or equal to 99.9995%; column flow rate: 2.5mL/min;
sample inlet temperature: 150 ℃; sample injection amount: 2.0 μl;
heating program: the initial temperature is 100 ℃, 1min is kept, 15 ℃/min is heated to 180 ℃, 1min is kept, 6 ℃/min is heated to 220 ℃, and 1min is kept;
a detector: a nitrogen-phosphorus detector; heater temperature: 300 ℃; air flow rate: 60mL/min; hydrogen gas flow rate: 3mL/min; tail blow flow (N) 2 ) Combination: 7.5mL/min.
Spacer purge mode: a standard; spacer purge flow: 3mL/min.
Preferably: step (3), standard curve regression equation is y= 33.236X-117.02, where X is concentration and Y is peak area.
The beneficial effects are that: the molecular structure of the acetohydroxamic acid contains N element, and can be measured by a gas chromatography nitrogen-phosphorus detector (NPD). The sample is extracted by ethanol solution, polyethylene glycol is used as dispersing agent, and is measured by a gas chromatograph nitrogen-phosphorus detector (NPD), the retention time is qualitative, and the external standard method is quantitative.
Compared with the prior art, the invention discloses a method for measuring the acetohydroxamic acid in wheat flour and flour treatment agent thereof, which has the technical effects that the method detects the acetohydroxamic acid based on gas chromatography technology, and extracts C by ethanol 18 Purifying by solid phase extraction column, filtering, detecting by gas chromatograph equipped with nitrogen-phosphorus detector, and quantifying by gas chromatograph retention time qualitative and external standard method. The method is simple and convenient, the method is simple,the sensitivity is high, and the method is very suitable for the measurement of the acetohydroxamic acid in the wheat flour and the flour treatment agent. The detection limit is 10mg/kg; the limit of quantification was 30mg/kg.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the standard curve of acetohydroxamic acid according to the present invention.
FIG. 2 is a diagram showing a gas chromatogram of the acetohydroxamic acid standard provided by the invention.
FIG. 3 is a graph showing the gas chromatography of acetohydroxamic acid in the wheat flour according to the present invention.
FIG. 4 is a graph showing the gas chromatography of acetohydroxamic acid in the flour treatment agent provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a method for measuring acetohydroxamic acid in wheat flour and a flour treatment agent thereof.
In the examples, the starting reagents used were analytically pure and the flours or flour treatment agents in the samples were of commercially available origin.
Reagent(s)
Absolute ethyl alcohol (C) 2 H 5 OH): chromatographic grade.
Polyethylene glycol [ 8000 for relative molecular mass, HO (CH) 2 CH 2 O)nH]。
Methanol (CH) 3 OH): chromatographic grade.
Standard substance
Acetohydroxamic acid standard (Acetohydroxamic acid, AHA): CAS number 546-88-3, molecular formula C 2 H 5 NO 2 The relative molecular weight 75.07, the purity is more than or equal to 98 percent, or the standard substance is authenticated by the country and granted to the standard substance certificate.
Standard stock solution of acetohydroxamic acid: accurately weighing 0.01016g of the standard product of acetohydroxamic acid, dissolving the standard product of acetohydroxamic acid in ethanol, and fixing the volume to 10mL to prepare the standard stock solution of acetohydroxamic acid with the concentration of 1.0 mg/mL. The solution is preserved in dark at 0-4 ℃ and can be used for 6 months.
Acetohydroxamic acid standard series working solution: accurately sucking 0.03, 0.05, 0.10, 0.20, 0.50, 1.00 and 2.00mL of the standard stock solution of the acetohydroxamic acid with the concentration of 3, 5, 10, 20, 50, 100 and 200 mug/mL respectively, and fixing the volume to a 10mL volumetric flask by using ethanol to obtain standard series working solutions with the concentration of 3, 5, 10, 20, 50, 100 and 200 mug/mL.
Material
0.22 μm filter membrane: an organic system.
C 18 The solid phase extraction column (250 mg/3 mL) was activated with 6mL of methanol and 6mL of ethanol prior to use.
Apparatus and device
Gas chromatograph: equipped with a Nitrogen Phosphorus Detector (NPD).
Analytical balance: the sensory amounts were 0.01mg and 0.01g, respectively.
Volumetric flask: 10mL.
Centrifuge tube: 15mL centrifuge tube with plug.
An ultrasonic cleaner.
Vortex mixer.
Centrifuge: the rotating speed is not lower than 8000r/min.
Pipetting gun: the measurement ranges are 200 mu L and 1mL respectively.
Sample preparation and preservation
Weighing about 200g of representative wheat flour or flour treating agent sample, uniformly mixing, storing in a clean wide-mouth container, drying and keeping in dark place for later use.
Example 1
Extraction of
1g (accurate to 0.01 g) of the sample is accurately weighed, placed in a 15mL centrifuge tube with a plug, and 0.4g (accurate to 0.01 g) of polyethylene glycol is added for uniform mixing. Accurately adding 10mL of absolute ethanol, shaking for 10min by vortex, extracting with ultrasound (power: 100 kHz) for 10min, and centrifuging at 4deg.C for 5min at 8000r/min. The supernatant was ready for use.
Purification of
Transfer 5mL of supernatant to activated C 18 In the solid phase extraction column, the initial 3mL effluent is discarded, the effluent is collected and passes through a 0.22 mu m organic filter membrane, 2-5 drops of initial filtrate are discarded, and the subsequent filtrate is taken and measured by a machine.
Reference conditions for gas chromatography
Chromatographic column: agilent 19091N-1131 HP-INNOWAX (40-270 ℃), 30 m.times.320 μm.times.0.25 μm, or equivalent;
carrier gas: the purity of the high-purity nitrogen is more than or equal to 99.9995%; column flow rate: 2.5mL/min.
Sample inlet temperature: 150 ℃; sample injection mode: not split; liner (ultra-high inert liner): agilent 5190-2293: 900. Mu.L; sample injection amount: 2.0. Mu.L.
Heating program: the initial temperature was 100℃for 1min,15℃per minute was raised to 180℃for 1min,6℃per minute was raised to 220℃for 1min.
A detector: nitrogen Phosphorus Detector (NPD); heater temperature: 300 ℃; air flow rate: 60mL/min; hydrogen gas flow rate: 3mL/min; tail blow flow (N2) combination: 7.5mL/min.
Spacer purge mode: a standard; spacer purge flow: 3mL/min.
Production of standard curve
Respectively injecting the standard series of acetohydroxamic acid working solutions into a gas chromatograph from low to high according to mass concentration, measuring corresponding peak areas, and drawing a standard curve by taking the mass concentration of the standard series of working solutions as an abscissa and the peak areas as an ordinate:
the standard solution with the concentration of 3, 5, 10, 20, 50, 100 and 200 mug/mL of acetohydroxamic acid is prepared by dilution with absolute ethyl alcohol, the concentration (X) is taken as an abscissa, the peak area (Y) is taken as an ordinate, a standard curve is drawn, and the result is shown in table 1 and figure 1.
TABLE 1
Standard curve regression equation is y= 33.236X-117.02 (R 2 0.9996), the acetohydroxamic acid has good linearity in the concentration range of 3-200 mug/mL, R 2 Reaching more than 0.995. However, considering that the content of the actual sample is unknown, the possible difference is large, and the response intensity of the gas chromatograph with different brands and models is different, in the actual sample measurement, different linear ranges can be selected according to the concentration of the sample or the response intensity of the compound, or the sample is properly diluted and then is subjected to the on-machine measurement, so that the more accurate quantitative effect is achieved.
Qualitative determination
The sample solution is injected into a gas chromatograph, the retention time of the detected substances in the sample solution and the retention time of the acetohydroxamic acid in the standard solution are within +/-2.5 percent, and the existence of the acetohydroxamic acid in the sample solution can be identified.
Measurement of sample solution
The sample solution was injected into a gas chromatograph to obtain the peak area of acetohydroxamic acid. And quantifying according to a standard curve by an external standard method to obtain the concentration of the acetohydroxamic acid in the sample solution. The concentration of the acetohydroxamic acid in the sample solution is within the linear range of the standard curve, and the concentration of the acetohydroxamic acid is determined by loading the diluted acetohydroxamic acid on the machine again when the concentration exceeds the linear range.
Blank test
The same procedure was conducted as in the case of the sample except that no sample was added or only a blank sample containing no acetohydroxamic acid was added as confirmed by gas chromatography.
Result calculation
The content of acetohydroxamic acid in the sample is calculated according to formula (1):
wherein:
the content of acetohydroxamic acid in the X-test sample is expressed in milligrams per kilogram (mg/kg);
c-concentration of acetohydroxamic acid in the sample solution in micrograms per milliliter (μg/mL) from the standard curve;
v-volume of measurement solution in milliliters (mL);
m-mass of sample in grams (g);
f-dilution factor;
1000-conversion factor.
The calculation result is expressed as an arithmetic average of two independent measurement results obtained under the condition of repeatability, and two significant digits are reserved.
Detection limit and quantitative limit
To determine the detection limit, a low concentration labeling (labeling level is 50 mg/kg) experiment was performed in the sample, 7 blank labeling experiments were repeated according to the sample analysis step, the standard deviation of the number of 7 parallel determinations was calculated, and the detection limit was calculated according to the formula (2). The standard deviation and the standard deviation of the labeled measurement results are shown in tables 2 and 3. The detection limit of wheat flour was calculated to be 9.37mg/kg and the detection limit of the flour treatment agent was calculated to be 5.81mg/kg. The detection limit of the acetohydroxamic acid in the method is tentatively set to 10mg/kg, and the detection limit of 3 times is set to 30mg/kg.
MDL＝t (n-1,0.99) ×S (2)
Wherein: MDL-method detection limit;
number of parallel determinations of n-samples;
the t-degree of freedom is n-1, the distribution of t (one-sided) when the confidence is 99%;
standard deviation of S-n replicates.
Wherein, when the degree of freedom is n-1, the t value when the confidence is 99% refers to Table 4.
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
Examples 2 to 13
Sample detection
6 representative samples of wheat flour and flour treatment agent were selected, and a total of 12 samples were tested according to the methods prescribed by the present standard, and the residual amounts of acetohydroxamic acid in the two samples of flour treatment agent were found to be 38.46 (see FIG. 4) and 37.25mg/kg, and none of the residual amounts of acetohydroxamic acid in the remaining samples were detected.
Experimental effect
Precision of
The absolute difference between the two independent measurements obtained under repetitive conditions must not exceed 10% of the arithmetic mean.
1g of a blank matrix sample is weighed, an acetohydroxamic acid standard (n=6) is respectively added at different levels, the addition levels are 50mg/kg and 100mg/kg, the blank matrix sample is treated according to a planned sample pretreatment method, and the blank matrix sample is measured on a machine, and the precision of the method is inspected. The results are shown in tables 5 and 6. The addition level of acetohydroxamic acid in the wheat flour matrix was 0.7% and 2.3% Relative Standard Deviation (RSD) at 50mg/kg and 100mg/kg, respectively; the Relative Standard Deviation (RSD) of acetohydroxamic acid in the flour treatment matrix was 1.8% and 3.0% at addition levels of 50mg/kg and 100mg/kg, respectively. The precision of the method at the planned addition level can meet the requirements of residual analysis.
TABLE 5 precision results for wheat flour substrates
Table 6 precision results for flour treatment matrices
Determination of the recovery of the addition of the mark
Taking wheat flour and flour treating agent without acetohydroxamic acid as blank matrix samples, taking 1g of the blank matrix samples, adding acetohydroxamic acid standard substances (n=6) at different levels, treating according to a planned sample pretreatment method, performing on-machine measurement, performing an addition recovery experiment, calculating average recovery rate, and examining the accuracy of the method, wherein the addition levels are 50mg/kg, 70mg/kg and 100mg/kg, and the results are shown in tables 7 and 8.
The recovery rates of the acetohydroxamic acid in the wheat flour matrix are respectively 81.1%, 92.4% and 106.2% at the addition levels of 50mg/kg, 70mg/kg and 100 mg/kg; the recovery rates of acetohydroxamic acid in the flour treatment agent matrix are 93.4%, 95.6% and 94.9% at the addition levels of 50mg/kg, 70mg/kg and 100mg/kg, respectively. The accuracy of the method at the proposed addition level is described as meeting the requirements of residual analysis.
TABLE 7 accuracy results for wheat flour matrices
Table 8 accuracy results of flour treatment matrices
Stability test
The prepared wheat flour matrix is added with the standard solution, continuous sample injection is carried out in 24 hours, and the measurement results are shown in Table 9, and fig. 2 and 3. 7 time points are selected within 24 hours, and the peak area of the matrix adding standard solution is compared, and the RSD value is 5.3%, so that the stability of the matrix adding standard solution within 24 hours is good.
TABLE 9 stability results of wheat flour matrix
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer 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 (5)
1. A method for determining acetohydroxamic acid in wheat flour and a flour treatment agent thereof, which is characterized by comprising the following steps:
(1) Sample extraction: adding polyethylene glycol into the sample, mixing uniformly, adding absolute ethyl alcohol, oscillating, performing ultrasonic extraction, and centrifuging to obtain a supernatant for later use;
(2) Purification analysis: transferring the supernatant obtained in step (1) to activated C 18
Collecting effluent liquid in the solid phase extraction column, filtering to obtain subsequent filtrate, and performing gas chromatographic analysis;
(3) Drawing a standard curve;
(4) Qualitative and quantitative analysis of the sample;
the mass volume ratio of the sample, the polyethylene glycol and the absolute ethyl alcohol in the step (1) is 1g:0.4g:10mL;
conditions of the gas chromatography described in step (2):
HP-INNOWAX column;
heating program: the initial temperature is 100 ℃, the temperature is kept for 1min, the temperature is raised to 180 ℃ at 15 ℃/min, the temperature is kept for 1min, the temperature is raised to 220 ℃ at 6 ℃/min, and the temperature is kept for 1min;
a detector: a nitrogen-phosphorus detector.
2. The method for measuring acetohydroxamic acid in wheat flour and a flour treatment agent according to claim 1, wherein the shaking vortex is carried out for 10min; the power of the ultrasonic wave is 100kHz, and the extraction time is 10min; the centrifugation conditions are as follows: 8000 of r/min at 4℃and 5min of centrifugation time.
3. The method for measuring acetohydroxamic acid in wheat flour and flour treatment agent according to claim 1, wherein the activating in step (2) comprises: activation with 6mL methanol, 6mL ethanol; the collecting effluent liquid: discarding the initial 3/5 volume of the effluent; the filtration: passing through 0.22 mu m organic system filter membrane; the subsequent filtrate: discarding 2-5 drops of primary filtrate to obtain subsequent filtrate.
4. The method for measuring acetohydroxamic acid in wheat flour and flour treatment agent according to claim 1, wherein the conditions of the gas chromatography in step (2) are as follows:
chromatographic column specification: 30m×320 μm×0.25 μm;
carrier gas: the purity of the high-purity nitrogen is more than or equal to 99.9995%; column flow rate: 2.5mL/min;
sample inlet temperature: 150. the temperature is lower than the temperature; sample injection amount: 2.0 Mu L;
heater temperature: 300. the temperature is lower than the temperature; air flow rate: 60mL/min; hydrogen gas flow rate: 3mL/min; tail blow flow N 2 Combination: 7.5 mL/min;
spacer purge mode: a standard; spacer purge flow: 3mL/min.
5. The method of claim 1, wherein in step (3), the standard curve regression equation is y= 33.236X-117.02, wherein X is concentration and Y is peak area.
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