Method for simultaneously detecting contents of seven saturated fatty acids in feed flavoring agent

Abstract

The invention relates to the technical field of quantitative detection of substances, in particular to a method for simultaneously detecting the content of seven saturated fatty acids in a feed flavoring agent. The method comprises the following steps: respectively preparing a to-be-detected sample solution and standard working solutions of seven saturated fatty acids, and carrying out chromatographic detection by adopting a liquid chromatograph with a differential refraction detector; according to the method, the seven saturated fatty acids can be accurately qualitatively and quantitatively detected simultaneously through a liquid chromatograph with a differential refraction detector by utilizing the specific generality and difference of the seven saturated fatty acids under the differential refraction detector and combining with pertinently selected liquid chromatography conditions, and the detection method is convenient and rapid in process, accurate and reliable in result and high in detection accuracy. Equipment preparation is common and easy to obtain, and the method is suitable for large-scale rapid determination of the content of seven saturated fatty acids.

Description

Technical Field

[0001] The present invention relates to the technical field of quantitative detection of substances, mainly to the quantitative detection technology of saturated fatty acids, and in particular to a method for simultaneously detecting the contents of seven saturated fatty acids in feed flavorings. Background Art

[0002] Heptanoic acid (English: Heptanoic Acid), also known as grape flower acid, poison celery acid, heptanoic acid, chemical formula is C7H 14 O2, molecular weight 130.18, CAS registration number 111-14-8, is a colorless, oily liquid with a fatty odor, which can be foul when impure. It is slightly soluble in water but soluble in ethanol, ether, dimethylformamide, and dimethyl sulfoxide. It is primarily used in the production of heptanoic acid esters, as a raw material for fragrances, antifungal drugs, chemical reagents, organic synthesis raw materials, and solvents. It is commonly used in baked goods, candies, jams, and gels.

[0003] Caprylic acid (English: n-Caprylic acid), also known as caprylic acid, is an organic compound and a saturated fatty acid with a molecular formula of C8H 16 O2, molecular weight 144.211, CAS registration number 124-07-2, colorless, transparent, oily liquid. It has a slightly unpleasant odor, which develops a fruity aroma upon dilution. It is slightly soluble in hot water but soluble in ethanol, benzene, and ether. It occurs naturally in nutmeg, lemongrass, apples, coconut oil, and wine. It is primarily used in the manufacture of dyes, pharmaceuticals, fragrances, plasticizers, and lubricants. It can also be used as a preservative and fungicide. It is produced by saponifying coconut oil, followed by acidification and fractional distillation.

[0004] Nonanoic acid (English: Nonanoic Acid), also known as n-nonanoic acid, has a molecular formula of C9H 18 O2, molecular weight 158.238, CAS registration number 112-05-0, is a colorless or pale yellow oily liquid with a faint fatty and coconut aroma. It is insoluble in water but soluble in organic solvents such as ethanol, chloroform, and ether. It is permitted as a flavoring in my country according to GB2760-86 and is primarily used to prepare coconut and berry flavors.

[0005] Capric acid, also known as capric acid, n-decanoic acid, and decanoic acid, is an organic compound and a saturated fatty acid with a molecular formula of C 10 H 20 O2, molecular weight 172.26, CAS registration number 334-48-5, is a white solid at room temperature with an unpleasant odor. It is insoluble in water but soluble in ethanol, dilute nitric acid, and most organic solvents. It is a byproduct of the hydrolysis of laurel oil, coconut oil, or litsea cubeba oil to produce lauric acid. It is primarily used to produce capric esters, and its esters are used as fragrances, wetting agents, plasticizers, and food additives.

[0006] Lauric acid (English: Laμric acid), also known as dodecanoic acid, is a saturated fatty acid with a molecular formula of C 12 H 24 O2. Molecular weight 200.318, CAS registration number 143-07-7, white needle-shaped crystals, insoluble in water, soluble in organic solvents such as methanol, ether, and chloroform, and slightly soluble in acetone and petroleum ether. It has a slight laurel oil aroma. Despite its name, lauric acid only accounts for 1-3% of laurel oil. It is primarily used in the manufacture of alkyd resins, chemical fiber oils, pesticides, synthetic fragrances, plastic stabilizers, and as an anti-corrosion additive for gasoline and lubricants.

[0007] Myristic acid (English: Myristic acid), also known as tetradecanoic acid, is a saturated fatty acid with a molecular formula of C 14 H 28 O2, molecular weight 228.37, CAS registration number 544-63-8, is a white to yellowish-white hard solid, occasionally a lustrous crystalline solid, or a white to yellowish-white powder, odorless. It is insoluble in water but soluble in anhydrous ethanol, methanol, ether, petroleum ether, benzene, and chloroform. It occurs naturally as a glyceride in vegetable oils such as cardamom oil (70%-80%), palm oil (1%-3%), and coconut oil (17%-20%). It can be used to formulate various flavorings.

[0008] Myristic acid, also known as hexadecanoic acid, hexadecanoic acid, and palmitic acid, is an organic substance and a saturated higher fatty acid with a molecular formula of C 16 H 32 Palmitic acid, molecular weight 256.424, CAS registration number 57-10-3, white flakes with a pearly sheen. Insoluble in water, soluble in ethanol, and readily soluble in ether, chloroform, and acetic acid. It is widely present in nature, with varying amounts of palmitic acid present in almost all oils and fats. It is used as a precipitant, chemical reagent, and waterproofing agent.

[0009] Currently, there are numerous methods for detecting these saturated fatty acids, including gas chromatography, gas chromatography-mass spectrometry (GC-MS), near-infrared reflectance spectroscopy, thin-layer chromatography, capillary electrophoresis, and liquid chromatography-mass spectrometry (LC-MS). Gas chromatography or GC-MS is the most commonly used method for detecting saturated fatty acids. However, because saturated fatty acids are unstable and easily degraded at high temperatures, they are prone to loss during analysis. Therefore, when detecting long-chain fatty acids (such as myristic acid and hexadecanoic acid), GC is generally not used directly. Instead, they are converted into fatty acid methyl esters (FAMs) and then indirectly determined using GC.

[0010] Chinese patent CN109655541B discloses a method for determining the content of myristic acid. In this method, the methylation of fatty acids is the most critical step in the detection of fatty acids by gas chromatography-mass spectrometry. Whether the fatty acid methylation is complete directly affects the accuracy of qualitative and relative content detection of fatty acids in the sample. Therefore, the accuracy of this detection method is affected by more factors, resulting in poor reliability and stability of its detection results. In addition, the steps are relatively complex, which is not conducive to large-scale promotion and application.

[0011] Chinese patent CN 113281425B discloses a method for detecting free fatty acids in polyoxyethylene (35) castor oil. The method uses liquid chromatography-mass spectrometry to detect polyoxyethylene (35) castor oil samples. Although the detection results are accurate and reliable, the method cannot detect seven saturated fatty acids simultaneously, the detection time is long, and the detection equipment cost is high, which is not conducive to large-scale promotion and application.

[0012] It can be seen that the existing quantitative detection methods of saturated fatty acids have the problems of poor reliability and stability of detection results, complex steps, high equipment costs and long detection time, and are not suitable for large-scale promotion and application. Summary of the Invention

[0013] The purpose of the present invention is to overcome the problems of existing quantitative detection methods for seven saturated fatty acids (heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid), such as poor reliability and stability of detection results, complex procedures, high equipment costs, and long detection time. A method for simultaneously detecting the content of seven saturated fatty acids in feed flavorings is proposed.

[0014] To achieve the above-mentioned object, the present invention proposes a method for simultaneously detecting the contents of seven saturated fatty acids in a feed flavoring, comprising the following steps: preparing a sample solution to be tested and standard working solutions of seven saturated fatty acids, respectively, and performing chromatographic detection using a liquid chromatograph with a differential refractive index detector (HPLC-RID); Among them, the chromatographic conditions of the liquid chromatograph are: Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: pH 2-4, a mixed aqueous solution of 75% by volume acetonitrile and 0.1% by volume triethylamine; Flow rate: 1.0 mL / min; Detector temperature: 35°C; Column temperature: 30±0.8℃.

[0015] The present invention discloses a method for simultaneously detecting the contents of seven saturated fatty acids in a feed flavoring. The method utilizes the unique similarities and differences of the seven saturated fatty acids under a differential refractive index detector (the retention times of the seven saturated fatty acids under the detection conditions vary greatly, making qualitative and quantitative distinction easy), combined with specifically selected liquid chromatography conditions, so that the seven saturated fatty acids can be simultaneously and accurately qualitatively and quantitatively detected by a liquid chromatograph equipped with a differential refractive index detector. The detection method is convenient and rapid, the results are accurate and reliable, the equipment is common and readily available, and the method is suitable for large-scale rapid determination of the contents of the seven saturated fatty acids.

[0016] Among them, preferably, in the standard working solutions of the seven saturated fatty acids, the concentrations of each saturated fatty acid include 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL and 100.0 μg / mL, respectively.

[0017] Preferably, the solvent in the standard working solution is ethanol; using ethanol as a solvent has a good dissolving effect, will not affect the chromatographic results, and is conducive to improving the accuracy of the test results.

[0018] Among them, preferably, the preparation method of the test sample solution is: weigh a certain amount of the test sample and dissolve it in ethanol, adjust the pH value to 2-4, fix the volume, and filter to obtain the test sample solution; the formulation method is simple and convenient for rapid detection.

[0019] Preferably, a phosphoric acid solution is used for pH adjustment; more preferably, the concentration of the phosphoric acid solution is 1 mol / L; using phosphoric acid to adjust the pH value can avoid interference with the detection results during the chromatography process.

[0020] Preferably, a 0.22 μm organic filter membrane is used for filtration; the filtration effect is good and the speed is fast, which is conducive to improving the detection accuracy.

[0021] Among them, preferably, phosphoric acid solution is used to adjust the pH value of the acetonitrile aqueous solution; more preferably, the concentration of the phosphoric acid solution is 1 mol / L; using phosphoric acid to adjust the pH value of the mobile phase can avoid interference with the detection results during the chromatography process.

[0022] Wherein, preferably, the specific steps of chromatographic detection include: A. Use a liquid chromatograph with a differential refractive index detector to detect the standard working solutions of the seven saturated fatty acids, and perform multi-point calibration based on the standard working solutions of the seven saturated fatty acids to draw standard curves for the seven saturated fatty acids; The sample solution to be tested is detected by a liquid chromatograph equipped with a differential refractive index detector to obtain a chromatogram of the sample to be tested; B. Based on the standard curves of the seven saturated fatty acids, the seven saturated fatty acids were qualitatively and quantitatively analyzed simultaneously according to the retention time and peak area on the chromatogram of the sample to be tested to obtain the test results.

[0023] Wherein, the standard curve is a curve of the relationship between the peak area of ​​saturated fatty acid and the concentration of the standard working solution in the saturated fatty acid standard working solution.

[0024] Wherein, preferably, in step B, the qualitative analysis includes: based on the principle that the retention time of the same saturated fatty acids is the same, qualitatively analyzing the seven saturated fatty acids according to the retention time on the chromatogram of the sample to be tested.

[0025] Preferably, the quantitative analysis includes: based on the qualitative analysis results and the standard curve of the corresponding saturated fatty acids, according to the peak area of ​​the corresponding saturated fatty acids on the chromatogram of the sample to be tested, obtaining the concentration of the corresponding saturated fatty acids in the sample solution to be tested; and then according to the concentration of the corresponding saturated fatty acids in the sample solution to be tested, respectively calculating the content of the corresponding saturated fatty acids in the sample to be tested.

[0026] Preferably, the calculation formula is: ; Where X is the content of the corresponding saturated fatty acid in the sample to be tested (%); C is the concentration of the corresponding saturated fatty acid in the sample solution to be tested obtained from the standard curve (mg / mL); V is the total volume of the sample solution to be tested (mL); and m is the weighed mass of the sample to be tested (g). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a chromatogram of seven saturated fatty acids in Example 1 of the present invention; Figure 2 This is a standard curve diagram of heptanoic acid in Example 1 of the present invention; Figure 3 This is a standard curve diagram of octanoic acid in Example 1 of the present invention; Figure 4 This is a standard curve diagram of nonanoic acid in Example 1 of the present invention; Figure 5 This is a standard curve diagram of capric acid in Example 1 of the present invention; Figure 6 This is a standard curve diagram of dodecanoic acid in Example 1 of the present invention; Figure 7 This is a standard curve diagram of myristic acid in Example 1 of the present invention; Figure 8 This is a standard curve diagram of hexadecanoic acid in Example 1 of the present invention; Figure 9 This is the chromatogram of sample 1 in Example 1 of the present invention; Figure 10This is the chromatogram of sample 2 in Example 1 of the present invention; Figure 11 This is the chromatogram of sample 3 in Example 1 of the present invention; Figure 12 This is the chromatogram of sample 4 in Example 1 of the present invention; Figure 13 This is the chromatogram of sample 5 in Example 1 of the present invention; Figure 14 This is the chromatogram of sample 6 in Example 1 of the present invention; Figure 15 This is the chromatogram of sample 7 in Example 1 of the present invention; Figure 16 This is a chromatogram of 7 saturated fatty acids in Comparative Example 1 of the present invention; Figure 17 This is a chromatogram of 7 saturated fatty acids in Comparative Example 2 of the present invention; Figure 18 This is a chromatogram of 7 saturated fatty acids in Comparative Example 3 of the present invention; Figure 19 This is a chromatogram of 7 saturated fatty acids in Comparative Example 4 of the present invention; Figure 20 This is a chromatogram of 7 saturated fatty acids in Comparative Example 5 of the present invention; Figure 21 This is a chromatogram of 7 saturated fatty acids in Comparative Example 6 of the present invention; Figure 22 This is a chromatogram of 7 saturated fatty acids in Comparative Example 7 of the present invention; Figure 23 This is a chromatogram of 7 saturated fatty acids in Comparative Example 8 of the present invention; Figure 24 This is a chromatogram of 7 saturated fatty acids in Comparative Example 9 of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the embodiments described are only some embodiments of the present disclosure, not all embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present disclosure.

[0029] Example 1

[0030] A method for simultaneously detecting the content of seven saturated fatty acids in feed flavorings, comprising the following steps: 1. Prepare the sample solution: Accurately weigh 5.0-10.0g (accurate to 0.0001g) of the sample to be tested (7 samples) into a 100mL volumetric flask, add ethanol, and sonicate to dissolve for 30 minutes. Adjust the pH to 3 with phosphoric acid (1mol / L). After cooling to room temperature, dilute to 100ml, shake well, and filter with a 0.22μm organic filter membrane. The filtrate is the sample solution to be tested. Prepare standard working solutions of seven saturated fatty acids: accurately weigh 0.2000 g of each of the seven saturated fatty acids (purity > 98%) into a 100 mL volumetric flask, add ethanol and sonicate until completely dissolved, then cool and dilute to obtain a standard stock solution (2.0 mg / mL); the standard stock solution is stored at 4°C with a shelf life of 1 month; accurately measure 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5 mL of the standard stock solution into a 100 mL volumetric flask, add ethanol to dilute to volume, and mix to obtain solutions with concentrations of 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively, which are the standard working solutions; 2. Use liquid chromatography with differential refractive index detector (HPLC-RID) to detect the standard working solutions of 7 saturated fatty acids respectively, and obtain the chromatogram of the standard working solution ( Figure 1 ), and based on the chromatogram of the standard working solution, the retention time and standard curve of the seven saturated fatty acids were obtained simultaneously (after multi-point calibration based on the standard working solution of the seven saturated fatty acids, the peak areas of the seven saturated fatty acids were used to perform linear regression on their corresponding mass concentrations. The curves corresponding to the linear regression equations were the standard curves of the seven saturated fatty acids; see Table 1, Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 shown); Table 1 Retention times and standard curves of seven saturated fatty acids Detection object Retention time (min) Curve equation Correlation coefficient r Heptanoic acid 4.097 Y=345.0847X-1306.53441 0.99967 bitter 4.758 Y=550.4384X-49.0615 0.99971 Nonanoic acid 5.716 Y=566.7928X-140.7799 0.99999 Decanoic acid 7.147 Y=575.2519X-3.0482 0.99999 Dodecanoic acid 12.482 Y=586.2218X+90.0669 0.99997 Myristate 24.371 Y=616.5928X-103.6450 0.99999 Hexadecanoic acid 50.552 Y=610.5034X+87.0281 0.99991 The sample solution was tested using a liquid chromatograph with a differential refractive index detector (HPLC-RID) to obtain a chromatogram of the sample ( Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 , Figure 15 ); The chromatographic conditions of the liquid chromatograph are: Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: pH 2.5, a mixed aqueous solution of 75% by volume acetonitrile and 0.1% by volume triethylamine; Flow rate: 1.0 mL / min; Detector temperature: 35°C; Column temperature: 30°C; Injection volume: 10 μL; 3. Based on the standard curves of the seven saturated fatty acids and the retention time and peak area on the chromatogram of the sample to be tested, the seven saturated fatty acids were qualitatively analyzed (based on the principle that the retention time of the same saturated fatty acid is the same, the seven saturated fatty acids were qualitatively analyzed according to the retention time on the chromatogram of the sample to be tested) and quantitatively analyzed (based on the qualitative analysis results and the standard curves of the corresponding saturated fatty acids, according to the peak area of ​​the corresponding saturated fatty acid on the chromatogram of the sample to be tested, the concentration of the corresponding saturated fatty acid in the sample solution to be tested was obtained; then, according to the concentration of the corresponding saturated fatty acid in the sample solution to be tested, the content of the corresponding saturated fatty acid in the sample to be tested was calculated), and the test results were obtained (see Table 2).

[0031] Table 2 Detection results of 7 saturated fatty acids in the sample to be tested in Example 1 Detection object Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Heptanoic acid 0.50% / / 0.41% / / 0.10% bitter / 0.10% / / 0.35% / 0.22% Nonanoic acid / / / / 0.40% / 0.34% Decanoic acid / 0.18% / / 0.53% 0.39% 0.18% Dodecanoic acid 0.51% 0.37% 0.81% 0.56% / 0.53% 0.08% Myristate / 0.29% 0.49% / / / 0.11% Hexadecanoic acid 0.47% / 0.42% / / 0.35% 0.22% The calculation formula is: ; Where X is the content of the corresponding saturated fatty acid in the sample to be tested (%); C is the concentration of the corresponding saturated fatty acid in the sample solution to be tested obtained from the standard curve (mg / mL); V is the total volume of the sample solution to be tested (mL); and m is the weighed mass of the sample to be tested (g).

[0032] Experimental Example 1: The detection limit, quantification limit, repeatability, precision and recovery rate of "a method for simultaneously detecting the contents of seven saturated fatty acids in feed flavorings" in Example 1 were verified.

[0033] Detection limit: Accurately measure 10 μL of a low-concentration solution of seven saturated fatty acids and inject it into the liquid chromatograph. Repeat the injection six times. Calculate the measured concentrations of the seven saturated fatty acids based on the standard curve. Then calculate the standard deviation of the six measured concentrations. Three times the standard deviation is the detection limit of the seven saturated fatty acids in this method (see Table 3 for the detection limit).

[0034] Limit of quantitation: Accurately measure 10 μL of 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, and 20.0 μg / mL solutions of seven saturated fatty acids and inject them into the liquid chromatograph six times. Calculate the concentrations of the seven saturated fatty acids based on the standard curve. Calculate the standard deviation of the six measured concentrations. Six times the standard deviation is the limit of quantitation for the acid in this method (see Table 3 for the limit of quantitation). Table 3 Detection limits and quantification limits of 7 saturated fatty acids Detection object Detection limit (μg / mL) Limit of quantification (μg / mL) Heptanoic acid 5.0 10 bitter 2.5 5 Nonanoic acid 2.5 5 Decanoic acid 2.5 5 Dodecanoic acid 2.5 5 Myristate 2.5 5 Hexadecanoic acid 5.0 10 Repeatability: Sample 5 was injected 6 times continuously. The results are shown in Table 4.

[0035] Table 4 Repeatability test results of sample 5 Object under test 1 2 3 4 5 6 bitter 0.35% 0.35% 0.37% 0.34% 0.36% 0.34% Nonanoic acid 0.40% 0.38% 0.38% 0.39% 0.39% 0.41% Decanoic acid 0.53% 0.54% 0.54% 0.54% 0.54% 0.59% Precision: Six replicates were performed on the homemade samples, and the theoretical values ​​of the contents of the seven analytes were all 4%; the results are shown in Tables 5, 6, 7, 8, 9, 10, and 11; Table 5 Heptanoic acid precision test results

[0036] Table 6 Caprylic acid precision test results

[0037] Table 7 Nonanoic acid precision test results

[0038] Table 8 Decanoic acid precision test results

[0039] Table 9 Dodecanoic acid precision test results

[0040] Table 10 Myristate precision test results

[0041] Table 11 Hexadecanoic acid precision test results

[0042] The results showed that according to the method and conditions of Example 1, the relative standard deviations of heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid in the seasoning sample were 3.27%, 1.75%, 0.34%, 0.19%, 0.81%, 0.31%, and 0.27%, respectively; all were less than 5%, indicating good precision test results.

[0043] Recovery: Recovery tests were conducted using various blank matrices, including silica, fine bran, sodium sulfate, and glucose. Spiked samples contained seven saturated fatty acids at a concentration of 160 μg / mL. Six replicates were run, and the recoveries were calculated. The results are shown in Table 12.

[0044] Table 12 Experimental results of recovery rates of seven acids

[0045] The results showed that the average recovery rates of the seven saturated fatty acids were between 99.42% and 101.94% according to the above method and conditions, indicating that the method has good precision and accuracy.

[0046] Comparative Example 1 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a 65% acetonitrile aqueous solution and the pH was adjusted to 2.5 using acetic acid; the chromatograms were as shown in FIG. Figure 16 shown.

[0047] The chromatographic results show that the peak of dodecanoic acid is good, the peak shapes of heptanoic acid, octanoic acid, nonanoic acid and decanoic acid are poor, and tetradecanoic acid and hexadecanoic acid are not detected within 60 minutes.

[0048] Comparative Example 2 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a 70% acetonitrile aqueous solution and the pH was adjusted to 2.5 using acetic acid; the chromatograms were as shown in FIG. Figure 17 shown.

[0049] The chromatographic results show that the peak shape of decanoic acid is good, but the peak shapes of heptanoic acid, octanoic acid, nonanoic acid and dodecanoic acid are not good, and tetradecanoic acid and hexadecanoic acid were not detected within 50 minutes.

[0050] Comparative Example 3 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a 70% acetonitrile aqueous solution with a pH of 2.5 adjusted with phosphoric acid; the chromatogram was as shown in FIG. Figure 18 shown.

[0051] The chromatographic analysis results show that myristate and hexadecanoic acid can be detected within 50 minutes, but the peak shape is not ideal.

[0052] Comparative Example 4 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a mixed aqueous solution of acetonitrile with a volume fraction of 0.1% triethylamine and a volume fraction of 65% acetonitrile, the pH value of which was adjusted to 2.5 using phosphoric acid; the chromatogram was as shown in FIG. Figure 19 shown.

[0053] The chromatographic results show that the peak shapes of the seven saturated fatty acids are good, but the peak elution time is too slow, and it takes 2 hours to complete the detection.

[0054] Comparative Example 5 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a mixed aqueous solution of 80% acetonitrile containing 0.1% triethylamine by volume and adjusted to pH 2.5 with phosphoric acid; the chromatogram was as shown in FIG. Figure 20 shown.

[0055] The chromatographic analysis results show that the peak shapes of the seven saturated fatty acids are good, but the elution time of heptanoic acid and octanoic acid is too fast, which is not conducive to qualitative analysis.

[0056] Comparative Example 6 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the mobile phase was a mixed aqueous solution of acetonitrile with a volume fraction of 75% and a pH value of 2.5 adjusted with phosphoric acid and containing 0.1% triethylamine; the chromatogram was as shown in FIG. Figure 21 shown.

[0057] The chromatographic results showed that the peak shapes of the seven saturated fatty acids were good and the detection was completed within 50 minutes.

[0058] Comparative Example 7 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that phosphoric acid was not used to adjust the pH value of the test samples; the chromatograms are shown in FIG. Figure 22 shown.

[0059] The analysis of the chromatographic results showed that the baseline of the sample solution to be tested was unstable.

[0060] Comparative Example 8 The same method as in Example 1 was used to detect the standard working solutions of seven saturated fatty acids, except that the pH value of the sample to be tested was adjusted to 2.5 using phosphoric acid; the chromatograms are shown in FIG. Figure 23 shown.

[0061] The chromatographic results show that the baseline of the sample solution to be tested is stable.

[0062] Comparative Example 9 The same method as in Example 1 was used to detect the standard working solutions of 7 saturated fatty acids, except that the chromatographic column was imported; the chromatogram was as shown in FIG. Figure 24 shown.

[0063] Analysis of the chromatographic results showed that the peak shape and elution time were basically consistent with those in Example 1, and the origin of the chromatographic column had no effect on the chromatographic results.

[0064] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0065] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for simultaneously detecting the contents of seven saturated fatty acids in feed flavorings, characterized in that: The method comprises the following steps: respectively preparing a sample solution to be tested and standard working solutions of seven saturated fatty acids, and performing chromatographic detection using a liquid chromatograph with a differential refractive index detector; Among them, the chromatographic conditions of the liquid chromatograph are: Chromatographic column: Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: pH 2-4, a mixed aqueous solution of 75% by volume acetonitrile and 0.1% by volume triethylamine; Flow rate: 1.0 mL / min; Detector temperature: 35°C; Column temperature: 30±0.8℃.

2. The method according to claim 1, characterized in that The solvent in the standard working solution is ethanol.

3. The method according to claim 1, characterized in that The method for preparing the test sample solution is as follows: weigh a certain amount of the test sample, dissolve it in ethanol, adjust the pH value to 2-4, fix the volume, and filter to obtain the test sample solution.

4. The method according to claim 3, characterized in that The pH value was adjusted using phosphoric acid solution.

5. The method according to claim 3, characterized in that Filter using a 0.22 μm organic filter membrane.

6. The method according to claim 1, characterized in that The pH value of the acetonitrile aqueous solution was adjusted with phosphoric acid solution.

7. The method according to any one of claims 1 to 6, characterized in that The specific steps of chromatographic detection include: A. Use a liquid chromatograph with a differential refractive index detector to detect the standard working solutions of the seven saturated fatty acids, and perform multi-point calibration based on the standard working solutions of the seven saturated fatty acids to draw standard curves for the seven saturated fatty acids; The sample solution to be tested is detected by a liquid chromatograph equipped with a differential refractive index detector to obtain a chromatogram of the sample to be tested; B. Based on the standard curves of the seven saturated fatty acids, the seven saturated fatty acids were qualitatively and quantitatively analyzed simultaneously according to the retention time and peak area on the chromatogram of the sample to be tested to obtain the test results.

8. The method according to claim 7, characterized in that In step B, the qualitative analysis includes: based on the principle that the same saturated fatty acids have the same retention time, the seven saturated fatty acids are qualitatively analyzed according to their retention times on the chromatogram of the sample to be tested.

9. The method according to claim 8, characterized in that Quantitative analysis includes: based on the qualitative analysis results and the standard curve of the corresponding saturated fatty acids, according to the peak area of ​​the corresponding saturated fatty acids on the chromatogram of the sample to be tested, obtaining the concentration of the corresponding saturated fatty acids in the sample solution to be tested; and then according to the concentration of the corresponding saturated fatty acids in the sample solution to be tested, respectively calculating the content of the corresponding saturated fatty acids in the sample to be tested.

10. The method according to claim 9, characterized in that The calculation formula is: ; Wherein, X is the content of the corresponding saturated fatty acid in the sample to be tested; C is the concentration of the corresponding saturated fatty acid in the sample solution to be tested obtained from the standard curve; V is the total volume of the sample solution to be tested; and m is the weighed mass of the sample to be tested.

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