Method for detecting content of menthol based on liquid chromatography of rapid and catalyst-free precolumn derivative

Abstract

The invention discloses a method for detecting the content of menthol based on liquid chromatography of a rapid and catalyst-free pre-column derivative, which comprises the following steps: firstly, carrying out pre-column nucleophilic substitution esterification reaction derivatization on pure menthol and a menthol-containing product to obtain a reaction solution of a menthol derivative capable of being absorbed from a near ultraviolet region to a visible light region; detecting and analyzing by using a high performance liquid chromatograph, so as to qualitatively and quantitatively detect the menthol derivative in the reaction solution, and further obtain the exact content of the effective component of the menthol in the detected product containing the menthol before the derivatization reaction. Compared with the traditional analysis method, the method has the advantages of low cost, practicability, rapidness, high sensitivity, no purification treatment in the reaction, no addition of a reaction catalyst, no appearance of other impurity peaks and the like.

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 based on quick, catalyst-free pre-column derivative liquid chromatography. 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 peppermint oil and Asian peppermint oil.

[0003] Since 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 medicines and chemical products. Therefore, menthol extracted from natural mint plants alone can no longer meet the growing industrial demand. In addition, the natural mint plant has a long maturity cycle, so 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 climate 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 in order to further optimize the menthol production and extraction process.

[0004] Many methods for detecting and analyzing menthol that have been reported include qualitative and quantitative analysis of menthol using a gas flame ionization detector, gas chromatography, liquid refractometer, liquid chromatography-mass spectrometry, and liquid fluorescence detector. At the same time, since menthol itself has no absorption peak signal in the light range of 200-800nm, it is necessary to perform a derivatization reaction on menthol before liquid phase detection. With the help of the original light-absorbing groups of the derivatization reagent, the menthol derivative has an optical response in the absorption region from near ultraviolet to visible light, and is used as a high-performance liquid chromatography detection medium for product menthol content detection sample testing. However, the usual derivatization reaction requires the addition of a catalyst and further purification treatment. Therefore, the above detection and analysis methods have the disadvantages of being expensive, highly polluting, and requiring purification treatment, which is not conducive to large-scale detection of menthol samples. Summary of the invention

[0005] In view of the above-mentioned problems of the background technology, the object of the present invention is to provide a method for detecting the content of menthol by liquid chromatography based on a rapid and catalyst-free pre-column derivative, 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 based on rapid, catalyst-free pre-column derivative liquid chromatography comprises the following steps:

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

[0009] Step 2) 1000 μL of the menthol acetonitrile solution and the aromatic acyl chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added to a 10 mL volumetric flask in sequence, acetonitrile was added to dilute the total volume to 10 mL, the volumetric flask was capped, and the mixture was heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction for derivatization, and then the reaction system was cooled to room temperature, and the reaction solution was fixed 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) According to the above step 2), 1000 μL of the acetonitrile solution of the menthol product and the acetonitrile solution of 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;

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

[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 of the menthol-containing 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 for the aromatic acid chloride or aromatic sulfonyl chloride: the pure menthol.

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

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

[0027] Figure 1 It is a simplified 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 menthol 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 cinnamic acid menthol ester prepared according to Example 8 of the present invention. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention is further described by the following examples. Obviously, the following examples are only 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, rather than to limit the scope of protection of the present invention.

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

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

[0034] Step 2) 1000 μL of the menthol acetonitrile solution and the aromatic acyl chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added to a 10 mL volumetric flask in sequence, acetonitrile was added to dilute the total volume to 10 mL, the volumetric flask was capped, and the mixture was heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction for derivatization, and then the reaction system was cooled to room temperature, and the reaction solution was fixed 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) According to the above step 2), 1000 μL of the acetonitrile solution of the menthol product and the acetonitrile solution of 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;

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

[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 of the menthol-containing 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 reaction derivatization process shown in the figure does not require any catalyst to be added in the pre-column nucleophilic substitution esterification reaction derivatization process, and is carried out only under simple and easy-to-implement experimental conditions such as heating. The obtained menthol derivatization product does not need to be separated from the reaction system, and does not need to be further purified. The separation and purification steps are removed during the reaction process, which can shorten the time of the entire test step, greatly saving about one-third of the time of the entire test step, and 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 by molar ratio of aromatic acid chloride or aromatic sulfonyl chloride:pure menthol is 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-naphthoylmethanesulfonyl 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 the above step 4), when a high performance liquid chromatograph is used, the liquid phase conditions are as follows: the reversed 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), acetonitrile is used to prepare the reaction solution 1 and the reaction solution 2 containing the menthol derivative prepared above into a test solution with a concentration of 0.1-3.0 mg / mL.

[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 in conjunction with specific embodiments.

[0052] 1. Preparation of menthol acetonitrile solution, acetonitrile solution of menthol 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 solution to prepare a 5 mg / mL menthol acetonitrile solution for later use;

[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 of the menthol-containing product for later use;

[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 solution 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 of the menthol-containing product for later use;

[0062] Accurately weigh 0.37 g of 2-naphthoyl chloride (based on a molar ratio of 2-naphthoyl chloride: 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 solution 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 of the menthol-containing product for later use;

[0067] Accurately weigh 0.44 g of 2-naphthylmethylsulfonyl chloride (based on a molar ratio of 2-naphthylmethylsulfonyl chloride: 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 solution 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 of the menthol-containing product for later use;

[0072] Accurately weigh 0.42 g of 4-methyl-3-nitrobenzoyl chloride (based on a molar ratio of 4-methyl-3-nitrobenzoyl chloride: 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 solution 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 of the menthol-containing product for later use;

[0077] Accurately weigh 0.36 g of 3-methoxy-4-methylbenzoyl chloride (based on a molar ratio of 3-methoxy-4-methylbenzoyl chloride: 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 solution 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 of the menthol-containing product for later use;

[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 solution 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 of the menthol-containing product for later use;

[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 solution 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 of the menthol-containing product for later use;

[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 is 34% (gas phase detection result).

[0094] 2. According to the above embodiments, the reaction solution 1 and the reaction solution 2 containing menthol derivatives were prepared:

[0095] The specific preparation steps are:

[0096] In a 10 mL volumetric flask, 1000 μL of the menthol acetonitrile solution and the aromatic acyl 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 fixed 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 product prepared above and 1000 μL of the acetonitrile solution of the aromatic acid chloride or aromatic sulfonyl chloride were added to another 10 mL volumetric flask in sequence, and the same experimental operation was performed to obtain a reaction solution 2 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 containing menthol product, and the acetonitrile solution of aromatic acid chloride or aromatic sulfonyl chloride are respectively prepared:

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

[0100]

[0101] The reaction solution 1 and the reaction solution 2 contain the derivative 2-naphthoic acid menthyl ester, and the structural formula of the derivative 2-naphthoic acid menthyl ester 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 menthol ester, and the structural formula of the derivative 4-methyl-3-nitrobenzoic acid menthol ester is as follows:

[0106]

[0107] The reaction solution 1 and the reaction solution 2 contain the derivative 3-methoxy-4-methyl benzoic acid menthol ester, and the structural formula of the derivative 3-methoxy-4-methyl benzoic acid menthol 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 menthol ester, and the structural formula of the derivative 4-chlorobenzoic acid menthol 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 ingredients 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 scale with HPLC acetonitrile, and shaken well as a reference solution;

[0118] (2) 200 μL of the reaction solution containing the derivative cinnamic acid menthyl ester prepared according to Example 8 above was selected and placed in a 10 mL volumetric flask, diluted to the scale line using HPLC acetonitrile, and shaken well as a standard solution;

[0119] (3) 200 μL of the reaction solution containing the derivative cinnamic acid menthyl ester prepared according to the above Example 8 was selected and placed in a 10 mL volumetric flask, diluted to the scale line 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 to prepare the samples for HPLC testing.

[0121] (5) respectively analyzing the three test samples described above by high performance liquid chromatography under the following liquid phase conditions, recording the chromatograms, and then calculating the exact content of the menthol active ingredient before the derivatization reaction in the tested menthol-containing product 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] Among them, S 0 : Peak area value of menthol derivatives obtained by testing the standard solution, S 1 : Peak area value of menthol derivatives obtained from the test solution, c 0 : The concentration of pure menthol in the standard solution (mg / mL), c 1 : The concentration of menthol in the test solution (mg / mL), m 0 : 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 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[t 1 / min]]> <![CDATA[t 2 / 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: t in Table 1 1 is the retention time of cinnamoyl chloride, t 2 is the retention time of menthol cinnamate, and S is the peak area of ​​menthol 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 according to Table 1 show that the liquid phase peak signal of excess cinnamoyl chloride appears at about 2.750 min, and the liquid phase peak signal of cinnamic acid menthyl ester appears at about 8.850 min; wherein, the peak area value of cinnamic acid menthyl ester obtained by the standard solution test is 2337642, which corresponds to 0.5 mg / mL of pure menthol (as described in the previous experimental operation), and the peak area value of cinnamic acid menthyl ester obtained by the test solution test is 2308493. According to calculation formula 1, the concentration of menthol in the test solution can be calculated to be about 0.494 mg / mL, and according to calculation formula 2, the mass fraction of menthol in the measured menthol-containing product is about 34%; therefore, the menthol content in the measured menthol-containing product is consistent with the menthol gas phase detection result content 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] According to the known amount of pure menthol and the determined amount of the product containing menthol, other errors are avoided by controlling the added aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution and the same reaction environment for the two, and performing pre-column nucleophilic substitution esterification derivatization. The reaction solution (containing menthol derivatives and excess aromatic acid chloride or aromatic sulfonyl chloride) after the reaction is fixed to the same solution volume and then liquid phase detection is performed. The peak area solubility of the obtained derivatives is compared to obtain the concentration of the menthol derivative of the product to be tested, and then further reduced to the menthol content before the derivatization reaction, the exact content of the menthol active ingredient before the derivatization reaction in the measured menthol-containing product can be obtained.

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

Claims

1. A method for detecting menthol content based on rapid, catalyst-free pre-column derivative liquid chromatography, characterized in that: The following steps are involved: Step 1) preparing a menthol acetonitrile solution, an acetonitrile solution containing a menthol product, and an acetonitrile solution of an aromatic acid chloride or an aromatic sulfonyl chloride; Step 2) 1000 μL of the menthol acetonitrile solution and the aromatic acyl chloride or aromatic sulfonyl chloride acetonitrile solution prepared above were added to a 10 mL volumetric flask in sequence, acetonitrile was added to dilute the total volume to 10 mL, the volumetric flask was capped, and the mixture was heated at 55-70° C. for 3 h to perform a pre-column nucleophilic substitution esterification reaction for derivatization, and then the reaction system was cooled to room temperature, and the reaction solution was fixed 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) According to the above step 2), 1000 μL of the acetonitrile solution of the menthol product and the acetonitrile solution of 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; Step 4) The aromatic acid chloride or aromatic sulfonyl chloride acetonitrile solution, the reaction solution containing menthol derivatives, and the reaction solution containing menthol derivatives are respectively prepared as test solutions using acetonitrile, and then each of the test solutions is detected and analyzed using a high performance liquid chromatograph using a commercially available conventional C-18 column as a chromatographic column, thereby qualitatively and quantitatively analyzing the menthol derivatives in the reaction solution, and then obtaining the exact content of the menthol active ingredient before the derivatization reaction in the measured menthol-containing product.

2. The preparation method of the method for detecting menthol content by liquid chromatography based on a quick, 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 of the menthol-containing 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 rapid, catalyst-free pre-column derivative according to claim 2, characterized in that: The amount of aromatic acid chloride or aromatic sulfonyl chloride and pure menthol used is calculated by molar ratio of aromatic acid chloride or aromatic sulfonyl chloride: pure menthol in a ratio of 1.2:

1.

4. The method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative according to claim 2, characterized in that: The types of aromatic acid chlorides or aromatic sulfonyl chlorides used 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.

5. The preparation method of the method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative according to claim 1, characterized in that: In the above step 4), when a high performance liquid chromatograph is used, the liquid phase conditions are as follows: the reversed 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.

6. The preparation method of the method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative according to claim 5, characterized in that: The reverse phase mobile phase system is a mixed solution composed of acetonitrile and water, which is calculated by volume percentage, namely, acetonitrile: water = 70-100%: 0-30%.

7. The preparation method of the method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative according to claim 6, characterized in that: The reverse phase mobile phase system is a mixed solution composed of acetonitrile and water, calculated by volume percentage, that is, acetonitrile: water = 80%: 20%.

8. The preparation method of the method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative 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.

9. The preparation method of the method for detecting menthol content by liquid chromatography based on rapid, catalyst-free pre-column derivative according to claim 1, characterized in that: In the above step 4), the high performance liquid chromatograph used is one of Agilent LC1260, Waters e2695, iChrom 5100, Agress1100, EClassical 3200, and Nexera LC-40.

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