Method for determining contents of linoleic acid and α-linolenic acid in fat emulsion injection

Abstract

A method for determining the contents of linoleic acid and α-linolenic acid in a fat emulsion injection. The method comprises: demulsifying a fat emulsion, subjecting fatty acids to methyl esterification, separating methyl ester compounds of linoleic acid and α-linolenic acid by using gas chromatography, and performing quantitative calculation to obtain the contents of linoleic acid and α-linolenic acid. By means of demulsifying the fat emulsion and subjecting linoleic acid and α-linolenic acid to methyl esterification, followed by separation of the resulting methyl ester compounds, the contents of linoleic acid and α-linolenic acid in the fat emulsion can be accurately and rapidly determined. Moreover, the method involves a simple pretreatment process and high precision, realizing an efficient, rapid and accurate method for detecting the contents of linoleic acid and α-linolenic acid in a fat emulsion injection.

Description

A method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection. Technical Field

[0001] This invention belongs to the field of pharmaceutical or food quality testing technology, specifically relating to a method for determining the content of linoleic acid and α-linolenic acid in fat emulsion injections. Background Technology

[0002] Linoleic acid's scientific name is 9,12-octadecadienoic acid, and its chemical formula is C60-12 ... 18 H 32 O2 is a colorless, oily liquid, insoluble in water but readily soluble in organic solvents such as petroleum ether and toluene. Since the human body cannot synthesize linoleic acid, it must be obtained through dietary supplementation or by forming specific precursors to maintain normal physiological functions; therefore, it is considered an essential fatty acid. α-Linolenic acid is cis-9, cis-12, cis-15-octadecanoic acid, with the chemical formula C2. 18 H 30 O2 is an essential fatty acid in the omega-3 series and must be obtained from external sources. A deficiency can lead to weakened immunity, fatigue, and other symptoms, especially in infants and adolescents. Long-term deficiency of alpha-linolenic acid during growth can severely impair normal intellectual development.

[0003] Linoleic acid and alpha-linolenic acid (ALA) are the main active ingredients in soybean oil, which is widely used in nutritional preparations. Monitoring the content of ALA and ALA can effectively monitor the quality level of soybean oil (or other products containing ALA and ALA) during the production process, and their content levels have a significant impact on drug efficacy. Currently, there is no method to accurately determine the content of ALA and ALA. Therefore, researching an efficient, accurate, and universally applicable method for detecting ALA and ALA content in fat emulsions is particularly important for providing strong technical support for product quality supervision of pharmaceuticals in my country.

[0004] Therefore, this patent application is filed. Summary of the Invention

[0005] The purpose of this patent application is to solve the problem that existing methods cannot accurately determine the content of linoleic acid and α-linolenic acid in fat emulsion injections, and to provide a method for determining the content of linoleic acid and α-linolenic acid in fat emulsion injections, which can efficiently, accurately and universally detect the content of linoleic acid and α-linolenic acid in fat emulsions.

[0006] This invention is achieved through the following technical solution:

[0007] The purpose of this invention is to provide a method for determining the content of linoleic acid and α-linolenic acid in fat emulsion injection. After demulsification of the fat emulsion, the fatty acids are methyl esterified, and the methyl ester compounds of linoleic acid and α-linolenic acid are separated by gas chromatography and quantitatively calculated to obtain the content of linoleic acid and α-linolenic acid.

[0008] This invention involves demulsifying the fat emulsion to ensure the release of linoleic acid and alpha-linolenic acid from the oil, facilitating subsequent separation using gas chromatography and improving separation efficiency. It also includes the methyl esterification of linoleic acid and alpha-linolenic acid. Since fatty acids have high boiling points and are difficult to vaporize, methyl esterification produces fatty acid methyl ester derivatives, lowering the boiling point and enabling rapid vaporization followed by gas chromatography analysis. This allows for accurate and rapid determination of the linoleic acid and alpha-linolenic acid content in the fat emulsion with high precision, providing a highly efficient, rapid, and accurate method for detecting the linoleic acid and alpha-linolenic acid content in fat emulsion injections.

[0009] In an optional embodiment, the fat emulsion is demulsified by ultrasonication using an organic reagent, wherein the organic reagent is tert-butyl methyl ether.

[0010] In this invention, tert-butyl methyl ether is used for demulsification, which is well miscible with fat emulsions and facilitates subsequent separation.

[0011] In an optional embodiment, fatty acids are methylated using a derivatizing agent, wherein the derivatizing agent is trimethylsulfonium hydroxide.

[0012] In an optional embodiment, the following steps are included:

[0013] Prepare the gas chromatograph;

[0014] An internal standard solution with an organic reagent as a solvent was added to the fat emulsion injection. After ultrasonic demulsification, the organic phase was taken for methyl esterification reaction, and the methyl ester compounds of linoleic acid and α-linolenic acid were analyzed by gas chromatography.

[0015] The contents of linoleic acid and α-linolenic acid in the sample were calculated using the internal standard method;

[0016] The organic reagent is tert-butyl methyl ether;

[0017] The methyl esterification reaction was carried out using trimethyl sulfone hydroxide.

[0018] In an optional embodiment, the gas chromatograph uses a capillary column with polyethylene glycol as the stationary phase, with an initial column temperature of 100°C, maintained for 5 minutes, then increased to 205°C at a rate of 15°C per minute and maintained for 18 minutes; the injection port temperature is 250°C; and the detector temperature is 275°C.

[0019] In an optional embodiment, the gas chromatograph has a flow rate of 1.0 ml / min and an injection volume of 2 μl.

[0020] In an optional embodiment, the internal standard solution is a methyl undecanoate solution.

[0021] In an optional embodiment, after ultrasonic demulsification, the product is dried with anhydrous sodium sulfate, and the dried organic clear liquid is subjected to a methyl esterification reaction. The methyl esterification reaction is carried out in a water bath at 70°C for 45 minutes.

[0022] In an optional embodiment, the reference standards used in the gas chromatography analysis are methyl linoleate and methyl α-linolenic acid.

[0023] In an optional embodiment, the fat emulsion injection is a fat emulsion with soybean oil as the oil phase, or other fat emulsions containing linoleic acid and α-linolenic acid.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] This invention develops a method for accurately determining the content of linoleic acid and α-linolenic acid in fat emulsion injections. The method features simple sample preparation steps, low detection cost, high accuracy and precision, strong specificity, and more stable and reliable detection results.

[0026] In this invention, fat emulsion is demulsified and methylated with linoleic acid and α-linolenic acid. By separating the methyl ester compounds, the content of linoleic acid and α-linolenic acid in fat emulsion can be accurately and rapidly determined. The pretreatment process is simple and has high precision, thus obtaining a method that can efficiently, quickly and accurately detect the content of linoleic acid and α-linolenic acid in fat emulsion injection. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0028] Figure 1 shows the gas chromatogram of the internal standard solution in the specificity determination.

[0029] Figure 2 shows the gas chromatogram of the methyl linoleate localization solution for specific determination.

[0030] Figure 3 shows the gas chromatogram of the methyl linoleate localization solution in the specific determination.

[0031] Figure 4 shows the gas chromatogram of the reference solution in the specificity determination.

[0032] Figure 5 shows the gas chromatogram of the test solution in the specificity determination. Detailed Implementation

[0033] The embodiments of this application will be described in further detail below with reference to the examples. The detailed description of the following embodiments is used to illustrate the principles of this application, but should not be used to limit the scope of this application, that is, this application is not limited to the described embodiments.

[0034] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0035] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0036] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0037] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0038] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0039] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0040] Example 1

[0041] Preparation of internal standard solution: Accurately weigh an appropriate amount of methyl undecanoate reference standard and dilute it with tert-butyl methyl ether to prepare a solution with a concentration of 1.210 mg / ml.

[0042] Preparation of the test solution: Accurately weigh an appropriate amount of fat emulsion injection (approximately equivalent to 0.4 g of soybean oil), place it in a stoppered test tube, add 10 ml of the internal standard solution prepared above, sonicate for 15 minutes, and cool to room temperature; add 1.5 g of anhydrous sodium sulfate, shake well, and let stand for 45 minutes; accurately measure 0.1 ml of the supernatant, place it in the sample vial of the gas chromatograph, add 50 μl of trimethyl sulfone hydroxide and 500 μl of tert-butyl methyl ether to trigger the entire reaction system, seal, heat in a 70°C water bath for 45 minutes, and cool to room temperature to obtain the test solution.

[0043] Preparation of reference solution: Accurately weigh 21.07 mg of methyl linoleate reference standard and 4.088 mg of methyl α-linolenic acid reference standard, place them in a stoppered test tube, accurately add 10 ml of the above internal standard solution, and shake well to obtain the solution.

[0044] 1. Instrumental chromatographic conditions

[0045] The chromatographic column was a capillary column with polyethylene glycol as the stationary phase (HP-INNOWAX 0.32mm*30m, 0.5μm or equivalent column efficiency); the initial column temperature was 100℃, maintained for 5 minutes, then increased to 205℃ at a rate of 15℃ per minute, and maintained for 18 minutes; the injection port temperature was 250℃; the detector temperature was 275℃; the split ratio was 10:1; the flow rate was 1.0 ml / min; and the injection volume was 2 μl.

[0046] 2. Content Calculation

[0047] ;

[0048] ;

[0049] ;

[0050] .

[0051] In the formula: Sample density (g / ml) = 0.9982 × Sample relative density, where 0.9982 is the density of water at 20℃.

[0052] 1.05 is the molecular weight ratio of methyl linoleate (methyl linolenic acid) to linoleic acid (linolenic acid).

[0053] Experimental example:

[0054] 1. Specificity determination

[0055] Accurately weigh 12.37 mg of methyl undecanoate, 11.28 mg of methyl linoleate, and 10.05 mg of methyl α-linolenic acid, and add 10 ml of tert-butyl methyl ether to prepare a positioning solution.

[0056] Take 10 ml of the prepared internal standard solution, sonicate for 15 minutes, cool to room temperature, add about 1.5 g of anhydrous sodium sulfate, shake well, let stand for 45 minutes, accurately measure 0.1 ml of the supernatant, place it in a sample vial, add 50 μl of trimethyl sulfonium hydroxide and 500 μl of tert-butyl methyl ether, seal, heat in a 70°C water bath for 45 minutes, cool to room temperature, and the blank solution is obtained.

[0057] Blank solution, each positioning solution, reference solution, and test solution were injected for analysis. The results are shown in Table 1 below, and the gas chromatograms are shown in Figures 1, 2, 3, 4, and 5, respectively.

[0058] Table 1: Linoleic acid and α-linolenic acid content in soybean oil - specificity

[0059]

[0060] As can be seen from Table 1 and Figures 1-5, under the determined chromatographic conditions, the resolution between the target peak and adjacent peaks is greater than 1.5, and the blank solution does not interfere with the determination of the internal standard and each fatty acid peak. This method has good specificity.

[0061] 2. Linear range

[0062] Mixed reference stock solution: Accurately weigh appropriate amounts of methyl linoleate reference standard and methyl α-linolenic acid reference standard, and dilute to volume with internal standard solution to prepare a solution containing 9.922 mg / ml of methyl linoleate and 1.861 mg / ml of methyl α-linolenic acid.

[0063] Mixed standard series working solutions: Measure 0.5, 1.0, 2.0, 3.0, and 4.0 mL of the prepared mixed reference stock solution into 10 mL volumetric flasks, respectively. Add internal standard solution to each flask and dilute to the mark. Mix well to obtain linear solutions 1-5. Accurately measure 1.0 mL of solution 3 into a dry 10 mL volumetric flask, add internal standard solution to the mark, and obtain linear solution 6.

[0064] Take the mixed standard series working solutions and inject them sequentially in order of increasing concentration. Plot the standard curves with the injection volume of each component as the abscissa (X) and the peak area as the ordinate (Y).

[0065] result:

[0066] For methyl linoleate in the range of 0.1988 mg / ml to 3.9688 mg / ml, the linear equation is y = 0.9108x - 0.0045, and the correlation coefficient r = 0.9999 > 0.9900; for methyl α-linolenic acid in the range of 0.0372 mg / ml to 0.7444 mg / ml, the linear equation is y = 0.9079x - 0.0017, and the correlation coefficient r = 0.9999 > 0.9900. The correlation coefficients both meet the requirements, proving that the method has good linearity.

[0067] 3. Recovery rate determination

[0068] Negative spiked recovery tests were performed at 80%, 100%, and 120% of the concentrations of methyl linoleate (2 mg / ml) and methyl α-linolenic acid (0.4 mg / ml) in the test solution. The samples were injected and analyzed under the prescribed chromatographic conditions, and the recovery was calculated as the measured amount / added amount. The recovery rates of methyl linoleate ranged from 97.16% to 99.54%, with an average recovery rate of 98.2% and an RSD of 0.9%; the recovery rates of methyl α-linolenic acid ranged from 94.44% to 98.91%, with an average recovery rate of 97.4% and an RSD of 1.4%, indicating good method accuracy.

[0069] 4. Precision determination

[0070] Six parallel test solutions were prepared from the same sample, and the contents of linoleic acid and α-linolenic acid in soybean oil were determined. The linoleic acid content in the six parallel samples was 48.86 mg / ml, with an RSD of 0.4%, and the α-linolenic acid content was 6.41 mg / ml, with an RSD of 0.5%, indicating that the method has good precision.

[0071] The instruments and reagents used in the embodiments of this invention are shown in the table below:

[0072] (1) Instrument Information

[0073]

[0074] (2) Reference Standard Information

[0075]

[0076] (3) Reagent and test solution information

[0077]

[0078] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for determining the contents of linoleic acid and α-linolenic acid in fat emulsion injection, characterized by, After demulsifying the fat emulsion, the fatty acids are methyl esterified. The methyl ester compounds of linoleic acid and α-linolenic acid are separated by gas chromatography and quantitatively calculated to obtain the content of linoleic acid and α-linolenic acid.

2. The method according to claim 1, wherein the contents of linoleic acid and α-linolenic acid in the fat emulsion injection are measured. Fat emulsions are demulsified using an organic reagent via ultrasonication, wherein the organic reagent is tert-butyl methyl ether.

3. The method according to claim 1, wherein the contents of linoleic acid and α-linolenic acid in the fat emulsion injection are measured. Fatty acids are methyl esterified using a derivatizing agent, wherein the derivatizing agent is trimethylsulfonium hydroxide.

4. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 1, characterized in that, Includes the following steps: Prepare the gas chromatograph; An internal standard solution with an organic reagent as a solvent was added to the fat emulsion injection. After ultrasonic demulsification, the organic phase was taken for methyl esterification reaction, and the methyl ester compounds of linoleic acid and α-linolenic acid were analyzed by gas chromatography. The contents of linoleic acid and α-linolenic acid in the sample were calculated using the internal standard method; The organic reagent is tert-butyl methyl ether; The methyl esterification reaction was carried out using trimethyl sulfone hydroxide.

5. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 4, characterized in that, The gas chromatograph uses a capillary column with polyethylene glycol as the stationary phase. The initial column temperature is 100°C, maintained for 5 minutes, and then increased to 205°C at a rate of 15°C per minute, maintained for 18 minutes. The injection port temperature is 250°C, and the detector temperature is 275°C.

6. A method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 4 or 5, characterized in that, The flow rate of the gas chromatograph was 1.0 ml / min, and the injection volume was 2 μl.

7. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 4, characterized in that, The internal standard solution is a methyl undecanoate solution.

8. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 4, characterized in that, After ultrasonic demulsification, the solution was dried with anhydrous sodium sulfate. The dried organic clear liquid was then subjected to a methyl esterification reaction, which was carried out in a water bath at 70°C for 45 minutes.

9. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 4, characterized in that, In the gas chromatography analysis, the reference standards used were methyl linoleate and methyl α-linolenic acid.

10. The method for determining the content of linoleic acid and α-linolenic acid in a fat emulsion injection according to claim 1, characterized in that, The fat emulsion injection is a fat emulsion with soybean oil as the oil phase, or other fat emulsions containing linoleic acid and α-linolenic acid.

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