Determination method of short-chain free fatty acid and application thereof

Abstract

The invention belongs to the technical field of food safety, and particularly relates to a short-chain free fatty acid determination method and application thereof. The determination method of the short-chain free fatty acid comprises the following steps: S1, extracting the short-chain free fatty acid; s2, derivatization reaction of short-chain free fatty acid: under the protection of argon, adding a dichloromethane solution containing 1.5 mL of a fluorescent derivatization reagent into a drying flask containing 6mg of an extract, stirring for 2 hours at room temperature under the condition of blue light, and performing HPLC (High Performance Liquid Chromatography) determination after the reaction is finished; and S3, determining the short-chain free fatty acid. The novel and efficient fluorescent derivatization reagent is used in the determination method, and the determination method can improve the sensitivity and the accuracy of the HPLC method for determining the short-chain free fatty acid.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety, and in particular relates to a method for determining short-chain free fatty acids and an application thereof. Background Art

[0002] Goat milk or goat milk products have high nutritional value, rich in protein, fat, vitamins, minerals and other ingredients, and the overall nutritional content is better than that of cow milk, closer to human milk, and easy to absorb, so it is called the "king of milk". At the same time, the lactose content in goat milk or goat milk products is low, which is a better choice for patients with lactose intolerance. However, the sales volume of goat milk or goat milk products is much lower than that of cow milk, which is mainly related to the mutton smell of goat milk or goat milk products, which is also one of the main factors restricting the development and utilization of goat milk products. A large number of studies have shown that the mutton smell in goat milk or goat milk products is related to the content of free fatty acids, among which short-chain free fatty acids capric acid, caprylic acid and capric acid are the main components of the mutton smell of goat milk or goat milk products. Therefore, the determination of these three short-chain free fatty acids is crucial to the study and control of the mutton smell of goat milk or goat milk products.

[0003] At present, the industry generally uses gas chromatography (GC) and gas chromatography-tandem mass spectrometry (GC-MS) to detect short-chain free fatty acids, but these determination methods have obvious shortcomings, including: ① Short-chain free fatty acids are volatile and easily lost during derivatization and determination, and the stability and accuracy of the determination results are poor; ② The content of short-chain free fatty acids is low, and there is a lack of easily ionized functional groups in the structure, resulting in low sensitivity of mass spectrometry analysis; ③ GC uses high-purity nitrogen as a carrier, which has high requirements for experimental conditions. Compared with GC, high-performance liquid chromatography (HPLC) can be carried out at lower temperatures, and the degree of thermal degradation and loss of free fatty acids is greatly reduced. It has higher sensitivity, accuracy and reproducibility, and has gradually been applied to the detection of free fatty acids. However, short-chain free fatty acids do not contain chromophores or fluorescent groups, and cannot be directly detected at trace levels using ultraviolet spectrophotometers or fluorometers.

[0004] Therefore, it is particularly important to develop a method that uses a new and efficient fluorescent derivatization reagent to improve the sensitivity and accuracy of HPLC determination of short-chain free fatty acids. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for determining short-chain free fatty acids, which uses a new and efficient fluorescent derivatization reagent and can improve the sensitivity and accuracy of HPLC determination of short-chain free fatty acids.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for determining short-chain free fatty acids, the method comprising the following steps:

[0008] S1. Extraction of short-chain free fatty acids:

[0009] To 0.5-1 g of the sample to be tested, 2 mL of internal standard (heptanoic acid derivative) 5-30 mg, 1 mL of H2SO4 solution (concentration of 2.5 mol / L), 0.2 g of NaCl and a chloroform and methanol solution with a volume ratio of 2:1 were added, and the solution was extracted three times (the volume of the chloroform and methanol solution was 25 mL), and each time was allowed to stand for 1.5 h. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under low temperature conditions to obtain an extract;

[0010] S2. Derivatization reaction of short chain free fatty acids:

[0011] Under argon protection, a dichloromethane solution containing 1.5 mL of fluorescent derivatization reagent (0.1-0.8 mmol) was added to a dry flask containing 6 mg of the extract, and stirred at room temperature for 2 h under blue light. After the reaction was completed, it was used for HPLC determination;

[0012] S3. Determination of short-chain free fatty acids:

[0013] S3.1. A mixed standard solution is prepared with heptanoic acid derivatives as internal standards and hexanoic acid, octanoic acid and decanoic acid derivatives as standard substances, wherein the mass ratio of hexanoic acid derivative to internal standard is 0.020 to 10.48, the mass ratio of octanoic acid derivative to internal standard is 0.0024 to 4.76, and the mass ratio of decanoic acid derivative to internal standard is 0.032 to 11.33;

[0014] S3.2.

[0015] The method for HPLC determination of hexanoic acid and octanoic acid was isocratic elution: 68% methanol;

[0016] The method for HPLC determination of decanoic acid is gradient elution: 0-20 min, 68% methanol; 21-27 min, 70% methanol; 28-35 min, 74% methanol; 36-52 min, 78% methanol; 53-59 min, 100% methanol;

[0017] Drawing a standard curve: using the injection amount ratio of each standard to the heptanoic acid derivative as the abscissa and the peak area ratio of each standard to the heptanoic acid derivative as the ordinate to draw a standard curve and obtain a linear regression equation;

[0018] The method for drawing the standard curves for hexanoic acid and octanoic acid was isocratic elution: 68% methanol;

[0019] The method for drawing the standard curve of decanoic acid is gradient elution: 0-20 min, 68% methanol; 21-27 min, 70% methanol; 28-35 min, 74% methanol; 36-52 min, 78% methanol; 53-59 min, 100% methanol;

[0020] S3.3. Use HPLC to determine the sample to be tested, obtain the peak area ratio of the component to be analyzed and the heptanoic acid derivative, substitute the peak area ratio into the standard curve, and calculate the content of hexanoic acid, octanoic acid and decanoic acid in the sample to be tested.

[0021] S3.3. Use HPLC to determine the sample to be tested, obtain the peak area ratio of the component to be analyzed and the heptanoic acid derivative, substitute the peak area ratio into the standard curve, and calculate the content of hexanoic acid, octanoic acid and decanoic acid in the sample to be tested.

[0022] Formula (I)

[0023] The calculation formula of the hexanoic acid content in the sample is shown in formula (I). Wherein, A is the peak area ratio of the hexanoic acid derivative to the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0024] Formula (II)

[0025] The calculation formula of the octanoic acid content in the sample is shown in formula (II). Wherein, B is the peak area ratio of the octanoic acid derivative to the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0026] Formula (III)

[0027] The calculation formula for the decanoic acid content in the sample is shown in formula (III). Wherein, C is the ratio of the peak area of ​​the decanoic acid derivative to the peak area of ​​the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0028] Preferably, the short-chain free fatty acids are caproic acid, caprylic acid and capric acid.

[0029] Preferably, the fluorescent derivatization reagent is 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester.

[0030] Preferably, the conditions for HPLC determination in step S3.2 are: flow rate of 1.0 mL / min, column temperature of 30° C., detection wavelength of 254 nm, and injection volume of 10 μL.

[0031] Preferably, the sample to be tested is goat milk, ham, flour or oil.

[0032] In a second aspect, a method for determining mutton smell-related substances in goat milk or goat milk products is provided, including the determination method described in the present invention.

[0033] Preferably, the mutton smell-related substances are short-chain free fatty acids, and the short-chain free fatty acids are caproic acid, caprylic acid and capric acid.

[0034] A third aspect provides an application of the determination method of the present invention in the determination of caproic acid.

[0035] In a fourth aspect, the invention provides an application of the determination method of the present invention in the determination of octanoic acid.

[0036] A fifth aspect provides application of the determination method described in the present invention in determining capric acid.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] (1) Short-chain free fatty acids do not contain chromophores or fluorescent groups and are difficult to be directly used for HPLC analysis. The present invention uses 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester as a new type of fluorescent derivatization reagent, which greatly improves the sensitivity of detection;

[0039] (2) Short-chain free fatty acids have a certain volatility, and inappropriate derivatization reaction conditions will cause losses, resulting in large errors in the test results. The fluorescent derivatization reagent used in the present invention can react with short-chain free fatty acids quickly and efficiently at room temperature and pressure, thereby improving the pre-column derivatization efficiency of short-chain free fatty acids; and reducing volatility losses, thereby improving the accuracy of the final test results. In addition, no catalyst needs to be added during the reaction, and the reaction system is simple, which is very suitable for conventional detection of short-chain free fatty acids;

[0040] (3) The present invention adopts the internal standard curve method, which eliminates the influence of external factors such as human operation, temperature, instruments, etc. during the extraction, derivatization and testing of free fatty acids, making the measurement results more precise and accurate;

[0041] (4) The present invention adopts gradient elution in HPLC analysis, which can realize the detection of capric acid in complex samples;

[0042] (5) The present invention investigates the linearity, detection limit, quantification limit, specificity test (blank), precision, stability, and accuracy (recovery rate) of the method, proving that the content determination method is scientific and effective and can quantitatively analyze caproic acid, caprylic acid, and capric acid in complex samples;

[0043] (6) The present invention combines pre-column derivatization with HPLC, and can achieve accurate quantification of short-chain free fatty acids through the internal standard curve method, with a quantification limit of 1.13 to 3.50 μg / mL and a detection limit of 0.53 to 1.75 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 For the preparation of standards (1, 2, 3) and internal standard (4).

[0045] Figure 2 is a specificity test chromatogram; wherein, a is the mixed standard solution spectrum, 1, 4, 2 and 3 correspond to hexanoic acid, internal standard, octanoic acid and decanoic acid, respectively; b is the extracted free fatty acids; c is the derivatization reaction solution.

[0046] Figure 3 The following are the spectra of short-chain free fatty acids in goat milk; a is the spectrum of the mixed standard solution under the conditions of method 1; b is the determination of caproic acid; c is the determination of octanoic acid; d is the spectrum of the mixed standard solution under the conditions of method 2; and e is the detection of decanoic acid. 1, 4, 2, and 3 correspond to caproic acid, internal standard, octanoic acid, and decanoic acid, respectively. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Experimental part:

[0049] High performance liquid chromatography: Agilent 1260, including quaternary gradient elution device, 100-position autosampler, and diode array detector.

[0050] Chromatographic conditions: Hypersil GOLD (150×4.6 mm, 5 μm, Thermo, USA) liquid chromatography column, flow rate constant at 1.0 mL / min, column temperature maintained at 30°C, detection wavelength at 254 nm, mobile phase of methanol and water, injection volume of 10 μL.

[0051] method:

[0052] Method 1 is isocratic elution: 68% methanol.

[0053] Method 2 is gradient elution: 0-20 min, 68% methanol; 21-27 min, 70% methanol; 28-35 min, 74% methanol; 36-52 min, 78% methanol; 53-59 min, 100% methanol.

[0054] Method 3 is gradient elution: 0-20 min, 68% methanol; 21-25 min, 72% methanol; 26-35 min, 76% methanol; 36-46 min, 80% methanol; 47-57 min, 100% methanol.

[0055] Reagents: The mobile phase of HPLC was chromatographically pure, and all reagents in chemical reactions were analytically pure.

[0056] Samples to be tested: goat milk comes from the Saanen Sheep Breeding Farm of Northwest Agriculture and Forestry University; ham samples are taken from "Jinluo Chicken Ham Sausage"; flour samples are taken from "Laoniu Flour" in Shaanxi; oil is taken from "primary extracted low-erucic acid rapeseed oil".

[0057] Preparation of fluorescent derivatization reagents, standards, internal standards and mixed standard solutions:

[0058] ① Synthesis of fluorescent derivatization reagent: A mixture of methyl 4-chlorophenylacetate (0.77mL, 5mmol) and p-toluenesulfonyl azide (1.3mL, 6mmol) was dissolved in 10mL acetonitrile, cooled with an ice bath, and then DBU (1mL, 7mmol) was added. The reaction mixture was stirred overnight and slowly warmed to room temperature. TLC detection reaction, after the reaction was complete, saturated salt water was added, the reaction was quenched with ammonium chloride, extracted with dichloromethane, dried with Na2SO4, and the solvent was removed with a rotary evaporator. Petroleum ether was selected as the eluent, and silica gel column chromatography was performed to obtain 1.03g 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester, with a yield of 98%.

[0059] ② Synthesis of standard and internal standard: Under argon protection, 1.5 mL of a dichloromethane solution of a fluorescent derivatization reagent 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester (42 mg, 0.2 mmol) was added to a dry flask containing hexanoic acid, octanoic acid, decanoic acid or heptanoic acid (0.2 mmol), and stirred at room temperature for 2 h under illumination (blue light). After the reaction was completed, silica gel column chromatography (petroleum ether: ethyl acetate = 70:1) was used to obtain the corresponding derivatives 1-4 ( Figure 1 ). The structure of the compound was confirmed by NMR and ESI-MS.

[0060] ③ Preparation of mixed standard solution: Use chromatographic grade methanol as solvent, heptanoic acid derivatives as internal standards, hexanoic acid, octanoic acid and decanoic acid derivatives as standards to prepare mixed standard solution, the mass ratio of the three standards to the internal standard is 0.002 to 12. Among them, the mass ratio of hexanoic acid derivatives to the internal standard is 0.020 to 10.48, the mass ratio of octanoic acid derivatives to the internal standard is 0.0024 to 4.76, and the mass ratio of decanoic acid derivatives to the internal standard is 0.032 to 11.33.

[0061] The calculation formula of the hexanoic acid content in the sample is shown in formula (I). Wherein, A is the peak area ratio of the hexanoic acid derivative to the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0062] Formula (I)

[0063] The calculation formula of the octanoic acid content in the sample is shown in formula (II). Wherein, B is the peak area ratio of the octanoic acid derivative to the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0064] Formula (II) The calculation formula for the decanoic acid content in the sample is shown in formula (III). Wherein, C is the ratio of the peak area of ​​the decanoic acid derivative to the peak area of ​​the internal standard; m s is the mass of the internal standard added to the sample (g); m is the mass of the sample (g).

[0065] Formula (III)

[0066] Example 1 Determination of short-chain free fatty acid content in goat milk samples

[0067] 1.1 Extraction of short-chain free fatty acids:

[0068] To 1 g of milk sample, add 2 mL of internal standard (mass: 30 mg), 1 mL of H2SO4 solution (concentration: 2.5 mol / L), 0.2 g of NaCl and a chloroform and methanol solution with a volume ratio of 2:1 (the volume of chloroform and methanol solution is 25 mL), extract three times, let stand for 1.5 h each time, combine the organic phases, dry over anhydrous Na2SO4, and concentrate under low temperature conditions to obtain an extract.

[0069] 1.2 Derivatization reaction of short-chain free fatty acids:

[0070] ① Under argon protection, add 1.5 mL of a dichloromethane solution containing a fluorescent derivatization reagent, 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester (0.6 mmol), into a dry flask containing 6 mg of the extract and stir at room temperature for 2 h under blue light. After the reaction, use it for qualitative and quantitative analysis of octanoic acid and decanoic acid.

[0071] ② Under argon protection, add 1.5 mL of a dichloromethane solution of a fluorescent derivatization reagent 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester (0.4 mmol) into a dry flask containing 5.9 mg of the extract and stir at room temperature for 2 h under blue light. After the reaction is completed, use it for qualitative and quantitative analysis of hexanoic acid.

[0072] 1.3 Drawing of standard working curve

[0073] The standard products 1 and 2 derived from caproic acid and octanoic acid were measured by method 1, and the standard product 3 derived from capric acid was measured by method 2, and the chromatograms were recorded. The standard curve was drawn with the injection amount ratio of each standard product to the internal standard as the abscissa and the peak area ratio of each standard product to the internal standard as the ordinate to obtain the linear regression equation (Table 1). The signal-to-noise ratio (S / N) of the main peak was recorded. When the S / N was about 3, it was the detection limit, and when the S / N was about 10, it was the quantification limit.

[0074] Table 1 Method linearity, correlation coefficient, detection limit and quantification limit determination

[0075]

[0076] 1.4 Specificity test (blank)

[0077] Method 3 was used to determine the extracted free fatty acids and their derivatives. Compared with the spectrum of the mixed standard solution, it was found that almost no substances were detected in the underivatized sample, while the peaks corresponding to caprylic acid and capric acid were clearly seen after the derivatization reaction ( Figure 2 ), thus proving the effectiveness of the derivatization reagent in the present invention.

[0078] 1.5 Determination and method validation of short-chain free fatty acids

[0079] ① Method 1 was used to determine caproic acid and octanoic acid, and method 2 was used to determine decanoic acid. Compared with the spectrum of the mixed standard solution, goat milk contains caproic acid, octanoic acid and decanoic acid ( Figure 3 );

[0080] ② Under the corresponding chromatographic conditions (flow rate of 1.0 mL / min, column temperature of 30° C., detection wavelength of 254 nm), sample injection analysis was performed at 0, 2, 4, and 6 h, and the contents of caproic acid, caprylic acid, and capric acid in the sample to be tested were calculated by formula (I). The quantitative analysis results of caproic acid, caprylic acid, and capric acid are shown in Table 2, indicating that the method has good repeatability;

[0081] ③ Take the corresponding samples (injection volume is 10 μL), add 11.7, 12.6 and 10.4 mg of standard 1-3 to the samples respectively, and the measured spiked recoveries are shown in Table 2.

[0082] Table 2 Determination of short-chain free fatty acid content in goat milk

[0083]

[0084] Example 2 Determination of short-chain free fatty acid content in ham

[0085] 2.1 Extraction of free fatty acids: Weigh 0.5 g of ham sample, place it in a 10 mL centrifuge tube, add 2 mL of dichloromethane and 5 mg of internal standard, mix well, sonicate for 20 min, and take the supernatant.

[0086] 2.2 Derivatization reaction of free fatty acids: According to the reaction conditions of 1.2 in Example 1, 1 mL of the extracted supernatant was reacted with 0.1 mmol of methyl 2-diazo-2-(4-chlorophenyl)acetate and then used for HPLC analysis.

[0087] 2.3 Determination results: Hexanoic acid and capric acid were not detected, and the content of caprylic acid was 0.023±0.0008g / 100g.

[0088] Example 3 Determination of short-chain free fatty acid content in flour

[0089] 3.1 Extraction of free fatty acids: Weigh 0.5 g of wheat flour, place it in a 10 mL centrifuge tube, add 5 mL of dichloromethane and 5 mg of internal standard, mix well, sonicate for 20 min, and take the supernatant.

[0090] 3.2 Derivatization reaction of free fatty acids: According to the reaction conditions of 1.2 in Example 1, 1 mL of the extracted supernatant was reacted with 0.1 mmol of methyl 2-diazo-2-(4-chlorophenyl)acetate and then used for HPLC analysis.

[0091] 3.3 Determination results: Hexanoic acid and capric acid were not detected, and the content of caprylic acid was 0.030±0.0034g / 100g.

[0092] Example 4 Determination of short-chain free fatty acid content in oil

[0093] 4.1 Derivatization reaction: According to the reaction conditions of 1.2 in Example 1, 1 mL of the oil was directly reacted with 0.1 mmol of methyl 2-diazo-2-(4-chlorophenyl)acetate and then used for HPLC analysis.

[0094] 4.2 Determination results: Hexanoic acid, caprylic acid and capric acid were not detected.

[0095] Example 5 Determination of short-chain free fatty acid content in goat milk

[0096] Compared with Example 1, the only difference is that in 1.2①, the content of 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester is 0.4 mmol, the extract is 5.9 mg, and the measurement results show that the content of caprylic acid is 0.92 g / 100 g, which is lower than the measurement result in Example 1, and a very small amount of capric acid is detected.

[0097] Example 6 Determination of short-chain free fatty acid content in goat milk

[0098] Compared with Example 1, the only difference is that in 1.2②, the content of 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester is 0.8 mmol, the extract is 6.2 mg, and the measurement results show that the content of octanoic acid is 1.28 g / 100 g, and the content of decanoic acid is 0.18 g / 100 g, which are lower than the measurement results in Example 1, indicating that the derivatization reaction conditions of Example 1 are the optimal conditions.

[0099] Example 7 Determination of short-chain free fatty acid content in goat milk

[0100] Compared with Example 1, the only difference is that in 1.2②, the amount of 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester is 0.6 mmol, the amount of the extract is 6.0 mg, and no caproic acid is detected.

[0101] Example 8 Determination of short-chain free fatty acid content in goat milk

[0102] Compared with Example 1, the only difference is that in 1.2②, 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester is 0.2 mmol, the extract is 5.4 mg, and a very small amount of hexanoic acid is detected, indicating that the derivatization reaction conditions of Example 1 are the optimal conditions.

[0103] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for determining short-chain free fatty acids, characterized in that: The determination method comprises the following steps: S1. Extraction of short-chain free fatty acids: To 0.5-1 g of the sample to be tested, add 2 mL of internal standard, 1 mL of H2SO4 solution, 0.2 g of NaCl and a chloroform-methanol solution with a volume ratio of 2:1, and extract three times, each time standing for 1.5 h, combine the organic phases, dry over anhydrous Na2SO4, and concentrate under low temperature conditions to obtain an extract; S2. Derivatization reaction of short chain free fatty acids: Under argon protection, a dichloromethane solution containing 1.5 mL of fluorescent derivatization reagent was added to a dry flask containing 6 mg of the extract, and stirred at room temperature for 2 h under blue light. After the reaction was completed, it was used for HPLC determination; S3. Determination of short-chain free fatty acids: S3.

1. A mixed standard solution is prepared with heptanoic acid derivatives as internal standards and hexanoic acid, octanoic acid and decanoic acid derivatives as standard substances, wherein the mass ratio of hexanoic acid derivative to internal standard is 0.020 to 10.48, the mass ratio of octanoic acid derivative to internal standard is 0.0024 to 4.76, and the mass ratio of decanoic acid derivative to internal standard is 0.032 to 11.33; S3.

2. The method for HPLC determination of hexanoic acid and octanoic acid was isocratic elution: 68% methanol; The method for HPLC determination of decanoic acid is gradient elution: 0-20 min, 68% methanol; 21-27 min, 70% methanol; 28-35 min, 74% methanol; 36-52 min, 78% methanol; 53-59 min, 100% methanol; Drawing a standard curve: using the injection amount ratio of each standard to the heptanoic acid derivative as the abscissa and the peak area ratio of each standard to the heptanoic acid derivative as the ordinate to draw a standard curve and obtain a linear regression equation; The method for drawing the standard curves for hexanoic acid and octanoic acid was isocratic elution: 68% methanol; The method for drawing the standard curve of decanoic acid is gradient elution: 0-20 min, 68% methanol; 21-27 min, 70% methanol; 28-35 min, 74% methanol; 36-52 min, 78% methanol; 53-59 min, 100% methanol; S3.

3. Use HPLC to determine the sample to be tested, obtain the peak area ratio of the component to be analyzed and the heptanoic acid derivative, substitute the peak area ratio into the standard curve, and calculate the content of hexanoic acid, octanoic acid and decanoic acid in the sample to be tested.

2. The measuring method according to claim 1, characterized in that The short-chain free fatty acids are caproic acid, caprylic acid and capric acid.

3. The measuring method according to claim 1, characterized in that The fluorescent derivatization reagent is 2-diazo-2-(4-chlorophenyl)acetic acid methyl ester.

4. The measuring method according to claim 1, characterized in that The conditions for HPLC determination in step S3.2 are: flow rate of 1.0 mL / min, column temperature of 30° C., detection wavelength of 254 nm, and injection volume of 10 μL.

5. The measuring method according to claim 1, characterized in that The sample to be tested is goat milk, ham, flour or oil.

6. A method for determining mutton-related substances in goat milk or goat milk products, characterized in that: The method comprises the assay method according to any one of claims 1 to 4.

7. The measuring method according to claim 6, characterized in that The mutton smell-related substances are short-chain free fatty acids, and the short-chain free fatty acids are caproic acid, caprylic acid and capric acid.

8. Use of the assay method according to any one of claims 1 to 4 or claim 7 in the determination of caproic acid.

9. Use of the assay method according to any one of claims 1 to 4 or claim 7 in the determination of octanoic acid.

10. Use of the determination method according to any one of claims 1 to 4 or claim 7 in the determination of capric acid.

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