A method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivatization

Abstract

The present invention discloses a method for detecting menthol content based on rapid, catalyst-free pre-column derivatization by liquid chromatography. Specifically, pure menthol and a menthol-containing product are first subjected to a pre-column nucleophilic substitution esterification reaction for derivatization to obtain a reaction solution of menthol derivatives having absorption in the near-ultraviolet to visible light region. The menthol derivatives in the reaction solution are then detected and analyzed using a high-performance liquid chromatograph, thereby qualitatively and quantitatively determining the menthol derivatives in the reaction solution and obtaining the exact content of the menthol active ingredient in the measured menthol-containing product before the derivatization reaction. Compared with traditional analytical methods, this method has the advantages of being inexpensive, practical, rapid, highly sensitive, requiring no purification treatment, requiring no reaction catalyst to be added, and resulting in no other impurity peaks.

Description

Technical Field

[0001] The invention belongs to the technical field of menthol content detection, and in particular relates to a method for detecting menthol content by liquid chromatography based on a quick and catalyst-free pre-column derivative. Background Art

[0002] Menthol, also known as menthol, is named 5-methyl-2-isopropyl-1-cyclohexanol by IUPAC. Menthol is widely used in daily chemical flavors, edible flavors, and medical and health products. Initially, menthol was extracted from the leaves or stems of the mint plant. The sources of natural menthol are mainly divided into European mint oil and Asian mint oil.

[0003] Menthol has a wide range of applications. It is not only used in the flavor and fragrance and food industries, but is also consumed in large quantities as a raw material for pharmaceuticals and chemical products. Therefore, menthol extracted solely from natural mint plants can no longer meet the growing industrial demand. In addition, the maturity cycle of natural mint plants is relatively long. Therefore, more and more manufacturers are increasingly relying on chemically synthesized menthol to meet market demand. First, chemical synthesis takes a short time and is not restricted by climatic and geographical factors. Therefore, chemically synthesized menthol is more accepted by menthol manufacturers. At the same time, it is also necessary to test the content of menthol products produced by natural extraction and chemical synthesis to further optimize the menthol production and extraction process.

[0004] Many reported methods for detecting and analyzing menthol include the use of flame ionization detection (FID), gas chromatography, liquid refractometers (LC-MS / MS), and liquid fluorescence detectors for qualitative and quantitative analysis. Furthermore, because menthol itself lacks an absorption peak signal in the 200-800 nm range, it must be derivatized prior to liquid chromatography detection. This utilizes the inherent absorbing groups of the derivatization reagent to impart an optical response in the near-ultraviolet to visible light region, allowing the menthol derivative to serve as a high-performance liquid chromatography (HPLC) detection medium for testing menthol content in product samples. However, these derivatization reactions typically require the addition of catalysts and further purification. Consequently, these methods often suffer from the disadvantages of being expensive, highly polluting, and requiring purification, making them unsuitable for large-scale menthol sample testing. Summary of the Invention

[0005] In view of the above-mentioned problems in the background technology, the present invention aims to provide a method for detecting the content of menthol by liquid chromatography based on a rapid and catalyst-free pre-column derivatization, by which the exact content of the menthol active ingredient in the measured menthol-containing product can be obtained.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivatization method comprises the following steps:

[0008] Step 1) preparing a menthol acetonitrile solution, an acetonitrile solution of a menthol-containing product, and an acetonitrile solution of an aromatic acid chloride or aromatic sulfonyl chloride;

[0009] Step 2) 1000 μL each of the menthol acetonitrile solution and the aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added sequentially to a 10 mL volumetric flask, diluted to a total volume of 10 mL by adding acetonitrile, capped the volumetric flask, and heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction. The reaction system was then cooled to room temperature, and the reaction solution was diluted to 10 mL to obtain a reaction solution 1 containing a menthol derivative having absorption in the near-ultraviolet to visible light region;

[0010] Step 3) As in step 2), 1000 μL of the acetonitrile solution of the menthol-containing product and 1000 μL of the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride prepared above were added to another 10 mL volumetric flask, and the same experimental procedures were repeated to obtain a second reaction solution containing a menthol derivative having absorption in the near-ultraviolet to visible region.

[0011] Step 4) The aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution, the menthol derivative-containing reaction solution 1, and the menthol derivative-containing reaction solution 2 prepared above are respectively prepared as test solutions using acetonitrile. Then, each of the test solutions is detected and analyzed using a high performance liquid chromatography (HPLC) using a commercially available conventional C-18 column as a chromatographic column, thereby qualitatively and quantitatively determining the menthol derivative in the reaction solution, thereby obtaining the exact content of the menthol active ingredient in the measured menthol-containing product before the derivatization reaction.

[0012] More preferably, the step 1) comprises the following steps:

[0013] Step 11) Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 5 mg / mL menthol acetonitrile solution for later use;

[0014] Step 12) Weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol product for later use;

[0015] Step 13) Weigh 0.30-0.44 g of aromatic acid chloride or aromatic sulfonyl chloride and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 6-8.8 mg / mL aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution for later use.

[0016] Preferably, the amount of the aromatic acid chloride or aromatic sulfonyl chloride and the pure menthol used is calculated in a molar ratio of 1.2:1.

[0017] Preferably, the aromatic acid chloride or aromatic sulfonyl chloride used is 2-naphthoyl chloride, 2-naphthomethylsulfonyl chloride, 4-methyl-3-nitrobenzoyl chloride, 3-methoxy-4-methylbenzoyl chloride, 4-nitrobenzenesulfonyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, or cinnamoyl chloride.

[0018] Preferably, in the above step 4), when a high performance liquid chromatograph is used, the liquid phase conditions are as follows: the reversed-phase mobile phase system is a mixture of acetonitrile and water or a mixture of methanol and potassium phosphate brine, the flow rate is controlled at 0.7-1.0 mL / min, the injection volume is 5-20 μL, the detection wavelength is 270-320 nm, and the column temperature is 25-30 ° C.

[0019] Preferably, the reverse phase mobile phase system consists of an aqueous phase and an organic phase.

[0020] Preferably, the reverse phase mobile phase system is a mixture of acetonitrile and water, calculated by volume percentage, that is, acetonitrile:water=70-100%:0-30%.

[0021] Preferably, the reverse phase mobile phase system is a mixture of acetonitrile and water, calculated by volume percentage, that is, acetonitrile:water=80%:20%.

[0022] Preferably, in the above step 4), the commercially available conventional C-18 column is C18 5μm250mm*4.6mm.

[0023] Preferably, in the above step 4), the high performance liquid chromatograph used is one of Agilent LC1260, Waterse2695, iChrom 5100, Agress1100, EClassical 3200, and Nexera LC-40.

[0024] The beneficial effects of the present invention are:

[0025] (1) The method of the present invention for detecting menthol content based on rapid, catalyst-free pre-column derivatization by liquid chromatography specifically comprises first subjecting pure menthol and a menthol-containing product to a pre-column nucleophilic substitution esterification reaction for derivatization to obtain a reaction solution containing menthol derivatives having absorption in the near-ultraviolet to visible light region, and then performing detection and analysis using a high performance liquid chromatograph to qualitatively and quantitatively determine the menthol derivatives in the reaction solution, thereby obtaining the exact content of the menthol active ingredient in the menthol-containing product before the derivatization reaction.

[0026] (2) Compared with traditional analytical methods, the method of the present invention for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivative can effectively obtain the content of derivatized menthol esters. It has the advantages of being inexpensive, practical, rapid, highly sensitive, requiring no purification treatment, requiring no reaction catalyst to be added, and lacking the appearance of other impurity peaks. The separation and purification steps are eliminated during the reaction process, which can shorten the entire testing step time and avoid unnecessary detection errors such as loss due to derivative transfer, thereby improving detection accuracy and efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the derivatization process of the pre-column nucleophilic substitution esterification reaction in the present invention;

[0028] Figure 2 is a chromatogram of the reference solution containing cinnamoyl chloride prepared in Example 8 of the present invention;

[0029] Figure 3 This is a chromatographic separation diagram of a standard solution containing the derivative menthyl cinnamate prepared according to Example 8 of the present invention;

[0030] Figure 4 This is a chromatographic separation diagram of the test solution containing the derivative menthyl cinnamate prepared according to Example 8 of the present invention. DETAILED DESCRIPTION

[0031] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention is further described by the following examples. Obviously, the following examples are only a part of the embodiments of the present invention, rather than all the embodiments; it should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, and are not used to limit the scope of protection of the present invention.

[0032] The present invention provides a method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivative, comprising the following steps:

[0033] Step 1) preparing a menthol acetonitrile solution, an acetonitrile solution of a menthol-containing product, and an acetonitrile solution of an aromatic acid chloride or aromatic sulfonyl chloride;

[0034] Step 2) 1000 μL each of the menthol acetonitrile solution and the aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added sequentially to a 10 mL volumetric flask, diluted to a total volume of 10 mL by adding acetonitrile, capped the volumetric flask, and heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction. The reaction system was then cooled to room temperature, and the reaction solution was diluted to 10 mL to obtain a reaction solution 1 containing a menthol derivative having absorption in the near-ultraviolet to visible light region;

[0035] Step 3) As in step 2), 1000 μL of the acetonitrile solution of the menthol-containing product and 1000 μL of the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride prepared above were added to another 10 mL volumetric flask, and the same experimental procedures were repeated to obtain a second reaction solution containing a menthol derivative having absorption in the near-ultraviolet to visible region.

[0036] Step 4) The aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution, the menthol derivative-containing reaction solution 1, and the menthol derivative-containing reaction solution 2 prepared above are respectively prepared as test solutions using acetonitrile. Then, each of the test solutions is detected and analyzed using a high performance liquid chromatography (HPLC) using a commercially available conventional C-18 column as a chromatographic column, thereby qualitatively and quantitatively determining the menthol derivative in the reaction solution, thereby obtaining the exact content of the menthol active ingredient in the measured menthol-containing product before the derivatization reaction.

[0037] The step 1) comprises the following steps:

[0038] Step 11) Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 5 mg / mL menthol acetonitrile solution for later use;

[0039] Step 12) Weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol product for later use;

[0040] Step 13) Weigh 0.30-0.44 g of aromatic acid chloride or aromatic sulfonyl chloride and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 6-8.8 mg / mL aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution for later use.

[0041] like Figure 1 The pre-column nucleophilic substitution esterification derivatization process shown does not require the addition of any catalyst and is carried out only under simple and easily achievable experimental conditions such as heating. The obtained menthol derivatization product does not need to be separated from the reaction system or further purified. The removal of the separation and purification steps during the reaction process can shorten the entire testing process, significantly saving about one-third of the entire testing process time. It also avoids unnecessary detection errors such as derivative transfer loss, thereby improving detection accuracy and efficiency.

[0042] Specifically, the amount of the aromatic acid chloride or aromatic sulfonyl chloride and the pure menthol used is calculated in a molar ratio of 1.2:1.

[0043] The aromatic acid chloride or aromatic sulfonyl chloride used is an acid chloride or sulfonyl chloride containing an aromatic group, and its types are 2-naphthoyl chloride, 2-naphthomethylsulfonyl chloride, 4-methyl-3-nitrobenzoyl chloride, 3-methoxy-4-methylbenzoyl chloride, 4-nitrobenzenesulfonyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, and cinnamoyl chloride.

[0044] In step 4), when a high performance liquid chromatograph is used, the liquid phase conditions are as follows: the reversed-phase mobile phase system is a mixture of acetonitrile and water or a mixture of methanol and potassium phosphate brine, the flow rate is controlled at 0.7-1.0 mL / min, the injection volume is 5-20 μL, the detection wavelength is 270-320 nm, and the column temperature is 25-30°C.

[0045] The reverse phase mobile phase system consists of an aqueous phase and an organic phase.

[0046] Preferably, the reverse phase mobile phase system is a mixture of acetonitrile and water, calculated by volume percentage, that is, acetonitrile:water=70-100%:0-30%.

[0047] More preferably, the reverse phase mobile phase system is a mixture of acetonitrile and water, calculated by volume percentage, that is, acetonitrile:water=80%:20%.

[0048] In the above step 4), the reaction solution 1 and the reaction solution 2 containing the menthol derivative prepared above are prepared into a test solution with a concentration of 0.1-3.0 mg / mL using acetonitrile.

[0049] The concentration range of the test solution is a concentration range selected for selecting a suitable liquid phase test concentration, so as to screen out the final actual concentration suitable for detection, and is inclusive of the concentration of the test solution in the following specific embodiments.

[0050] In the above step 4), the commercially available conventional C-18 column is C18 5μm250mm*4.6mm; the high performance liquid chromatograph used is one of Agilent LC1260, Waters e2695, iChrom 5100, Agress1100, EClassical 3200, and Nexera LC-40.

[0051] The present invention will be further described below with reference to specific embodiments.

[0052] 1. Preparation of menthol acetonitrile solution, acetonitrile solution of menthol-containing product, aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution:

[0053] Example 1

[0054] The specific preparation steps are:

[0055] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution.

[0056] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0057] Accurately weigh 0.42 g of 4-bromobenzoyl chloride (based on a molar ratio of 4-bromobenzoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an 8.4 mg / mL 4-bromobenzoyl chloride acetonitrile solution for later use.

[0058] Example 2

[0059] The specific preparation steps are:

[0060] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0061] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0062] Accurately weigh 0.37 g of 2-naphthoyl chloride (based on a molar ratio of 2-naphthoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 7.4 mg / mL 2-naphthoyl chloride acetonitrile solution for later use.

[0063] Example 3

[0064] The specific preparation steps are:

[0065] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0066] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0067] Accurately weigh 0.44 g of 2-naphthylmethylsulfonyl chloride (based on a molar ratio of 2-naphthylmethylsulfonyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an 8.8 mg / mL 2-naphthylmethylsulfonyl chloride acetonitrile solution for later use.

[0068] Example 4

[0069] The specific preparation steps are:

[0070] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0071] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0072] Accurately weigh 0.42 g of 4-methyl-3-nitrobenzoyl chloride (based on a molar ratio of 4-methyl-3-nitrobenzoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an 8.4 mg / mL 4-methyl-3-nitrobenzoyl chloride acetonitrile solution for later use.

[0073] Example 5

[0074] The specific preparation steps are:

[0075] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0076] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0077] Accurately weigh 0.36 g of 3-methoxy-4-methylbenzoyl chloride (based on a molar ratio of 3-methoxy-4-methylbenzoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 7.2 mg / mL 3-methoxy-4-methylbenzoyl chloride acetonitrile solution for later use.

[0078] Example 6

[0079] The specific preparation steps are:

[0080] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0081] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0082] Accurately weigh 0.43 g of 4-nitrobenzenesulfonyl chloride (based on a molar ratio of 4-nitrobenzenesulfonyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an 8.6 mg / mL 4-nitrobenzenesulfonyl chloride acetonitrile solution for later use.

[0083] Example 7

[0084] The specific preparation steps are:

[0085] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0086] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0087] Accurately weigh 0.34 g of 4-chlorobenzoyl chloride (based on a molar ratio of 4-chlorobenzoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 6.8 mg / mL 4-chlorobenzoyl chloride acetonitrile solution for later use.

[0088] Example 8

[0089] The specific preparation steps are:

[0090] Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile to prepare a 5 mg / mL menthol acetonitrile solution for later use.

[0091] Accurately weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol-containing product, which is set aside;

[0092] Accurately weigh 0.32 g of cinnamoyl chloride (based on a molar ratio of cinnamoyl chloride to pure menthol of 1.2:1) and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 6.4 mg / mL cinnamoyl chloride acetonitrile solution for later use.

[0093] In Examples 1-8 of the present invention, the menthol content in the menthol-containing product was 34% (gas phase detection result).

[0094] 2. Reaction solution 1 and reaction solution 2 containing menthol derivatives were prepared according to the above examples:

[0095] The specific preparation steps are:

[0096] In a 10 mL volumetric flask, 1000 μL each of the menthol acetonitrile solution and the aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added in sequence, and acetonitrile was added to dilute the total volume to 10 mL. The volumetric flask was capped and heated at 65° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction for derivatization. The reaction system was then cooled to room temperature, and the reaction solution was diluted to 10 mL to obtain a reaction solution 1 containing a menthol derivative having absorption in the near-ultraviolet to visible light region.

[0097] According to the above steps, 1000 μL of the acetonitrile solution of the menthol-containing product and 1000 μL of the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride prepared above were added to another 10 mL volumetric flask in sequence, and the same experimental operation was performed to obtain a reaction solution II containing a menthol derivative having absorption in the near-ultraviolet to visible light region.

[0098] According to the above preparation steps and the menthol acetonitrile solution, the acetonitrile solution of the menthol-containing product, and the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride in Examples 1-8, the following were respectively prepared:

[0099] The reaction solution 1 and the reaction solution 2 contain the derivative 4-bromobenzoic acid menthyl ester, and the structural formula of the derivative 4-bromobenzoic acid menthyl ester is as follows:

[0100]

[0101] The reaction solution 1 and the reaction solution 2 contain the derivative 2-menthyl naphthoate, and the structural formula of the derivative 2-menthyl naphthoate is as follows:

[0102]

[0103] The reaction solution 1 and the reaction solution 2 contain the derivative 2-naphthyl methanesulfonate, and the structural formula of the derivative 2-naphthyl methanesulfonate is as follows:

[0104]

[0105] The reaction solution 1 and the reaction solution 2 contain the derivative 4-methyl-3-nitrobenzoic acid menthyl ester, and the structural formula of the derivative 4-methyl-3-nitrobenzoic acid menthyl ester is as follows:

[0106]

[0107] The reaction solution 1 and the reaction solution 2 contain the derivative 3-methoxy-4-methyl benzoic acid menthyl ester, and the structural formula of the derivative 3-methoxy-4-methyl benzoic acid menthyl ester is as follows:

[0108]

[0109] The reaction solution 1 and the reaction solution 2 contain the derivative 4-nitrobenzenesulfonic acid menthyl ester, and the structural formula of the derivative 4-nitrobenzenesulfonic acid menthyl ester is as follows:

[0110]

[0111] The reaction solution 1 and the reaction solution 2 contain the derivative 4-chlorobenzoic acid menthyl ester, and the structural formula of the derivative 4-chlorobenzoic acid menthyl ester is as follows:

[0112]

[0113] The reaction solution 1 and the reaction solution 2 contain the derivative cinnamic acid menthol ester, and the structural formula of the derivative cinnamic acid menthol ester is as follows:

[0114]

[0115] 3. Detection and analysis of the exact content of menthol active ingredient in menthol-containing products before derivatization reaction:

[0116] It specifically includes the following steps:

[0117] (1) 20 μL of the cinnamoyl chloride acetonitrile solution prepared in Example 8 was placed in a 10 mL volumetric flask, diluted to the mark with HPLC acetonitrile, and shaken well as a reference solution;

[0118] (2) 200 μL of the reaction solution containing the derivative menthol cinnamate prepared according to Example 8 above was placed in a 10 mL volumetric flask, diluted to the mark with HPLC acetonitrile, and shaken to prepare a standard solution;

[0119] (3) 200 μL of the reaction solution containing the derivative menthol cinnamate prepared according to Example 8 was placed in a 10 mL volumetric flask, diluted to the mark with HPLC acetonitrile, and shaken to obtain the test solution;

[0120] (4) Take 1000 μL of the reference solution, standard solution, and test solution respectively and filter them through a 0.22 μm organic filter membrane as the samples to be tested by high performance liquid chromatography.

[0121] (5) The three test samples described above were analyzed by high performance liquid chromatography under the following liquid chromatography conditions, and the chromatograms were recorded. The exact content of the menthol active ingredient before the derivatization reaction in the tested menthol-containing product was then calculated based on the peak area values ​​of the menthol derivatives obtained from the three test samples;

[0122] Calculation formula 1:

[0123] Calculation formula 2:

[0124] Wherein, S0: peak area value of menthol derivatives obtained from the test of standard solution, S1: peak area value of menthol derivatives obtained from the test solution, c0: content concentration of pure menthol in standard solution (mg / mL), c1: content concentration of menthol in test solution (mg / mL), m0: mass of pure menthol (g), m1: mass of menthol-containing product (g), "0.98": reaction progress value (complete reaction is 1), w%: mass fraction of menthol in menthol-containing product;

[0125] The liquid phase conditions are as follows:

[0126] Chromatographic column: C18 5μm (250mm*4.6mm); reversed-phase mobile phase system: acetonitrile:water = 80:20 (v / v); flow rate: 1.0mL / min; detection wavelength: 286nm; column temperature: 30℃; injection volume: 20μL.

[0127] The chromatogram results of the above test are shown in Table 1 below:

[0128] Test samples <![CDATA[t1 / min]]> <![CDATA[t2 / min]]> S Reference solution 2.749 —— —— Standard solution 2.753 8.854 2337642 Test solution 2.747 8.850 2308493

[0129] Table 1

[0130] Note: In Table 1, t1 is the retention time of cinnamoyl chloride, t2 is the retention time of menthyl cinnamate, and S is the peak area of ​​menthyl cinnamate content.

[0131] like Figure 2 As shown in the chromatogram and according to Table 1, the liquid phase peak retention time of 2.749min is cinnamoyl chloride;

[0132] like Figure 3 As shown in the chromatographic separation diagram, and according to Table 1, the liquid phase peak retention time of 2.753min is excessive cinnamoyl chloride, the liquid phase peak retention time of 8.854min is menthol cinnamate, and its peak area value is 2337642;

[0133] like Figure 4 As shown in the chromatographic separation diagram, and according to Table 1, the liquid phase peak retention time of 2.747min is excess cinnamoyl chloride, the liquid phase peak retention time of 8.850min is menthol cinnamate, and its peak area value is 2308493.

[0134] In summary, the data results obtained in Table 1 show that the liquid phase peak signal of excess cinnamoyl chloride appears at around 2.750 min, and the liquid phase peak signal of menthyl cinnamate ester appears at around 8.850 min. Among them, the peak area value of 2337642 of menthyl cinnamate ester obtained from the standard solution test corresponds to 0.5 mg / mL of pure menthol (as shown in the experimental operation description above). According to the peak area value of 2308493 of menthyl cinnamate ester obtained from the test solution test, the menthol content concentration in the test solution can be calculated according to calculation formula 1 to be approximately 0.494 mg / mL. According to calculation formula 2, the mass fraction of menthol in the measured menthol-containing product is calculated to be approximately 34%. Therefore, the menthol content in the measured menthol-containing product is consistent with the menthol content detected by gas phase detection in the menthol-containing product prepared and used in Example 8 of the present invention, indicating that the detection method of the present invention is feasible.

[0135] Based on the known amount of pure menthol and the determined amount of the menthol-containing product, a pre-column nucleophilic substitution esterification reaction is performed for derivatization by controlling the added aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution and maintaining the same reaction environment for both to avoid other errors. The reaction solution (containing the menthol derivative and excess aromatic acid chloride or aromatic sulfonyl chloride) is then diluted to the same solution volume and subjected to liquid chromatography detection. The peak area solubility of the obtained derivative is compared to obtain the concentration of the menthol derivative in the product to be tested. The concentration of the menthol derivative can then be further reduced to the menthol content before the derivatization reaction to obtain the exact content of the menthol active ingredient in the menthol-containing product to be tested before the derivatization reaction.

[0136] The implementation methods of the present invention are not limited to the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivative, characterized in that: The following steps are involved: Step 1) preparing a menthol acetonitrile solution, an acetonitrile solution of a menthol-containing product, and an acetonitrile solution of an aromatic acid chloride or aromatic sulfonyl chloride; the aromatic acid chloride or aromatic sulfonyl chloride used is cinnamoyl chloride; the amount of the aromatic acid chloride or aromatic sulfonyl chloride and pure menthol used is calculated in a molar ratio of 1.2:1; Step 2) 1000 μL each of the menthol acetonitrile solution and the aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added sequentially to a 10 mL volumetric flask, diluted to a total volume of 10 mL by adding acetonitrile, capped the volumetric flask, and heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction. The reaction system was then cooled to room temperature, and the reaction solution was diluted to 10 mL to obtain a reaction solution 1 containing a menthol derivative having absorption in the near-ultraviolet to visible light region; Step 3) As in step 2), 1000 μL of the acetonitrile solution of the menthol-containing product and 1000 μL of the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride prepared above were added to another 10 mL volumetric flask, and the same experimental procedures were repeated to obtain a second reaction solution containing a menthol derivative having absorption in the near-ultraviolet to visible region. Step 4) The aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution, the menthol derivative-containing reaction solution 1, and the menthol derivative-containing reaction solution 2 prepared above are respectively prepared as test solutions using acetonitrile. Then, each of the test solutions is detected and analyzed using a high performance liquid chromatography (HPLC) using a commercially available conventional C-18 column as a chromatographic column, thereby qualitatively and quantitatively determining the menthol derivative in the reaction solution, thereby obtaining the exact content of the menthol active ingredient in the measured menthol-containing product before the derivatization reaction; the reversed-phase mobile phase system when using the HPLC is acetonitrile:water = 80:20 (v / v), and the detection wavelength is 286 nm.

2. The method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivative according to claim 1, characterized in that: The step 1) comprises the following steps: Step 11) Weigh 0.25 g of pure menthol and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 5 mg / mL menthol acetonitrile solution for later use; Step 12) Weigh 0.75 g of the menthol-containing product and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare an acetonitrile solution containing the menthol product for later use; Step 13) Weigh 0.30-0.44 g of aromatic acid chloride or aromatic sulfonyl chloride and dissolve it in a volumetric flask containing 50 mL of acetonitrile solution to prepare a 6-8.8 mg / mL aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution for later use.

3. The method for detecting menthol content by liquid chromatography based on a rapid, catalyst-free pre-column derivative according to claim 1, characterized in that: In the above step 4), when using a high performance liquid chromatograph, the liquid phase conditions are as follows: controlling the flow rate to be 0.7-1.0 mL / min, the injection volume to be 5-20 μL, and the column temperature to be 25-30°C.

4. The method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivatization according to claim 1, characterized in that: In the above step 4), the commercially available conventional C-18 column is C18 5μm 250mm*4.6mm.

5. The method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivatization according to claim 1, characterized in that: In the above step 4), the high performance liquid chromatograph used is one of Agilent LC1260, Waterse2695, iChrom 5100, Agress1100, EClassical3200, and Nexera LC-40.

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