Separation method and determination method of plasticizer in ethyl ester type fish oil

Abstract

The invention discloses a separation method and a determination method of a plasticizer in ethyl ester type fish oil, and relates to the technical field of food. The separation method of the plasticizer in the ethyl ester type fish oil is a solid-phase extraction method and comprises the following steps: activating a solid-phase extraction column, loading a sample, leaching and eluting; a reagent used for leaching is first alkane or a compound of the first alkane and a first reagent in a volume ratio of (99.0-99.9): (0.1-1.0); a reagent used for elution is a compound of first alkane and a first reagent in a volume ratio of (50-95): (5-50); the first alkane is at least one of normal hexane, cyclohexane, normal heptane and isooctane; the first reagent is at least one of ethyl acetate, ethanol, acetone, tetrahydrofuran and isopropanol. By selecting leaching and elution reagents, the plasticizer can be separated from the fish oil, so that the food safety is improved, and the content of the plasticizer in the fish oil can be accurately measured.

Description

Technical Field

[0001] The present invention relates to the field of food technology, in particular to a method for separating and determining a plasticizer in ethyl ester fish oil. Background Art

[0002] Ethyl ester fish oil is an important functional health food based on fats and oils. Its main components are ethyl eicosapentaenoic acid (EPA) and ethyl docosahexaenoic acid (DHA), which have been shown to lower triglycerides and reduce the incidence of cardiovascular and cerebrovascular diseases. Plasticizers, also known as phthalates, are environmental hormones that can mimic hormones in the body and interfere with normal hormone metabolism, thereby inducing carcinogenic, teratogenic, and mutagenic effects, posing a serious threat to human health. Currently, many countries have mandated the regulation of phthalates in fat-based foods.

[0003] Testing the plasticizer content in ethyl ester fish oil requires separating it first. The currently commonly used method for separating phthalate plasticizers in food is generally a combination of liquid-liquid extraction and solid-phase extraction. However, this method is only applicable to glyceride-type oil samples. It cannot effectively separate ethyl ester fish oil, resulting in the inability to subsequently measure the plasticizer content in ethyl ester fish oil. Summary of the Invention

[0004] The main purpose of the present invention is to provide a method for separating and determining plasticizers in ethyl ester fish oil, so as to solve the problem that it is difficult to separate plasticizers in ethyl ester fish oil in the prior art.

[0005] In order to achieve the above object, according to a first aspect of the present invention, a method for separating plasticizers from ethyl ester fish oil is provided, the method comprising the following steps:

[0006] S1, activating the solid phase extraction column to obtain an activated solid phase extraction column;

[0007] S2, loading the sample onto the activated solid phase extraction column to obtain a loaded solid phase extraction column;

[0008] S3, eluting the solid phase extraction column after the sample is loaded to obtain a washed solid phase extraction column;

[0009] S4, eluting the washed solid phase extraction column to obtain a separation liquid containing the plasticizer;

[0010] Among them, in S3, the reagent used for elution is the first alkane or a compound of the first alkane and the first reagent in a volume ratio of (99.0-99.9): (0.1-1.0); in S4, the reagent used for elution is a compound of the first alkane and the first reagent in a volume ratio of (50-95): (5-50); the above-mentioned first alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane; the first reagent is at least one of ethyl acetate, ethanol, acetone, tetrahydrofuran, and isopropanol.

[0011] Furthermore, in the above S3, the elution includes a first elution and a second elution, the reagent used in the first elution is the first alkane, and the reagent used in the second elution is a mixture of the first alkane and the first reagent in a volume ratio of (99.0-99.9): (0.1-1.0).

[0012] Furthermore, the volume ratio of the reagent used in the first elution to the sample added is (0.5-1.5):1; the volume ratio of the reagent used in the second elution to the sample added is (10-20):1; the volume ratio of the reagent used in the elution to the sample added is (3-7):1.

[0013] Furthermore, in the above S1, the activation is: washing the solid phase extraction column with the first reagent and the first alkane in sequence.

[0014] Furthermore, the filler of the solid phase extraction column is a material with silica gel as a matrix.

[0015] According to a second aspect of the present invention, a method for determining plasticizers in ethyl ester fish oil is provided, comprising the following steps:

[0016] Prepare internal standard stock solution and reference substance stock solution;

[0017] Prepare standard solution with internal standard stock solution, reference substance stock solution and second alkane;

[0018] Prepare a blank solution with the internal standard stock solution and the second alkane;

[0019] The test sample is mixed with the internal standard stock solution to obtain a mixed solution, and the mixed solution is diluted with the second alkane to obtain a test sample solution;

[0020] The test solution, blank solution and standard solution are separated and concentrated by solid extraction method to obtain test solution concentrate, blank solution concentrate and standard solution concentrate;

[0021] The standard concentrate was tested by gas chromatography-mass spectrometry, and a standard curve was drawn with the concentration of the plasticizer in the standard concentrate as the abscissa and the ratio of the peak area of ​​the plasticizer to the peak area of ​​the internal standard as the ordinate;

[0022] The test sample concentrate and blank concentrate were tested by gas chromatography-mass spectrometry, and the concentration of plasticizer in the test sample concentrate and the concentration of plasticizer in the blank concentrate were calculated according to the standard curve; the content of plasticizer in the test sample was calculated according to formula (I):

[0023]

[0024] Where:

[0025] C: plasticizer concentration in the test sample concentrate, ng / ml;

[0026] V: dilution volume of the test sample, ml;

[0027] W: weight of test sample, mg;

[0028] M: plasticizer content of the test sample, ppm;

[0029] The plasticizer is phthalate, and the internal standard is deuterated phthalate;

[0030] The reference stock solution is a solution obtained by dissolving the plasticizer in the second alkane; the internal standard stock solution is a solution obtained by dissolving the internal standard in the second alkane;

[0031] The standard solutions are a group of solutions with the volume fractions of the reference substance stock solutions being 5%, 7.5%, 12%, 15%, 22.5%, and 30%, respectively, and the volume fraction of the internal standard stock solutions being the same;

[0032] The second alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane;

[0033] The solid phase extraction method is the separation method mentioned according to the first aspect of the present invention.

[0034] Furthermore, the concentrations of the internal standard stock solutions in the test solution, blank solution, and standard solution are the same.

[0035] Furthermore, when gas chromatography-mass spectrometry is used for testing, the ion source temperature is 300-330° C.; and / or the acquisition mode is multiple reaction monitoring mode; and the above-mentioned concentration is nitrogen blow concentration.

[0036] Further, the above-mentioned phthalate includes one or more of the following materials: di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, diisononyl phthalate, dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-methoxy)ethyl phthalate, di(4-methyl-2-pentyl)phthalate, di(2-ethoxy)ethyl phthalate, dipentyl phthalate, dihexyl phthalate, butyl benzyl phthalate, di(2-butoxy)ethyl phthalate, dicyclohexyl phthalate, di(α-ethylhexyl) phthalate, diphenyl phthalate, di-n-octyl phthalate, dinonyl phthalate; and / or

[0037] The above-mentioned deuterated isotope phthalate includes one or more of the following materials: dibutyl phthalate-d4, di(2-ethyl)hexyl phthalate-d4, diisononyl phthalate-d4, dimethyl phthalate-d4, diethyl phthalate-d4, diisobutyl phthalate-d4, di(2-methoxy)ethyl phthalate-d4, di(4-methyl-2-pentyl)phthalate-d4 d4, di(2-ethoxy)ethyl phthalate-d4, dipentyl phthalate-d4, dihexyl phthalate-d4, butyl benzyl phthalate-d4, di(2-butoxy)ethyl phthalate-d4, dicyclohexyl phthalate-d4, di(α-ethylhexyl) phthalate-d4, diphenyl phthalate-d4, di-n-octyl phthalate-d4, and dinonyl phthalate-d4.

[0038] Furthermore, the concentration of each material in the deuterated isotope phthalate in the internal standard stock solution is 0.3-1 μg / mL; and / or, the concentration of each material in the phthalate in the reference stock solution is 0.2-6 μg / mL; and / or, the volume fraction of the internal standard stock solution in the test solution is 20%-30%.

[0039] By applying the technical solution of the present invention, only solid phase extraction is used to separate the plasticizer in ethyl ester fish oil, and the solid phase extraction conditions are screened. The separation effect is good, the separation method is simple, the steps are few, and it is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a gas phase mass spectrum of a blank concentrated solution in an embodiment of the present invention;

[0041] Figure 2 This is a gas phase mass spectrum of the standard concentrated solution-1 in the embodiment of the present invention;

[0042] Figure 3This is a gas phase mass spectrum of the standard concentrated solution-4 in the embodiment of the present invention;

[0043] Figure 4 This is the gas phase mass spectrum of the concentrated solution of the test sample in Example 4 of the present invention. DETAILED DESCRIPTION

[0044] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0045] As described in the background of the present invention, the prior art has the problem of difficulty in separating plasticizers from ethyl ester fish oils. To address this problem, in a typical embodiment of the present invention, a method for separating plasticizers from ethyl ester fish oils is provided, comprising the following steps:

[0046] S1, activating the solid phase extraction column and discarding the effluent to obtain an activated solid phase extraction column;

[0047] S2, loading the sample onto the activated solid phase extraction column to obtain a loaded solid phase extraction column;

[0048] S3, eluting the solid phase extraction column after the sample is loaded to obtain a washed solid phase extraction column;

[0049] S4, eluting the washed solid phase extraction column to obtain a separation liquid containing the plasticizer;

[0050] Among them, in S3, the reagent used for elution is the first alkane or a compound of the first alkane and the first reagent in a volume ratio of (99.0-99.9): (0.1-1.0); in S4, the reagent used for elution is a compound of the first alkane and the first reagent in a volume ratio of (50-95): (5-50); the above-mentioned first alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane; the first reagent is at least one of ethyl acetate, ethanol, acetone, tetrahydrofuran, and isopropanol.

[0051] Solid-phase extraction utilizes the interaction between the solid-phase material and the compounds to be separated to achieve separation and enrichment. The compounds to be separated will interact with the solid-phase material and thus be enriched on the solid-phase material; then, by using special reagents for elution and washing, the compounds can be desorbed from the solid-phase material to obtain enriched compounds. The solid phase extraction column has a high polarity, and the plasticizer can be fixed after the sample is loaded onto the solid phase extraction column. The solid phase extraction column has a weak adsorption effect on ethyl ester fish oil with relatively low polarity. The present invention first uses a first alkane or a compound with a volume ratio of the first alkane to the first reagent of (99.0-99.9): (0.1-1.0) for elution. According to the principle of like dissolves like, the fish oil can be washed out. Subsequently, the compound with a volume ratio of the first alkane to the first reagent of (50-95): (5-50) is used for elution. The plasticizer fixed on the solid phase extraction column can be eluted for testing. The above method is simple, uses less equipment, and can quickly separate the plasticizer in the ethyl ester fish oil.

[0052] In a preferred embodiment of the present invention, in S3, the elution comprises a first elution and a second elution. The reagent used in the first elution is the first alkane, and the reagent used in the second elution is a mixture of the first alkane and the first reagent in a volume ratio of (99.0-99.9):(0.1-1.0). The two elutions fully remove the fish oil, so that the eluted liquid, in addition to the eluent, primarily consists of the plasticizer, facilitating subsequent detection and analysis.

[0053] In a preferred embodiment of the present invention, the volume ratio of the reagent used in the first elution to the sample added is (0.5-1.5):1; the volume ratio of the reagent used in the second elution to the sample added is (10-20):1; the volume ratio of the reagent used in the elution to the sample added is (3-7):1.

[0054] The above-mentioned limitation on the amount of reagents used in the elution and washing processes can fully elute the fish oil without removing the plasticizer fixed in the solid phase extraction column, which is conducive to the subsequent accurate measurement of the plasticizer content in the ethyl ester fish oil.

[0055] In a preferred embodiment of the present invention, in S1, activation comprises sequentially washing the solid phase extraction column with the first reagent and the first alkane. This activation allows for the packing to be wetted, facilitating subsequent loading of the test sample into the solid phase extraction column and removing impurities from the column.

[0056] In a preferred embodiment of the present invention, the filler of the solid phase extraction column is a material based on silica gel. This filler has a large difference in the fixing force of fish oil and plasticizer in ethyl ester fish oil products, and can effectively separate the plasticizer and fish oil.

[0057] In another typical embodiment of the present invention, a method for determining plasticizers in ethyl ester fish oil is provided, comprising the following steps:

[0058] Prepare internal standard stock solution and reference substance stock solution;

[0059] Prepare standard solution with internal standard stock solution, reference substance stock solution and second alkane;

[0060] Prepare a blank solution with the internal standard stock solution and the second alkane;

[0061] The test sample is mixed with the internal standard stock solution to obtain a mixed solution, and the mixed solution is diluted with the second alkane to obtain a test sample solution;

[0062] The test solution, blank solution and standard solution are separated and concentrated by solid extraction method to obtain test solution concentrate, blank solution concentrate and standard solution concentrate;

[0063] The standard concentrate was tested by gas chromatography-mass spectrometry, and a standard curve was drawn with the concentration of the plasticizer in the standard concentrate as the abscissa and the ratio of the peak area of ​​the plasticizer to the peak area of ​​the internal standard as the ordinate;

[0064] The test sample concentrate and blank concentrate were tested by gas chromatography-mass spectrometry, and the concentration of plasticizer in the test sample concentrate and the concentration of plasticizer in the blank concentrate were calculated according to the standard curve; the content of plasticizer in the test sample was calculated according to formula (I):

[0065]

[0066] Where:

[0067] C: plasticizer concentration in the test sample concentrate, ng / ml;

[0068] V: dilution volume of the test sample, ml;

[0069] W: weight of test sample, mg;

[0070] M: plasticizer content of the test sample, ppm;

[0071] The plasticizer is phthalate, and the internal standard is deuterated phthalate;

[0072] The reference stock solution is a solution obtained by dissolving the plasticizer in the second alkane; the internal standard stock solution is a solution obtained by dissolving the internal standard in the second alkane;

[0073] The standard solutions are a group of solutions with the volume fractions of the reference substance stock solutions being 5%, 7.5%, 12%, 15%, 22.5%, and 30%, respectively, and the volume fraction of the internal standard stock solutions being the same;

[0074] The second alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane;

[0075] The solid phase extraction method is the separation method mentioned in the aforementioned typical embodiment.

[0076] The above determination method refers to the national standard GB5009.271-2016 "Determination of Phthalate Esters in Foods". Before conducting gas chromatography-mass spectrometry testing, the test sample is first treated with solid-phase extraction to make the main component of the test sample concentrate plasticizer. This treatment method is simple and has a good separation effect, which is conducive to the subsequent quality control of the plasticizer content in ethyl ester fish oil.

[0077] In a preferred embodiment of the present invention, the concentration of the internal standard stock solution in the test solution, blank solution, and standard solution is the same. Testing at the same concentration can reduce test errors and improve the accuracy of test results.

[0078] In a preferred embodiment of the present invention, when gas chromatography-mass spectrometry is used for testing, the ion source temperature is 300-330°C; and / or the acquisition mode is the multiple reaction monitoring mode (MRM); and the above-mentioned concentration is nitrogen blow concentration. For the plasticizers in fish oil to be detected by the present invention, when the ion source temperature is within the above-mentioned range, the detection sensitivity is higher; in addition, the multiple reaction monitoring mode is a highly selective detection mode that can reduce interference and improve sensitivity. Nitrogen blow concentration is simple to operate and can concentrate multiple groups of samples at the same time, with high efficiency, and is suitable for industrial applications.

[0079] Typically, but not limiting, phthalates include one or more of the following: di-n-butyl phthalate, di-(2-ethyl)hexyl phthalate, diisononyl phthalate, dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-methoxy)ethyl phthalate, di(4-methyl-2-pentyl)phthalate, di- phthalate, dipentyl phthalate, dihexyl phthalate, butyl benzyl phthalate, di(2-butoxy)ethyl phthalate, dicyclohexyl phthalate, di(α-ethylhexyl) phthalate, diphenyl phthalate, di-n-octyl phthalate, dinonyl phthalate; and / or, deuterated isotope phthalates include one of the following materials or Various: dibutyl phthalate-d4, di(2-ethyl)hexyl phthalate-d4, diisononyl phthalate-d4, dimethyl phthalate-d4, diethyl phthalate-d4, diisobutyl phthalate-d4, di(2-methoxy)ethyl phthalate-d4, di(4-methyl-2-pentyl)phthalate-d4, di(2- phthalate-d4, dipentyl phthalate-d4, dihexyl phthalate-d4, butyl benzyl phthalate-d4, di(2-butoxy)ethyl phthalate-d4, dicyclohexyl phthalate-d4, di(α-ethylhexyl) phthalate-d4, diphenyl phthalate-d4, di-n-octyl phthalate-d4, and dinonyl phthalate-d4.

[0080] In a preferred embodiment of the present invention, the phthalate is at least one of the following: DBP, DEHP, and DINP, and the deuterated phthalate is at least one of the following: DBP-d4 and DEHP-d4. The method disclosed in the present invention can be used to detect these materials with improved responsiveness and higher sensitivity.

[0081] In a preferred embodiment of the present invention, the concentration of each deuterated phthalate ester in the internal standard stock solution is 0.3-1 μg / mL; and / or the concentration of each deuterated phthalate ester in the reference stock solution is 0.2-6 μg / mL; and / or the volume fraction of the internal standard stock solution in the test solution is 20%-30%. The concentrations of the materials in the internal standard stock solution and the reference stock solution are defined based on the content of the corresponding components in conventional ethyl ester fish oil.

[0082] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0083] Some of the materials used in the examples and comparative examples are as follows:

[0084] Solid phase extraction column: silica gel matrix column, PAS / Silica composite filler glass column (1000 mg, 6 ml);

[0085] Ethyl ester fish oil: from Xinchang Pharmaceutical Factory, Zhejiang Medicine Co., Ltd.

[0086] Prepare the solution:

[0087] DBP-d4 stock solution: Weigh an appropriate amount of DBP-d4 reference substance and add it to n-hexane to prepare a DBP-d4 solution with a concentration of 100 μg / mL;

[0088] DEHP-d4 stock solution: Weigh an appropriate amount of DEHP-d4 reference substance and add it to n-hexane to prepare a solution with a DEHP-d4 concentration of 100 μg / mL;

[0089] DBP stock solution: Weigh an appropriate amount of DBP reference substance and add it to n-hexane to prepare a DBP solution with a concentration of 200 μg / mL;

[0090] DEHP stock solution: Weigh an appropriate amount of DEHP reference substance and add it to n-hexane to prepare a solution with a DEHP concentration of 200 μg / mL;

[0091] DINP stock solution: Weigh an appropriate amount of DINP reference substance and add it to n-hexane to prepare a DINP solution with a concentration of 200 μg / mL;

[0092] Internal standard stock solution: Accurately measure 0.5 mL of DBP-d4 stock solution and DEHP-d4 stock solution respectively, place them in a 100 mL volumetric flask, add n-hexane to dilute to the mark, and shake well;

[0093] Reference substance stock solution: Accurately measure 0.1 mL of DBP stock solution, 0.5 mL of DEHP stock solution, and 6 mL of DINP stock solution, respectively, place them in a 100 mL volumetric flask, dilute to the mark with n-hexane, and shake well;

[0094] Blank solution: Accurately measure 2.5 mL of internal standard stock solution, place it in a 10 mL volumetric flask, add n-hexane to dilute to the mark, and shake well;

[0095] Standard solutions: Accurately measure 0.5 mL, 0.75 mL, 1.2 mL, 1.5 mL, 2.25 mL, and 3 mL of the reference stock solution, respectively, and then mix them with 2.5 mL of the internal standard stock solution. Finally, dilute them to 10 mL with n-hexane to obtain standard solutions with different concentrations of the reference stock solution. These solutions are recorded as standard solution-1, standard solution-2, standard solution-3, standard solution-4, standard solution-5, and standard solution-6, respectively.

[0096] Test solution: Accurately weigh 1 g of ethyl ester fish oil and place it in a 10 mL volumetric flask. Add 2.5 mL of internal standard stock solution, dilute to the mark with n-hexane, and shake well.

[0097] Method for separating plasticizers from ethyl ester fish oil

[0098] Example 1

[0099] An embodiment of a method for separating plasticizers from ethyl ester fish oil of the present invention comprises the following steps:

[0100] (1) Activation: The solid phase extraction column was cleaned and activated with 10 mL of ethyl acetate and 10 mL of n-hexane in sequence, and the effluent was discarded to obtain the activated solid phase extraction column;

[0101] (2) Sample loading: Pipette 1 mL of the above test solution onto the solid phase extraction column;

[0102] (3) Washing: Wash the SPE column once with 1 mL of n-hexane and discard the effluent; then wash the SPE column a second time with 15 mL of a mixture of n-hexane and ethyl acetate (volume ratio: 99.4:0.6) to remove the fish oil;

[0103] (4) Elution: Add 5 mL of a mixture of n-hexane and ethyl acetate in a volume ratio of 90:10 to the solid phase extraction column to elute the plasticizer, and collect the effluent, which is the separation liquid containing the plasticizer.

[0104] Example 2

[0105] An embodiment of a method for separating plasticizers from ethyl ester fish oil of the present invention comprises the following steps:

[0106] (1) Activation: The solid phase extraction column was cleaned and activated with 10 mL of ethyl acetate and 10 mL of n-hexane in sequence, and the effluent was discarded to obtain the activated solid phase extraction column;

[0107] (2) Sample loading: Pipette 1 mL of the above test solution onto the solid phase extraction column;

[0108] (3) Washing: Use 0.5 mL of n-hexane to wash the solid phase extraction column once, and discard the effluent; then wash the solid phase extraction column twice with 10 mL of a mixture of n-hexane and ethyl acetate (volume ratio 99:1) to wash away the fish oil;

[0109] (4) Elution: Add 3 mL of a mixture of n-hexane and ethyl acetate in a volume ratio of 95:5 to the solid phase extraction column to elute the plasticizer, and collect the effluent, which is the separation liquid containing the plasticizer.

[0110] Example 3

[0111] An embodiment of a method for separating plasticizers from ethyl ester fish oil of the present invention comprises the following steps:

[0112] (1) Activation: The solid phase extraction column was cleaned and activated with 10 mL of ethyl acetate and 10 mL of n-hexane in sequence, and the effluent was discarded to obtain the activated solid phase extraction column;

[0113] (2) Sample loading: Pipette 1 mL of the above test solution onto the solid phase extraction column;

[0114] (3) Washing: Use 1.5 mL of n-hexane to wash the solid phase extraction column once, and discard the effluent; then wash the solid phase extraction column twice with 20 mL of a mixture of n-hexane and ethyl acetate with a volume ratio of 99.9:0.1 to wash away the fish oil;

[0115] (4) Elution: Add 7 mL of a mixture of n-hexane and ethyl acetate in a volume ratio of 50:50 to the solid phase extraction column to elute the plasticizer, and collect the effluent, which is the separation liquid containing the plasticizer.

[0116] Comparative Example 1

[0117] A method for separating plasticizers from ethyl ester fish oil, comprising the following steps:

[0118] (1) Activation: The solid phase extraction column was cleaned and activated with 10 mL of ethyl acetate and 10 mL of n-hexane in sequence, and the effluent was discarded to obtain the activated solid phase extraction column;

[0119] (2) Sample loading: Pipette 1 mL of the above test solution onto the solid phase extraction column;

[0120] (3) Washing: Wash the SPE column once with 1 mL of n-hexane and discard the effluent; then wash the SPE column a second time with 15 mL of a mixture of n-hexane and ethyl acetate (volume ratio: 99.4:0.6) to remove the fish oil;

[0121] (4) Elution: Add 5 mL of a mixture of n-hexane and ethyl acetate in a volume ratio of 40:60 to the solid phase extraction column to elute the plasticizer, and collect the effluent, which is the separation liquid containing the plasticizer.

[0122] Determination of plasticizers in ethyl ester fish oil

[0123] Example 4

[0124] An embodiment of a method for determining plasticizers in ethyl ester fish oil of the present invention comprises the following steps:

[0125] (1) The separated solution obtained in Example 1 was concentrated by nitrogen blowing to 1 mL to obtain a test sample concentrate;

[0126] (2) The blank solution and the standard solution were treated in the same manner as in Example 1, and then concentrated to 1 mL using nitrogen blowdown to obtain blank concentrate and standard concentrates (standard concentrate-1, standard concentrate-2, standard concentrate-3, standard concentrate-4, standard concentrate-5, and standard concentrate-6).

[0127] (3) Testing the standard concentrate by gas chromatography-mass spectrometry, plotting a standard curve with the concentration of the plasticizer in the standard concentrate as the abscissa and the ratio of the peak area of ​​the plasticizer to the peak area of ​​the internal standard as the ordinate;

[0128] (4) The above-mentioned test sample concentrate and blank concentrate were tested by gas chromatography-mass spectrometry. The concentration of plasticizer in the test sample concentrate and the concentration of plasticizer in the blank concentrate were calculated according to the standard curve. The content of plasticizer in the test sample was calculated according to formula (I):

[0129]

[0130] Where:

[0131] C: plasticizer concentration in the test sample concentrate, ng / ml;

[0132] V: dilution volume of the test sample, ml;

[0133] W: weight of test sample, mg;

[0134] M: Plasticizer content of the test sample, ppm.

[0135] The gas chromatography conditions are as follows:

[0136] Chromatographic column: 5% phenyl-methylpolysiloxane, 30 m × 0.25 mm × 0.25 μm;

[0137] Inlet temperature: 280°C;

[0138] Injection volume: 1 μL;

[0139] Carrier gas: high purity helium;

[0140] Flow rate: 1.0 mL / min;

[0141] Temperature program: the initial column temperature was 60 °C, maintained for 1 min, then increased to 220 °C at 100 °C / min, maintained for 5 min, and then increased to 300 °C at 5 °C / min, maintained for 1 min.

[0142] Mass spectrometry conditions:

[0143] Ion source: EI source;

[0144] Ion source temperature: 320°C;

[0145] Quadrupole temperature: 180°C;

[0146] Transmission line temperature: 300°C;

[0147] Acquisition mode: multiple reaction monitoring (MRM);

[0148] The MRM parameters of each substance are shown in Table 1:

[0149] Table 1

[0150]

[0151] Performance Testing

[0152] 1) Specificity test

[0153] The blank concentrate, standard concentrate-1, standard concentrate-4, and test sample concentrate were tested using the above-mentioned gas chromatography-mass spectrometry method; Figure 1 is the gas chromatography mass spectrum of the blank concentrate. Figure 2 is the gas phase mass spectrum of standard concentrated solution-1, Figure 3 is the gas phase mass spectrum of standard concentrated solution-4, Figure 4 The GC-MS spectrum of the test sample concentrate is shown in Figure 1. As can be seen, there is no interference with the DBP, DEHP, and DINP peaks in both the blank and test solutions. In the standard concentrate, the signal-to-noise ratios for all three components are high, making them suitable for detection using the aforementioned method.

[0154] 2) Linear range test

[0155] DBP reference substance, DEHP reference substance, DINP reference substance and internal standard DBP-d4 reference substance and DEHP-d4 reference substance were accurately weighed and diluted with n-hexane to prepare a series of linear sample solutions. The concentration of internal standard DBP-d4 and DEHP-d4 in each sample solution was 125 ng / mL. According to the method disclosed in Example 4, solid phase extraction was first used for separation, and then concentrated to obtain the corresponding concentrated solution. The solution was then added to a gas chromatograph-mass spectrometer, and the peak area was recorded. A standard curve was plotted with the concentration of the target substance (ng / mL) in the linear solution as the abscissa and the ratio of the target substance peak area to the internal standard peak area as the ordinate. The results are shown in Table 2:

[0156] Table 2

[0157]

[0158] The above test results show that the linear regression equations and regression coefficients of DBP, DEHP, and DINP all meet the requirements, and the linear regression is significant. The above results further illustrate that the determination method disclosed in the present invention is suitable for determining the content of these plasticizers in ethyl ester fish oil.

[0159] 3) Recovery test

[0160] 50% spiked test sample: Accurately weigh 500 mg of ethyl ester fish oil and place it in a 5 mL volumetric flask. Add 1.25 mL of the internal standard stock solution and 375 μL of the reference stock solution, respectively. Dilute to the mark with n-hexane, shake well, and prepare three replicates.

[0161] 100% spiked test sample: Accurately weigh 500 mg of ethyl ester fish oil and place it in a 5 mL volumetric flask. Add 1.25 mL of the internal standard stock solution and 750 μL of the reference stock solution, respectively. Dilute to the mark with n-hexane, shake well, and prepare three replicates.

[0162] 200% spiked test sample: Accurately weigh 500 mg of ethyl ester fish oil and place it in a 5 mL volumetric flask. Add 1.25 mL of the internal standard stock solution and 1.5 mL of the reference stock solution, respectively. Dilute to the mark with n-hexane, shake well, and prepare three replicates.

[0163] According to the method disclosed in Example 4, the spiked sample was first subjected to solid phase extraction and concentration, and then the concentrate was injected into a gas chromatograph-mass spectrometer to measure the recovery rate of the spiked sample. The test results are shown in Table 3:

[0164] Table 3

[0165]

[0166] As shown in Table 3, the spiked recoveries of DBP, DEHP, and DINP at three concentration levels were 101.1%, 93.7%, and 88.0%, respectively, and the RSDs were 6.4%, 2.9%, and 3.9%, respectively, which met the requirements for the determination of trace impurities and indicated that this method had high accuracy.

[0167] 4) Precision test

[0168] Accurately weigh 500 mg of fish oil and place it in a 5 mL volumetric flask. Add 1.25 mL of the internal standard stock solution and 750 μL of the reference substance stock solution. Dilute to the mark with n-hexane, shake well, and prepare 6 parallel portions.

[0169] According to the method disclosed in Example 4, the spiked sample was first subjected to solid phase extraction and concentration, and then the concentrate was injected into a gas chromatograph mass spectrometer for determination.

[0170] The six 100% spiked solutions were tested, and the RSDs for DBP, DEHP, and DINP were 2%, 1.7%, and 6.1%, respectively. The RSDs were all less than 10%, indicating good measurement accuracy and repeatability.

[0171] 5) Limit of quantification and limit of detection test

[0172] Quantitation limit solution: Accurately measure 0.75 mL of reference substance stock solution and 2.5 mL of internal standard stock solution, place them in a 10 mL volumetric flask, add n-hexane to dilute to the mark, and shake well;

[0173] Detection limit solution: Accurately measure 0.5 mL of the reference stock solution and 2.5 mL of the internal standard stock solution, place them in a 10 mL volumetric flask, dilute to the mark with n-hexane, and shake well;

[0174] According to the method disclosed in Example 4, the above-mentioned quantitative limit solution and detection limit solution were subjected to solid phase extraction and concentration treatment, respectively, to obtain a quantitative limit concentrate and a detection limit concentrate. The quantitative limit concentrate was sampled for 6 consecutive injections. The RSD and signal-to-noise ratio test results of the ratios of the peak areas of DBP, DEHP, and DINP to the internal standard peak area are shown in Tables 4-5; the detection limit concentrate was sampled for 3 consecutive injections. The signal-to-noise ratios of DBP, DEHP, and DINP are shown in Table 6:

[0175] Table 4

[0176]

[0177] Table 5

[0178]

[0179] Table 6

[0180]

[0181]

[0182] 6) Actual sample testing

[0183] The test results of Example 4 are shown in Table 7. Two batches of ethyl ester fish oil were tested using the same method as in Example 4. The test results are shown in Table 7:

[0184] Table 7

[0185]

[0186] The above-mentioned actual test results show that this method can measure the content of DBP, DEHP and DINP in fish oil with high accuracy and good repeatability.

[0187] Example 1 in step (1) of Example 4 was replaced by Example 2, Example 3 and Comparative Example 1, respectively, and the contents of plasticizers DBP, DEHP and DINP were calculated. The results were recorded in Table 8.

[0188] Table 8

[0189]

[0190] The eluent used in Comparative Example 1 was unable to completely separate the plasticizer and fish oil. Therefore, the fish oil contained in the collected effluent interfered with the detection of the plasticizer, making it impossible to detect the plasticizer content. DBP, DEHP, and DINP were all detected in Examples 1-3, with similar test results. This result demonstrates that the methods disclosed in the present examples can effectively separate plasticizers from ethyl ester fish oil.

[0191] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for separating plasticizers from ethyl ester fish oil, characterized in that: The steps include: S1, activating the solid phase extraction column to obtain an activated solid phase extraction column; S2, loading the sample onto the activated solid phase extraction column to obtain a loaded solid phase extraction column; S3, eluting the solid phase extraction column after the sample is loaded to obtain a washed solid phase extraction column; S4, eluting the washed solid phase extraction column to obtain a separation liquid containing the plasticizer; In S3, the reagent used for elution is the first alkane or a compound of the first alkane and the first reagent in a volume ratio of (99.0-99.9): (0.1-1.0); in S4, the reagent used for elution is a compound of the first alkane and the first reagent in a volume ratio of (50-95): (5-50); the first alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane; the first reagent is at least one of ethyl acetate, ethanol, acetone, tetrahydrofuran, and isopropanol.

2. The method for separating plasticizers from ethyl ester fish oil according to claim 1, wherein: In S3, the elution includes a first elution and a second elution, the reagent used in the first elution is the first alkane, and the reagent used in the second elution is a mixture of the first alkane and the first reagent in a volume ratio of (99.0-99.9): (0.1-1.0).

3. The method for separating plasticizers from ethyl ester fish oil according to claim 2, wherein: The volume ratio of the reagent used in the first rinsing to the sample added is (0.5-1.5):1; the volume ratio of the reagent used in the second rinsing to the sample added is (10-20):1; the volume ratio of the reagent used in the elution to the sample added is (3-7):

1.

4. The method for separating plasticizers from ethyl ester fish oil according to any one of claims 1 to 3, characterized in that: In S1, the activation is: washing the solid phase extraction column with the first reagent and the first alkane in sequence.

5. The method for separating plasticizer from ethyl ester fish oil according to claim 4, characterized in that: The filler of the solid phase extraction column is a material with silica gel as a matrix.

6. A method for determining plasticizer in ethyl ester fish oil, characterized in that: The steps include: preparing an internal standard stock solution and a reference substance stock solution; preparing a standard solution with the internal standard stock solution, the reference substance stock solution and a second alkane; and preparing a blank solution with the internal standard stock solution and the second alkane; Mixing the test sample with the internal standard stock solution to obtain a mixed solution, and diluting the mixed solution with a second alkane to obtain a test sample solution; The test solution, the blank solution, and the standard solution are separated and concentrated by solid extraction to obtain a test solution concentrate, a blank solution concentrate, and a standard solution concentrate; The standard concentrate is tested by gas chromatography-mass spectrometry, and a standard curve is drawn with the concentration of the plasticizer in the standard concentrate as the abscissa and the ratio of the peak area of ​​the plasticizer to the peak area of ​​the internal standard as the ordinate; The test sample concentrate and the blank concentrate were tested by gas chromatography-mass spectrometry, and the concentration of the plasticizer in the test sample concentrate and the concentration of the plasticizer in the blank concentrate were calculated according to the standard curve; the content of the plasticizer in the test sample was calculated according to formula (I): Where: C: plasticizer concentration in the test sample concentrate, ng / ml; V: dilution volume of the test sample, ml; W: weight of test sample, mg; M: plasticizer content of the test sample, ppm; The plasticizer is phthalate, and the internal standard is a deuterated isotope phthalate; The reference substance stock solution is a solution obtained by dissolving the plasticizer in the second alkane; the internal standard stock solution is a solution obtained by dissolving the internal standard substance in the second alkane; The standard solutions are a group of solutions in which the volume fractions of the reference substance stock solutions are 5%, 7.5%, 12%, 15%, 22.5%, and 30%, respectively, and the volume fraction of the internal standard stock solutions are the same; The second alkane is at least one of n-hexane, cyclohexane, n-heptane, and isooctane; The solid phase extraction method is the separation method according to any one of claims 1 to 5.

7. The measuring method according to claim 6, wherein The concentrations of the internal standard stock solution in the test solution, the blank solution, and the standard solution are the same.

8. The measuring method according to claim 6 or 7, characterized in that When the gas chromatography-mass spectrometry method is used for testing, the ion source temperature is 300-330° C.; and / or the acquisition mode is the multiple reaction monitoring mode; The concentration is nitrogen blowing concentration.

9. The measuring method according to claim 8, characterized in that The phthalate esters include one or more of the following materials: di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, diisononyl phthalate, dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di(2-methoxy)ethyl phthalate, di(4-methyl-2-pentyl)phthalate, di(2-ethoxy)ethyl phthalate, dipentyl phthalate, dihexyl phthalate, butyl benzyl phthalate, di(2-butoxy)ethyl phthalate, dicyclohexyl phthalate, di(α-ethylhexyl) phthalate, diphenyl phthalate, di-n-octyl phthalate, dinonyl phthalate; and / or The deuterated isotope phthalate includes one or more of the following materials: di-n-butyl phthalate-d4, di-(2-ethyl)hexyl phthalate-d4, diisononyl phthalate-d4, dimethyl phthalate-d4, diethyl phthalate-d4, diisobutyl phthalate-d4, di-(2-methoxy)ethyl phthalate-d4, di-(4-methyl-2-pentyl)phthalate-d4, di-n-butyl ... d4, di(2-ethoxy)ethyl phthalate-d4, dipentyl phthalate-d4, dihexyl phthalate-d4, butyl benzyl phthalate-d4, di(2-butoxy)ethyl phthalate-d4, dicyclohexyl phthalate-d4, di(α-ethylhexyl) phthalate-d4, diphenyl phthalate-d4, di-n-octyl phthalate-d4, and dinonyl phthalate-d4.

10. The measuring method according to claim 9, characterized in that The concentration of each material of the deuterated isotope phthalate in the internal standard stock solution is 0.3-1 μg / mL; and / or the concentration of each material of the phthalate in the reference substance stock solution is 0.2-6 μg / mL; and / or the volume fraction of the internal standard stock solution in the test solution is 20%-30%.

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