Post-column derivatization detection method of ethyl lauroyl arginate

Abstract

The invention discloses a post-column derivatization detection method of ethyl lauroyl arginate, which comprises the following steps: pre-treating food, extracting ethyl lauroyl arginate hydrochloride with an acetonitrile aqueous solution, filtering with an aqueous filter membrane, separating with a high performance liquid chromatography column, taking mercaptan and o-phthalaldehyde as derivatization reagents, and carrying out post-column derivatization detection on ethyl lauroyl arginate under an alkaline condition, so as to obtain the ethyl lauroyl arginate. And carrying out a derivative reaction to generate a fluorescent derivative, carrying out derivative fluorescence detection, and quantitatively determining the content of the ethyl lauroyl arginate hydrochloride. The method has the characteristics of rapidness, high sensitivity and low detection limit. The method is suitable for determination of trace ethyl lauroyl arginate hydrochloride in dairy products, fruit products, meat products, aquatic products, beverages or gum-based candies. And a detection method is provided for the detection of trace ethyl cinnamyl arginate hydrochloride.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and particularly relates to a method for high performance liquid chromatography (HPLC) separation - chemical derivation preparation - fluorescence spectrum inspection of ethyl lauroyl arginate. The method of the present invention can be used in the fields of cosmetics, drug and food detection. Background Art

[0002] Ethyl lauroyl arginate hydrochloride (LAE), also known as Ethyl lauroyl arginate HCL, alias ethyl lauroyl arginine hydrochloride, has the molecular formula C 20 H 41 ClN4O3, molecular weight 421.01754, CAS number 60372 - 77 - 2, LogP value 1.43 at 20 °C, chemical formula: as shown in Formula 1:

[0003]

[0004] Ethyl lauroyl arginate hydrochloride is a new type of antibacterial agent. Ethyl lauroyl arginate hydrochloride has strong antibacterial effects on Gram - negative and Gram - positive cocci, yeasts and molds. Its mechanism of action lies in that ethyl lauroyl arginate hydrochloride is a cationic surfactant, which can affect the negatively charged membrane proteins and enzymatic mechanisms inside the bacterial cell membrane, change the cell membrane permeability, and achieve the effect of inhibiting the growth of microorganisms or inactivating microorganisms.

[0005] Ethyl lauroyl arginate hydrochloride has excellent safety. Its intermediate metabolites and final products in the human body are all natural endogenous substances. It has been proven by a large number of experiments to be a low - toxicity, safe and highly efficient preservative, and has a wide range of applications in the food and cosmetics industries, with broad market prospects and potential commercial value.

[0006] In 2005, the US FDA signed a no - objection letter, announcing that ethyl lauroyl arginate hydrochloride passed the generally recognized as safe (GRAS) food certification. At the same time, the US Department of Agriculture also allowed its use in meat and poultry products.

[0007] In 2009, the European Union first approved ethyl lauroyl arginate hydrochloride as a preservative for use in cosmetics.

[0008] In 2013, the Pest Management Regulatory Agency of Health Canada released a proposal regarding ethyl lauroyl arginate hydrochloride as a preservative for various standardized and non - standardized foods.

[0009] In 2014, the European Union issued a Commission Regulation approving ethyl lauroyl arginate hydrochloride as a preservative for use in heat - processed meat products (except emulsified sausages, smoked sausages and liver pâté).

[0010] In 2016, the EU newly permitted ethyl lauroyl arginate hydrochloride to be used as a preservative in mouthwash.

[0011] Although ethyl lauroyl arginate hydrochloride has been approved as a preservative for food and cosmetics abroad, it has not been approved as a food preservative in China yet. Therefore, there is no corresponding detection standard. Summary of the Invention

[0012] To solve the above technical problems, the present invention provides a post-column derivation detection method for ethyl lauroyl arginate, which includes the following steps: pretreat the food, extract ethyl lauroyl arginate hydrochloride with an acetonitrile aqueous solution, filter through a water-based filter membrane, separate by a high-performance liquid chromatography column, use thiol and o-phthalaldehyde as derivatization reagents, carry out a derivatization reaction at 62°C - 68°C under alkaline conditions to generate a fluorescent derivative, and perform fluorescence detection of the derivative to quantitatively determine the content of ethyl lauroyl arginate hydrochloride. This method is simple to operate, has good repeatability, and high detection sensitivity. To achieve the above invention purpose, the technical solution adopted by the present invention is as follows:

[0013] First step, pretreat the food and extract ethyl lauroyl arginate hydrochloride.

[0014] Further, the food includes dairy products, fruit products, meat products, aquatic products, beverages or chewing gums.

[0015] Furthermore, if the food is: dairy products, fruit products, meat products, aquatic products or beverages, the pretreatment includes the following steps:

[0016] (1) Weigh dairy products, fruit products, meat products, aquatic products or beverages, crush them, mix with a 50% acetonitrile solution at a liquid-to-material ratio of 1:5 g / mL, shake, centrifuge, and take the upper layer solution containing acetonitrile. Repeat the extraction 2 times to obtain an extract;

[0017] (2) Add a 50% acetonitrile solution to the extract to make up the volume to 2.5 times the volume of the 50% acetonitrile solution in step (1) to obtain a made-up solution;

[0018] (3) Weigh the made-up solution and an acetonitrile-saturated n-hexane solution, mix them at a volume ratio of 5:2, centrifuge, and filter the supernatant through a 0.22 μm water-based filter membrane for separation by a high-performance liquid chromatography column.

[0019] Furthermore, if the food is: chewing gum, the pretreatment includes the following steps:

[0020] (1) Weigh the shredded chewing gum base candies. According to the liquid-to-solid ratio of 1:5:2.5 g / mL, first add 50% acetonitrile solution, then add n-hexane, shake well, and take the lower layer solution containing acetonitrile to obtain the first extract. For the upper layer solution containing n-hexane, add the same volume of 50% acetonitrile solution again, shake well, and take the lower layer solution containing acetonitrile to obtain the second extract. Combine the first extract and the second extract to obtain the extract.

[0021] (2) Add 50% acetonitrile solution to the extract to make the volume up to 2.5 times the volume of the 50% acetonitrile solution added in step (1) to obtain the volume-fixed solution.

[0022] (3) Filter the volume-fixed solution through a 0.22 μm aqueous filter membrane for separation by a high-performance liquid chromatography column.

[0023] Furthermore, the method for preparing the 50% acetonitrile solution is as follows: Pipette 50 mL of acetonitrile into a 100 mL volumetric flask, add 100 μL of formic acid, make the volume up to the mark with water, and mix well.

[0024] The method for preparing the n-hexane solution saturated with acetonitrile is as follows: Add acetonitrile to n-hexane until saturation and stratification occurs.

[0025] In the present invention, since the chewing gum base candy is solid, before extracting ethyl lauroyl arginate hydrochloride, it is necessary to dissolve it with n-hexane first to make the solid become a molten state. At the same time, use n-hexane to remove non-polar compounds such as fat, which is convenient for acetonitrile to fully extract ethyl lauroyl arginate hydrochloride and reduce the impurities in the extract.

[0026] For dairy products, fruit products, meat products, aquatic products or beverages, their textures are relatively soft. Only need to use a tissue homogenizer to homogenize or mix them, and then ethyl lauroyl arginate hydrochloride can be fully extracted with acetonitrile. The purpose of using the n-hexane solution saturated with acetonitrile for further extraction is to remove non-polar compounds such as fat with n-hexane and reduce the impurities in the extract.

[0027] This method is also applicable to the detection of ethyl lauroyl arginate hydrochloride in cosmetics. Since the texture of cosmetics is relatively soft, the extraction method of ethyl lauroyl arginate hydrochloride is the same as that of dairy products, etc.

[0028] The second step is separation by a high-performance liquid chromatography column. The chromatographic conditions are as follows:

[0029] High-performance liquid chromatography column: octadecylsilica gel chromatographic column; column inner diameter: 150 mm × 4.6 mm, particle size 5 μm, or equivalent.

[0030] Mobile phase: A 10 mM ammonium acetate solution containing 0.1% formic acid and acetonitrile, with a volume ratio of 1:1; Preparation of the 10 mM ammonium acetate solution containing 0.1% formic acid: Accurately weigh 0.7708 g of ammonium acetate into a 1000 mL volumetric flask, add 1 mL of formic acid, and make up to the mark with water.

[0031] Detection wavelength: Excitation wavelength 340 nm, emission wavelength 455 nm.

[0032] In the third step, using thiol and o-phthalaldehyde as derivatization reagents, under alkaline conditions, a derivatization reaction is carried out at 62 - 68 °C to generate a fluorescent derivative, and the fluorescence of the derivative is detected to quantitatively determine the content of ethyl lauroyl arginate hydrochloride.

[0033] Further, the alkaline condition is an H3BO3-NaOH buffer solution system with a pH of 8.6 - 11.

[0034] Even further, the alkaline condition is preferably an H3BO3-NaOH buffer solution system with a pH of 10.0. Weigh 19.1 g of borax, dissolve it in 980 mL of water, adjust the pH to 10.0 with sodium hydroxide solution, and dilute to 1000 mL with water, and mix evenly.

[0035] Further, the molar concentration ratio of ethyl lauroyl arginate hydrochloride to o-phthalaldehyde is 1:1 - 100; the molar ratio of o-phthalaldehyde to mercaptoethanol is 1:3 - 10.

[0036] Selection of derivatization reagents in the present invention:

[0037] As mentioned above, ethyl lauroyl arginate hydrochloride, chemical formula: as shown in Formula 1:

[0038]

[0039] It can be seen from Formula 1 that there is a primary amino group at the end of this compound. The nitrogen atom of the amine contains two unpaired electrons, so it has nucleophilicity and is prone to substitution reactions with electrophilic reagents such as halogenated hydrocarbons, acyl chlorides, carbonyl groups, and acids. The inventors of the present invention tried suitable derivatization reagents: For example:

[0040] 1. Derivatization of amines and amino acids - ultraviolet-visible light reaction, selectable derivatization reagents:

[0041] 1) Sulfonyl chlorides:

[0042] Suitable for the derivatization of primary amines and secondary amines. The reaction temperature is generally 70 °C, the pH is about 8, the reaction time is 1 h, the absorption wavelength of the product is 254 nm, and the molar absorption coefficient is 10000. The product is extracted with chloroform, and the excess reagent is removed by partitioning with n-hexane.

[0043] 2) Acyl chlorides:

[0044] It is applicable to the analysis of primary amines and secondary amines, and also to polyamines. The reaction pH is 8. The primary amine reacts for 1 h at 50 °C, and the secondary amine refluxes for 12 h. Therefore, it is not suitable for post-column derivation. The absorption wavelength of its product is 254 nm, and the molar absorption coefficient is greater than 10,000. Polyamines are generally detected at 280 nm. The product is extracted with chloroform, etc., and the excess reagent can be removed by partitioning with n-hexane.

[0045] 3) Nitrohalobenzenes

[0046] Such as 2,4-dinitrofluorobenzene (DNFB), which is mainly used for the analysis of primary amines and secondary amines. The reaction pH is 9, the reaction is rapid, the absorption wavelength of the reaction product is 350 nm, and the molar absorption coefficient is 10,000. 2,4,6-trinitrobenzenesulfonic acid can also be used, and the absorption wavelength of the product is 254 nm.

[0047] 2. Fluorescence derivatization method, optional derivatization reagents:

[0048] 1) o-Phthalaldehyde (OPA)

[0049] Under alkaline conditions, OPA reacts with primary amines in the presence of thiols (such as ethyl mercaptan, mercaptoethanol) to form fluorescent derivatives. Under the conditions of pH 10 (borax buffer solution), room temperature and excess reagent, the reaction can be completed in only 1 - 2 min. The product has strong fluorescence, the excitation wavelength is 340 nm, and the emission wavelength is 455 nm. It can also be detected with an ultraviolet detector, the determination wavelength is 230 nm, and the sensitivity is 5×10 -12 .

[0050] 2) Sulfonyl chloride

[0051] Dansyl chloride (DNS-Cl) can react with primary amines, secondary amines, phenols and amino acids under alkaline conditions to form strongly fluorescent substances. The derivation is generally carried out in an acetone-water medium. The optimal pH for the reaction at room temperature is 9.5 - 10. Increasing the pH can accelerate the reaction rate, but also accelerate the hydrolysis of dansyl chloride; the reaction is incomplete when the pH is lower than 8. The reaction time generally requires 20 - 40 min, and the excitation wavelength of the product is 350 - 360 nm, and the emission wavelength is 490 - 540 nm.

[0052] Judging from the experimental results, the post-column derivation reaction requires a rapid reaction and a short time. Sulfonyl chlorides need to react at 70 °C and pH 8 for 1 h, and acyl chlorides need to react with primary amines at 50 °C for 1 h and with secondary amines for refluxing for 12 h. Although the reaction of DNFB is relatively fast, it still cannot meet the reaction requirement of 1 - 2 minutes for post-column derivation under the mobile phase conditions. Therefore, it is not suitable for post-column derivation. Therefore, the present invention selects o-phthalaldehyde (OPA) for post-column derivation, and its reaction principle is as follows:

[0053]

[0054] In the fourth step, fluorescence quantitative detection is carried out using a fluorescence detector.

[0055] The excitation wavelength for fluorescence detection is 335 - 340 nm, and the emission wavelength is 445 - 455 nm.

[0056] The present invention has the following advantages over the prior art:

[0057] 1. Ethyl lauroyl arginate hydrochloride (LAE) has been approved for use as a food and cosmetic preservative abroad. However, LAE has not been approved as a food preservative in China yet, so there is also a lack of corresponding detection standards.

[0058] Since the approved usage amount of LAE is relatively large abroad, high performance liquid chromatography (HPLC) combined with a DAD detector is mainly used for direct detection. However, this method has an obvious limitation: the sensitivity of the DAD detector is limited. Although it can meet the detection requirements for higher content of LAE, it cannot accurately detect trace amounts of LAE in foods or cosmetics. Currently, there is a lack of effective detection methods for low content of LAE both at home and abroad.

[0059] To address this technical problem, the present invention proposes an innovative detection scheme: a combined method of high performance liquid chromatography (HPLC) separation - chemical derivatization preparation - fluorescence spectroscopy inspection is used to quantitatively determine the content of LAE in foods, cosmetics or pharmaceuticals. This method is characterized by rapidity and high sensitivity.

[0060] 2. The present invention has also developed a set of pretreatment methods applicable to various foods, including dairy products, fruit products, meat products, aquatic products, beverages, and chewing gums, etc. This set of pretreatment methods has the advantages of simplicity, high extraction rate, reduction of LAE loss, and less impurities, laying a good foundation for subsequent accurate detection. Description of the Drawings

[0061] Figure 1 It is the post-column derivation - high performance liquid chromatogram of 200 μg / L ethyl lauroyl arginate hydrochloride.

[0062] Figure 2 It is the post-column derivation - high performance liquid chromatogram of the limit of quantification (4 μg / L) of ethyl lauroyl arginate hydrochloride.

[0063] Figure 3 It is the high performance liquid chromatogram of the pH = 10 buffer emulsified sauce in Example 1.

[0064] Figure 4 It is the high performance liquid chromatogram of the chewing gum in Example 2.

[0065] Figure 5It is the liquid chromatogram of the pH = 8.6 buffer emulsion sauce in Example 3.

[0066] Figure 6 It is the liquid chromatogram of the pH = 11 buffer emulsion sauce in Example 4.

[0067] Figure 7 It is the liquid chromatogram of the roasted eel strips in Example 5.

[0068] Figure 8 It is the liquid chromatogram of the raw emulsion sauce at a derivatization temperature of 50 °C in Example 6.

[0069] Figure 9 It is the liquid chromatogram of the emulsion sauce with an excitation wavelength of Ex = 330 and an emission wavelength of Em = 445 in Example 7. Detailed implementation mode

[0070] The present invention will be further described below in conjunction with the accompanying drawings and examples.

[0071] 1. Reagents and materials

[0072] Unless otherwise specified, all reagents are of analytical grade, and the water is the first-grade water specified in GB / T 6682. Methanol (CH3OH): Chromatographically pure. Acetonitrile (CH3CN): Chromatographically pure. Formic acid (HCOOH): Chromatographically pure. n-Hexane (C6H14): Chromatographically pure. Sodium hydroxide (NaOH). Ammonium acetate (CH3COONH4): Chromatographically pure. o-Phthalaldehyde (C8H6O 2) . Mercaptoethanol (C2H6OS).

[0073] Borax solution (0.05 mol / L, pH 10.0): Weigh 19.1 g of borax (Na2B4O7·10H2O), dissolve it in 980 mL of water, adjust the pH to 10.0 with sodium hydroxide solution, and dilute it to 1000 mL with water and mix well.

[0074] n-Hexane saturated with acetonitrile: Add acetonitrile to n-hexane until saturated.

[0075] 10 mmol / L ammonium acetate solution containing 0.1% formic acid: Accurately weigh 0.7708 g of ammonium acetate into a 1000 mL volumetric flask, add 1 mL of formic acid, and make up to the mark with water.

[0076] 50% acetonitrile solution: Pipette 60 mL of acetonitrile into a 100 mL volumetric flask, add 100 μL of formic acid, and make up to the mark with water and mix well.

[0077] o-Phthalaldehyde solution: Weigh 3 g of o-phthalaldehyde into a 100 mL volumetric flask, add 10 mL of methanol to dissolve it, and make up to the mark with water to prepare a 300 mg / L solution.

[0078] OPA reagent: Weigh 10 mL of o-phthalaldehyde solution, add 0.01 mL of mercaptoethanol, dilute to 1000 mL with sodium borate buffer solution, mix well, pour into a brown bottle, and store in the dark at room temperature for 1 week.

[0079] Standard: Ethyl lauroyl arginate hydrochloride (C 20 H 41 ClN4O3, CAS: 60372-77-2): Purity ≥ 98%.

[0080] Standard stock solution: Weigh an appropriate amount of the above standard into a 10 mL volumetric flask respectively, and prepare a standard stock solution with a concentration of 200 mg / L using acetonitrile. Store it in the dark at -18°C or below, and the validity period is 6 months.

[0081] Mixed standard working solution: Pipette an appropriate amount of the standard stock solution, and prepare a mixed standard solution with a concentration of 1.00 mg / L using acetonitrile. Store it at -18°C or below, and the validity period is 3 months.

[0082] Matrix mixed standard working solution: Accurately pipette a certain amount of the mixed standard solution, and gradually dilute it with 50% acetonitrile solution to prepare mixed standard working solutions with concentrations of 4.00 μg / L, 10.00 μg / L, 20.00 μg / L, 50.0 μg / L, 100.0 μg / L, and 200 μg / L. The mixed standard working solution should be prepared and used immediately.

[0083] Inorganic microporous filter membrane: Diameter 13 mm, pore size 0.22 μm.

[0084] 2. Instruments and equipment

[0085] Liquid chromatograph: Equipped with a fluorescence detector.

[0086] Post-column derivatizer.

[0087] Vortex mixer.

[0088] Centrifuge: Rotation speed not less than 9500 r / min.

[0089] Analytical balance: Sensitivity 0.1 mg and 0.001 g.

[0090] Ultra-pure water instrument.

[0091] Tissue homogenizer.

[0092] 3. Sample preparation and storage:

[0093] Liquid samples and powdered solid samples should be mixed evenly respectively. For semi-solid samples, take the solid-liquid coexisting substances for homogenization and mixing. Solid samples should be mixed evenly by means of crushing with an electric stirrer, sealed, and the prepared samples should be stored at -18°C or below for standby.

[0094] 4. Sample extraction

[0095] a) Dairy products, fruit products, meat products, aquatic products, beverages

[0096] Weigh 2 g (accurate to 0.01 g) of the sample into a 50 mL centrifuge tube, add 10 mL of 50% acetonitrile solution, shake for extraction for 5 min, centrifuge at 9500 r / min for 5 min, transfer the filtrate to a 25 mL volumetric flask, add 10 mL of 50% acetonitrile solution and repeat the extraction operation as above once. Combine the extraction solutions into the 25 mL volumetric flask, make up the volume to the mark with 50% acetonitrile solution, and mix well. Take 5 mL of the extraction solution into a centrifuge tube, add 2 mL of acetonitrile-saturated n-hexane, vortex for 2 min, centrifuge at 9500 r / min for 5 min, discard the n-hexane, and filter through a 0.22 μm water-based filter membrane. The filtrate is used for high performance liquid chromatography analysis.

[0097] b) Gum base candies

[0098] Use scissors to cut the gum base candies into thin strips. Weigh 2 g (accurate to 0.01 g) of the cut gum base candy sample into a 50 mL separatory funnel, add 10 mL of 50% acetonitrile solution and shake vigorously for about 1 min, then add 5 mL of n-hexane, continue to shake until the gum base candies are completely dissolved, let it stand for liquid separation for about 5 min, and put the lower layer solution into a 25 mL volumetric flask. Continue to add 10 mL of 50% acetonitrile solution to the separatory funnel, gently shake for about 10 s, let it stand for liquid separation for about 1 min, and put the lower layer solution into the same volumetric flask. Make up the volume to the mark with 50% acetonitrile solution, shake well and filter through a 0.22 μm water-based filter membrane. The filtrate is used for chromatographic analysis.

[0099] 5. The reference conditions for high performance liquid chromatography are as follows:

[0100] a) Chromatographic column: C18 column, 150 mm × 4.6 mm (inner diameter), particle size 5 μm, or equivalent.

[0101] b) Detection wavelength: Excitation wavelength 340 nm, emission wavelength 455 nm.

[0102] c) Mobile phase: 10 mM ammonium acetate solution containing 0.1% formic acid and acetonitrile, volume ratio 1:1.

[0103] d) Flow rate: 0.8 mL / min.

[0104] e) Column temperature: 30 °C.

[0105] f) Injection volume: 10 μL.

[0106] 6. Post-column derivation

[0107] a) OPA reagent, flow rate 0.8 mL / min.

[0108] b) Reactor temperature: 65 °C.

[0109] 7. Preparation of Derivatization Solution

[0110] The fluorescence intensity changes of the system in H3BO3-NaOH buffer solution with pH 8.6 - 11 were studied in the presence of 2.3×10 -5 mol / L OPA and 2.3×10 -6 mol / L LAE. The experimental results showed that below pH 10.0, the light intensity increased with the increase of pH, and the fluorescence intensity reached the maximum at pH 10.0 and decreased above pH 10.0. Therefore, the optimal pH condition was determined to be 10.0.

[0111] When the concentration of LAE was 2.3×10 －6 mol / L, the effect of 2.3×10 －6 -2.3×10 －4 mol / L o-phthalaldehyde on the fluorescence intensity was studied. The experiment found that when the concentration of o-phthalaldehyde was 2.3×10 －5 mol / L, the relative fluorescence intensity of the system reached the maximum. Therefore, the optimal concentration of o-phthalaldehyde was selected as 2.3×10 －5 mol / L.

[0112] According to the previous optimization results, when the concentrations of LAE and o-phthalaldehyde were 2.3×10 －6 mol / L and 2.3×10 － 5 mol / L respectively, the effect of the addition amount of mercaptoethanol on the fluorescence intensity was investigated in this experiment. Generally, the usage ratio of OPA to mercaptoethanol is 1:3, and the effect of β-mercaptoethanol concentration on the fluorescence intensity in the range of 6.9×10 －5 -2.3×10 －4 mol / L was studied. The fluorescence intensity increased with the increase of β-mercaptoethanol concentration in the range of 6.9×10 －5 -1.2×10 －4 mol / L. When the concentration of β-mercaptoethanol exceeded 1.2×10 －4 mol / L, the fluorescence intensity no longer increased. Therefore, 1.2×10 －4 mol / L was selected for the subsequent experiment.

[0113] 8. Preparation of Standard Curve

[0114] Inject the working solutions of the mixed standard series curves into the high performance liquid chromatograph in order of increasing concentration, and measure the peak areas. Use the concentration of the standard solution as the abscissa and the peak area as the ordinate to plot the standard curve. The post-column derivation-high performance liquid chromatogram of ethyl lauroyl arginate is shown in Figure 1 and Figure 2 . Among them, Figure 1 , the post-column derivation-high performance liquid chromatogram of ethyl lauroyl arginate hydrochloride at 200 μg / L, Figure 2 the post-column derivation-high performance liquid chromatogram of ethyl lauroyl arginate hydrochloride with a quantification limit of 4 μg / L.

[0115] 9. Determination of the test solution

[0116] Inject the test solution to obtain the corresponding peak area, and calculate the concentration of ethyl lauroyl arginate in the test solution according to the standard curve.

[0117] 10. Blank sample

[0118] Do not weigh the sample, and prepare a blank sample according to the steps for dairy products, fruit products, meat products, aquatic products, beverages and chewing gums.

[0119] 11. Expression of the analysis result

[0120] The content of ethyl lauroyl arginate in the test sample is calculated according to the following formula:

[0121]

[0122] Where:

[0123] X—the content of the analyte in the sample, in milligrams per kilogram (mg / kg);

[0124] c—the concentration of the analyte in the test solution calculated from the standard working curve, in micrograms per liter (μg / L);

[0125] V—the volume of the fixed volume, in milliliters (mL);

[0126] m—the mass of the sample, in grams (g);

[0127] 1000—the unit conversion factor;

[0128] The calculation result is rounded to three significant figures.

[0129] Example 1:

[0130] 1. Samples of emulsified sauces containing ethyl lauroyl arginate purchased overseas were tested. The initial test samples were supra-linear and were diluted to within the linear range with 50% dilution. They were processed according to the treatment methods for a) dairy products, fruit products, meat products, aquatic products, and beverages in the above-mentioned 4. Sample extraction, and the filtrate was used for high-performance liquid chromatography analysis.

[0131] 2. The reference conditions for high-performance liquid chromatography are as follows:

[0132] a) Chromatographic column: C18 column, 150 mm × 4.6 mm (inner diameter), particle size 5 μm, or equivalent.

[0133] b) Detection wavelength: Excitation wavelength 340 nm, emission wavelength 455 nm.

[0134] c) Mobile phase: 10 mM ammonium acetate solution containing 0.1% formic acid and acetonitrile, volume ratio 1:1.

[0135] d) Flow rate: 0.8 mL / min.

[0136] e) Column temperature: 30 °C.

[0137] f) Injection volume: 10 μL.

[0138] 3. Post-column derivation

[0139] a) OPA reagent, flow rate 0.8 mL / min.

[0140] The OPA reagent here, according to the aforementioned solution preparation, is: Weigh 10 mL of o-phthalaldehyde solution, add 0.01 mL of mercaptoethanol, dilute to 1000 mL with sodium borate buffer solution, mix well, fill into a brown bottle, and store at room temperature away from light, and it can be stored for 1 week.

[0141] b) Reactor temperature: 65 °C.

[0142] 4. The sample was diluted 25 times, and the result of detecting ethyl lauroyl arginate was 23.8 mg / kg. The chromatogram of the sample is shown in Figure 3 .

[0143] Example 2:

[0144] Replace the sample in Example 1 with chewing gum containing ethyl lauroyl arginate purchased overseas. The sample was tested, and the initial test sample was supra-linear. It was processed according to the treatment method for b) chewing gum in the above-mentioned 4. Sample extraction, and the filtrate was used for high-performance liquid chromatography analysis. Other conditions were the same as in Example 1. The result of detecting ethyl lauroyl arginate was 1.39 mg / kg. The chromatogram of the sample is shown in Figure 4 .

[0145] Example 3:

[0146] Replace the H3BO3-NaOH buffer solution in Example 1 with a pH of 8.6, and keep other conditions the same as in Example 1. The detected result of ethyl lauroyl arginate is 15.9 mg / kg. The sample chromatogram is shown in Figure 5 。

[0147] Example 4:

[0148] Replace the H3BO3-NaOH buffer solution in Example 1 with a pH of 11, and keep other conditions the same as in Example 1. The detected result of ethyl lauroyl arginate is 18.3 mg / kg. The sample chromatogram is shown in Figure 6 。

[0149] Example 5:

[0150] Replace the sample in Example 1 with grilled eel strips and crush them. Keep other conditions the same as in Example 1. The detected result of ethyl lauroyl arginate is 0.899 mg / kg. The sample chromatogram is shown in Figure 7 。

[0151] Example 6:

[0152] Change the derivatization reaction temperature in Example 1 to 50 °C, and keep other conditions the same as in Example 1. The detected result of ethyl lauroyl arginate is 9.27 mg / kg. The sample chromatogram is shown in Figure 8 。

[0153] Example 7:

[0154] Change the excitation wavelength in Example 1 to 330 nm and the emission wavelength to 445 nm. Keep other conditions the same as in Example 1. The detected result of ethyl lauroyl arginate is 18.7 mg / kg. The sample chromatogram is shown in Figure 9 。

[0155] The above are the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above content. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A post-column derivation detection method for ethyl lauroyl arginate, characterized in that: It includes the following steps: pre-treat the food, extract ethyl lauroyl arginate hydrochloride with an acetonitrile aqueous solution, filter through a water-based filter membrane, separate through a high-performance liquid chromatography column, use thiol and o-phthalaldehyde as derivatization reagents, carry out a derivatization reaction at 62°C - 68°C under alkaline conditions to generate a fluorescent derivative, and perform fluorescence detection of the derivative to quantitatively determine the content of ethyl lauroyl arginate hydrochloride.

2. The post-column derivation detection method of ethyl lauroyl arginate according to claim 1, wherein: The food includes dairy products, fruit products, meat products, aquatic products, beverages or chewing gums.

3. The post-column derivation detection method of ethyl lauroyl arginate according to claim 2, characterized in that: The pre-treatment of the dairy products, fruit products, meat products, aquatic products or beverages includes the following steps: (1) Weigh the dairy products, fruit products, meat products, aquatic products or beverages, crush them, mix with a 50% acetonitrile solution at a liquid-to-material ratio of 1:5 g / mL, shake, centrifuge, and take the upper layer solution containing acetonitrile. Repeat the extraction 2 times to obtain an extract; (2) Add a 50% acetonitrile solution to the extract to make the volume constant, and make the volume constant to 2.5 times the volume of the 50% acetonitrile solution in step (1) to obtain a constant-volume solution; (3) Weigh the constant-volume solution and an acetonitrile-saturated n-hexane solution, mix them at a volume ratio of 5:2, centrifuge, and filter the supernatant through a 0.22 μm water-based filter membrane for separation by a high-performance liquid chromatography column.

4. The post-column derivation detection method of ethyl lauroyl arginate according to claim 2, wherein: The pre-treatment of the chewing gum includes the following steps: (1) Weigh the shredded chewing gum, at a liquid-to-material ratio of 1:5:2.5 g / mL, first add a 50% acetonitrile solution, then add n-hexane, shake, and take the lower layer solution containing acetonitrile to obtain a first extract; for the upper layer containing n-hexane layer, add the same volume of 50% acetonitrile solution again, shake, and take the lower layer solution containing acetonitrile to obtain a second extract. Combine the first extract and the second extract to obtain an extract; (2) Add a 50% acetonitrile solution to the extract to make the volume constant, and make the volume constant to 2.5 times the volume of the 50% acetonitrile solution added first in step (1) to obtain a constant-volume solution; (3) Filter the constant-volume solution through a 0.22 μm water-based filter membrane for separation by a high-performance liquid chromatography column.

5. The post-column derivation detection method of ethyl lauroyl arginate according to claim 3 or 4, characterized in that: The preparation method of the 50% acetonitrile solution is: transfer 50 mL of acetonitrile into a 100 mL volumetric flask, add 100 μL of formic acid, make the volume constant with water to the scale, and mix evenly; The preparation method of the acetonitrile-saturated n-hexane solution is: add acetonitrile to n-hexane until saturation and stratification, and take the upper layer solution.

6. The post-column derivation detection method of ethyl lauroyl arginate according to claim 1, characterized in that: The high-performance liquid chromatography column: octadecyl silica gel chromatography column; Mobile phase: a 10 mM ammonium acetate solution containing 0.1% formic acid and acetonitrile, with a volume ratio of 1:

1.

7. The post-column derivation detection method of ethyl lauroyl arginate according to claim 1, wherein: The alkaline condition is an H3BO3-NaOH buffer solution system with a pH of 8.6 - 11.

8. The post-column derivation detection method of ethyl lauroyl arginate according to claim 7, characterized in that: The alkaline condition is an H3BO3-NaOH buffer solution system with a pH of 10.

0. Weigh 19.1 g of borax, dissolve it in 980 mL of water, adjust the pH to 10.0 with a sodium hydroxide solution, and dilute it to 1000 mL with water and mix evenly.

9. The post-column derivation detection method of ethyl lauroyl arginate according to claim 1, characterized in that: The molar concentration ratio of ethyl lauroyl arginate hydrochloride to o-phthalaldehyde is 1:1 - 100; the molar ratio of o-phthalaldehyde to 2-mercaptoethanol is 1:3 - 10.

10. The post-column derivation detection method of ethyl lauroyl arginate according to claim 1, characterized in that: The excitation wavelength for the fluorescence detection is 335 - 340 nm, and the emission wavelength is 445 - 455 nm.

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