CN113281421A - Efficient determination method for formaldehyde in starch food - Google Patents

Abstract

The invention discloses a high-efficiency determination method of formaldehyde in starch food, which comprises the following steps: (1) preparing a standard solution; (2) pretreating a sample; (3) preparing a reference substance standard solution; (4) establishing a formaldehyde standard reference substance database; (5) and (4) measuring the content. By adopting an ultra-high performance liquid chromatography-ion hydrazine mass spectrometry combined method, a formaldehyde standard reference substance database is established, and by spectrum library retrieval, the comparison and confirmation of the EPI spectrogram of the positive suspicious sample and the standard spectrum library can be carried out, so that false positives can be effectively eliminated. In the mass concentration range of 0.01 mu g/mL-1 mu g/mL, the method has good linear relation, the correlation coefficient r is more than 0.995, the detection limit is 0.05mg/kg, the normalized average recovery rate is 91.7 percent, and the RSD is 2.08 percent. The method has the advantages of simple pretreatment, high sensitivity and strong specificity, and can accurately perform qualitative and quantitative determination on the illegally added formaldehyde in the sample.

Description

Efficient determination method for formaldehyde in starch food

Technical Field

The invention relates to the field of food detection, in particular to a high-efficiency determination method for formaldehyde in starch food.

Background

Formaldehyde, also known as Formaldehyde, is known under the English name Formaldehyde. Formaldehyde has common toxic effects, and is easy to damage the central nervous system, sensory organs, liver, kidney and the like; also has severe irritation, mutagenesis, and carcinogenesis effects; high concentrations can cause laryngeal spasm, pneumonia, pulmonary edema and death. Animal experiments prove the carcinogenicity of formaldehyde, and epidemiological researches find that people who are contacted with formaldehyde for a long time can cause cancers of nasal cavities, mouths and throats, cancers of digestive systems, lung cancers, skin cancers, leukemia and the like.

Formaldehyde is an illegal additive prohibited from being used in food by the national plain text regulations and cannot be detected in food, but formaldehyde can be whitened and changed in color, so that formaldehyde is often fumigated by illegal vendors or directly added into food such as rice flour, wheaten food, mushrooms or bean products, and the presence of formaldehyde is detected in many foods to different degrees. The illegal addition of formaldehyde in food preservation leads to serious standard exceeding of formaldehyde, and the physical health of people is greatly damaged.

At present, liquid chromatography is mainly adopted for detecting formaldehyde in rice flour or wheat flour. Although the liquid chromatography is simple and convenient, after the formaldehyde is derived, in the process of liquid chromatography analysis, more interferents exist, the situation of false positive appears, and great difficulty is brought to the confirmation of the formaldehyde.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for efficiently measuring formaldehyde in starch food, which has the advantages of simple pretreatment, high sensitivity, strong specificity and accurate and illegal addition of formaldehyde to a sample.

According to one aspect of the invention, the invention provides a method for efficiently measuring formaldehyde in starch food, which is characterized by comprising the following steps:

(1) preparation of Standard solutions

Acetonitrile-saturated n-hexane: adding 100mL of n-hexane into 100mL of acetonitrile, fully oscillating, standing for layering, and taking supernatant;

buffer solution: weighing 5.28g of sodium acetate, adding water for dissolution, adding 2.0mL of glacial acetic acid, and adding water to a constant volume of 1L;

2, 4-dinitrophenylhydrazine solution: weighing 600mg of 2, 4-dinitrophenylhydrazine hydrochloride, dissolving with acetonitrile, and fixing the volume to 1L;

the derived liquid: measuring a buffer solution and a 2, 4-dinitrophenylhydrazine solution in a volume ratio of 1: 1, uniformly mixing;

intermediate solution of standard solution: accurately transferring 1mL of formaldehyde standard solution with the concentration of 100 mu g/mL, and diluting acetonitrile to a constant volume of 10mL to obtain a standard solution intermediate solution with the concentration of 10 mu g/mL;

(2) sample pretreatment

Preparing a sample to be tested: weighing 2g of a sample, adding 10mL of derivative solution, fully and uniformly mixing, placing in a constant temperature environment of 60 ℃ for shaking for 1h, taking out, cooling to room temperature, carrying out centrifugal separation for 5min, and taking supernatant to pass through a 0.22 mu m organic membrane to obtain a sample to be tested;

(3) preparation of Standard solutions of controls

Accurately transferring 10 mu L, 50 mu L, 100 mu L, 500 mu L and 1000 mu L to 10mL of standard solution intermediate solution into volumetric flasks respectively, adding a buffer solution to 5mL, then adding 5mL of 2, 4-dinitrophenylhydrazine solution, uniformly mixing, shaking for 1h in a constant temperature environment at 60 ℃, taking out and cooling to room temperature, carrying out centrifugal separation for 5min, and taking supernatant to pass through a 0.22 mu m organic membrane to obtain a standard solution reference sample;

(4) establishing a formaldehyde standard reference substance database

Injecting a standard solution reference sample into an ultra-high performance liquid chromatography-ion trap composite mass spectrometer, and establishing a formaldehyde standard reference sample database;

(5) determination of content

And adding the sample to be detected into a liquid chromatogram-ion trap composite mass spectrometer, retrieving through a spectrum library, and comparing and confirming the EPI spectrogram of the positive suspicious sample with a standard spectrum library to obtain a detection result.

In some embodiments, the sample in step (2) is rice, rice flour, pea flour, or other starch food, and the sample is in the form of large granules and is required to be thoroughly mashed and mixed.

In some embodiments, the liquid chromatography conditions in steps (4) and (5) are:

a chromatographic column: acquisty UPLC BEH C181.7 μm, 2.1 mm. times.100 mm; sample introduction amount: 5 mu L of the solution; column temperature: 40 ℃, mobile phase: a is water: b is methanol; gradient elution, flow rate: 0.25 mL/min; the gradient elution procedure was: 0-2 min, 90% A; 2.5min, 70% A; 3-5 min, 10% A; 5.5-8 min, 90% A.

In some embodiments, the mass spectrometry conditions in steps (4) and (5) are:

electrospray negative ion mode (ESI-); scanning mode: multiple Reaction Monitoring (MRM) → information association data acquisition (IDA) → enhanced ion scanning (EPI); scanning range: 50-210 m/z; capillary voltage: 4500V, ion source temperature: 500 ℃, curtain air (CUR) flow: 30 mL/min; collision gas (CAD) selection of "High"; atomizing gas (GS1) flow: 55mL/min, flow of auxiliary gas (GS 2): 55 mL/min.

The invention has the beneficial effects that: by adopting an ultra-high performance liquid chromatography-ion hydrazine mass spectrometry combined method, a formaldehyde standard reference substance database is established, and by spectrum library retrieval, the comparison and confirmation of the EPI spectrogram of the positive suspicious sample and the standard spectrum library can be carried out, so that false positives can be effectively eliminated. In the mass concentration range of 0.01 mu g/mL-1 mu g/mL, the method has good linear relation, the correlation coefficient r is more than 0.995, the detection limit is 0.05mg/kg, the normalized average recovery rate is 91.7 percent, and the RSD is 2.08 percent. The method has the advantages of simple pretreatment, high sensitivity and strong specificity, and can accurately perform qualitative and quantitative determination on the illegally added formaldehyde in the sample.

Drawings

FIG. 1 is a standard curve diagram of formaldehyde-2, 4-dinitrophenylhydrazone in the high-efficiency determination method of formaldehyde in starch food according to the present invention;

FIG. 2 is a second-order mass spectrum of formaldehyde-2, 4-dinitrophenylhydrazone;

FIG. 3 is a total ion flow diagram of formaldehyde (formaldehyde-2, 4-dinitrophenylhydrazone) reference substance, rice flour blank matrix sample and sample labeling.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings.

An embodiment of the method for efficiently measuring formaldehyde in starch food comprises the following steps:

materials and reagents: formaldehyde standard solution: 100 μ g/mL, Beijing northern great institute of metrological measures; 2, 4-dinitrophenylhydrazine hydrochloride: chemical agents of the national drug group, ltd; acetonitrile and n-hexane are pure by high performance liquid chromatography: merck, Germany; ammonium sulfate, sodium acetate, glacial acetic acid as analytical purities: chemical agents of the national drug group, ltd; the experimental water is first-grade water treated by an ultrapure water instrument; organic microporous filter membrane: 0.22 μm, available from Jinteng laboratory Co., Ltd, Tianjin; a chromatographic column: acquisty UPLC BEH C181.7 μm, 2.1 mm. times.100 mm, Volterra, USA; scale colorimetric cylinder: 10 mL; plastic centrifuge tube: 50 mL; volumetric flask: 25 mL.

Instruments and equipment: 4000Q-TRAP ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion TRAP composite mass spectrometer: AB SCIEX, USA; a constant-temperature water bath oscillator: heidolph Inc.; a mashing machine: germany Leichi company; vortex appearance: IKA corporation; millipore ultra pure water machine, Elix5(Millipore corporation, usa); a desk-top high-speed centrifuge: hunan instruments laboratory Instrument development Ltd; constant temperature water bath shaker, WNE10(MEMMERT, usa).

(1) Preparation of Standard solutions

Acetonitrile-saturated n-hexane: adding 100mL of n-hexane into 100mL of acetonitrile, fully oscillating, standing for layering, and taking supernatant;

buffer solution: weighing 5.28g of sodium acetate, adding water for dissolution, adding 2.0mL of glacial acetic acid, and adding water to a constant volume of 1L;

2, 4-dinitrophenylhydrazine solution: weighing 600mg of 2, 4-dinitrophenylhydrazine hydrochloride, dissolving with acetonitrile, and fixing the volume to 1L;

the derived liquid: measuring a buffer solution and a 2, 4-dinitrophenylhydrazine solution in a volume ratio of 1: 1, uniformly mixing;

intermediate solution of standard solution: accurately transferring 1mL of formaldehyde standard solution with the concentration of 100 mu g/mL, and diluting acetonitrile to a constant volume of 10mL to obtain a standard solution intermediate solution with the concentration of 10 mu g/mL;

(2) sample pretreatment

Preparation of a sample: and (3) fully mashing and uniformly mixing the sample by using a mashing machine, and refrigerating and storing at 0-4 ℃ for determination.

Preparing a sample to be tested: accurately weighing 2g of sample, adding 10mL of derivative solution, fully mixing uniformly, placing in a constant temperature water bath oscillator at 60 ℃ for shaking in a water bath for 1h, taking out every 20min, and mixing uniformly. After the water bath shaking is finished, taking out and cooling to room temperature, placing the mixture into a centrifugal machine for centrifugal separation for 5min at 8000r/min, and taking supernate to pass through a 0.22 mu m organic film to obtain a sample to be detected;

preparation of a blank sample: a blank sample was prepared in the same manner as in the preparation of the sample to be tested, except that no sample was taken.

(3) Preparation of Standard solutions of controls

Accurately transferring 10 mu L, 50 mu L, 100 mu L, 500 mu L and 1000 mu L of standard solution intermediate solution into a 10mL volumetric flask, adding a buffer solution to 5mL, adding 5mL of 2, 4-dinitrophenylhydrazine solution, uniformly mixing, placing in a constant temperature water bath oscillator at 60 ℃ for shaking for 1h in a water bath, taking out and uniformly mixing once every 20min, taking out and cooling to room temperature after shaking in the water bath, centrifugally separating for 5min at 8000r/min, taking the supernatant, and passing through an organic membrane with the thickness of 0.22 mu m to obtain a standard solution control sample;

(4) establishing a formaldehyde standard reference substance database

And (3) adding the standard solution reference sample into an ultra-high performance liquid chromatography-tandem triple quadrupole/linear ion trap composite mass spectrometer for detection to obtain a second-level mass spectrogram of the formaldehyde-2, 4-dinitrophenylhydrazone reference sample, and referring to fig. 2. Adding the collected second-order mass spectrogram of the formaldehyde-2, 4-dinitrophenylhydrazone reference substance into a spectrum library, and recording the retention time of the compound. And establishing a formaldehyde standard reference substance database.

The liquid chromatography conditions were:

a chromatographic column: acquisty UPLC BEH C181.7 μm, 2.1 mm. times.100 mm; sample introduction amount: 5 mu L of the solution; column temperature: 40 ℃, mobile phase: a is water: b is methanol; gradient elution, flow rate: 0.25 mL/min; the gradient elution procedure was: 0-2 min, 90% A; 2.5min, 70% A; 3-5 min, 10% A; 5.5-8 min, 90% A. The gradient elution percentage is volume ratio.

The mass spectrum conditions are as follows:

electrospray negative ion mode (ESI-); scanning mode: multiple Reaction Monitoring (MRM) → information association data acquisition (IDA) → enhanced ion scanning (EPI); scanning range: 50-210 m/z; capillary voltage: 4500V, ion source temperature: 500 ℃, curtain air (CUR) flow: 30 mL/min; collision gas (CAD) selection of "High"; atomizing gas (GS1) flow: 55mL/min, flow of auxiliary gas (GS 2): 55 mL/min. The parent ion (Q1), the daughter ion (Q3), the declustering voltage (DP), and the Collision Energy (CE) of formaldehyde-2, 4-dinitrophenylhydrazone are shown in Table 1 below:

Q1(Da)	Q3(Da)	DP(V)	CE(V)
				208.9	162.9	-70	-12
208.9	75.8	-70	-19

parent ion: the compound molecule obtains an ion formed by an electron; daughter ions: fragment ions from the parent ions are bombarded by electrons in the mass spectrum.

Linear range and detection limit

The method for adding the target compound into the blank rice flour sample is adopted, the detection limit of the method is 0.05mg/kg according to the sampling amount of 2.00g, the constant volume of the sample of 10mL and 0.01 mu g/mL serving as the lowest detection concentration. In the mass concentration range of 0.01 mu g/mL-1 mu g/mL, the linear relation of the method is good, and the linear equation is as follows: y 3.27e +004X +6.3e +005, X: the mass concentration is expressed in. mu.g/mL. The correlation coefficient r is 0.9968. The standard curve in the linear range is referred to in fig. 1.

And (3) precision test: and taking a reference substance solution of 0.1 mu g/mL, repeatedly injecting the sample for 6 times to perform MRM determination, and quantifying by an external standard method to obtain the RSD of 3.56%.

Test for recovery with addition of standard

Blank rice flour is used as a sample (the background value is 0), experiments are repeated for 3 times at each level of 0.5mg/kg addition level, the recovery rate of the target object is 89.5% -93.1%, the precision RSD is 2.08%, and the results are shown in table 2.

TABLE 2 Formaldehyde-2, 4-dinitrophenylhydrazone recovery by addition of standard and precision

(5) Determination of content

Injecting the sample to be detected into an ultra-high performance liquid chromatography-ion trap composite mass spectrometer, retrieving through a spectrum library, and comparing and confirming the EPI spectrogram of the positive suspicious sample with a standard spectrum library to obtain a detection result. For the screening of samples, when the concentration of the compound reaches the detection limit and an MRM chromatographic peak appears, the retention time is consistent with that of a reference substance, the extracted secondary mass spectrogram is compared with the reference substance in a spectral library, and the matching value given by software is combined to realize the confirmation of the compound.

The sample to be detected can be rice, rice flour, pea powder or other starch foods, and when the sample is large-particle-shaped, the sample needs to be fully mashed and uniformly mixed.

Total ion current comparative experiment

Under the same mass spectrum conditions, formaldehyde-2, 4-dinitrophenylhydrazone reference substance with the concentration of 0.1 mug/mL, a blank matrix sample of rice flour (the background value does not contain formaldehyde) and a matrix labeled sample are respectively measured, and the total ion flow diagram is shown in figure 3. Comparing the blank sample total ion flow diagram with the matrix marking total ion flow diagram, the results show that under the mass spectrum condition, endogenous substances in the rice flour have no interference on the determination of formaldehyde, and the target substance has fast peak-out time (2.8min) and strong specificity.

The invention establishes a method for measuring formaldehyde in rice flour by combining ultra-high performance liquid chromatography with ion hydrazine mass spectrometry, adopts a multi-reaction monitoring (MRM) → information associated data acquisition (IDA) → enhancer ion scanning (EPI) scanning mode, establishes a formaldehyde standard reference substance database, and compares a positive suspicious sample EPI spectrogram with a standard spectrogram library through spectrogram library retrieval to confirm, thereby effectively eliminating false positives. In the mass concentration range of 0.01 mu g/mL-1 mu g/mL, the method has good linear relation, the correlation coefficient r is more than 0.995, the detection limit is 0.05mg/kg, the normalized average recovery rate is 91.7 percent, and the RSD is 2.08 percent. The method has the advantages of simple pretreatment, high sensitivity, good repeatability and strong specificity, and can accurately perform qualitative and quantitative determination on the illegally added formaldehyde in the sample.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.

Claims (4)

1. A method for efficiently measuring formaldehyde in starch food is characterized by comprising the following steps:

(1) preparation of Standard solutions

Acetonitrile-saturated n-hexane: adding 100mL of n-hexane into 100mL of acetonitrile, fully oscillating, standing for layering, and taking supernatant;

buffer solution: weighing 5.28g of sodium acetate, adding water for dissolution, adding 2.0mL of glacial acetic acid, and adding water to a constant volume of 1L;

2, 4-dinitrophenylhydrazine solution: weighing 600mg of 2, 4-dinitrophenylhydrazine hydrochloride, dissolving with acetonitrile, and fixing the volume to 1L;

the derived liquid: measuring a buffer solution and a 2, 4-dinitrophenylhydrazine solution in a volume ratio of 1: 1, uniformly mixing;

intermediate solution of standard solution: accurately transferring 1mL of formaldehyde standard solution with the concentration of 100 mu g/mL, and diluting acetonitrile to a constant volume of 10mL to obtain a standard solution intermediate solution with the concentration of 10 mu g/mL;

(2) sample pretreatment

Preparing a sample to be tested: weighing 2g of a sample, adding 10mL of derivative solution, fully and uniformly mixing, placing in a constant temperature environment of 60 ℃ for shaking for 1h, taking out, cooling to room temperature, carrying out centrifugal separation for 5min, and taking supernatant to pass through a 0.22 mu m organic membrane to obtain a sample to be tested;

(3) preparation of Standard solutions of controls

Accurately transferring 10 mu L, 50 mu L, 100 mu L, 500 mu L and 1000 mu L to 10mL of standard solution intermediate solution into volumetric flasks respectively, adding a buffer solution to 5mL, then adding 5mL of 2, 4-dinitrophenylhydrazine solution, uniformly mixing, shaking for 1h in a constant temperature environment at 60 ℃, taking out and cooling to room temperature, carrying out centrifugal separation for 5min, and taking supernatant to pass through a 0.22 mu m organic membrane to obtain a standard solution reference sample;

(4) establishing a formaldehyde standard reference substance database

Injecting a standard solution reference sample into an ultra-high performance liquid chromatography-ion trap composite mass spectrometer, and establishing a formaldehyde standard reference sample database;

(5) determination of content

Injecting the sample to be detected into an ultra-high performance liquid chromatography-ion trap composite mass spectrometer, retrieving through a spectrum library, and comparing and confirming the EPI spectrogram of the positive suspicious sample with a standard spectrum library to obtain a detection result.

2. The method as claimed in claim 1, wherein the sample is rice, rice flour, pea flour or other starch food, and if the sample is in the form of large granules, the sample is thoroughly crushed and mixed.

3. The method for efficiently measuring formaldehyde in starch food as claimed in claim 2, wherein the liquid chromatography conditions in the steps (4) and (5) are as follows:

chromatographic column, Acquity UPLC BEH C181.7 μm, 2.1mm × 100 mm; sample introduction amount: 5 mu L of the solution; column temperature: 40 ℃, mobile phase: a is water: b is methanol; gradient elution, flow rate: 0.25 mL/min; the gradient elution procedure was: 0-2 min, 90% A; 2.5min, 70% A; 3-5 min, 10% A; 5.5-8 min, 90% A.

4. The method for efficiently determining formaldehyde in starch-based food according to claim 1, 2 or 3, wherein the mass spectrometry conditions in the steps (4) and (5) are as follows:

electrospray negative ion mode (ESI-); scanning mode: multiple Reaction Monitoring (MRM) → information association data acquisition (IDA) → enhanced ion scanning (EPI); scanning range: 50-210 m/z; capillary voltage: 4500V, ion source temperature: 500 ℃, curtain air (CUR) flow: 30 mL/min; collision gas (CAD) selection of "High"; atomizing gas (GS1) flow: 55mL/min, flow of auxiliary gas (GS 2): 55 mL/min.

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