CN111610270A

CN111610270A - Method for detecting organic pollutants in wheat flour based on high performance liquid chromatography-diode array method

Info

Publication number: CN111610270A
Application number: CN202010500246.0A
Authority: CN
Inventors: 郑红; 周传静; 于文江; 尹丽丽; 刘艳明; 薛霞; 王骏; 张喜琦
Original assignee: Shandong Institute for Food and Drug Control
Current assignee: Shandong Institute for Food and Drug Control
Priority date: 2020-06-04
Filing date: 2020-06-04
Publication date: 2020-09-01
Anticipated expiration: 2040-06-04
Also published as: CN111610270B

Abstract

The invention provides a method for detecting organic pollutants in wheat flour based on a high performance liquid chromatography-diode array method, and belongs to the technical field of detection and analysis. According to the invention, the methanol is adopted to extract the benzohydroxamic acid in the sample, and the liquid chromatography-diode array detection method is further established, so that the optimized liquid chromatography method has strong interference removing capability, is simple to operate, has high sensitivity and good accuracy, is stable and reliable, is suitable for measuring the benzohydroxamic acid in the wheat flour, and provides technical support for establishing relevant standards and monitoring risk of supervision departments, thereby having good value of practical application.

Description

Method for detecting organic pollutants in wheat flour based on high performance liquid chromatography-diode array method

Technical Field

The invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting organic pollutants in wheat flour based on a high performance liquid chromatography-diode array method.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The benzohydroxamic acid is easy to dissolve in water, methanol and the like, and because two oxygen atoms and nitrogen atoms with lone pair electrons exist in the polar group of the benzohydroxamic acid molecule and the two atoms are close to each other, the benzohydroxamic acid is easy to be separated from Cu²⁺、Ni²⁺、Fe³ ⁺The metal ions form a stable heterocyclic complex, which is an important mineral dressing agent and is usually used as an effective collector for difficult-to-separate minerals such as calamine, wolframite, scheelite, cassiterite and the like. However, the benzohydroxamic acid contains a benzene ring structure and N element, so that the benzohydroxamic acid is difficult to degrade organic matters, and potential risks and hazards are brought to human beings and ecological environment. Wheat flour is one of the staple foods most frequently eaten by people in northern China and is also a main raw material source of many processed foods such as baked foods. Recently, the report that the benzohydroxamic acid is not a food raw material is mixed into wheat flour is reported, so that the establishment of a method for measuring the benzohydroxamic acid in the wheat flour and the monitoring of the method are very important.

The currently reported methods for measuring the bendroxyximic acid are still relatively few, and the detection methods for the bendroxyximic acid in the reports mainly include an infrared spectroscopy method, an ultraviolet photometry, a direct spectrophotometry, a reversed-phase high-performance liquid chromatography method and the like, but the inventors find that the detection methods have many problems of false positive, low detection sensitivity, large analysis error and the like, and meanwhile, the reports for detecting the bendroxyximic acid in wheat flour are not reported in the prior art, so that the method needs to consider various factors to select a proper detection method so as to realize the detection of the bendroxyximic acid in the wheat flour.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for detecting organic pollutants in wheat flour based on a high performance liquid chromatography-diode array method.

In order to achieve the technical purpose, the technical scheme of the invention is as follows:

in a first aspect of the present invention, there is provided a method for detecting organic contaminants in wheat flour based on a high performance liquid chromatography-diode array method, the method comprising:

and (3) adding a sample to be detected into a solvent for dissolving, carrying out pretreatment, and detecting by adopting a high performance liquid chromatography-diode array method.

Wherein the sample to be detected is wheat flour;

the organic pollutant is benzohydroxamic acid.

Such solvents include, but are not limited to, water and methanol, and combinations thereof; methanol is preferred because benzohydroxamic acid is a weak acid and is readily soluble in water and methanol. According to the principle of similarity and compatibility, the recovery rates of three extracting agents of water, methanol/water (5:5, v: v) and methanol are compared, the recovery rate of the benzohydroxamic acid in the wheat flour is over 90% under the three extracting agents, but the methanol is finally selected as the optimal extracting agent in consideration of more polar impurities extracted by aqueous solution.

The pretreatment method specifically comprises the following steps: adding methanol into a sample to be detected, carrying out vortex, ultrasonic extraction and centrifugation, and taking an upper organic layer filtering membrane to obtain a liquid to be detected for on-machine detection.

The liquid chromatography conditions were: a chromatographic column: waters Xbridge C18 chromatography column (4.6 mm. times.250 mm, 3.5 μm); mobile phase: a is potassium dihydrogen phosphate solution (pH 3.7), B is methanol; detection wavelength: 228 nm; flow rate 1.0mL/min, sample volume: 10 mu L of the solution; the column temperature is 40 ℃; gradient elution.

The gradient elution procedure is shown in the following table:

in a second aspect of the present invention, the above detection method is applied in:

a) controlling the quality of the flour;

b) and (5) monitoring organic pollutants of the flour.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme establishes a liquid chromatography method of benzohydroxamic acid in wheat flour. After the target compound is extracted by methanol, the target compound is directly measured on a machine, and the pretreatment operation is simple. Experiments prove that the technical scheme has good linear relation (r is r) within the range of 0.1-10 mu g/mL²> 0.999); the detection limit of the method is 0.1mg/kg, and the quantification limit is 0.5 mg/kg; the recovery rate of the product is 93.7-102.7% and the RSD is 1.1-3.8% after repeated determination for 6 times under three addition levels of LOQ, 2LOQ and 10 LOQ.

Therefore, the optimized liquid chromatography method has strong interference removing capability, the detection method is simple to operate, high in sensitivity, good in accuracy, stable and reliable, and suitable for measuring the benzohydroxamic acid in the wheat flour, and meanwhile, technical support is provided for establishing relevant standards and monitoring risks of supervision departments.

Drawings

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

FIG. 1a is a chromatogram peak of a standard solution with methanol-phosphate as a mobile phase in an example of the present invention;

FIG. 1b is a chromatogram peak of a standard solution with methanol-ammonium acetate as a mobile phase in an example of the present invention;

FIG. 1c shows the chromatographic peak of a standard solution with methanol-water as the mobile phase in an example of the present invention;

FIG. 2 is a UV spectrum of benzohydroxamic acid and interferents in an embodiment of the present invention;

FIG. 3a is a chromatogram peak of a standard solution with 5% methanol as the mobile phase in an example of the present invention;

FIG. 3b is a chromatogram peak of a standard solution with 10% methanol as the mobile phase in an example of the present invention;

FIG. 3c is a chromatogram peak of a standard solution with 20% methanol as the mobile phase in an example of the present invention;

FIG. 3d is a chromatogram peak of a standard solution with 30% methanol as the mobile phase in an example of the present invention;

FIG. 3e is a chromatogram peak of a standard solution with 40% methanol as the mobile phase in an example of the present invention;

FIG. 4 is a graph showing the calibration curve of mellitic acid in the example of the present invention;

FIG. 5a is an HPLC chromatogram of negative wheat flour in an example of the present invention;

FIG. 5b is an HPLC chromatogram of wheat flour added at a quantitative limit (0.5mg/kg) in the examples of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, the currently reported methods for measuring p-hydroxamic acid are still relatively few, and the methods for detecting p-hydroxamic acid in the prior art mainly include infrared spectroscopy, ultraviolet photometry, direct spectrophotometry, reversed-phase high-performance liquid chromatography, but the above detection methods have many problems such as false positive, low detection sensitivity, large analysis error, and the like, and meanwhile, no report for detecting p-hydroxamic acid in wheat flour exists in the prior art.

In view of the above, in one embodiment of the present invention, there is provided a method for detecting organic contaminants in wheat flour based on high performance liquid chromatography-diode array method, the method comprising:

Wherein the sample to be detected is wheat flour;

the organic pollutant is benzohydroxamic acid.

In yet another embodiment of the present invention, the solvent includes, but is not limited to, water and methanol, and combinations thereof; methanol is preferred because benzohydroxamic acid is a weak acid and is readily soluble in water and methanol. According to the principle of similarity and compatibility, the recovery rates of three extracting agents of water, methanol/water (5:5, v: v) and methanol are compared, the recovery rate of the benzohydroxamic acid in the wheat flour is over 90% under the three extracting agents, but the methanol is finally selected as the optimal extracting agent in consideration of more polar impurities extracted by aqueous solution.

In another embodiment of the present invention, the pretreatment method specifically comprises: adding methanol into a sample to be detected, carrying out vortex, ultrasonic extraction and centrifugation, and taking an upper organic layer filtering membrane to obtain a liquid to be detected for on-machine detection.

In another embodiment of the present invention, the mass-to-volume ratio of the sample to be tested to methanol is 1 g: 3-6 mL, preferably 1 g: 5 mL.

Carrying out vortex treatment for 0.5-3 min, preferably for 1 min;

ultrasonic extraction is carried out for 10-20 min, preferably for 15 min;

the centrifugation conditions were: centrifuging for 2-8 min at a speed of 5000-10000 r/min, preferably centrifuging for 5min at a speed of 9000 r/min;

in yet another embodiment of the present invention, the filter is an organic filter with a pore size of 0.22. mu.m.

In yet another embodiment of the present invention, the liquid chromatography conditions are: a chromatographic column: waters Xbridge C18 chromatography column (4.6 mm. times.250 mm, 3.5 μm); mobile phase: a is potassium dihydrogen phosphate solution (pH 3.7), B is methanol; detection wavelength: 228 nm; flow rate 1.0mL/min, sample volume: 10 mu L of the solution; the column temperature is 40 ℃; gradient elution.

In yet another embodiment of the present invention, the gradient elution procedure is as follows:

in another embodiment of the present invention, the quantification is performed by external standard method.

In another embodiment of the present invention, the above detection method is applied to:

a) controlling the quality of the flour;

b) and (5) monitoring organic pollutants of the flour.

Wherein the flour is wheat flour;

the organic pollutant is benzohydroxamic acid.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

1. Materials and methods

1.1 instruments and reagents

Model S6000 high performance liquid chromatograph equipped with diode array detector (hua spectral science and technology limited, china); BT 125D electronic balance (Sartorius, germany); model MS3 vortex mixer (IKA, Germany); SB-800DTD ultrasonic cleaning apparatus (Ningbo Xinzhi Biotech Co., Ltd.); Mili-Q ultra-pure water machine (Millipore, USA).

Benzohydroxamic acid (available from CATO, USA); methanol (chromatographically pure); potassium dihydrogen phosphate (KH)₂PO₄) Analytically pure, group of national medicine; phosphoric acid (H)₃PO₄) Analytically pure, group of Chinese medicine.

1.2 Standard solution preparation

Accurately weighing the standard substance of the benzohydroxamic acid, dissolving the standard substance of the benzohydroxamic acid by using methanol, fixing the volume, preparing a standard stock solution with the concentration of 1mg/mL, and storing the standard stock solution at the temperature of minus 18 ℃ in a dark place. Accurately transferring the standard stock solution of the benzohydroxamic acid, and diluting the stock solution with methanol to prepare a mixed standard intermediate solution with the concentration of 10 mu g/mL. Then, the mixture was diluted with methanol to prepare a series of standard working solutions having concentrations of 0.1. mu.g/mL, 0.2. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 5. mu.g/mL, and 10. mu.g/mL.

1.3 sample pretreatment

Accurately weighing 2g (accurate to 0.001g) of sample, placing in a 50mL centrifuge tube with a plug, accurately adding 10mL methanol, vortexing for 1min, ultrasonically extracting for 15min, centrifuging for 5min at 9000r/min, collecting the upper organic layer, filtering with 0.22 μm organic filter membrane, and determining with a liquid chromatograph.

1.4 liquid chromatography conditions

Chromatographic column Waters Xbridge C18 chromatographic column (4.6 mm. times.250 mm, 3.5 μm); mobile phase: a is potassium dihydrogen phosphate solution (pH 3.7), B is methanol; detection wavelength: 228 nm; flow rate 1.0mL/min, sample volume: 10 mu L of the solution; the column temperature is 40 ℃; and (3) an elution mode: the gradient elution is shown in Table 1.

Table 1 liquid chromatography method mobile phase gradient elution procedure

2 results and discussion

2.1 optimization of pretreatment conditions

Benzohydroxamic acid is a weak acid, readily soluble in water and methanol. According to the principle of similarity and compatibility, the recovery rates of three extracting agents of water, methanol/water (5:5, v: v) and methanol are compared in experiments, and the recovery rates of the benzohydroxamic acid in the wheat flour under the three extracting agents are all over 90 percent, but the methanol is selected as the final extracting agent considering that polar impurities extracted by an aqueous solution are more.

2.2 optimization of liquid chromatography conditions

Since benzohydroxamic acid is a weak acid, its PKa is 8.89, and both molecular and ionic morphologies exist. When the mobile phase is pH > PKa +2, namely pH > 10.89, the mobile phase exists mainly in an ion form, is easily distributed in the mobile phase, the retention property is weak, and in most common C18 chromatographic columns (pH 2-8), the pH is high, and silica gel is easily dissolved. When the pH value is less than PKa-2, namely when the pH value is less than 6.89, the molecular form exists mainly, and the molecular form is easy to distribute in a stationary phase, and the retention property is strong.

2.2.1 selection of Mobile phase

This example first attempted to compare the chromatographic peak profiles (fig. 1a, 1b, 1C) and retention times, peak heights and peak areas (table 2) of three mobile phases, methanol-phosphate (pH 3.7), methanol-ammonium acetate (unadjusted pH, pH 6.8) and methanol-water, using an X-bridge C18 column under the same gradient elution conditions (organic phase: aqueous phase: 20:80, v/v). As can be seen from the above figures and tables, the retention times under the three mobile phase conditions are substantially consistent, since after water dissolution of carbon dioxide in air, the pH of all three mobile phases is less than 7, and the benzohydroxamic acid exists in a molecular state. However, the peak shape is greatly different, and gradually worsened according to the order of methanol-phosphate (pH 3.7), methanol-ammonium acetate (pH 6.8 without adjusting pH), and methanol-water, leading to gradually reduced peak height and seriously affecting the sensitivity of the compound. Then, an attempt was made to adjust the pH of ammonium acetate to 3.7 in accordance with the phosphate, and no improvement was found in the peak shape, so methanol-phosphate was finally selected as the final mobile phase.

Table 2 retention time, peak height, peak area of the three mobile phase compositions under the same gradient elution conditions (organic phase: aqueous phase: 20:80, v/v)

2.2.2 selection of gradient conditions

After 10mg/kg of target compound is added into wheat flour and treated according to 1.3, the qualitative and quantitative determination that the benzohydroxamic acid is influenced by the existence of potential interferent under the elution of the mobile phase gradient (methanol: phosphate is 20:80, v/v) is examined, and the wheat flour is found to have a relatively obvious interference peak, the ultraviolet spectral characteristics of the interference peak are obviously different from those of the target benzohydroxamic acid (figure 2), and the target compound and the interference peak can not be completely separated. Therefore, it is attempted to increase the separation degree of the interferents and the hydroxamic acid by adjusting the ratio of the organic phase (5% to 40%), and as shown in FIGS. 3a to 3e, it was found that when the ratio of methanol is 5% and 30%, the two compounds are completely separated, but only when the ratio of methanol is 5%, the separation degree of the target and the interferents is optimal, so that 5% methanol was finally selected as the final elution condition. Finally, the separation effect of an Atlantis T3 chromatographic column is tried, and the fact that although no peak influence occurs in wheat flour and a flour modifier is found, the retention capacity of the Atlantis T3 chromatographic column is strong, so that the peak of the target compound appears at 24.00min and is obviously later than that of X-bridge C18(15.68min), the peak shape is widened, the sensitivity is reduced, the running time is prolonged, and the detection efficiency is reduced.

2.3 Linear Range, calibration Curve and correlation coefficient of the method

Performing high performance liquid chromatography detection on the standard working solution prepared in the step 1.2, drawing a standard curve by taking the peak area of the standard working solution as the ordinate and the concentration of the standard solution as the abscissa, wherein the result shows that the HPLC-PDA method is used for measuring the concentration of the standard working solution within the range of 0.1-10 mu g/mL and r is²Both are greater than 0.999 and the linearity is good, see figure 4.

2.4 detection and quantitation limits

The detection limit and the quantitative limit of the method are determined by adding the target compound into the blank wheat flour. The mass concentration corresponding to the 3 times signal-to-noise ratio of the chromatographic peak response value is taken as the detection limit of the method, and the lowest point of the curve concentration capable of being accurately quantified is taken as the quantification limit of the method, which is shown in table 3.

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

3.4 method recovery and precision

The target compound with the quantitative limit of 2 times, the quantitative limit of 10 times is added into the negative wheat flour to carry out the standard adding recovery test, and the accuracy and precision of the method are verified. Each concentration level was measured in parallel 6 times and averaged to calculate recovery and precision. The results are shown in Table 3. HPLC-PDA chromatograms with addition levels at the limit of quantitation are shown in FIGS. 5a-5 b.

3.5 sample determination

The method has good applicability to the determination of the commercial 20 wheat flour on the benzohydroxamic acid, and no positive sample is detected.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for detecting organic pollutants in wheat flour based on a high performance liquid chromatography-diode array method is characterized by comprising the following steps:

adding a sample to be detected into a solvent for dissolving, carrying out pretreatment, and detecting by adopting a high performance liquid chromatography-diode array method;

wherein the sample to be detected is wheat flour;

the organic pollutant is benzohydroxamic acid.

2. The method of claim 1, wherein the solvent comprises water and methanol, and combinations thereof; methanol is preferred.

3. The method according to claim 1, wherein the pre-treatment method is specifically: adding methanol into a sample to be detected, carrying out vortex, ultrasonic extraction and centrifugation, and taking an upper organic layer filtering membrane to obtain a liquid to be detected for on-machine detection.

4. The method of claim 3, wherein the mass to volume ratio of the sample to be tested to methanol is 1 g: 3-6 mL, preferably 1 g: 5 mL.

5. A method according to claim 3, wherein the vortexing is performed for 0.5 to 3min, preferably 1 min.

6. The method of claim 3, wherein the ultrasonic extraction is performed for 10-20 min, preferably 15 min.

7. The method of claim 3, wherein the centrifugation conditions are: centrifuging at 5000-10000 r/min for 2-8 min, preferably at 9000r/min for 5 min.

8. The method of claim 3, wherein the filter membrane is an organic filter membrane.

9. The method of claim 1, wherein the liquid chromatography conditions are: a chromatographic column: waters xbridge c18 column, 4.6mm × 250mm, 3.5 μm; mobile phase: a is potassium dihydrogen phosphate solution (pH 3.7), B is methanol; detection wavelength: 228 nm; flow rate 1.0mL/min, sample volume: 10 mu L of the solution; the column temperature is 40 ℃; gradient elution;

preferably, the gradient elution procedure is as follows:

preferably, the quantification is performed by external standard methods.

10. Use of the assay method of any one of claims 1-9 in:

a) controlling the quality of the flour;

b) monitoring organic pollutants of the flour;

preferably, the flour is wheat flour;

preferably, the organic contaminant is benzohydroxamic acid.