CN114660189A

CN114660189A - Method for detecting ethyl maltol in edible oil

Info

Publication number: CN114660189A
Application number: CN202011550242.XA
Authority: CN
Inventors: 肖荣辉; 潘坤; 于喆; 刘晓丽; 冯敏
Original assignee: Yihai Kerry Jinlongyu Oils and Grains Industries Co Ltd
Current assignee: Yihai Kerry Jinlongyu Oils and Grains Industries Co Ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2022-06-24

Abstract

The invention provides a method for detecting ethyl maltol in edible oil, which comprises a qualitative and quantitative test method.

Description

Method for detecting ethyl maltol in edible oil

Technical Field

The invention belongs to the field of analysis and detection of food additives, and particularly relates to an analysis method of chemical substances possibly contained or added in edible oil, and more particularly relates to a qualitative and quantitative detection method of ethyl maltol in vegetable oil.

Background

Ethyl maltol (CAS NO:4940-11-8), usually as a white needle or as a white crystalline powder. As a flavor modifier and a flavoring agent, the flavor enhancer is more and more widely applied, is a good flavor synergist for tobacco, food, beverage, essence, fruit wine, daily cosmetics and the like, has obvious effect on improving and enhancing the flavor of the food, and plays a role in sweetening sweet food.

As a food flavor, ethyl maltol is not found in natural substances at present, and is obtained by a chemical synthesis method, so that the ethyl maltol belongs to an artificially synthesized flavor and fragrance. Although ethyl maltol has certain safety, and is a synthetic spice for food allowed by GB 2760-. However, GB 2760 supplement 2014 appendix B.1 clearly stipulates that the plant oil and fat can not be added with food spice and essence. Therefore, ethyl maltol is actually an illegal addition to edible vegetable oil and is not detected.

Further, methods for detecting the content of ethyl maltol in food substances exist, the existing methods for detecting the compounds mainly focus on ultraviolet spectroscopy, liquid chromatography, gas chromatography and liquid chromatography-mass spectrometry, and the methods are often used together with solid phase extraction methods to achieve the purposes of purifying a sample matrix and improving the detection accuracy. For example:

at present, GB 5009.250-2016 (national food safety Standard-determination of ethyl maltol in food) stipulates that a detection method of ethyl maltol in food is high performance liquid chromatography, the detection limit of the method is 0.5mg/Kg, but the application range of the method does not include edible oil products;

the food supplement detection method BJS (2017.08, China) < determination of ethyl maltol in edible vegetable oil > is used for a supervision and spot inspection project of ethyl maltol in edible oil; the method is a high performance liquid chromatography-triple quadrupole mass spectrometry combined method; the detection limit of the method is 25 mug/Kg, and instruments and equipment adopted by the method are expensive and cannot be widely used and popularized; because the components of the grease sample are complex, the situation that the detection is interfered by background substances exists.

Currently, for the detection method of p-ethyl maltol using gas chromatography in combination with mass spectrometry, for example:

citation 1 discloses a method for determining maltol and ethyl maltol in blend oil by gas chromatography-mass spectrometry using solid phase extraction as an extraction and separation method. After a sample is dissolved by n-hexane, an amino solid phase extraction column is adopted for separation and purification, and the applicability of the method is investigated from 3 aspects of detection limit, recovery rate and precision by selecting proper monitoring ions in combination with high resolution and high sensitivity of gas. In the detection result, the detection limit of ethyl maltol was 0.35 mg/kg. Within the measuring range of 0-0.5g/kg, the recovery rate of the added standard is 95.2-107%, and the precision is 0.9-1.2%.

Citation 2 discloses a method for identifying adulterated peanut oil, which comprises the following steps: extracting and pretreating, analyzing the extracted aromatic substances by using a gas chromatograph-mass spectrometer, processing an experimental map by using Xcalibur software to obtain related data, and qualitatively analyzing the peanut essence by using ethyl maltol and glyceryl triacetate as characteristic substances.

However, in the cited document 1, the sample needs to be subjected to solid phase extraction and then to the GC-MS test, the convenience of detection and the detection accuracy (detection limit) for ethyl maltol are still insufficient, and in the cited document 2, only qualitative analysis is referred to and the feasibility of quantitative detection is not disclosed.

In addition, in the detection of other vegetable pesticide residues, there is a case where a target pesticide residue is quantitatively detected by using a detection means combining gas chromatography and mass spectrometry.

For example, cited document 3 uses 4 common vegetables as materials, and uses a gas chromatography/mass spectrometry combined method to study the influence of the polarity of a chromatographic column and a scanning mode on the determination of the residual amount of 5 pyrethroid pesticides in the vegetables. The detection limit of pyrethroid pesticide in vegetable can be reduced by using capillary column with stronger polarity (such as DB-17 MS). On the basis of determining characteristic ions of each pyrethroid pesticide, selective ion monitoring Scanning (SIM) is adopted, so that the detection limit of the pyrethroid pesticides in vegetables can be obviously reduced, and the detection limit range of 5 pyrethrins in 4 vegetables is 0.00253-0.19100 mg/kg. Although the cited reference 3 has a low detection limit, it is not known whether or not a similar detection can be performed in the edible oil, particularly in consideration of the presence of a large amount of interfering substances in the vegetable oil.

It can be seen that there is no economical and effective qualitative and quantitative test method for detecting ethyl maltol possibly present in edible oils, especially vegetable oils, and the research on the quantitative uncertainty possibly caused by the substance interference factors present in the oils and fats is still insufficient, so there is still a need for further improvement of the detection method.

Cited documents:

cited document 1: chinese food additive, 2017, No. 6, page 209-213,

cited document 2: the content of the CN106526031A is determined,

citation 3: research on the detection of the pesticide residue in vegetable by gas chromatography-mass spectrometry, Zhang Jing et al, Anhui agricultural science, 2011.

Disclosure of Invention

Problems to be solved by the invention

In view of the above problems or disadvantages in the prior art, the present invention is to provide a reliable method for detecting ethyl maltol additive in edible oil, especially vegetable oil, and to solve the problems of inaccurate quality and inaccurate quantity caused by interference of background substances.

In addition, another object of the present invention is to provide a method for detecting ethyl maltol possibly existing in edible oil, which comprises performing quantitative detection by using a low-polarity chromatographic column in combination with a mass spectrometer, and if the limit value of the quantitative detection or the lower limit value of the concentration (i.e. safety threshold) in the current safety standard is exceeded, performing quantitative or qualitative detection (re-detection) by using a high-polarity chromatographic column in combination with a mass spectrometer. Such methods are particularly useful for efficiently conducting large-scale, screening assays.

In addition, the detection method provided by the invention is simple in process, economical, convenient and fast, and easy for large-scale industrial application.

Means for solving the problems

After long-term intensive research by the present inventors, it was found that the above technical problems can be solved by the following methods:

[1] the invention firstly provides a method for detecting ethyl maltol in edible oil, which is a quantitative detection method and comprises the following quantitative steps:

preparing a sample to be detected through edible oil to be detected, and preparing n standard samples based on a standard addition method, wherein n is an integer greater than or equal to 3, preferably, n is an integer from 3 to 10;

a pretreatment step, namely enriching the polar substances in the sample to be detected and the standard sample to obtain a detection sample; preferably, the enrichment is to extract the sample to be detected and the standard sample by using a polar solvent, and the polar solvent is selected from acetonitrile or methanol;

detecting, namely testing each detection sample by combining gas chromatography and mass spectrometry, preparing a working curve based on a standard addition method so as to determine the content of the ethyl maltol in the sample to be detected,

wherein the gas chromatograph comprises a strongly polar chromatography column, and the mass spectrometer employs a feature selective ion monitoring Scanning (SIM) mode of operation; preferably, the strongly polar chromatography column is selected from a polyethylene glycol based chromatography column, a polar group modified polyethylene glycol chromatography column or a polar group modified polysiloxane chromatography column.

[2] The method according to [1], wherein in the step of detecting, a weak polarity column is further used in series with the strong polarity column of gas chromatography before and/or after it.

[3] The method according to [1] or [2], wherein the weakly polar chromatography column comprises phenyl-modified dimethylpolysiloxane; and/or the mass spectrum is a single quadrupole mass spectrum.

[4] The method according to any one of [1] to [3], wherein the step of detecting, the temperature-raising program of the gas chromatography comprises raising the temperature at a rate of 10 ℃/min or less to 100 ℃ or less and holding the temperature for 15min or less; and then heating to 230-260 ℃ at a speed of 30-40 ℃/min, and keeping for 2-10 min.

[5]According to [1]]～[4]The method of any one of the preceding claims, wherein the method relies on a linear correlation coefficient R of a standard addition method working curve²Greater than 0.998; and/or the method has a detection limit of 5 mu g/Kg for ethyl maltol.

[6] Furthermore, the invention also provides a method for detecting ethyl maltol in edible oil, in particular a simple qualitative detection method, which comprises the following qualitative steps:

preparing a sample to be detected by using the edible oil to be detected, and preparing a standard sample with known ethyl maltol concentration;

a pretreatment step, namely enriching the polar substances in the sample to be detected to obtain a detection sample; preferably, the enrichment is to extract the sample to be tested by using a polar solvent, wherein the polar solvent is selected from acetonitrile or methanol;

a step of detecting, in which the standard sample and the detection sample are respectively tested by a combination of gas chromatography and mass spectrometry, and recording:

i) retention time t1 of ethyl maltol gas chromatographic peak in the standard sample, and abundance ratio of characteristic ion peak in mass spectrum;

ii) detecting the retention time t2 of a suspected ethyl maltol gas chromatographic peak in the sample and the abundance ratio of a characteristic ion peak in the mass spectrum,

wherein, the gas chromatogram comprises a strong polarity chromatographic column, and the mass spectrum adopts a characteristic selection ion monitoring Scanning (SIM) working mode; preferably, the strongly polar chromatography column is selected from a polyethylene glycol based chromatography column, a polar group modified polyethylene glycol chromatography column or a polar group modified polysiloxane chromatography column, and/or the mass spectrum is a single quadrupole mass spectrum;

a step of judging whether the sample to be tested contains the ethyl maltol or not through the comparison of the t1 and the t2 and the comparison of the abundance ratio of the characteristic ion peaks in the mass spectrum if necessary,

[7] the method according to [6], wherein in the step of detecting, the temperature-raising program of the gas chromatography comprises raising the temperature at a rate of 10 ℃/min or less to 100 ℃ or less and holding the temperature for 15min or less; then, the temperature is increased to 230-260 ℃ at a speed of 30-40 ℃/min and is kept for 2-10 min.

[8] The method according to [6] or [7], wherein in the judging step, if the deviation between t1 and t2 is less than or equal to +/-0.05 min, whether the sample to be tested contains the ethyl maltol or not is determined by comparing abundance ratios of characteristic ion peaks of mass spectra.

[9] The method of [7] or [8], wherein the comparison of the abundance ratios of characteristic ion peaks in the mass spectrum comprises comparing the ratios of 139,125 and 97 ion peak relative intensities to 140 ion peak relative intensities of the detection sample with the ratios of 139,125 and 97 ion peak relative intensities to 140 ion peak relative intensities in a standard sample, respectively.

In some specific embodiments, the method of [6] to [9] above may employ the steps of:

a. preparing a standard sample (200 mu g/Kg of standard sample) with known ethyl maltol content;

b. carrying out gas chromatography-mass spectrometry detection on the standard sample, and recording the retention time t1 of ethyl maltol in gas chromatography and the relative intensities of 139,125 and 97 ion peaks and 140 ion peaks in mass spectrum (reference abundance ratio);

c. pretreating a sample to be detected to obtain a detection sample, carrying out gas chromatography-mass spectrometry detection on the detection sample under the same condition as b, and recording the retention time t2 of ethyl maltol in gas chromatography and the relative intensities of 139,125 and 97 ion peaks and the relative intensity of 140 ion peaks (detection abundance ratio) in mass spectrum;

d. t1 and t2, the reference abundance ratio and the detected abundance ratio were compared.

[10] The invention further provides a method for detecting ethyl maltol in edible oil, which comprises the following steps:

preparing a sample to be detected through edible oil to be detected, and preparing a standard sample containing ethyl maltol with the concentration being a safety standard threshold concentration;

detecting, namely testing the detection sample and the standard sample by combining gas chromatography and mass spectrometry, wherein a weak-polarity chromatographic column is used in the gas chromatography, and the mass spectrometry adopts a characteristic selection ion monitoring Scanning (SIM) working mode; preferably, the weakly polar chromatography column is selected from phenyl-modified dimethylpolysiloxane, and/or the mass spectrum is a single quadrupole mass spectrum;

and judging, if the result of the detecting step shows that the detection concentration of the ethyl maltol in the sample to be detected is greater than the safety standard threshold concentration, replacing a weak polarity chromatographic column in the gas chromatogram with a strong polarity chromatographic column or connecting the weak polarity chromatographic column and the strong polarity chromatographic column in series, and carrying out the test by combining with the mass spectrum so as to carry out quantitative or qualitative detection on the sample to be detected again.

[11] The method according to [10], wherein in the determining step, if the peak area corresponding to the mass spectrometry quantitative ion 140 ion in the detection sample is larger than the peak area corresponding to the standard sample quantitative ion 140 ion, the detection concentration of the ethyl maltol in the sample to be detected is considered to be larger than the safety standard threshold concentration.

[12] The method according to [10] or [11], characterized in that in the step of detecting, the temperature-raising program of the gas chromatography comprises raising the temperature to not more than 160 ℃ at a speed of 3-7 ℃/min and keeping the temperature for not more than 15 min; then heating to 190-210 ℃ at the speed of 8-12 ℃/min, and keeping for 0.5-2 min; and then heating to 230-255 ℃ at a speed of 25-35 ℃/min and keeping for 1-3 min.

[13] The method according to any one of [10] to [12], wherein the safety standard threshold concentration is 25 μ g/Kg.

[14] The method according to any one of [10] to [13], wherein the weak polarity chromatography column is connected in series with the strong polarity chromatography column, including connecting the weak polarity chromatography column in series before and/or after the strong polarity chromatography column.

In some specific embodiments, the method of [10] to [14] above may employ the steps of:

a. determining a safety standard threshold concentration, configuring a standard sample (for example, a standard sample of 25 μ g/Kg) containing ethyl maltol with the threshold concentration, detecting by using a gas chromatography-mass spectrometer, and recording a peak area (reference peak area) of a chromatographic peak corresponding to the ion 140 to be quantified by mass spectrometry;

b. taking a sample to be detected, pretreating to obtain a detection sample, carrying out gas chromatography-mass spectrometry detection under the same test condition as a, and recording the peak area (detection peak area) of a chromatographic peak corresponding to the mass spectrum quantitative ion 140;

c. and comparing the reference peak area with the detection peak area, if the detection peak area is higher than the reference peak area, replacing a weak-polarity chromatographic column in the gas chromatogram with a strong-polarity chromatographic column or connecting the weak-polarity chromatographic column with the strong-polarity chromatographic column in series, and carrying out a test by combining with the mass spectrum so as to carry out quantitative or qualitative detection on the sample to be detected again.

[15] The method according to any one of [10] to [14], wherein the re-performing of quantitative or qualitative detection comprises using the detection method according to any one of [1] to [5] above.

ADVANTAGEOUS EFFECTS OF INVENTION

Through the implementation of the technical scheme, the invention can obtain the following technical effects:

1) the method adopts a strong polarity chromatographic column or a method of connecting a weak polarity chromatographic column and the strong polarity chromatographic column in series, and can avoid the situation of co-outflow peak in the gas chromatographic separation process of the ethyl maltol under the optimal chromatographic condition, thereby completely separating the ethyl maltol and interfering substances. Meanwhile, the content of ethyl maltol in the edible oil can be accurately determined by adopting a characteristic selection ion monitoring scanning mode (SIM) for monitoring and adding an external standard method-gas chromatography/mass spectrometry combined method through standards, and the method is accurate in quantification, sensitive, accurate and good in reproducibility. In some specific embodiments, the ethyl maltol has a good and symmetrical peak shape and does not generate a tailing peak phenomenon by using a strong polarity chromatographic column or a method of connecting a weak polarity chromatographic column and the strong polarity chromatographic column in series.

2) The results of the linear test and the detection limit test show that the linear relation between the peak area and the mass concentration in a certain concentration range is good (R²>0.998)；

3) The detection limit of the detection method of the invention is low, and in some specific embodiments, the detection limit can be as low as 5 mug/Kg.

4) The detection method has good recovery rate and Relative Standard Deviation (RSD).

5) In some embodiments of the invention, a method for qualitatively detecting ethyl maltol in a sample to be tested is provided by using a combination of chromatography including a strong polarity chromatographic column and mass spectrometry.

6) In other preferred embodiments of the invention, the weak polarity chromatography is used in combination with the mass spectrometry, and if the quantitative detection result of a certain sample to be detected exceeds the safety standard threshold concentration, the strong polarity chromatography column retest method is adopted, which is beneficial to the rapid detection and evaluation of large-scale samples to be detected.

Drawings

FIG. 1: the detection system of the gas chromatography-mass spectrometry combination in the embodiment of the invention detects the result of the standard sample;

FIG. 2 is a schematic diagram: working curve prepared by adding standard sample in the embodiment 1 of the invention;

FIG. 3: in the embodiment 1 of the invention, a gas chromatogram map is a superposition graph when the heavy rapeseed oil labeled sample and the sample to be detected (actual oil sample which is not labeled) are detected;

FIG. 4: in the embodiment 2 of the invention, a gas chromatogram map is superimposed when the refined fourth-stage rapeseed oil is detected by adding a standard sample and a sample to be detected (actual oil sample without adding a standard);

FIG. 5: the gas chromatograms of fig. 3 and 4 are superimposed.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention. All documents described in this specification are incorporated herein by reference.

Unless defined otherwise, 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.

In the present specification, a numerical range represented by "a value to B value" or "a value to B value" means a range including the end point value A, B.

In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In the present invention, the "safety standard" is used to mean a standard existing in a local, regional, national, regional or international organization in relation to food quality detection or monitoring, for the minimum detected amount of ethyl maltol in food and edible oil.

Reference throughout this specification to "some particular/preferred embodiments," "other particular/preferred embodiments," "some particular/preferred aspects," "other particular/preferred aspects," or the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

The terms "standard sample", "spiked sample", "sample to be tested" and "test sample" according to the present invention have the following meanings, respectively:

standard samples: i.e., a sample that has a defined amount or concentration of the target analyte and that does not contain components that interfere with detection of the target analyte or that does not contain components other than the non-interfering essential solvent and the target analyte;

adding a standard sample: using a sample obtained by mixing a standard sample and a sample to be detected, calculating the concentration of a target analyte in the mixed sample according to the amount of the target analyte in the added standard sample by using the mixed sample, and regarding the concentration as the concentration of the target analyte in the added standard sample;

a sample to be tested: the sample to be detected is a sample actually sampled, and in the invention, the sample to be detected also refers to an oil sample actually required to be detected;

detecting a sample: the method refers to a sample actually detected on a computer, namely a sample obtained by pre-treating a standard sample and a sample to be detected, and the sample can be directly detected on the computer.

The terms "comprises" and "comprising," and any variations thereof, in the description and claims of this invention and the above-described drawings are intended to cover non-exclusive inclusions. For example, a process, method, or system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.

< first aspect >

In a first aspect of the present invention, there is provided a reliable quantitative detection method for ethyl maltol additive which may be present in edible oil, especially vegetable oil, and which can solve the problem of inaccurate quantification due to interference of background substances, has a low detection limit and excellent detection accuracy, and can perform good quantitative measurement.

More specifically, in the first aspect of the present invention, a test is performed by a combination of gas chromatography and mass spectrometry based on a standard addition method, and a working curve is prepared based on the standard addition method to determine the ethyl maltol content in the sample to be tested.

Ethyl maltol and vegetable oil

As mentioned above, ethyl maltol is a commonly used flavoring agent in the food industry, but since it is prohibited for use in edible oils, a reliable and accurate detection method is necessary for the edible oil to identify whether the flavoring agent has been illegally added.

The edible oils of the present invention, in some embodiments of the invention, are generally referred to as vegetable oils.

For these vegetable oils, include, but are not limited to: one or more of rice oil, sunflower seed oil, palm kernel oil, peanut oil, rapeseed oil, soybean oil, linseed oil, cottonseed oil, safflower seed oil, perilla seed oil, tea seed oil, hemp seed oil, jojoba oil, olive oil, cocoa bean oil, Chinese tallow seed oil, almond oil, tung seed oil, rubber seed, corn germ oil, wheat germ oil, sesame seed oil, evening primrose seed oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, linseed oil, glass endive seed oil, sea buckthorn seed oil, tomato seed oil, macadamia nut oil, and coconut oil.

In addition, the edible oil of the invention also comprises mixed oil or blend oil formed by the various vegetable oils according to any proportion.

Further, the production method of these vegetable oils is not particularly limited in the present invention, and can be obtained by physical pressing or by chemical leaching.

In addition, the edible oil of the present invention, in some specific embodiments, comprises unused edible oil, and in other embodiments, can also be used or heated at high temperature. That is, the detection method provided by the invention not only can be used for fresh edible oil, but also can be applied to edible oil used at high temperature.

GC-MS detection based on standard addition method

The detection method of the present invention is a quantitative detection method based on a standard addition method.

The standard addition method is a method of adding a standard solution with a certain concentration gradient and a known concentration into a sample to be tested to prepare a plurality of standard adding samples, and determining certain characteristics of the standard adding samples (and blank samples) to prepare a working curve through a fitted straight line. This method is particularly useful for testing samples for the presence of interfering substances.

In some embodiments of the present invention, n spiked samples are obtained simultaneously with the test sample by diluting a standard sample containing ethyl maltol of known concentration with the edible oil to be tested.

There is no particular limitation on the number of spiked samples, and it is understood that a greater number of spiked samples will likely improve the reliability of the test results, but may also lead to more complex processing for linear fitting. In some specific embodiments of the present invention, n is an integer of 3 or more, preferably an integer of 3 to 10, and more preferably an integer of 4 to 6.

Further, the concentration of ethyl maltol in the spiked sample is not particularly limited, but from the viewpoint of the linear range and the detection limit, in some preferred embodiments of the present invention, the concentration of the spiked sample may be 10. mu.g/kg to 500. mu.g/kg, more preferably 15. mu.g/kg to 350. mu.g/kg, and still more preferably 15. mu.g/kg to 300. mu.g/kg. For example, the concentration of these spiked samples may be 15. mu.g/kg, 25. mu.g/kg, 30. mu.g/kg, 35. mu.g/kg, 40. mu.g/kg, 45. mu.g/kg, 50. mu.g/kg, 55. mu.g/kg, 80. mu.g/kg, 100. mu.g/kg, 120. mu.g/kg, 140. mu.g/kg, 160. mu.g/kg, 180. mu.g/kg, or 200. mu.g/kg, etc.

(step of pretreatment)

The pretreatment step of the invention is the pre-separation of components in samples, including samples to be detected and labeled samples.

In fats and oils, polar components (strongly polar components and weakly polar components) and nonpolar components are present in large amounts. In a particular embodiment of the invention, the sample is subjected to a pre-separation treatment of polar and non-polar components by extraction with a solvent.

For such solvents, various polar solvents may be used, and in some preferred embodiments, acetonitrile or methanol may be used as the solvent to mix with each sample. Since acetonitrile or methanol belongs to a strong polar solvent, polar substances in a sample enter the strong polar solvent such as acetonitrile and the like to be extracted. It should be noted that ethanol, chloroform, and DMSO are not used in this pretreatment step because they are easily miscible with oils.

In the pretreatment step of the present invention, an auxiliary means may be used for the preliminary separation of components in fats and oils. For such an auxiliary means, typically, centrifugation can be used. In some preferred embodiments, the rotation speed of the centrifugal separation treatment can be more than 4000r/min, and the centrifugal time can be 3-8 min.

Through the extraction and the centrifugal treatment in the pretreatment steps, the polar components in the sample are enriched, at least part or all of the nonpolar components are separated and removed, and the interference of part of background substances is eliminated.

(gas chromatography)

The enriched component or extract obtained by the pretreatment is separated by gas chromatography. By optimizing the control conditions of the gas chromatography, polar components (especially strong polar components) in the oil sample can be separated from the ethyl maltol with weak polarity, and the interference of another part of background substances is eliminated, so that the quantitative detection of the ethyl maltol is possible.

The sample injection method of the gas chromatography is not particularly limited, and the sample may be manually injected or may be injected by using an autosampler. In some embodiments, an autosampler, non-split sample injection is used.

The gas chromatograph of the present invention includes at least one high polarity chromatographic column, and the high polarity chromatographic column separates various chemical substances with different polarities from a sample, and the chemical substances are detected by mass spectrometry as described later.

In another specific embodiment of the present invention, a low polarity chromatographic column connected in series with the high polarity chromatographic column is further included before the high polarity chromatographic column, and in this case, during detection, various chemical substances with different polarities can be pre-separated by the low polarity chromatographic column, and after pre-separation, the chemical substances are re-separated by the high polarity chromatographic column. In the invention, a mode of connecting the weak polarity chromatographic column and the strong polarity chromatographic column in series is used, so that the ethyl maltol and other interference components can be better separated.

In another specific embodiment of the present invention, the strong polarity column is followed by a weak polarity column in series with the strong polarity column. In this case, in the detection, the component is separated by using a strong polarity column, and then the separation is continued by using a weak polarity column. The present invention also recognizes that such a column attachment also facilitates better separation of ethyl maltol from other interfering components.

Further, there is no particular limitation on the other column arrangement method in which at least one strong polarity column is present, as long as the technical effects of the present invention are not impaired. For example, a weak polarity column may be disposed before and after a strong polarity column; and a weak polarity chromatographic column is connected in parallel with the strong polarity chromatographic column.

The "strongly polar column" and "weakly polar column" for the purposes of the present invention may be selected according to commercially available columns conventional in the art.

In some specific embodiments of the present invention, the so-called "strongly polar chromatography column" may be selected from polyethylene glycol based chromatography columns, polar group modified polyethylene glycol chromatography columns, or polar group modified polysiloxane chromatography columns, and the like. More specifically, the so-called "strongly polar chromatography column" may be selected from the group consisting of the columns sold under the trade names HP-INNOWAX MS, PEG20M, BP-10MS, RSL-1701MS, DB-1701MS, HP-1701MS and CPISL-19 MS.

In addition, for the so-called "weakly polar chromatography column", it may be selected from phenyl-modified dimethylpolysiloxanes, in some preferred cases with a phenyl content of not more than 8%, preferably not more than 5%. More specifically, the so-called "weak polarity chromatography column" may be selected from those known under the trade name: DB-5 MS; HP-5MS, TG-5 MS; SE-54 MS; RTX-5 MS; DB-1MS and HP-1MS chromatographic column.

In general, the "strong polarity column" and the "weak polarity column" described above are commercially available from companies such as agilent, seemer, and the like.

In the case of a strong polarity and a weak polarity column in series using the above-described commercially available columns, a glass cone capillary tube may be used in some embodiments to connect the weak polarity column (e.g., DB-5MS) to the strong polarity column (e.g., DB-1701 MS). For example, the outlet of the low polarity chromatographic column is inserted into the two-way inlet of the glass tapered capillary, one end of the high polarity chromatographic column is inserted into the two-way outlet of the glass tapered capillary, and the outlet of the high polarity chromatographic column is connected to the mass spectrometer. In other connection modes, such as the mode of connecting the strong polarity chromatographic column and the weak polarity chromatographic column in parallel, the switching of the detection channels can be realized through a multi-way valve bank preferably.

In addition, the total length of the column of the present invention (including the total length of the strong polarity column and one or two of the preceding and following weak polarity columns, which may be connected in series therewith) may be not more than 100m, preferably not more than 80m, and further preferably not more than 70m, from the viewpoints of convenience of the apparatus and reliability of detection. In one embodiment, the low polarity column is in series with the high polarity column, the column length is 60 meters, in which case the column length is set on the instrument software to be 60 meters. And (5) starting the instrument, and completing connection after the self-inspection is qualified.

Regarding the operating temperature of the gas chromatography, the present inventors believe that sufficient heating is employed in the gas chromatography separation to facilitate the separation of the strongly polar material from the less polar ethyl maltol.

In some specific embodiments of the present invention, a temperature control manner of temperature increase in stages can be adopted in the gas chromatography detection. Firstly, a certain initial temperature is obtained by a chromatographic column, such as an initial temperature of more than 50 ℃, the temperature can be further increased to be not more than 100 ℃ by using a slower temperature increasing rate, and the temperature is kept for not more than 15min, wherein the temperature increasing rate can be not more than 10 ℃/min; and then, the temperature is increased to 230-260 ℃ through a relatively rapid temperature increase rate, the temperature increase rate can be 30-40 ℃/min, and the temperature is kept for 2-10 min at the temperature increase end point.

In some preferred embodiments of the present invention, the gas chromatography of the present invention may employ the following temperature-controlled manner: setting the initial temperature of the chromatographic column to be 55-65 ℃, and keeping the temperature for 3-5 min (for example, 3.5min, 4min and the like); further heating to a temperature not higher than 100 deg.C at a speed of 2.5-5 deg.C/min (e.g. 3 deg.C/min, 3.5 deg.C/min, 4 deg.C/min, etc.), and maintaining for a time not higher than 15min (e.g. 8-13 min, etc.); then, the temperature is increased at a rate of 25-35 deg.C/min (e.g., 28 deg.C/min, 30 deg.C/min, 32 deg.C/min, etc.) to 230-255 deg.C (e.g., 240 deg.C, 250 deg.C, etc.), and the temperature is maintained for 3-8 min (e.g., 5min, 7min, etc.).

Through the temperature control mode, strong polar substances can better permeate into a solid phase structure of the strong polar chromatographic column, and then the substances can be separated from the ethyl maltol with weak polarity.

Further, the carrier gas (mobile phase) of the gas chromatograph of the present invention is selected from inert gases such as helium and the like.

Other elements or modes of operation of the gas chromatograph are not particularly limited, and reference may be made to technical means or modes of operation that are conventional in the art.

(Mass Spectrometry)

The invention uses mass spectrometry to carry out mass spectrometry detection on the gas chromatography isolate.

In some embodiments of the invention, a feature-selective ion-monitoring Scanning (SIM) mode of operation is used as the mass spectrometer detector. For scannable characteristic ion peaks, at least 140 ion peaks are included as quantitative ions; other scannable ion peaks may include 139,125 and 97 ion peaks as qualitative ions, which are primarily related to the relative peak intensities of the ion peaks in the mass spectrometric detection results.

Furthermore, the ionization mode of the mass spectrometric detection of the present invention is not particularly limited, and in some preferred embodiments, bombardment ionization may be performed using an electron bombardment ionization source (EI).

In some preferred embodiments of the invention, single quadrupole mass spectrometry is used.

Detection reliability

According to the gas chromatography-mass spectrometry test method, the linear correlation coefficient R of the working curve of the standard addition method is used²Greater than 0.998 and a detection limit for ethyl maltol of 5 μ g/kg (3 fold signal to noise ratio calculation) for the method. More specifically:

the test method based on the strong polarity chromatographic column comprises the following steps: the ethyl maltol content is in good linear relation within the range of 10-200 mu g/Kg, and the linear equation is that Y is 8.145 multiplied by 10³X+1.992×10⁴The correlation coefficient is 0.9984; the method detects that the recovery rate of ethyl maltol is 95-110% and the RSD is 8%;

based on a series connection method of a weak polarity chromatographic column (front) and a strong polarity chromatographic column (back): the content of ethyl maltol is in a good linear relation within 15-200 mu g/Kg, the linear equation is 655.57X +7993.07, and the correlation coefficient is 0.9990; the method determines that the standard recovery rate of ethyl maltol is 82-95% and the RSD is 2.2-9.8%.

< second aspect >

In the second aspect of the invention, a simple and reliable qualitative detection method for ethyl maltol additive possibly existing in edible oil, especially vegetable oil, is provided, and the problem of inaccurate qualitative detection caused by background substance interference can be solved.

It should be noted that the qualitative detection method provided by the present invention may be performed before the quantitative detection in the first aspect, and the qualitative detection method that can be used before the quantitative detection in the first aspect is not limited to the method disclosed in this aspect of the present invention. For such a test result, it is first determined whether the final mass spectrometric peak represents the presence of ethyl maltol to reject the effect of interfering substances.

More specifically, in the second aspect of the present invention, using the combined gas chromatography and mass spectrometry test system and conditions disclosed in the first aspect, the sample to be tested and a standard sample with a known concentration are respectively subjected to gas chromatography or a combined gas chromatography and mass spectrometry test, and whether the sample to be tested contains ethyl maltol is determined.

Firstly, preparing a sample to be detected through edible oil to be detected, and preparing a standard sample of ethyl maltol with known (arbitrary) concentration;

further carrying out a pretreatment step, enriching the polar substances in the sample to be detected and obtaining a detection sample; preferably, the enrichment is to extract the sample to be tested by using a polar solvent, and the polar solvent is selected from acetonitrile or methanol;

the qualitative detection step is mainly designed based on the following two ideas:

the i) th point was determined by gas chromatography on the retention time of the characteristic peak of ethyl maltol. Specifically, the method comprises the following steps: the retention time of ethyl maltol in the standard sample is first determined by performing the gas chromatography (or GC-MS) assay of the invention as described above using a standard sample of known arbitrary concentration. Further, under the same gas chromatography conditions, a test sample obtained by pretreating a sample to be tested is tested, and if the retention time of a suspected ethyl maltol peak in the gas chromatography is the same as that of a standard sample or the deviation is not more than ± 0.05min, the following qualitative test related to the point ii) can be performed.

Point ii) the (qualitative) determination of the presence or absence of ethyl maltol in the sample by mass spectrometry ion peak abundance ratio. Generally, when the same gas chromatography-mass spectrometry instrument is used for detecting substances, the abundance ratio of each characteristic peak is determined in each detection. For example, when the detection is performed by using a gas chromatography-mass spectrometer applicable to the present invention, and the detection is performed by using samples of known different concentrations (or standard samples of known concentrations), although the relative intensities of the 140 ion peak and the 139,125 and 97 ion peaks vary in each detection result in the case of different concentrations, the ratio of the 139 ion peak to the 140 ion peak, the ratio of the 125 ion peak to the 140 ion peak, and the ratio of the 97 ion peak to the 140 ion peak are substantially the same in the detection results of mass spectra of samples of different concentrations.

Considering the above points i) and ii), the present invention can qualitatively determine whether the mass spectrum result detected by the gas chromatography-mass spectrometer represents ethyl maltol by the following method:

using a standard sample of ethyl maltol with a known (arbitrary) concentration, detecting under defined conditions (which can be referred to the disclosure of the first aspect of the invention), using a gas chromatography-mass spectrometry combination, and recording the retention time of the characteristic peak of ethyl maltol in the gas chromatography. Further, under the determined conditions, detecting the detection sample after the pretreatment of the sample to be detected, and if the retention time of the gas chromatography characteristic peak of the detection sample is the same as that of the standard sample or the deviation is less than or equal to +/-0.05 min, carrying out the following further qualitative detection;

② using a standard sample containing ethyl maltol in a known (arbitrary) concentration (which may be the same as the standard sample used in the above i), detecting using gas chromatography-mass spectrometry under certain conditions (the certain conditions may refer to the disclosure of the first aspect of the invention of the above text), and obtaining the ratio of the relative intensities of 139 ion peak to 140 ion peak, the ratio of 125 ion peak to 140 ion peak, and the ratio of 97 ion peak to 140 ion peak of the standard sample as qualitative control standards. The relative intensity ratios herein have equivalent physical significance to the abundance ratios described above. And further performing gas chromatography-mass spectrometry detection on the detection sample after pretreatment of the sample to be detected under the same conditions to obtain a mass spectrometry detection result, determining the relative intensity ratio of 139,125 and 97 ion peaks to the relative intensity of 140 ion peaks in the result, comparing the ion abundance ratio with the qualitative control standard, and if the deviation of each ion abundance ratio and the qualitative control standard meets the following requirements (see the following table 1), determining that the sample to be detected contains the ethyl maltol.

TABLE 1 maximum permissible deviation of ion abundance ratio (compared to standard sample ion abundance)

Abundance ratio (% with 140 ion peak as base peak)	Tolerance deviation
		＞50	±10％
20～50	±15％
		10～20	±20％
≤10	±50％

It should be noted that, in some preferred embodiments, step (ii) may be performed in a single measurement with step (i). For example, for the above-mentioned standard sample containing ethyl maltol at a known (arbitrary) concentration, detection using gas chromatography-mass spectrometry combination can be performed under certain conditions, and retention time of characteristic peak of ethyl maltol in gas chromatography and standard abundance ratio of each ion in mass spectrum are recorded. The sample to be detected can be subjected to gas chromatography-mass spectrometry detection under the same conditions after the sample to be detected is obtained after pretreatment, so that retention time and abundance ratio of each ion are obtained. And comparing the retention time and each ion abundance ratio of the sample to be tested with those of the standard sample by the method described above to determine whether the sample to be tested contains ethyl maltol or whether the object to be quantitatively analyzed is ethyl maltol.

In conclusion, the GC-MS detection method for ethyl maltol in edible oil provided by the second aspect of the invention is a simple, convenient, economic and reliable qualitative test method, and provides a new solution for food safety detection.

< third aspect >

In a third aspect of the invention, another reliable method for detecting ethyl maltol additives that may be present in edible oils, especially vegetable oils, is provided.

Specifically, such a detection method includes:

a pretreatment step, namely enriching the polar substances in the sample to be detected to obtain a detection sample;

and a judging step, if the result of the detecting step shows that the detected concentration of the ethyl maltol in the sample to be detected is greater than the safety standard threshold concentration, replacing a weak polarity chromatographic column in the gas chromatogram with a strong polarity chromatographic column or connecting the weak polarity chromatographic column and the strong polarity chromatographic column in series, and carrying out a test by combining with the mass spectrum so as to carry out quantitative or qualitative detection judgment (re-detection) on the sample to be detected again.

In the judging step, in some specific embodiments of the present invention, a gas chromatography-mass spectrometry test is performed on a standard sample containing ethyl maltol at a safe standard threshold concentration level (under certain conditions, such as the operating conditions of gas chromatography and mass spectrometry to obtain the area of the peak corresponding to 140 ions of the standard sample; and then the sample to be detected is detected under the same detection condition, and the area of the peak corresponding to 140 ions of the sample to be detected is obtained. If the area of the 140 ion peak of the sample to be detected is not higher than that of the standard sample, the content of the ethyl maltol possibly existing in the sample to be detected is not higher than the safety standard threshold concentration, namely, the gas chromatography-mass spectrometry recheck with a strong polarity chromatographic column is not needed, otherwise, the qualitative or quantitative recheck is needed to be carried out on the sample.

The detection method of the third aspect of the present invention is mainly based on the following considerations:

when the content of the ethyl maltol possibly existing in the edible oil is detected by combining gas chromatography and mass spectrometry, when a weak-polarity chromatographic column is used in the gas chromatography, the detection efficiency is faster than that of the case of using a strong-polarity chromatographic column. However, although there is an advantage in terms of detection efficiency, there may be insufficient concern about separation of some interfering substances from ethyl maltol when a test sample is separated using a weakly polar chromatography column. Therefore, theoretically, when the combined detection of gas chromatography and mass spectrometry is performed by using a weak-polarity chromatographic column, the detection value of quantitative detection is usually higher than the actual content of ethyl maltol in the sample to be detected.

In light of the above considerations, the present invention provides methods that are particularly well suited for efficient (large scale) screening of problem products. The method comprises the following steps of carrying out quantitative detection on a sample to be detected by combining gas chromatography and mass spectrometry based on a weak-polarity chromatographic column:

i) if the detected value of the sample to be detected does not exceed the safety standard threshold concentration, the sample to be detected meets the requirement of the safety standard;

ii) if the detected value of the sample to be detected exceeds the safety standard threshold concentration, it indicates that the sample to be detected may have a problem (because some interfering substances may not be distinguished based on the result of gas chromatography-mass spectrometry of the weak polarity chromatographic column), in such a case, the weak polarity chromatographic column needs to be replaced by a strong polarity chromatographic column or connected in series with the strong polarity chromatographic column (the connection of the weak polarity chromatographic column and the strong polarity chromatographic column includes the connection of the weak polarity chromatographic column before and/or after the strong polarity chromatographic column), and then the sample to be detected needs to be subjected to gas chromatography-mass spectrometry detection, such detection can be quantitative detection or qualitative detection (determined according to specific needs), and a specific detection method, such as described below, can still be performed according to the method disclosed in the < first aspect > of the present invention.

Therefore, it can be seen that when the method of the present aspect is faced with a large number of samples to be tested, and few of these samples have problems, efficient screening can be performed.

Gas chromatography-mass spectrometry detection based on weak polarity chromatographic column

The sample preparation step and the pretreatment step of the detection method of the present aspect can be carried out with reference to < first aspect > of the present invention.

The weak polarity chromatography column in the present aspect may be selected in accordance with the weak polarity chromatography column disclosed in < first aspect >. Also, in a preferred embodiment, a phenyl-modified dimethylpolysiloxane having a phenyl content of not more than 8% is used.

In addition, as the temperature raising method of the gas chromatography when the weak polarity column is used, a temperature control method of raising the temperature in stages can be adopted. In some specific embodiments, the temperature raising procedure of the gas chromatography comprises raising the temperature to no more than 160 ℃ at a speed of 3-7 ℃/min, and keeping the temperature for no more than 15 min; then heating to 190-210 ℃ at the speed of 8-12 ℃/min, and keeping for 0.5-2 min; and then heating to 230-255 ℃ at a speed of 25-35 ℃/min and keeping for 1-3 min.

More specifically, for example, the initial column temperature of the column is set to 58 to 62 ℃ and maintained for 1 to 3 min; further, heating to a temperature not exceeding 160 ℃ (e.g. 155 ℃, 150 ℃ and the like) at a speed of 3-7 ℃/min (e.g. 4 ℃/min, 5 ℃/min and the like), and keeping the temperature for not exceeding 15min (e.g. 7-10 min, 8-12 min and the like); then heating to 190-210 deg.C (e.g. 200 deg.C) at 8-12 deg.C/min (e.g. 9 deg.C/min, 10 deg.C/min, etc.), and maintaining for 0.5-2 min (e.g. 1min, 1.5min, etc.); and finally heating to 230-255 deg.C (e.g. 240 deg.C, 250 deg.C, etc.) at 25-35 deg.C/min (e.g. 30 deg.C/min, etc.), and maintaining for 1-3 min (e.g. 1.5min, 2min, 2.5min, etc.).

Through the temperature control mode, strong polar substances can better permeate into a solid phase structure of the chromatographic column, and then the substances can be separated from the ethyl maltol with weak polarity.

For other operating conditions for gas chromatography-mass spectrometry, such as operating conditions for mass spectrometry, etc., reference may be made to the disclosure of < first aspect > above.

Further, the "safety standard" in the present invention may be a safety standard specified by a local, regional, national, regional or international organization as described above, for example, the content of ethyl maltol in a subject product tolerated in such a safety standard is not more than 5 μ g/kg to 25 μ g/kg, for example, not more than 10 μ g/kg, not more than 15 μ g/kg or not more than 20 μ g/kg, and the like. In some preferred embodiments of the invention, the limit of detection of ethyl maltol in edible vegetable oil (BJS 201708) was 25 μ g/kg (i.e. safety standard threshold concentration).

Examples

Hereinafter, the present invention will be further described by way of examples.

Testing instrument：

Gas chromatography-mass spectrometry, Sammer Feishell science, USA, Trace 1310-ISQ.

Refrigerated centrifuge, sigma, 2-16 KL.

Quartz two-way for gas chromatography column connection, american sermer fly, C-QSC 10.

Raw material：

The samples of the aromatic rapeseed oil and the refined fourth-grade rapeseed oil are from Yihai (Guanghan) grain and oil feed Co.

Determination of the reference retention time and abundance ratio of the detection system:

1) standard sample:

a standard sample of ethyl maltol at a concentration of 200. mu.g/Kg was prepared using acetonitrile solvent.

2) Gas chromatography-mass spectrometry test system:

gas chromatography conditions:

and (3) sample introduction mode: gas chromatograph injection port temperature: 250 ℃; no shunt sampling; sample introduction amount: 1 μ L. Separating ethyl maltol with TG-1701MS (specification: 30m 0.25mm 0.25um) strong polarity chromatographic column, and measuring;

the temperature rising procedure is as follows: the initial column temperature is 60 ℃, the temperature is kept for 4min, the temperature is raised to 100 ℃ at the speed of 3 ℃/min, the temperature is kept for 10min, and then the temperature is raised to 250 ℃ at the speed of 30 ℃/min, and the temperature is kept for 5 min.

Chromatographic carrier gas: high purity helium (> 99.999%) flow rate: 1.0 mL/min.

Mass spectrum conditions:

selective ion Scanning (SIM) monitoring; an ionization mode: electron impact ionization source (EI); ionization energy: 70 eV; the temperature of the transmission line is 300 ℃; the ion source temperature is 300 ℃; solvent retardation: and (5) 10min. Determining the character by retention time and ion abundance ratio; the quantitative ions were 140 ions, and the qualitative ions were: 139. 125, 97 ions.

The retention time and the base abundance ratio of the matrix spectral ions were determined by testing the standard samples using the gas chromatography-mass spectrometer system described above, see fig. 1a and 1b, where the gas chromatography retention time was 28.91 min.

Example 1 (gas chromatography-mass spectrometry quantitative determination method based on strong polarity chromatography column)：

In the embodiment, after a target object is separated by a strong-polarity chromatographic column, single quadrupole mass spectrometry is used for quantitative analysis; the method comprises the following steps:

a. preparation of labeled sample

Diluting a standard sample by using an oil sample to be detected (strong fragrant rapeseed oil), and preparing a standard sample for a working curve of a standard addition method; the oil sample to be detected is rapeseed oil, and the standard sample is prepared into standard samples with the concentrations of 15 microgram/Kg, 25 microgram/Kg, 50 microgram/Kg, 100 microgram/Kg and 200 microgram/Kg by using the oil sample to be detected as a matrix diluent;

b. pretreatment

After the standard sample and the oil sample to be detected are mixed uniformly, 2g +/-0.01 g of the standard sample and the oil sample to be detected are weighed accurately into a 15mL glass centrifuge tube, 2mL of chromatographic grade acetonitrile is added, the mixture is swirled for 5min, the centrifuge tube sample is placed into a centrifuge, the centrifuge tube sample is centrifuged for 5min at the rotating speed of 4000r/min and at the temperature of 4 ℃, a pipette is used for sucking and collecting 100 mu L of supernatant, the supernatant is placed into an inner insertion tube, a bottle cap is covered, and the mixture is placed on a gas chromatography-mass spectrometer for sample introduction and analysis;

c. gas chromatography conditions:

and (3) sample introduction mode: gas chromatograph injection port temperature: 250 ℃; no-shunt sample introduction; sample introduction amount: 1 μ L. Separating ethyl maltol with TG-1701MS (specification: 30m 0.25mm 0.25um) strong polarity chromatographic column, and measuring;

the temperature-raising program is: the initial column temperature is 60 ℃, the temperature is kept for 4min, the temperature is raised to 100 ℃ at the speed of 3 ℃/min, the temperature is kept for 10min, and then the temperature is raised to 250 ℃ at the speed of 30 ℃/min, and the temperature is kept for 5 min.

d. Mass spectrum conditions:

selective ion Scanning (SIM) monitoring; an ionization mode: electron impact ionization source (EI); ionization energy: 70 eV; the temperature of the transmission line is 300 ℃; the ion source temperature is 300 ℃; solvent retardation: determining by retention time and ion abundance ratio; the quantitative ions were 140 ions, and the qualitative ions were: 139. 125, 97 ions.

e. The testing process comprises the following steps:

before quantitative analysis, a detection sample obtained by pretreating a sample to be detected is detected, the chromatographic peak time and the mass spectrum ion abundance ratio are used as qualitative bases for judgment, and the retention time and the reference ion abundance in the graph 1 are compared to judge that the sample to be detected contains the ethyl maltol.

And respectively injecting the pretreated standard working curve sample solution (standard sample solution) and the detection sample obtained after pretreatment of the sample solution to be detected into a gas chromatography-mass spectrometer for testing.

Taking the peak area as a vertical coordinate and the standard addition content (mu g/Kg) as a horizontal coordinate, drawing a quantitative curve by adopting a standard addition method, and then carrying out quantitative determination on the sample to be measured by adopting an external standard method. The linear equation is obtained as Y-8.145 × 10³X+1.992×10⁴The correlation coefficient was 0.9984 (see fig. 2).

Meanwhile, preparing standard addition samples with different concentrations by using a sample to be tested, and verifying according to the process to obtain recovery rate and parallelism data; to examine the accuracy and precision of the method. Recovery of 95-110% and an RSD of 8% were obtained.

FIG. 3 shows that the peak pattern of ethyl maltol is not interfered in the gas chromatogram of the test sample pretreated by using the sample to be tested in example 1 (simultaneously, the chromatogram of the standard sample of 100. mu.g/Kg is superimposed), and the quantitative test result shows that the ethyl maltol content of the oil sample to be tested is 7. mu.g/Kg (10 times of signal-to-noise ratio).

Reference example 1:

the conditions were the same as in example 1 except that the temperature raising program of the gas chromatography column in example 1 was replaced with the following temperature raising program:

the initial column temperature is 60 ℃, the temperature is kept for 0min, the temperature is increased to 120 ℃ at the speed of 5 ℃/min, the temperature is kept for 0min, and then the temperature is increased to 250 ℃ at the speed of 35 ℃/min, and the temperature is kept for 5 min.

The preliminary quantification result is about 100 mu g/Kg, mass spectrogram analysis shows that the abundance ratio of the mass spectrogram at the width of 1/2 peak around the peak is inconsistent with that of a standard sample, M/Z125 ions completely disappear (original 25% abundance), which indicates that the co-outflow peak exists, the peaks are not completely separated, and other compounds interfere.

Example 2

The detection result of the detection method of the embodiment 1 on the four-stage rapeseed oil sample refined in the factory oil extraction workshop is shown in figure 4, which is a superposition spectrum of an actual sample spectrum and a standard working curve point (a standard addition point of 25 mug/Kg), and is shown in a TG-1701MS chromatographic column; the rapeseed oil was refined to be completely unpeaked without any interference, and the result was 0 (no ethyl maltol was present).

In addition, FIG. 5 is a superposition of the detection profiles of the various samples of FIGS. 3 and 4, which also reflects the validity and reliability of the test method of the present invention.

It should be noted that, although the technical solutions of the present invention are described by specific examples, those skilled in the art can understand that the present disclosure should not be limited thereto.

Having described embodiments of the present disclosure, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Industrial applicability

The gas chromatography-mass spectrometry detection method can be industrially used for reliably detecting the ethyl maltol and the content thereof in the edible oil.

Claims

1. A method for detecting ethyl maltol in edible oil is characterized by comprising the following quantitative steps:

the gas chromatography comprises a strong polarity chromatographic column, and the mass spectrum adopts a characteristic selection ion monitoring Scanning (SIM) working mode; preferably, the strongly polar chromatography column is selected from a polyethylene glycol based chromatography column, a polar group modified polyethylene glycol chromatography column or a polar group modified polysiloxane chromatography column.

2. The method of claim 1, further characterized by one or more of the following features:

in the step of detecting, a weak polarity chromatographic column is also used before and/or after the strong polarity chromatographic column of the gas chromatograph and is connected with the strong polarity chromatographic column in series;

in the step of detection, the temperature rise program of the gas chromatography comprises the steps of raising the temperature to be not more than 100 ℃ at the speed of less than 10 ℃/min and keeping the temperature for not more than 15 min; then heating to 230-260 ℃ at a speed of 30-40 ℃/min, and keeping for 2-10 min;

the method is based on the linear correlation coefficient R of the working curve of the standard addition method²Greater than 0.998; and/or the method has a detection limit of 5 mug/Kg for ethyl maltol.

3. The method of claim 2, wherein the weakly polar chromatography column comprises phenyl-modified dimethylpolysiloxane; and/or the mass spectrum is a single quadrupole mass spectrum.

4. A method for detecting ethyl maltol in edible oil is characterized by comprising the following qualitative steps:

a pretreatment step, namely enriching the polar substances in the sample to be detected to obtain a detection sample; preferably, the enrichment is to extract the sample to be tested by using a polar solvent, and the polar solvent is selected from acetonitrile or methanol;

a step of detection, in which the standard sample and the detection sample are tested by a combination of gas chromatography and mass spectrometry, and recording:

and a step of judging, namely determining whether the sample to be tested contains the ethyl maltol or not through the comparison of t1 and t2 and the comparison of abundance ratios of characteristic ion peaks in the mass spectrum if necessary.

5. The method of claim 4, further characterized by one or more of the following features:

in the judging step, if the deviation between the t1 and the t2 is less than or equal to +/-0.05 min, whether the sample to be detected contains the ethyl maltol or not is determined continuously through comparison of abundance ratios of characteristic ion peaks of mass spectra.

6. The method of claim 4 or 5, wherein the comparison of the abundance ratios of characteristic ion peaks in the mass spectrum comprises comparing the ratios of 139,125 and 97 ion peak relative intensities to 140 ion peak relative intensities of the test sample with the ratios of 139,125 and 97 ion peak relative intensities to 140 ion peak relative intensities in a standard sample, respectively.

7. A method for detecting ethyl maltol in edible oil is characterized by comprising the following steps:

and judging, if the result of the detecting step shows that the detection concentration of the ethyl maltol in the sample to be detected is greater than the safety standard threshold concentration, replacing a weak polarity chromatographic column in a gas chromatograph with a strong polarity chromatographic column or connecting the weak polarity chromatographic column with the strong polarity chromatographic column in series, and testing by combining with the mass spectrum so as to carry out quantitative or qualitative detection on the sample to be detected again.

8. The method according to claim 7, wherein in the step of determining, if the peak area corresponding to the mass spectrometric quantification ion 140 ion in the test sample is larger than the peak area corresponding to the standard sample quantification ion 140 ion, the detected concentration of ethyl maltol in the test sample is considered to be larger than the safety standard threshold concentration.

9. The method according to claim 7 or 8, wherein in the step of detecting, the temperature rise program of the gas chromatograph comprises raising the temperature to not more than 160 ℃ at a speed of 3-7 ℃/min and keeping the temperature for not more than 15 min; then heating to 190-210 ℃ at the speed of 8-12 ℃/min, and keeping for 0.5-2 min; and then heating to 230-255 ℃ at a speed of 25-35 ℃/min and keeping for 1-3 min.

10. A method according to any of claims 7 to 9, characterized in that the method has one or more of the following features:

the safety standard threshold concentration is 25 mug/Kg;

the series connection mode of the weak polarity chromatographic column and the strong polarity chromatographic column comprises that the weak polarity chromatographic column is connected in series in front of and/or behind the strong polarity chromatographic column;

the re-performing of the quantitative or qualitative detection comprises using the detection method according to any one of claims 1 to 3.