CN112763605A

CN112763605A - Detection method and application of tocopherol content

Info

Publication number: CN112763605A
Application number: CN202011553676.5A
Authority: CN
Inventors: 杨国文
Original assignee: Sichuan Xinmeijia Biomedicine Co ltd
Current assignee: Sichuan Xinmeijia Biomedicine Co ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-05-07

Abstract

The invention discloses a detection method and application of tocopherol content, and relates to the field of analytical chemistry. The detection method of tocopherol content adopts gas chromatography for detection, and takes n-heptane as a solvent of a working standard solution and a sample solution; the chromatographic conditions include: chromatographic column HP-5; the split ratio is 1: 18-22; temperature programming: the initial temperature is 185-195 ℃, the temperature is raised at 3.5-4.5 ℃/min, the final temperature is 290-310 ℃, and the temperature is kept for 1-2 min. Sample inlet temperature: 315-325 ℃; the temperature of the detector is as follows: 315-325 ℃. Compared with the existing GC-MS or GC-headspace sample injection detection, the method has the advantages of lower cost, simplicity, feasibility, high detection efficiency and low detection limit, and compared with the common gas chromatography detection, the method provided by the invention can be used for detecting the tocopherol in the fish oil even if the content of the tocopherol in the sample is extremely low, and is suitable for detecting the tocopherol in the fish oil.

Description

Detection method and application of tocopherol content

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a method for detecting tocopherol content and application thereof.

Background

In order to ensure the stability of fish oil products, tocopherol is added to fish oil products on the market to ensure that the products are not oxidized, and in order to prevent the tocopherol in the products from being excessively/unintentionally added, a method for measuring the content of the tocopherol in the products is needed, and the tocopherol is fat-soluble product, is dissolved in the fish oil after being added, and the content of the tocopherol is difficult to detect.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for detecting the content of tocopherol and application thereof.

The invention is realized by the following steps:

the first method, provided by the embodiment of the invention, is a method for detecting tocopherol content, wherein gas chromatography is adopted for detection, and n-heptane is used as a solvent of a working standard solution and a sample solution;

the chromatographic conditions include:

a chromatographic column: HP-5;

sample injector: split-flow sample injection is carried out, wherein the split-flow ratio is 1: 18-22;

temperature programming:

sample inlet temperature: 315-325 ℃;

detector temperature: 315-325 ℃.

In an optional embodiment, the carrier gas is nitrogen gas when the gas chromatography is used for detection, and the flow rate of the carrier gas is 0.9-1.1 ml/min.

In an alternative embodiment, the chromatographic instrument is Shimadzu GC-2014C;

in an alternative embodiment, the detector is a FID.

In an optional embodiment, before starting the temperature programming, the method further comprises the step of preserving the heat at 185-195 ℃ for 1-2 min.

In an alternative embodiment, the needle wash solvent is n-heptane when the gas chromatography assay is performed.

In an alternative embodiment, the gas chromatography is performed with a sample volume of 1 μ l.

In an alternative embodiment, the sample introduction requirements include:

1 needle of blank solution is fed, the base line is required to be stable, and no impurity peak exists;

the RSD% of the working standard solution for feeding 3 needles is required to be not more than 10%;

the RSD% of the detection result is required to be not more than 10% by feeding the test solution of the 3-pin sample;

the tocopherol in the sample solution and the tocopherol in the standard solution are kept for the same time.

In an optional embodiment, the method further comprises calculating the detection result of the gas chromatography, wherein the calculation formula is as follows:

the result is (M)_{Sign board}*A_{Sample (A)}/A_{Sign board})/M_{Sample (A)}

Wherein:

M_{sign board}Represents the concentration of tocopherol in the working standard solution;

A_{sign board}Represents the peak area of tocopherol in the working standard solution;

A_{sample (A)}Represents the peak area of tocopherol in the sample solution;

M_{sample (A)}Indicating the concentration of the sample in the sample solution.

In an alternative embodiment, the sample is fish oil.

The detection method provided by the invention is applied to detection of the content of tocopherol in fish oil products.

The invention has the following beneficial effects:

because the gas chromatography is adopted for direct sample injection, the detection cost is lower compared with the existing GC-MS or GC-headspace sample injection detection; compared with the common gas chromatography detection, the method provided by the invention can be used for detecting the tocopherol content in the sample even if the tocopherol content is extremely low, and is suitable for detecting the tocopherol in the fish oil.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a line graph;

FIG. 2 is a graph of sample peak area versus time;

FIG. 3 is a graph of standard peak area versus time.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The method for detecting tocopherol content and the application thereof provided by the embodiment of the invention are specifically described below.

The detection method for the tocopherol content provided by the embodiment of the invention adopts gas chromatography for detection, and takes n-heptane as a solvent of a working standard solution and a sample solution.

Working standard solution: and (3) taking a certain amount (generally 10-20 mg) of the alpha-tocopherol standard substance to a 10ml volumetric flask, dissolving the alpha-tocopherol standard substance to a scale with n-heptane, taking 1ml to a 10ml volumetric flask of the solution, and dissolving the alpha-tocopherol standard substance to the scale with n-heptane to obtain the alpha-tocopherol solution.

Sample solution: a certain amount of sample (generally 200-300 mg) is taken into a 10ml volumetric flask, and is dissolved by n-heptane to reach a constant volume to a scale mark. Preferably, the sample is fish oil.

The chromatographic conditions include:

a chromatographic column: HP-5;

carrier gas: nitrogen gas;

flow rate of carrier gas: 0.9-1.1 ml/min;

a detector: FID;

sample injector: split-flow sample injection with a split-flow ratio of 1:18 to 22;

needle washing solvent: n-heptane;

temperature programming:

sample inlet temperature: 315-325 ℃;

detector temperature: 315-325 ℃;

sample introduction volume: 1 μ l.

Preferably, the most common conditions typically used in detection are:

flow rate of carrier gas: 1.0 ml/min; initial column temperature: 190 ℃; keeping the initial column temperature for 1 min; the heating rate is 4 ℃/min; final temperature after temperature rise: 300 ℃; detector temperature: 320 ℃; sample inlet temperature: 320 ℃; the split ratio is as follows: 1:20.

It should be noted that, in the subsequent method verification test, the chromatographic conditions are not mentioned as the above-mentioned common conditions.

In order to ensure that the tocopherol content can be measured more accurately, the sample introduction requirement is as follows:

the RSD% of the working standard solution for three needles is required to be not more than 10%;

the RSD% of the detection result is required to be not more than 10% by entering the test solution of the three-needle sample;

After the detection is finished, the method also comprises the step of calculating the detection result of the gas chromatography, wherein the calculation formula is as follows:

the result is (M)_{Sign board}*A_{Sample (A)}/A_{Sign board})/M_{Sample (A)}

Wherein:

M_{sign board}Represents the concentration of tocopherol in the working standard solution (mg/ml)

A_{Sign board}Shows the area of the peak of tocopherol in the working standard solution

A_{Sample (A)}Represents the peak area of tocopherol in the sample solution

M_{Sample (A)}Represents the concentration (mg/ml) of the sample (i.e., fish oil) in the sample solution.

Examples of the experiments

The feasibility of the method provided by the present invention was verified in the following manner.

First, acceptance criteria

Reagent and instrument

1. Reagent

Name (R)	Rank of	Manufacturer of the product	Batch number
				N-heptane	HPLC	TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd.	20130308
Tocopherol	Standard article	EDQM	T1550000

Sample 4638EE batch number: 130219-4638EE-01 production date: 19/2/2013

2. Instrument for measuring the position of a moving object

GC-2014C gas chromatograph, one-ten-thousandth electronic balance (1/10,000 balance), one-hundred-thousandth electronic balance (1/100,000 balance).

Third, verify the project

1. Specificity

1.1 solution preparation

Blank solvent: n-heptane.

Standard solution: taking 14.99mg of tocopherol standard substance to a 10ml volumetric flask, using n-heptane solvent to reach the scale, taking 1ml to a 10ml volumetric flask, and using n-heptane solvent to reach the scale.

Sample solution: a sample of 250.01mg was taken to a 10ml volumetric flask and dissolved in n-heptane to a constant volume.

No tocopherol sample solution added: a sample of 250.03mg was taken to a 10ml volumetric flask and dissolved in n-heptane to a constant volume.

1.2 sample introduction and results

A needle blank solvent, a standard solution, a sample solution and a tocopherol-free sample solution were added, respectively, and the results are shown in Table 1 below.

TABLE 1

Name (R)	Retention time	Peak separation situation
			Blank solvent	/	/
Tocopherol standard substance	27.318min	/
			Sample solution	/	Retention time 27.318min noneAppearance of peak
Adding tocopherol	27.299min	The separation degree from the front peak is far more than 1.5

And (4) conclusion: the tocopherol peak is not coincident with the peaks of other substances in the sample, the separation degree of the tocopherol peak and other nearby peaks is far more than 1.5, and a blank solvent and a sample solution have no interference on detection of the tocopherol, so that the method has specificity on detection of the tocopherol.

2. Precision of the system

2.1 preparation of the solution

Standard solution: taking 14.99mg of the alpha-tocopherol standard substance to a 10ml volumetric flask, using an n-heptane solvent to reach the scale, taking 1ml to a 10ml volumetric flask, and dissolving the solution with n-heptane to reach the scale.

2.2 sample introduction and results

6 needles of the standard solution were continuously fed, and the obtained peak area RSD% and the results are shown in Table 2 below.

TABLE 2

Needle insertion	Peak area
		1	89347
2	90491
		3	89556
4	91468
		5	93024
6	86964
		Mean value of	90142
RSD％	2.29％

And (4) conclusion: the system precision meets the requirements.

3. Limit of detection

3.1 solution preparation

Standard solution: accurately weighing 14.99mg of alpha-tocopherol to 10ml of volumetric flask, dissolving with n-heptane to fix the volume to a scale, taking 1ml to 5ml of the solution in the volumetric flask, and dissolving with n-heptane to fix the volume to the scale to obtain the concentration of 0.2998 mg/ml.

3.2 sample introduction and results

The detection limit of the method is determined by diluting the standard solution until the ratio of the peak height of the tocopherol to the peak height of the baseline is 3: 1. The results obtained are shown in Table 3 below.

TABLE 3

And (4) conclusion: the detection limit was 0.00383744 mg/ml.

4. Limit of quantification

4.1 solution preparation

Standard solution: accurately weighing 14.99mg to 10ml of alpha-tocopherol in a volumetric flask, dissolving with n-heptane to fix the volume to a scale, taking 1ml to 5ml of the solution in the volumetric flask, dissolving with n-heptane to fix the volume to the scale to obtain the solution with the concentration of 0.2998mg/ml, and diluting the standard solution until the ratio of the peak height of the tocopherol to the peak height of a baseline is 10:1, namely the quantitative limit of the method.

4.2 sample introduction and results

The results obtained by continuously feeding 6 needles of the standard solution are shown in the following table.

TABLE 4

And (4) conclusion: the limit of quantitation RSD is satisfactory, and the limit of quantitation is 0.0095936 mg/ml.

5. Precision (repeatability and intermediate precision)

5.1 repeatability

5.1.1 solution preparation

Sample test solution: accurately weighing 6 parts of fish oil (fish oil) sample 250mg to 10ml in a volumetric flask, dissolving with n-heptane and fixing the volume to a scale to obtain the fish oil (fish oil) sample. The weighing data for each fish oil are shown in table 6 below.

5.1.2 test results

The test solutions of the above samples were tested separately, and the results are shown in Table 5 below.

TABLE 5 repeatability

And (4) conclusion: according to the results obtained by analyst 1, the RSD is less than 10%, and the method is good in reproducibility.

5.2 intermediate precision

5.2.1 preparation of solution

Sample test solution: accurately weighing 6 parts of fish oil sample by different experimenters on different days, namely weighing the fish oil sample in a volumetric flask of 250mg to 10ml, dissolving the fish oil sample by using n-heptane, and fixing the volume to a scale to obtain the fish oil. The weighing data for each fish oil are shown in table 7 below.

5.2.2 test results

The test solutions of the above samples were tested separately, and the results are shown in Table 6 below.

TABLE 6 intermediate precision

5.3 repeatability compared to results obtained with intermediate precision, see Table 7 below:

TABLE 7 comparative results

And (4) conclusion: according to the above results, the method was excellent in intermediate precision, and RSD was less than 10%.

6. Linearity

6.1 preparation of solution

Standard a solution: accurately weighing 14.99mg of alpha-tocopherol to 10ml of volumetric flask, dissolving with n-heptane to fix the volume to a scale, taking 1ml to 5ml of the solution in the volumetric flask, and dissolving with n-heptane to fix the volume to the scale to obtain the concentration of 0.2998 mg/ml.

Standard B solution: accurately weighing 14.99mg of alpha-tocopherol to 10ml of volumetric flask, dissolving the alpha-tocopherol with n-heptane to a constant volume to a scale, taking 1ml of the solution to 10ml of the volumetric flask, and dissolving the alpha-tocopherol with n-heptane to a constant volume to obtain the alpha-tocopherol with the concentration of 0.1499 mg/ml.

Standard C solution: precisely transferring 1ml of the standard substance A solution into a 5ml volumetric flask, dissolving with n-heptane and fixing the volume to a scale, thereby obtaining the concentration of 0.05996 mg/ml.

Standard D solution: precisely transferring 2ml of the standard substance C solution into a 5ml volumetric flask, dissolving with n-heptane and fixing the volume to a scale, thereby obtaining the concentration of 0.023984 mg/ml.

Standard E solution: precisely transferring 2ml of the standard substance D solution into a 5ml volumetric flask, dissolving with n-heptane and fixing the volume to a scale, thereby obtaining the concentration of 0.0095936 mg/ml.

Standard F solution: precisely transferring 2ml of the standard substance E solution into a 5ml volumetric flask, dissolving with n-heptane and fixing the volume to a scale, thereby obtaining the concentration of 0.00383744 mg/ml.

6.2 sample introduction and results

The different standard substance solutions are injected into a needle, the mass concentration of the tocopherol is used as an abscissa (X), the peak area of the tocopherol is used as an ordinate (Y), binary linear regression analysis is carried out, the linearity and the coefficient are calculated, and the obtained result is shown in the following table 8.

TABLE 8

The linear plot is shown in fig. 1.

From table 9 and fig. 1, it can be concluded: the method has good linearity²Greater than 0.998.

7. Accuracy recovery rate

7.1 preparation of solution

Sample stock solution: 4803.76mg of fish oil were weighed precisely into a 100ml volumetric flask, dissolved with n-heptane and brought to volume.

120% of sample spiking solution: accurately transferring 5.0ml of sample stock solution into a 10ml volumetric flask, adding 1.2ml of standard solution (internal standard lot number: BY130820-01, concentration: 1.598mg/ml), and dissolving with n-heptane to constant volume to scale. 3 parts of the compound is prepared by the same method.

100% of sample spiking solution: accurately transferring 5.0ml of sample stock solution into a 10ml volumetric flask, adding 1.0ml of standard solution (internal standard lot number: BY130820-01, concentration: 1.598mg/ml), and dissolving with n-heptane to constant volume to scale. 3 parts of the compound is prepared by the same method.

80% of the sample spiking solution: accurately transferring 5.0ml of sample stock solution into a 10ml volumetric flask, adding 0.8ml of standard solution (internal standard lot number: BY130820-01, concentration: 1.598mg/ml), and dissolving with n-heptane to a constant volume to obtain the finished product. 3 parts of the compound is prepared by the same method.

Sample test solution: accurately transferring 5.0ml of the sample stock solution into a 10ml volumetric flask, dissolving with n-heptane and fixing the volume to the scale to obtain the product.

Standard working solution: taking 1.0ml to 10ml volumetric flask of standard stock solution (internal standard lot number: BY130820-01, concentration: 1.598mg/ml), dissolving with n-heptane and fixing to volume to scale to obtain the final product.

7.2 sample introduction

1-needle feeding of blank solution

And 3 needles of standard working solution are added.

The solution was tested into 3 pin samples.

Respectively adding 120%, 100% and 80% of sample labeling test solution into 1 needle

7.3 calculation formula:

recovery ═ (determination of total tocopherol amount-amount of tocopherol in the sample)/amount known to add standard 100%.

7.4 results

The results obtained are shown in Table 9 below.

TABLE 9

And (4) conclusion: according to the results, the method has good accuracy, the recovery rate is between 90% and 110%, and the recovery rate RSD is less than 10%.

8. Range of

The analysis results show that according to the linear relation: the mass concentration of the analyte in the sample is in the range of 0.01534976-1.1992% (wherein, the tocopherol is 0.00383744 mg/ml-0.2998 mg/ml), and the analysis method can obtain good linear relation.

9. Durability

9.1 solution preparation

Standard solution: accurately weighing 14.99mg of alpha-tocopherol into a 10ml volumetric flask, dissolving with n-heptane to fix the volume to a scale, taking 1ml of the solution into the 10ml volumetric flask, and dissolving with n-heptane to fix the volume to the scale, thereby obtaining the concentration of 0.1499 mg/ml.

Sample solution: accurately weighing 3 parts of fish oil sample, dissolving with n-heptane, and metering to 10ml volumetric flask to obtain the fish oil. (the weight of fish oil weighed under each condition is shown in Table 11).

9.2 Condition setting

The durability of the method was determined by examining the effect of changes in carrier gas flow rate and initial temperature, detector temperature, injection port temperature, split ratio on the amount of sample content measured, under the following set conditions.

Investigation of carrier gas flow velocity	0.9	1.0	1.1
				Investigation of initial column temperature	185℃	190℃	195℃
Investigation of detector temperature	315℃	320℃	325℃
				Investigation of sample inlet temperature	315℃	320℃	325℃
Split ratio	1:18	1:20	1:22

The results of the above conditions are shown in the tables below. It should be noted that, when an investigation experiment is performed to examine one of the factors, the factor is used as a variable, and other chromatographic conditions are common conditions. For example: when the flow rate of the carrier gas is inspected, the initial column temperature, the detector temperature, the injection port temperature and the split ratio are all common conditions.

9.3 sample introduction and results

The results obtained with 3 pins of standard solution and 3 pins of sample test solution under the above conditions are shown in the tables below.

Watch 10

TABLE 11 inspection of carrier gas flow Rate

TABLE 12 Detector temperature investigation

Table 13 initial column temperature investigation

TABLE 14 sample inlet temperature investigation

TABLE 15 Split ratio examination

TABLE 16 comparison of durability under each condition with that under normal condition

And (4) conclusion: according to the above results, the durability of the method was good, and the RSD of the results was less than 10% under each condition.

10. Stability study of sample and Standard solutions

And (3) respectively inspecting the stability of the standard substance stock solution and the sample test solution, and judging the stability of the standard substance stock solution and the sample test solution through the change of the peak area.

TABLE 17 stability Studies of sample solutions

The peak area of the sample as a function of time is shown in FIG. 2.

From table 17 fig. 2 it can be concluded: the sample has stability within 26 hours, and the peak area RSD is less than 10%.

Table 18 stability study of stock solutions of standards

The plot of standard peak area versus time is shown in FIG. 3.

From table 19 and fig. 3, it can be concluded: the standard solution has stability within 26 hours, and the peak area RSD is less than 10%.

In conclusion, the detection method for the tocopherol content provided by the invention adopts the gas chromatography for detection, and can accurately detect the tocopherol content in the fish oil due to the selection of proper chromatographic conditions; compared with the existing detection methods such as GC-MS, HPLC and the like, the gas chromatography adopted in the invention has the characteristics of simplicity, easiness and low cost. The detection method provided by the invention is suitable for detecting the content of the tocopherol in the fish oil.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The detection method of tocopherol content is characterized in that gas chromatography is adopted for detection, and n-heptane is used as a solvent of a working standard solution and a sample solution;

the chromatographic conditions include:

a chromatographic column: HP-5;

temperature programming:

sample inlet temperature: 315-325 ℃;

detector temperature: 315-325 ℃.

2. The method for detecting the tocopherol content according to claim 1, wherein a carrier gas is nitrogen gas and the flow rate of the carrier gas is 0.9 to 1.1ml/min during detection by gas chromatography.

3. The method for detecting the tocopherol content according to claim 1, wherein the chromatographic instrument is Shimadzu GC-2014C;

preferably, the detector is a FID.

4. The method for detecting the tocopherol content according to claim 1, wherein before the temperature programming is started, the temperature is maintained at 185-195 ℃ for 1-2 min.

5. The method for detecting tocopherol content according to claim 1, wherein the needle-washing solvent used in the detection by gas chromatography is n-heptane.

6. The method for detecting tocopherol content according to claim 1, wherein the gas chromatography is used for detection in a sample volume of 1 μ l.

7. The method for detecting tocopherol content according to claim 1, wherein the sample injection requirement includes:

8. The method for detecting tocopherol content according to claim 1, further comprising calculating the detection result of gas chromatography by the following formula:

the result is (M)_{Sign board}*A_{Sample (A)}/A_{Sign board})/M_{Sample (A)}

Wherein:

A_{sample (A)}Represents the peak area of tocopherol in the sample solution;

9. The method for detecting tocopherol content according to claim 1, wherein the sample is fish oil.

10. Use of the method of any one of claims 1 to 9 for detecting tocopherol content in a fish oil product.