CN114660189A - Method for detecting ethyl maltol in edible oil - Google Patents

Abstract

The present invention provides a method for detecting ethyl maltol in edible oil, which includes qualitative and quantitative testing methods.

Description

Method for detecting ethyl maltol in edible oil

technical field

The invention belongs to the field of analysis and detection of food additives, in particular to a method for analyzing chemical substances that may be contained or added in edible oil, and more specifically to a method for qualitative and quantitative detection of ethyl maltol in vegetable oil.

Background technique

Ethyl maltol (CAS NO: 4940-11-8), usually white needle-like or white crystalline powder. As a flavor modifier and flavor enhancer, it is more and more widely used. It is a good flavor synergist for tobacco, food, beverage, essence, fruit wine, daily cosmetics, etc. It has a significant effect on improving and enhancing the flavor of food. Plays a sweetening effect on sweets.

As a food spice, ethyl maltol has not been found in natural substances at present, and it must be obtained by chemical synthesis, so it belongs to a kind of artificially synthesized flavor and spice. Although, ethyl maltol has certain safety, and it is a synthetic flavor for food allowed by GB 2760-2014 "National Food Safety Standard for the Use of Food Additives", which can be used in tobacco, food, beverages, flavors, alcohol, etc. Fragrance. However, GB 2760-2014 Appendix B.1 clearly stipulates that food flavors and essences shall not be added to vegetable oils and fats. Therefore, ethyl maltol is actually illegally added in edible vegetable oil and cannot be detected.

Further, there are already detection methods for the content of ethyl maltol in food substances, and the methods for detecting such compounds are mainly concentrated in ultraviolet spectroscopy, liquid chromatography, gas chromatography and liquid chromatography. Combined with solid phase extraction, it can purify the sample matrix and improve the detection accuracy. E.g:

At present, GB 5009.250-2016 "National Food Safety Standard - Determination of Ethyl Maltol in Food" stipulates that the detection method for ethyl maltol in food is high performance liquid chromatography, and the detection limit of this method is 0.5mg/Kg, However, the scope of application of this method does not include edible oil and fat products;

The promulgated food supplement testing method BJS (2017.08, China) "Determination of Ethyl Maltol in Edible Vegetable Oil" is used for the supervision and sampling of ethyl maltol in edible oils and fats; this method is high performance liquid chromatography - triple quadruple Rod mass spectrometry method; the detection limit of the method is 25μg/Kg, the equipment used in this method is expensive, and it cannot be widely used and promoted; due to the complex composition of oil samples, background substances may interfere with the detection.

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

Citation 1 discloses a method for the determination of maltol and ethyl maltol in blended oil by GC-MS using solid phase extraction as the extraction and separation method. After the sample was dissolved in n-hexane, it was separated and purified by an amino solid phase extraction column. Combined with the high resolution and high sensitivity of gas quality, the method was investigated from the three aspects of detection limit, recovery rate and precision by selecting appropriate monitoring ions. applicability. In the test results, the detection limit of ethyl maltol was 0.35 mg/kg. In the determination range of 0-0.5g/kg, the recovery of standard addition was 95.2%-107%, and the precision was 0.9%-1.2%.

Reference 2 discloses a method for identifying adulterated peanut oil, which includes: extraction pretreatment and analysis of the extracted aromatic substances by using a gas chromatography-mass spectrometry (GC-MS) instrument, and obtaining relevant data by using Xcalibur software to process the experimental atlas, and using B. The qualitative analysis of peanut flavor was carried out with maltol and triacetin as characteristic substances.

However, in Citation 1, the sample needs to undergo solid-phase extraction and then GC-MS testing. The convenience of detection and the detection accuracy (detection limit) for ethyl maltol are still not sufficient. Citation 2 only Involves qualitative analysis without disclosing the feasibility of quantitative detection.

In addition, in the detection of other vegetable pesticide residues, there are also detection methods combined with gas chromatography and mass spectrometry to quantitatively detect the target pesticide residues.

For example, Citation 3 used four common vegetables as materials, and applied gas chromatography/mass spectrometry to study the effects of column polarity and scanning method on the determination of five pyrethroid pesticide residues in vegetables. The detection limit of pyrethroid pesticides in vegetables can be lowered by using a capillary column with stronger polarity (such as DB-17MS). On the basis of determining the characteristic ions of each pyrethroid pesticide, the detection limit of pyrethroid pesticides in vegetables can be significantly reduced by using selected ion monitoring scanning (SIM), and the detection limit range of 5 kinds of pyrethroid pesticides in 4 kinds of vegetables At 0.00253-0.19100 mg/kg. Although Citation 3 has a lower detection limit, it is not known whether a similar detection can be performed in edible oil, especially considering the presence of a large amount of interfering substances in vegetable oil.

It can be seen that there is no economical and effective qualitative and quantitative test method for the detection of ethyl maltol that may exist in edible oil, especially vegetable oil. Deterministic studies are still not sufficient, therefore, there is still a need for further improvements in detection methods.

Citation:

Citation 1: "China Food Additives", 2017, No. 6, pp. 209-213,

Citation 2: CN106526031A,

Citation 3: Study on the detection of pyrethroid pesticide residues in vegetables by gas chromatography-mass spectrometry, Zhang Jing et al., "Anhui Agricultural Science", 2011.

SUMMARY OF THE INVENTION

Invention to solve problem

In view of the existing problems or deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a reliable detection method for the ethyl maltol additive that may exist in edible oil, especially vegetable oil, and can solve the background The problem of inaccurate qualitative and quantitative inaccuracy caused by substance interference, this method has low detection limit and excellent detection accuracy, and can perform good qualitative and quantitative measurements.

In addition, another object of the present invention also is to provide a method for detecting ethyl maltol that may exist in edible oil. Quantitative detection limit or concentration lower limit (ie safety threshold), then use strong polar chromatographic column coupled with mass spectrometry for quantitative or qualitative detection (recheck). Such methods are particularly suitable for efficient large-scale, screening tests.

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

solution to the problem

Through the long-term intensive research of the present inventor, it is found that the above-mentioned technical problems can be solved by the following methods:

[1]. The present invention first provides a method for detecting ethyl maltol in edible oil, which is a quantitative detection method, comprising the following quantitative steps:

In the sample preparation step, the sample to be tested is prepared by the edible oil to be tested, and n spiked samples are prepared based on the standard addition method, where n is an integer greater than or equal to 3, preferably, the n is an integer from 3 to 10;

In the step of pretreatment, the polar substances in the sample to be tested and the spiked sample are enriched to obtain the test sample; preferably, the enrichment is to extract the sample to be tested and the spiked sample with a polar solvent, The polar solvent is selected from acetonitrile or methanol;

In the step of detection, each of the detection samples is tested by the combination of gas chromatography and mass spectrometry, and a working curve is prepared based on the standard addition method to determine the content of ethyl maltol in the sample to be tested,

Wherein, the gas chromatography includes a strong polar chromatographic column, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode; preferably, the strong polar chromatographic column is selected from polyethylene glycol-based chromatographic columns, polar A polar group-modified polyethylene glycol column or a polar group-modified polysiloxane column.

[2]. The method according to [1], wherein, in the step of detecting, a weakly polar chromatographic column is also used in series before and/or after the strong polar chromatographic column of the gas chromatography.

[3]. The method according to [1] or [2], wherein the weakly polar chromatographic column comprises phenyl-modified dimethylpolysiloxane; and/or the mass spectrometry is single-quaternary Rod mass spectrometry.

[4]. The method according to any one of [1] to [3], wherein, in the step of detecting, the temperature rise program of the gas chromatography includes raising the temperature to not more than 100° C. at a rate of 10° C./min or less , and keep the temperature for no more than 15min; then raise the temperature to 230-260°C at 30-40°C/min, and keep it for 2-10min.

[5]. The method according to any one of [1] to [4], wherein the linear correlation coefficient R ² of the working curve of the method according to the standard addition method is greater than 0.998; and/or the method for ethyl malt The detection limit for phenol was 5 μg/Kg.

[6]. Further, the present invention also provides a method for detecting ethyl maltol in edible oil, especially a simple and convenient qualitative detection method, comprising the following qualitative steps:

The sample preparation step is to prepare the sample to be tested by the edible oil to be tested, and prepare a standard sample with a known concentration of ethyl maltol;

In the step of pretreatment, the polar substances in the sample to be tested are enriched to obtain the sample to be tested; preferably, the enrichment is to use a polar solvent to extract the sample to be tested, and the polar solvent is selected from acetonitrile or methanol;

In the detection step, the standard sample and the detection sample are respectively tested by the combination of gas chromatography and mass spectrometry, and record:

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

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

Wherein, the gas chromatography includes a strong polar chromatographic column, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode; preferably, the strong polar chromatographic column is selected from polyethylene glycol-based chromatographic columns, A polar group-modified polyethylene glycol chromatography column or a polar group-modified polysiloxane chromatography column, and/or the mass spectrometer is a single quadrupole mass spectrometer;

The judgment step is to determine whether the sample to be tested contains ethyl maltol through the comparison of t1 and t2, and if necessary, through the comparison of the abundance ratio of the characteristic ion peaks in the mass spectrum,

[7]. The method according to [6], wherein, in the step of detecting, the temperature rise program of the gas chromatography comprises: raising the temperature to no more than 100°C at a speed of 10°C/min or less, and maintaining the temperature for no more than 15min; Then the temperature is raised to 230-260°C at 30-40°C/min, and maintained for 2-10min.

[8]. The method according to [6] or [7], wherein, in the step of judging, if the deviation between t1 and t2 is less than or equal to ±0.05min, continue to pass the abundance of characteristic ion peaks of mass spectrometry A comparison of the ratios determines whether the sample to be tested contains ethyl maltol.

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

In some specific embodiments, the methods of the above [6] to [9] may adopt the following steps:

a. Prepare a standard sample with a known content of ethyl maltol (200μg/Kg standard sample);

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

c. Pre-treat the sample to be tested to obtain a test sample, perform GC-MS detection on the test sample under the same conditions as b, and record the retention time t2 of ethyl maltol in gas chromatography and 139, 125 and 97 ions in mass spectrometry Peak relative intensity and 140 ion peak relative intensity (detection abundance ratio);

d. Compare t1 and t2, the reference abundance ratio and the detection abundance ratio.

[10]. Further, the present invention also provides a method for detecting ethyl maltol in edible oil, which comprises the steps:

The preparation step of the sample is to prepare the sample to be tested by the edible oil to be tested, and prepare a standard sample containing ethyl maltol whose concentration is the safety standard threshold concentration;

In the step of pretreatment, the polar substances in the sample to be tested are enriched to obtain the sample to be tested; preferably, the enrichment is to use a polar solvent to extract the sample to be tested, and the polar solvent is selected from acetonitrile or methanol;

In the step of detection, the detection sample and the standard sample are respectively tested by the combination of gas chromatography and mass spectrometry, in which a weakly polar chromatographic column is used in the gas chromatography, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode ; Preferably, the weakly polar chromatographic column is selected from phenyl-modified dimethyl polysiloxane, and/or the mass spectrometer is a single quadrupole mass spectrometer;

The step of judging, if the result of the step of described detection shows that the detected concentration of ethyl maltol in the described sample to be tested is greater than the safety standard threshold concentration, then the weak polar chromatographic column in the gas chromatography is replaced with a strong polar chromatographic column. The column or the weak polarity chromatographic column is connected in series with the strong polarity chromatographic column, and the test is combined with the mass spectrometer to perform quantitative or qualitative detection of the sample to be tested again.

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

[12]. The method according to [10] or [11], characterized in that, in the step of detecting, the temperature rise program of the gas chromatography comprises: raising the temperature to no more than 160°C at a rate of 3-7°C/min, And keep the temperature for no more than 15min; then raise the temperature to 190-210°C at 8-12°C/min, and keep for 0.5-2min; then heat up to 230-255°C at 25-35°C/min, and keep for 1-3min.

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

[14]. The method according to any one of [10] to [13], wherein the series connection of the weakly polar chromatographic column and the strong polar chromatographic column comprises, before and/or the strong polar chromatographic column A weakly polar chromatographic column is connected in series.

In some specific embodiments, the methods of the above [10] to [14] may adopt the following steps:

a. Determine the threshold concentration of the safety standard, and configure a standard sample containing ethyl maltol at the threshold concentration (for example, a standard sample of 25 μg/Kg), and use the GC-MS system for detection, and record the corresponding mass spectrometer quantitative ion 140 The peak area of the chromatographic peak (reference peak area);

b. Take the sample to be tested to obtain the test sample through pretreatment, and perform GC/MS detection under the same test conditions as a, and record the peak area (detection peak area) of the chromatographic peak corresponding to the mass spectrometer quantitative ion 140;

c. Compare the reference peak area with the detection peak area. If the detection peak area is higher than the reference peak area, replace the weakly polar chromatographic column in the gas chromatography with a strong polar chromatographic column or replace the weakly polar chromatographic column with a The strong polar chromatographic column is connected in series, and the test is performed with the mass spectrometer to perform quantitative or qualitative detection of the sample to be tested again.

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

effect of invention

Through the implementation of the above-mentioned technical solutions, the present invention can obtain the following technical effects:

1) the present invention adopts strong polarity chromatographic column or weak polarity chromatographic column and strong polarity chromatographic column series connection method in the preferred chromatographic condition can make ethyl maltol avoid the co-elution peak situation in the gas chromatographic separation process, therefore can Complete separation of ethyl maltol and interfering substances. At the same time, the content of ethyl maltol in edible oils and fats can be accurately determined by adopting feature selected ion monitoring scanning mode (SIM) monitoring and standard addition of external standard method-gas chromatography/mass spectrometry method. The method is quantitatively accurate, sensitive, accurate and Good reproducibility. In some specific embodiments, using a strong polar chromatographic column or a method of connecting a weak polar chromatographic column and a strong polar chromatographic column in series, the ethyl maltol peak shape is good and symmetrical, and no tailing peak phenomenon occurs.

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

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

4) The detection method of the present invention has good recovery rate and relative standard deviation (RSD).

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

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

Description of drawings

Fig. 1: in the embodiment of the present invention, the detection system of gas chromatography-mass spectrometry is used for the detection result of standard sample;

Figure 2: Working curve prepared by adding standard samples in Example 1 of the present invention;

Fig. 3: Gas chromatogram superposition diagram when the spiked sample of coriander seed oil in the embodiment of the present invention and the sample to be tested (actual oil sample not spiked) are detected;

Fig. 4: Gas chromatogram superimposed figure when the standard sample of refined grade 4 rapeseed oil and the sample to be tested (actual oil sample without standard addition) are detected in the embodiment of the present invention 2;

Figure 5: Overlay of the gas chromatograms of Figures 3 and 4.

Detailed ways

Embodiments of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications can be made within the scope of the claimed invention, and embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples are also included in the present invention. within the technical scope of the invention. In addition, all the documents described in this specification are incorporated in this specification as a reference.

Unless otherwise defined, technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In this specification, the numerical range represented by "numerical value A - numerical value B" or "numerical value A - numerical value B" means the range including the numerical values A and B at the endpoints.

In this specification, the meaning expressed by "may" includes both the meaning of performing a certain processing and not performing a certain processing. 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 use of "safety standards" refers to the standards for the minimum detection amount of ethyl maltol in food and edible oil existing in local, regional, national, regional or international organizations related to food quality testing or monitoring .

In this specification, "some specific/preferred embodiments", "some other specific/preferred embodiments", "some specific/preferred technical solutions", "some other specific/preferred technical solutions", etc. mentioned in this specification means that a particular element (eg, feature, structure, property, and/or characteristic) described in relation to this embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments present in other embodiments. Additionally, it should be understood that the described elements may be combined in any suitable manner in the various embodiments.

The terms "standard sample", "spiked sample", "test sample" and "test sample" in the present invention have the following meanings respectively:

Standard sample: that is, a sample with a determined content or concentration of the target analyte, and the sample does not contain any components that interfere with the detection of the target analyte, or is not added except for non-interfering essential solvents and target analytes. samples of other ingredients;

Spiked sample: a sample obtained by mixing a standard sample and a sample to be tested, the mixed sample is calculated by the amount of the target analyte in the added standard sample to calculate the concentration of the target analyte in the mixed sample, and the concentration Considered the concentration of the target analyte in the spiked sample;

Sample to be tested: refers to the object sample to be detected, such a sample to be tested is the sample actually sampled, and in the present invention, it also refers to the oil sample that actually needs to be detected;

Test sample: refers to the sample that is actually tested on the machine, that is, the sample obtained after the pretreatment of the spiked sample and the sample to be tested, the sample can be directly tested on the machine.

The term "comprising" and any variations thereof in the description and claims of the present invention and the above drawings are intended to cover non-exclusive inclusions. For example, a process, method or system, product or device comprising a series of steps or units is not limited to the listed steps or units, but optionally also includes unlisted steps or units, or optionally also includes Other steps or units inherent in these processes, methods, products or devices.

<Aspect 1>

In the first aspect of the present invention, a reliable quantitative detection method for the ethyl maltol additive that may exist in edible oil, especially vegetable oil, is provided, and the problem of inaccurate quantification caused by interference of background substances can be solved, The method has a low detection limit and excellent detection accuracy, enabling good quantitative measurements.

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

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 from being used in edible oil, in order to identify whether the flavoring agent is illegally added in edible oil, Reliable and precise detection methods are necessary.

The edible oil of the present invention, in some specific embodiments of the present invention, generally refers to various vegetable oils.

For these vegetable oils, including but not limited to: rice oil, sunflower oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, soybean oil, linseed oil, cottonseed oil, safflower oil, perilla seed oil, tea Seed Oil, Hemp Seed Oil, Jojoba Oil, Olive Oil, Cocoa Oil, Tallow Seed Oil, Almond Oil, Almond Oil, Tung Oil, Rubber Seed Oil, Corn Germ Oil, Wheat Germ Oil, Sesame Seed Oil one or more of , evening primrose seed oil, hazelnut oil, pumpkin seed oil, walnut oil, grapeseed oil, flax seed oil, borage seed oil, sea buckthorn seed oil, tomato seed oil, macadamia nut oil, and coconut oil kind.

In addition, the edible oil of the present invention also includes the mixed oil or blended oil formed by the above-mentioned various vegetable oils in any proportion.

Further, the present invention does not specifically limit the production method of these vegetable oils, and they can be obtained by physical pressing or chemical leaching.

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

The standard addition method is to add a standard solution of known concentration with a certain concentration gradient to the sample to be tested to prepare multiple spiked samples, and by measuring a certain characteristic of these spiked samples (and blank samples), through the fitting method. A method for preparing a working curve from a straight line. This method is particularly useful for testing samples for the presence of interfering substances.

In some specific embodiments of the present invention, by using the edible oil to be tested to dilute a standard sample containing ethyl maltol with a known concentration, while the sample to be tested is obtained, n spiked samples are also obtained.

There is no special limit on the number of spiked samples. It is understood that the more the spiked samples are, the more reliable the test results will be, but at the same time, it may also bring more complicated processing to the linear fitting. In some specific embodiments of the present invention, n is an integer greater than or equal to 3, preferably an integer of 3-10, more preferably an integer of 4-6.

Further, there is no particular restriction on the concentration of ethyl maltol in the spiked sample, but from the perspective of linear range and detection limit, in some preferred embodiments of the present invention, the concentration of the spiked sample can be 10 μg/kg ~500 μg/kg, more preferably 15 μg/kg to 350 μg/kg, more preferably 15 μg/kg to 300 μg/kg. For example, these spiked samples can be at concentrations of 15 μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55 μg/kg, 80 μg/kg, 100 μg/kg, 120μg/kg, 140μg/kg, 160μg/kg, 180μg/kg or 200μg/kg, etc.

(Pre-processing step)

The pretreatment step of the present invention is to pre-separate the components in the sample, including the sample to be tested and the sample for which the standard is added.

In oils and fats, there are a large number of polar components (strong polar components and weak polar components) and non-polar components. In a specific embodiment of the present invention, the polar and non-polar components are pre-separated by extracting the sample with a solvent.

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

In addition, in the pretreatment step of the present invention, auxiliary means can also be used for pre-separation of components in oil and fat. For such auxiliary means, typically centrifugation can be used. In some preferred embodiments, the rotational speed of the centrifugal separation treatment can be above 4000 r/min, and the centrifugation time can be 3-8 min.

Through the extraction and centrifugation in the above pretreatment steps, the polar components in the sample are enriched, at least part or all of the non-polar components are separated and removed, and the interference of some background substances is excluded.

(Gas chromatography)

In the present invention, the enriched components or extracts obtained from the pretreatment are separated by gas chromatography. By optimizing the control conditions of gas chromatography, the polar components (especially the strong polar components) from the oil sample can be separated from the weakly polar ethyl maltol, and the interference of another part of the background material can be excluded, thereby It provides the possibility for the quantitative detection of ethyl maltol.

There is no particular restriction on the injection method of the gas chromatograph, and the injection can be performed manually or by using an autosampler. In some embodiments, an autosampler, splitless injection is used.

In the gas chromatography of the present invention, at least one strong polar chromatographic column is included, and various chemical substances with different polarities in the test substance are separated by the strong polar chromatographic column, and further separated by the mass spectrometry described later. detection.

In another specific embodiment of the present invention, before the strong polar chromatographic column, it further includes a weak polar chromatographic column connected in series with the strong polar chromatographic column. The chromatographic column is used to pre-separate various chemical substances with different polarities. In the present invention, the use of a series connection of a weak polar chromatographic column and a strong polar chromatographic column is beneficial to better separation of ethyl maltol and other interfering components.

In another specific embodiment of the present invention, after the strong polar chromatographic column, a weak polar chromatographic column connected in series with the strong polar chromatographic column is also included. In this case, when performing detection, the components are separated using a strong-polarity column, and then continue to be separated by a weaker-polarity column. The present invention also believes that such a connection mode of the chromatographic column is also conducive to better separation of ethyl maltol and other interfering components.

In addition, there is no particular limitation on the arrangement of other chromatographic columns in which at least one highly polar chromatographic column exists, as long as the technical effects of the present invention are not hindered. For example, a weakly polar chromatographic column is arranged before and after a strong polar chromatographic column; a weakly polar chromatographic column is connected in parallel with the strong polar chromatographic column, etc.

The "strongly polar chromatographic column" and "weakly polar chromatographic column" described in the present invention can be selected according to conventional commercially available chromatographic columns in the art.

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

In addition, for the so-called "weakly polar chromatographic column", it can be selected from phenyl-modified dimethyl polysiloxane, and in some preferred cases, the content of phenyl group is not more than 8%, preferably not more than 5%. More specifically, the so-called "weakly polar chromatographic column" can be selected from the chromatographic columns under the trade names: DB-5MS; HP-5MS, TG-5MS; SE-54MS; RTX-5MS; DB-1MS and HP-1MS .

Generally, the above-mentioned "strongly polar chromatographic column" and "weakly polar chromatographic column" are commercially available from companies such as Agilent and Thermo Fisher.

When using the above-mentioned commercially available chromatographic columns to connect strong polar and weak polar chromatographic columns in series, in some specific embodiments, a glass conical capillary union can be used to connect weak polar chromatographic columns (such as DB-5MS) with Connect to a highly polar column (eg DB-1701MS). For example, insert the outlet of the weak polar chromatographic column into the two-way inlet of the glass conical capillary, insert one end of the strong polar chromatographic column into the two-way outlet of the glass conical capillary, and connect the outlet of the strong polar chromatographic column to the mass spectrometer detector. In other connection modes, such as the mode of parallel connection of strong polarity and weak polarity chromatographic columns, preferably, a multi-port valve group can be used to realize the conversion of detection channels.

In addition, for the total length of the chromatographic column of the present invention (including the total length of the strong polar chromatographic column and the total length of one or two weakly polar chromatographic columns that may be connected in series with it), the convenience of the equipment and the reliability of detection Considering the aspect, it can be no more than 100m, preferably no more than 80m, and more preferably no more than 70m. In one embodiment, the weak polarity chromatographic column is connected in series with the strong polarity chromatographic column, and the length of the chromatographic column is 60 meters. In this case, the length of the chromatographic column is set to 60 meters on the instrument software. Turn on the instrument and complete the connection after passing the self-test.

Regarding the operating temperature of the gas chromatography, the present invention considers that in the separation of the gas chromatography, sufficient heating is used to separate the strongly polar substances from the weakly polar ethyl maltol.

In some specific embodiments of the present invention, in the gas chromatography detection, a temperature control mode of staged heating may be adopted. First, the chromatographic column obtains a certain initial temperature, such as an initial temperature above 50 °C, and further, a slower heating rate can be used to heat up to no more than 100 °C, and the temperature is kept for no more than 15 minutes. Such a heating rate can be no more than 100 °C. 10°C/min; then, the temperature is increased to 230-260°C through a relatively rapid heating rate, such a heating rate can be 30-40°C/min, and the temperature is maintained for 2-10min at the heating end point.

In some preferred embodiments of the present invention, the gas chromatography of the present invention can adopt the following temperature control methods: the initial temperature of the chromatographic column is set to 55-65° C. and kept for 3-5 minutes (for example, 3.5 minutes, 4 minutes, etc.); and then Raise the temperature to no more than 100°C at a rate of 2.5-5°C/min (for example, 3°C/min, 3.5°C/min, 4°C/min, etc.), and keep the temperature for no more than 15min (for example, 8-13min, etc.); Increase the temperature at a rate of ~35°C/min (for example, 28°C/min, 30°C/min, 32°C/min, etc.) to 230-255°C (for example, 240°C, 250°C, etc.), and hold for 3-8 minutes (for example, 5min, 7min, etc.) Wait).

Through the above temperature control method, the strong polar substances can better penetrate into the solid phase structure of the strong polar chromatographic column, and then these substances can be separated from the weakly polar ethyl maltol.

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

There is no particular limitation on other elements or operation modes of the gas chromatography, and reference may be made to conventional technical means or operation modes in the art.

(mass spectrometry)

The present invention uses mass spectrometry to perform mass spectrometry detection on gas chromatographic isolates.

In some specific embodiments of the present invention, as the mass spectrometer detector, a Feature Selected Ion Monitoring Scan (SIM) working mode is adopted. For the scannable characteristic ion peaks, at least 140 ion peaks are included as quantitative ions; for other scannable ion peaks, 139, 125 and 97 ion peaks can be included as qualifier ions, which are mainly related to the relative peaks of the ion peaks in the mass spectrometry detection results. intensity related.

In addition, there is no particular limitation on the ionization mode of mass spectrometry detection of the present invention, and in some preferred embodiments, bombardment ionization can be performed using an electron impact ionization source (EI).

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

Detection reliability

According to the GC/MS test method of the present invention, the linear correlation coefficient R ² of the working curve according to the standard addition method is greater than 0.998, and the detection limit of the method for ethyl maltol is 5 μg/kg (3 times the signal to noise). than the calculation). More specifically:

In the test method based on strong polar chromatographic column: the content of ethyl maltol has a good linear relationship in the range of 10-200μg/Kg, the linear equation is Y=8.145×10 ³ X+1.992×10 ⁴ , and the correlation coefficient is 0.9984; The recovery rate of ethyl maltol measured by this method is 95-110%, and the RSD is 8%;

Based on the tandem method of weakly polar chromatographic column (front) and strong polar chromatographic column (back): the content of ethyl maltol has a good linear relationship within the range of 15-200 μg/Kg, the linear equation is Y=655.57X+7993.07, and the correlation coefficient is 0.9990; the recovery rate of ethyl maltol measured by this method is 82-95%, and the RSD is 2.2-9.8%.

<Second aspect>

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

It should be noted that the qualitative detection method provided by the present invention can be implemented before the quantitative detection in the first aspect. In addition, the qualitative detection method that can be used before the quantitative detection in the first aspect is not limited to this aspect of the present invention. public method. For such detection results, it is first necessary to determine whether the final mass spectral detection peak represents the presence of ethyl maltol, in order to exclude the influence of interfering substances.

More specifically, in the second aspect of the present invention, using the combined testing system and conditions of gas chromatography and mass spectrometry disclosed in the first aspect, by subjecting the sample to be tested and the standard sample of known concentration to gas chromatography, respectively Or the method of gas chromatography and mass spectrometry is used for detection, and it is judged whether the sample to be tested contains ethyl maltol.

First, prepare a sample to be tested by the edible oil to be tested, and prepare a standard sample with a known (arbitrary) concentration of ethyl maltol;

Further pre-processing steps are performed to enrich the polar substances in the sample to be tested to obtain a sample to be tested; preferably, the enrichment is to extract the sample to be tested with a polar solvent, and the polar solvent is selected from the group consisting of Acetonitrile or methanol;

The steps of qualitative detection are mainly designed based on the following two ideas:

The i) point is determined by the retention time of the characteristic peak of ethyl maltol in gas chromatography. Specifically: first, the gas chromatography (or gas chromatography-mass spectrometry) detection described above in the present invention is carried out by using a standard sample of known arbitrary concentration, and the retention time of ethyl maltol in the standard sample is determined. Then, under the same gas chromatographic conditions, the test sample obtained by the pretreatment of the sample to be tested is detected, if the retention time of the suspected ethyl maltol peak in the gas chromatography is the same as the retention time of the standard sample or the deviation is less than or equal to ± 0.05min , the relevant qualitative detection of the following point ii) can be carried out.

At point ii), the presence or absence of ethyl maltol in the sample is (qualitatively) determined by the abundance ratio of the ion peaks of the mass spectrum. Generally speaking, when a substance is detected by the same gas chromatography-mass spectrometry instrument, the abundance ratio of each characteristic peak is determined for each detection. For example, when a gas chromatography-mass spectrometry instrument suitable for the present invention is used for detection, and samples with known different concentrations (or standard samples with known concentrations) are used for detection, although the concentrations are different, including The relative intensities of the 140 ion peak and the 139, 125, and 97 ion peaks vary in intensity from each detection result, however, the ratio of the relative intensities of the 139 ion peak to the 140 ion peak and the ratio of the relative intensities of the 125 ion peak to the 140 ion peak The relative intensities of the 97 ion peak and the 140 ion peak are basically the same in the mass spectrometry detection results of samples with different concentrations.

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

① Using a standard sample of ethyl maltol with a known (arbitrary) concentration, under certain conditions (for the certain conditions, please refer to the content disclosed in the first aspect of the invention in the above text), use gas chromatography-mass spectrometry to carry out Detect and record the retention time of the characteristic peak of ethyl maltol in gas chromatography. And then under the above-determined conditions, the detection sample after the pretreatment of the sample to be tested is detected, if the retention time of the gas chromatographic characteristic peak of the detection sample is the same as the retention time of the standard sample or the deviation is less than or equal to ± 0.05min, it can be carried out. The following further qualitative testing;

② Use a standard sample containing ethyl maltol with a known (arbitrary) concentration (which can be the same as the standard sample used in i) above), under certain conditions (for the determined conditions, please refer to the above-mentioned invention chapter 1). Content disclosed on the one hand), use gas chromatography-mass spectrometry for detection, and obtain the ratio of the relative intensities of the 139 ion peak to the 140 ion peak, the ratio of the relative intensities of the 125 ion peak to the 140 ion peak, and the ratio of the relative intensities of the 125 ion peak to the 140 ion peak of the standard sample. The ratio of the relative intensities of the ion peak to the 140 ion peak was used as a qualitative control standard. The ratio of relative intensities here has the same physical meaning as the ratio of abundances described above. Further, under the same conditions, the pre-treated sample to be tested is subjected to GC-MS detection, and the mass spectrometry detection result is obtained. The ion abundance ratio is compared with the above qualitative reference standard. If the deviation of each ion abundance ratio from the qualitative reference standard meets the following requirements (see Table 1 below), it means that the test sample is determined to contain ethyl maltol.

Table 1 Maximum allowable deviation of ion abundance ratio (compared with standard sample ion abundance)

Abundance ratio (%, based on 140 ion peak) allowable deviation >50 ±10% 20～50 ±15% 10～20 ±20% ≤10 ±50%

It should be noted that, in some preferred embodiments, step ② may be performed with step ① in one measurement. For example, for the above-mentioned standard sample containing ethyl maltol with known (arbitrary) concentration, it can be detected by gas chromatography-mass spectrometry under certain conditions, and the retention of the characteristic peak of ethyl maltol in the gas chromatography can be recorded. time and the standard abundance ratio of each ion in the recorded mass spectrum. The same can be done for the sample to be tested. After the test sample is obtained after pretreatment, the test sample is subjected to gas chromatography-mass spectrometry under the same conditions to obtain the retention time and the abundance ratio of each ion. Then with reference to the method described above, the retention time of the sample to be tested and each ion abundance ratio are compared with the standard sample to determine whether the sample to be tested contains ethyl maltol or whether the object to be quantitatively analyzed is Ethyl maltol.

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

<The third aspect>

In the third aspect of the present invention, another reliable detection method for ethyl maltol additive that may be present in edible oil, especially vegetable oil, is provided.

Specifically, such detection methods include:

The preparation step of the sample is to prepare the sample to be tested by the edible oil to be tested, and prepare a standard sample containing ethyl maltol whose concentration is the safety standard threshold concentration;

The step of pretreatment is to enrich the polar substances in the sample to be tested to obtain the sample to be tested;

In the step of detection, the detection sample and the standard sample are respectively tested by the combination of gas chromatography and mass spectrometry, in which a weakly polar chromatographic column is used in the gas chromatography, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode ; Preferably, the weakly polar chromatographic column is selected from phenyl-modified dimethyl polysiloxane, and/or the mass spectrometer is a single quadrupole mass spectrometer;

The step of judging, if the result of the step of described detection shows that the detected concentration of ethyl maltol in the described sample to be tested is greater than the safety standard threshold concentration, then the weak polar chromatographic column in the gas chromatography is replaced with a strong polar chromatographic column. Column or connect the weakly polar chromatographic column and the strong polar chromatographic column in series, and use the mass spectrometer for testing, so as to perform quantitative or qualitative detection and judgment (re-test) again on the sample to be tested.

In the step of judging, in some specific embodiments of the present invention, (under certain conditions, such as the working conditions of gas chromatography and mass spectrometry mentioned below) The standard sample containing ethyl maltol is tested by GC-MS to obtain the area of the peak corresponding to the 140 ion of the standard sample; and then the sample to be tested is detected under the same detection conditions to obtain the corresponding peak of the 140 ion of the sample to be tested. area of the peak. If the area of the 140 ion peak of the sample to be tested is not higher than the above-mentioned standard sample, it means that the content of ethyl maltol that may be present in the sample to be tested is not higher than the threshold concentration of the safety standard, that is, there is no need to use a column containing a strong polarity. GC/MS re-examination, otherwise the sample needs to be re-examined qualitatively or quantitatively.

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

In the detection of possible ethyl maltol content in edible oil by gas chromatography coupled with mass spectrometry, when a weakly polar column is used in gas chromatography, it has a faster rate than when a strong polar column is used. detection efficiency. However, despite the advantages in detection efficiency, there may be concerns that the separation of some interfering substances from ethyl maltol may be insufficient when using weakly polar columns to separate the detection samples. Therefore, theoretically, when a weakly polar chromatographic column is used for combined detection by gas chromatography and mass spectrometry, the quantitative detection value is usually higher than the actual content of ethyl maltol in the sample to be tested.

In view of the above considerations, the present invention provides a method that is particularly suitable for efficient (large-scale) screening of problematic products. In this method, the sample to be tested is quantitatively detected by the combination of gas chromatography and mass spectrometry based on a weakly polar chromatographic column:

i) If the detected value of the sample to be tested does not exceed the threshold concentration of the safety standard, it means that the sample to be tested meets the requirements of the safety standard;

ii) If the detected value of the sample to be tested exceeds the threshold concentration of the safety standard, it means that there may be a problem with the sample to be tested (because the GC-MS results based on the weakly polar chromatographic column may not be able to distinguish some interfering substances), in such a In this case, the weak polarity chromatographic column needs to be replaced with a strong polarity chromatographic column or the weak polarity chromatographic column and the strong polarity chromatographic column are connected in series (the weak polarity chromatographic column and the strong polarity chromatographic column are connected in series including: , before and/or after the strong polar chromatographic column is connected in series with a weak polar chromatographic column), then the sample to be tested is detected by GC-MS, such detection can be quantitative detection or qualitative detection (depending on specific needs) and determination), the specific detection method, 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 this aspect faces a large number of samples to be tested, and there are few problems in these samples, efficient screening can be performed.

GC-MS detection based on weakly polar chromatographic column

The sample preparation steps and pretreatment steps of the detection method of this aspect can be performed with reference to the <first aspect> of the present invention.

The weakly polar chromatographic column in this aspect can be selected according to the weakly polar chromatographic 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 for the heating method of gas chromatography when using a weakly polar column, a temperature control method of stepwise heating can be adopted. In some specific embodiments, the temperature rise program of the gas chromatography includes: heating at a rate of 3-7°C/min to no more than 160°C, and maintaining the temperature for no more than 15min; then heating to 190-210°C at a rate of 8-12°C/min, And keep for 0.5-2min; then heat up to 230-255℃ at 25-35℃/min, and keep for 1-3min.

More specifically, for example, the initial column temperature of the chromatographic column is set at 58 to 62° C. and held for 1 to 3 minutes; further, the temperature is increased to 3 to 7° C./min (for example, 4° C./min, 5° C./min, etc.) Do not exceed 160°C (such as 155°C, 150°C, etc.), and keep the temperature for no more than 15min (such as 7-10min, 8-12min, etc.); min, etc.) to 190-210°C (for example, 200°C, etc.), and keep for 0.5-2min (for example, 1min, 1.5min, etc.); 230-255°C (for example, 240°C, 250°C, etc.), and keep for 1-3min (for example, 1.5min, 2min, 2.5min, etc.).

Through the above temperature control method, the strong polar substances can better penetrate into the solid phase structure of the chromatographic column, and then these substances can be separated from the weakly polar ethyl maltol.

For other operating conditions of GC-MS, such as operating conditions of mass spectrometry, etc., reference can be made to the content disclosed in the foregoing <First Aspect>.

In addition, the "safety standard" in the present invention, as mentioned above, may be a safety standard specified by a local, regional, national, regional or international organization, such as the content of ethyl maltol in the target product tolerated in such a safety standard It is not more than 5 μg/kg to 25 μg/kg, such as not more than 10 μg/kg, not more than 15 μg/kg, or not more than 20 μg/kg, and the like. In some preferred embodiments of the present invention, according to "Determination of Ethyl Maltol in Edible Vegetable Oil (BJS 201708)", the detection limit of ethyl maltol in edible oil is 25 μg/kg (that is, the safety standard threshold concentration) .

Example

Hereinafter, the present invention will be further illustrated by examples.

Test equipment :

Gas chromatography-mass spectrometer, Thermo Fisher Scientific, Trace 1310-ISQ.

Refrigerated centrifuge, sigma, 2-16KL.

Quartz union for gas chromatography column connection, Thermo Fisher Scientific, C-QSC10.

Raw materials :

The concentrated rapeseed oil and refined grade 4 rapeseed oil samples were all from Yihai (Guanghan) Grain, Oil and Feed Co., Ltd.

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

1) Standard sample:

A standard sample with a concentration of 200 μg/Kg of ethyl maltol was prepared using acetonitrile solvent.

2) GC/MS test system:

Gas chromatography conditions:

Injection method: gas chromatograph inlet temperature: 250°C; splitless injection; injection volume: 1 μL. Using TG-1701MS (specification: 30m*0.25mm*0.25um) strong polar chromatographic column to separate and determine ethyl maltol;

The heating program was as follows: the initial column temperature was 60 °C, maintained for 4 min, increased to 100 °C at 3 °C/min, held for 10 min, and then increased to 250 °C at 30 °C/min, maintained for 5 min.

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

MS conditions:

Selected ion scanning (SIM) monitoring; ionization mode: electron bombardment ionization source (EI); ionization energy: 70 eV; transmission line temperature: 300 °C; ion source temperature: 300 °C; solvent delay: 10 min. Qualitative by retention time and ion abundance ratio; quantitative ions are 140 ions, and qualitative ions are: 139, 125, and 97 ions.

Use the above GC/MS system to test the standard sample to determine the retention time and the reference abundance ratio of the base mass ion, see Figure 1a and Figure 1b, where the gas chromatography retention time is 28.91min.

Embodiment 1 (quantitative detection method by GC-MS based on strong polar chromatographic column) :

In this embodiment, after the target substance is separated by a strong polar chromatographic column, single quadrupole mass spectrometry is used for quantitative analysis; the following steps are included:

a. Configuration of spiked samples

The standard sample is diluted with the oil sample to be tested (flavor rapeseed oil), and the spiked sample for the working curve of the standard addition method is prepared; the oil sample to be tested is rapeseed oil, and the spiked sample is diluted with the oil sample to be tested as the matrix The concentration of 15μg/Kg, 25μg/Kg, 50μg/Kg, 100μg/Kg, 200μg/Kg spiked samples;

b. Pretreatment

After mixing each of the above-mentioned spiked samples and the oil sample to be tested, accurately weigh 2g ± 0.01g of each into a 15ml glass centrifuge tube, add 2ml of chromatographic grade acetonitrile, vortex for 5min, and place the centrifuge tube sample in a centrifuge for 4000r/min rotation speed and 4℃ conditions, centrifuge for 5min, collect 100μL of the supernatant liquid with a pipette, cover the bottle cap in the inner tube, and then analyze it on a gas chromatography-mass spectrometer;

c. Gas chromatography conditions:

Injection method: gas chromatograph inlet temperature: 250°C; splitless injection; injection volume: 1 μL. Using TG-1701MS (specification: 30m*0.25mm*0.25um) strong polar chromatographic column to separate and determine ethyl maltol;

The heating program was as follows: the initial column temperature was 60 °C, maintained for 4 min, increased to 100 °C at 3 °C/min, held for 10 min, and then increased to 250 °C at 30 °C/min, maintained for 5 min.

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

d. Mass spectrometry conditions:

Selected ion scanning (SIM) monitoring; ionization mode: electron bombardment ionization source (EI); ionization energy: 70eV; transmission line temperature 300℃; ion source temperature 300℃; solvent delay: 10min. Qualitative by retention time and ion abundance ratio; Quantitative ions are 140 ions, and qualitative ions are: 139, 125, and 97 ions.

e. Test process:

Before quantitative analysis, the test sample obtained by the pretreatment of the sample to be tested is tested, and the chromatographic peak time and the mass spectrum ion abundance ratio are used as the qualitative basis for judgment. It is judged that the sample to be tested contains ethyl maltol.

The pre-treated standard working curve sample solution (spiked sample solution) and the test sample obtained after the pre-treatment of the sample solution to be tested were injected into the gas chromatography-mass spectrometer for testing.

Taking the peak area as the ordinate and the standard addition content (μg/Kg) as the abscissa, after drawing the quantitative curve by the standard addition method, the sample to be tested was quantitatively determined by the external standard method. The obtained linear equations are Y=8.145×10 ³ X+1.992×10 ⁴ respectively, and the correlation coefficient is 0.9984 (see FIG. 2 ).

At the same time, the samples to be tested were used to prepare standard addition samples of different concentrations, and the verification was carried out according to the above process to obtain the recovery rate and parallelism data; to examine the accuracy and precision of the method. A recovery of 95-110% was obtained with an RSD of 8%.

Figure 3 shows the gas chromatogram of the test sample after the pretreatment of the test sample in Example 1 (the chromatogram of the 100 μg/Kg spiked sample is superimposed at the same time), the peak shape of ethyl maltol has no interference and is quantitative The test results showed that the ethyl maltol content of the oil sample to be tested was 7 μg/Kg (10 times the signal-to-noise ratio).

Reference example 1:

Except that the temperature rise program of the gas chromatographic column of Example 1 was replaced with the following temperature rise program, other conditions were the same as those of Example 1:

The initial column temperature was 60 °C, held for 0 min, heated to 120 °C at 5 °C/min, held for 0 min, and then heated to 250 °C at 35 °C/min, held for 5 min.

The preliminary quantitative result is about 100μg/Kg. After the analysis of the mass spectrum, it is found that the abundance ratio of the mass spectrum at the left and right 1/2 peak width of the peak is inconsistent with the standard sample, and the M/Z125 ion has completely disappeared (the original 25% abundance) , indicating that there are co-elution peaks, the peaks are not completely separated, and there is interference from other compounds.

Example 2

Use the detection method of Example 1 to detect the results of refining the fourth-grade rapeseed oil sample in the oil pressing workshop of the factory. Figure 4 is the superimposed chromatogram of the actual sample chromatogram and the standard working curve point (25 μg/Kg standard addition point), and the TG-1701MS chromatographic column ; Explain that the refined quaternary rapeseed oil is completely without peaks, without any interference, and the result is 0 (there is no ethyl maltol).

In addition, Figure 5 superimposes the detection patterns of various samples in Figure 3 and Figure 4, which also reflects the validity and reliability of the testing method of the present invention.

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

Various embodiments of the present disclosure have been described above, and the foregoing descriptions are exemplary, not exhaustive, and not limiting of the disclosed embodiments. Numerous 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 was chosen to best explain the principles of the embodiments, the practical application or improvement over the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Industrial Availability

The GC/MS detection method of the present invention can be used for reliable detection of ethyl maltol and its content in edible oil in industry.

Claims (10)

1. a method for detecting ethyl maltol in edible oil, is characterized in that, comprises following quantitative step: In the sample preparation step, the sample to be tested is prepared by the edible oil to be tested, and n spiked samples are prepared based on the standard addition method, where n is an integer greater than or equal to 3, preferably, the n is an integer from 3 to 10; In the step of pretreatment, the polar substances in the sample to be tested and the spiked sample are enriched to obtain the test sample; preferably, the enrichment is to extract the sample to be tested and the spiked sample with a polar solvent, The polar solvent is selected from acetonitrile or methanol; In the step of detection, each of the detection samples is tested by the combination of gas chromatography and mass spectrometry, and a working curve is prepared based on the standard addition method to determine the content of ethyl maltol in the sample to be tested, Wherein, the gas chromatography includes a strong polar chromatographic column, and the mass spectrometry adopts a feature-selected ion monitoring scan (SIM) working mode; preferably, the strong polar chromatographic column is selected from polyethylene glycol-based chromatographic columns, Polar group modified polyethylene glycol column or polar group modified polysiloxane column. 2. The method according to claim 1, wherein the method also has one or more of the following features: In the step of detecting, before and/or after the strong polar chromatographic column of the gas chromatography, a weak polar chromatographic column is also used in series with it; In the step of detecting, the heating program of the gas chromatograph includes: raising the temperature to no more than 100°C at a speed below 10°C/min, and maintaining the temperature for no more than 15min; then heating up to 230-260°C at 30-40°C/min, And keep it for 2-10min; The linear correlation coefficient R ² of the working curve of the method according to the standard addition method is greater than 0.998; and/or the detection limit of the method for ethyl maltol is 5 μg/Kg. 3. The method according to claim 2, wherein the weakly polar chromatographic column comprises phenyl-modified dimethylpolysiloxane; and/or the mass spectrometer is a single quadrupole mass spectrometer. 4. a method for detecting ethyl maltol in edible oil, is characterized in that, comprises following qualitative step: The sample preparation step is to prepare the sample to be tested by the edible oil to be tested, and prepare a standard sample with a known concentration of ethyl maltol; In the step of pretreatment, the polar substances in the sample to be tested are enriched to obtain the sample to be tested; preferably, the enrichment is to use a polar solvent to extract the sample to be tested, and the polar solvent is selected from acetonitrile or methanol; In the detection step, the standard sample and the detection sample are respectively tested by the combination of gas chromatography and mass spectrometry, and record: i) The retention time t1 of the ethyl maltol gas chromatographic peak in the standard sample, and the abundance ratio of the characteristic ion peaks in the mass spectrum; ii) detecting the retention time t2 of the suspected ethyl maltol gas chromatographic peak in the sample, and the abundance ratio of the characteristic ion peak in the mass spectrometer, Wherein, the gas chromatography includes a strong polar chromatographic column, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode; preferably, the strong polar chromatographic column is selected from polyethylene glycol-based chromatographic columns, A polar group-modified polyethylene glycol chromatography column or a polar group-modified polysiloxane chromatography column, and/or the mass spectrometer is a single quadrupole mass spectrometer; In the judgment step, whether the sample to be tested contains ethyl maltol is determined by comparing t1 with t2, and if necessary, by comparing the abundance ratio of characteristic ion peaks in the mass spectrometer. 5. The method of claim 4, wherein the method further has one or more of the following features: In the step of detecting, the heating program of the gas chromatograph includes: raising the temperature to no more than 100°C at a speed below 10°C/min, and maintaining the temperature for no more than 15min; then heating up to 230-260°C at 30-40°C/min, And keep it for 2-10min; In the judging step, if the deviation between t1 and t2 is less than or equal to ±0.05min, continue to determine whether the sample to be tested contains ethyl maltol by comparing the abundance ratio of characteristic ion peaks of the mass spectrum. 6. The method according to claim 4 or 5, wherein the comparison of the abundance ratios of characteristic ion peaks in the mass spectrometry comprises comparing the relative intensities of the 139, 125 and 97 ion peaks of the detection sample to the 140 ion peak The ratio of the intensities is compared to the ratio of the relative intensities of the 139, 125 and 97 ion peaks to the relative intensity of the 140 ion peak in the standard sample, respectively. 7. a method for detecting ethyl maltol in edible oil, is characterized in that, comprises the steps: The sample preparation step is to prepare the sample to be tested by using the edible oil to be tested, and prepare a standard sample containing ethyl maltol with a concentration of a safety standard threshold concentration; In the step of pretreatment, the polar substances in the sample to be tested are enriched to obtain the sample to be tested; preferably, the enrichment is to extract the sample to be tested with a polar solvent, and the polar solvent is selected from acetonitrile or methanol; In the step of detection, the detection sample and the standard sample are respectively tested by the combination of gas chromatography and mass spectrometry, in which a weakly polar chromatographic column is used in the gas chromatography, and the mass spectrometer adopts a feature-selected ion monitoring scan (SIM) working mode ; Preferably, the weakly polar chromatographic column is selected from phenyl-modified dimethyl polysiloxane, and/or the mass spectrometer is a single quadrupole mass spectrometer; The step of judging, if the result of the step of described detection shows that the detected concentration of ethyl maltol in the described sample to be tested is greater than the safety standard threshold concentration, then the weak polar chromatographic column in the gas chromatography is replaced with a strong polar chromatographic column. The column or the weak polarity chromatographic column is connected in series with the strong polarity chromatographic column, and the test is combined with the mass spectrometer to perform quantitative or qualitative detection of the sample to be tested again. 8. The method according to claim 7, wherein, in the step of judging, if the peak area corresponding to the mass spectrometry quantitative ion 140 ion in the detection sample is greater than the peak area corresponding to the standard sample quantitative ion 140 ion, Then it is considered that the detected concentration of ethyl maltol in the sample to be tested is greater than the safety standard threshold concentration. 9. method according to claim 7 or 8 is characterized in that, in the step of described detection, the temperature rise program of gas chromatography comprises, with 3～7 ℃/min speed warming up to no more than 160 ℃, and keep the temperature no more than 15min; then heat up to 190-210°C at 8-12°C/min and hold for 0.5-2min; then heat up to 230-255°C at 25-35°C/min and hold for 1-3min. 10. The method according to any one of claims 7 to 9, wherein the method has one or more of the following features: The safety standard threshold concentration is 25 μg/Kg; The series connection mode of the weak-polarity chromatographic column and the strong-polarity chromatographic column includes connecting the weak-polarity chromatographic column in series before and/or after the strong-polarity chromatographic column; The re-performing quantitative or qualitative detection includes using the detection method described in any one of claims 1-3.

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