A method for simultaneous determination of six components in Wanjinxiang refreshing gel

Abstract

The invention discloses a method for simultaneously determining 6 components in Wanjinxiang refreshing gel, belongs to the field of drug analysis technology, and solves the problem that there is no method in the prior art that can simultaneously determine multiple components in Wanjinxiang refreshing gel. The invention adopts gas chromatography, and naphthalene is used as an internal standard for determination. The chromatographic conditions are: a polyethylene glycol capillary column is used, nitrogen or an inert gas is used as a carrier gas, the injection port temperature is 230-260°C, and the detector temperature is 230-260°C; the detector type is a hydrogen flame ionization detector; program temperature rise: initial temperature 30-50°C, maintained for 1-5min, 5-20°C / min rises to 210-260°C, and maintained for 8-15min. The invention is scientifically designed and ingeniously conceived. The invention creatively adopts low-priced naphthalene as an internal standard to establish a method for determining the content of Wanjinxiang refreshing gel, and can simultaneously determine 6 index components. The method is simple and easy to operate, has good repeatability, and high sensitivity, and provides a basis for the quality control of Wanjinxiang refreshing gel.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for simultaneously determining six components in Wan Jin Xiang refreshing gel. Background Art

[0002] Wan Jin Xiang refreshing gel is made of Artemisia selengensis oil, eucalyptol and menthol as the main ingredients, which has the effects of sterilization, anti-inflammation, swelling and pain relief, and prevention of mosquito bites. Artemisia selengensis is a herb widely used in ethnic minority areas such as Li, Miao and Zhuang in China and Southeast Asian countries. Artemisia selengensis oil is the essence extracted from Artemisia selengensis by modern processing technology. It tastes spicy, slightly bitter and slightly cold, and has the effects of clearing away heat and detoxification, relieving itching, anti-inflammation and antioxidant. It mainly contains borneol, camphor, β-caryophyllene, linalool and other ingredients.

[0003] Wanjinxiang Shushuang Gel has complex ingredients, and for traditional Chinese medicine with complex ingredients, only the joint control of multiple index ingredients can effectively guarantee the quality of the medicine. In the prior art, there is little research on the quality standard of Wanjinxiang Shushuang Gel, and there is no method that can simultaneously measure multiple ingredients in Wanjinxiang Shushuang Gel. Therefore, providing a detection method for Wanjinxiang Shushuang Gel that can simultaneously measure multiple ingredients therein to achieve the purpose of ensuring the quality of the medicine has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0004] The purpose of the present invention is to provide a method for simultaneously determining six components in the Herb Refreshing Gel, so as to solve the problem that there is no method in the prior art that can simultaneously determine multiple components in the Herb Refreshing Gel.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The invention provides a method for simultaneously determining six components in Wanjinxiang refreshing gel, which adopts gas chromatography and uses naphthalene as an internal standard for determination. The chromatographic conditions are: a polyethylene glycol capillary column is used, nitrogen or an inert gas is used as a carrier gas, the injection port temperature is 230-260° C., the detector temperature is 230-260° C., and the detector type is a hydrogen flame ionization detector;

[0007] Program temperature rise: initial temperature 30-50℃, hold for 1-5min, increase to 210-260℃ at 5-20℃ / min, hold for 8-15min.

[0008] In the method of the present invention, due to the complex composition of the sample to be detected, the advantages of the internal standard method and the external standard method are compared, and it is found that the internal standard method is more suitable for the detection of the sample. In the prior art, the internal standard substance generally adopts meta-xylene, n-propyl alcohol, n-butyl alcohol, phthalic acid, diethyl malonate. The applicant has found through experiments that when the above-mentioned substances are used as internal standard substances, they cannot be effectively separated from the index components. The applicant has unexpectedly found through a large number of experiments that when naphthalene is used as the internal standard substance, the retention time is suitable, and it can be effectively separated from the adjacent components, so naphthalene is selected as the internal standard substance.

[0009] In some embodiments of the present invention, the capillary column has a specification of 30m×0.32mm, 0.25μm. In one embodiment of the present invention, the capillary column is an Agilent DB-WAX capillary column (30m×0.32mm, 0.25μm).

[0010] In some embodiments of the present invention, during measurement, the system constant pressure is 4.65-4.80 psi; preferably 4.73 psi.

[0011] In some embodiments of the present invention, the injection port temperature is 240°C and the detector temperature is 240°C.

[0012] In some embodiments of the present invention, the split ratio is 10:1.

[0013] In some embodiments of the present invention, during the programmed temperature increase, the initial temperature is 40°C, maintained for 2 minutes, increased to 230°C at 10°C / min, and maintained for 10 minutes.

[0014] Some embodiments of the present invention include the following steps:

[0015] Step 1. Prepare an internal standard solution: weigh an appropriate amount of naphthalene, add methanol to dissolve it, and prepare an internal standard solution with a concentration of 50-200 μg / mL;

[0016] Step 2. Prepare a reference solution: weigh an appropriate amount of a menthol reference substance, a camphor reference substance, a eucalyptol reference substance, a linalool reference substance, a borneol reference substance, and an oleanol reference substance, respectively, dissolve them in methanol to prepare a stock solution of each reference substance, then take the stock solution of each reference substance and the internal standard solution, place them in the same container, mix them evenly, and prepare a reference solution; in the reference solution, the content of each component is: menthol 10-30 μg / mL, camphor 1-3 μg / mL, eucalyptol 2-4 μg / mL, linalool 20-80 μg / mL, borneol 4-12 μg / mL, oleanol 1-3 μg / mL, naphthalene 8-30 μg / mL;

[0017] Step 3. Preparation of the test solution: Pipette 0.1-0.3 g of Wan Jin Xiang refreshing gel and 0.2-0.4 mL of internal standard solution into the same container, dilute with methanol and make up to 10 mL, shake well, and obtain the solution;

[0018] Accurately pipette 0.1-0.5uL of internal standard solution, reference solution and test solution respectively, inject into gas chromatograph for determination.

[0019] In some embodiments of the present invention, the concentration of the internal standard solution is 100 μg / mL.

[0020] In some embodiments of the present invention, the content of each component in the reference solution is: menthol 15-20 μg / mL, camphor 2.0-2.5 μg / mL, eucalyptol 2.5-3 μg / mL, linalool 45-48 μg / mL, borneol 7-9 μg / mL, oleanol 1.2-2 μg / mL, and naphthalene 14.5-16.5 μg / mL.

[0021] In some embodiments of the present invention, in step 3, 0.2 g of Wan Jin Xiang refreshing gel and 0.35 mL of internal standard solution are respectively taken into the same container, diluted with methanol and the volume is adjusted to 10 mL.

[0022] The carrier gases described in the present invention are all high-purity gases with a purity of >99.999%.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention is scientifically designed and ingeniously conceived. The invention creatively uses low-priced naphthalene as an internal standard to establish a method for determining the content of Wan Jin Xiang Shu Shuang Gel, and can simultaneously determine 6 index components. The method of the invention is simple and easy to implement, has good repeatability and high sensitivity, and provides a basis for the quality control of Wan Jin Xiang Shu Shuang Gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Attached Figure 1 The chromatogram of the test solution of Example 1;

[0026] Attached Figure 2 It is the chromatogram of the reference substance solution of Example 1;

[0027] Attached Figure 3 This is the chromatogram of the negative sample lacking eucalyptol in Example 1;

[0028] Attached Figure 4 The chromatogram is of the menthol-deficient negative sample of Example 1;

[0029] Attached Figure 5 The chromatogram of the negative sample lacking Artemisia selengensis oil of Example 1 is shown in FIG.

[0030] Attached Figure 6 The chromatogram is a negative sample of the main Blumea balsamifera oil lacking eucalyptol and menthol in Example 1.

[0031] Attached Figure 7 This is the sample chromatogram with a split ratio of 20:1.

[0032] Attached Figure 8 This is the sample chromatogram with the chromatographic column being INNOWAX;

[0033] Attached Fig. 9 The chromatogram of the sample with the chromatographic column being HP-5;

[0034] Attached Fig.10 This is the sample chromatogram with the chromatographic column being DB-1701;

[0035] Attached Fig.11 is the chromatogram with diethyl malonate as internal standard;

[0036] Attached Fig.12 is the sample chromatogram with m-xylene as internal standard;

[0037] Attached Fig.13 is the sample chromatogram with n-propanol as internal standard;

[0038] Attached Fig.14 is the sample chromatogram with n-butanol as internal standard;

[0039] Attached Fig.15 The sample chromatogram of Comparative Example 1 is shown in FIG. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following embodiments.

[0041] The instruments involved in the embodiments of the present invention are as follows: Agilent 7890B, Agilent 6890B gas chromatograph, Discovery DV215CD analytical balance (Ohaus Instruments (Shanghai) Co., Ltd.).

[0042] Reagents and drugs: Wanjinxiang Shushuang Gel was provided by Guizhou Hongyu Pharmaceutical Co., Ltd. Eucalyptol (batch number wkq22011706, purity ≥98%), dianthin (batch number wkq21092408, purity ≥98%), camphor (batch number wkq21112403, purity ≥98%), linalool (batch number wkq21052711, purity ≥98%) reference substances were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; menthol (batch number 110728-201707) and borneol (batch number 110881-200706) reference substances were purchased from Sinopharm Chemical Reagent Co., Ltd.; naphthalene (batch number 20080515, purity ≥99.7%) reference substances were purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. Methanol (batch number 20210906) was of analytical grade and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0043] Example 1

[0044] This embodiment discloses a method for simultaneously determining six components in Wan Jin Xiang refreshing gel of the present invention, which specifically comprises the following steps:

[0045] 1. Preparation of internal standard solution: Take an appropriate amount of naphthalene, accurately weigh it, add 10 mL of methanol to make an internal standard solution with a concentration of 0.446 mg / mL, and store it at 2-8°C for later use.

[0046] 2. Preparation of reference solution: Weigh appropriate amount of menthol reference substance, camphor reference substance, eucalyptol reference substance, linalool reference substance, borneol reference substance and dianthusol reference substance, dissolve them in methanol respectively, and prepare reference substance stock solutions with concentrations of 0.2904, 0.0138, 0.1020, 0.0169, 0.0498 and 0.0102 mg / mL respectively. Accurately pipette 1.60, 1.59, 1.66, 1.60, 1.60 and 1.60 mL of each and 0.35 mL of internal standard solution to obtain (the mass concentrations of each component are 0.0465, 0.0022, 0.0170, 0.0027, 0.0080, 0.00163 and 0.0156 mg / mL respectively), and store them at 2-8°C for use.

[0047] Table 1

[0048] Concentration of reference substance stock solution mg / mL Volume mL Concentration in reference solution μg / mL Menthol 0.2904 1.6 46.5 Camphor 0.0138 1.59 2.2 Eucalyptus 0.102 1.66 16.9 Linalool 0.0169 1.6 2.7 Borneol 0.0498 1.6 8.0 Caryophyllene 0.0102 1.6 1.6 Internal Standard 0.0976 0.35 3.4

[0049] 3. For the test solution, accurately pipette 0.2 g of Wan Jin Xiang refreshing gel and 0.35 mL of internal standard solution, make up to 10 mL with methanol, shake well, and store at 2-8 °C for future use.

[0050] 4. Preparation of negative sample solutions: Take the negative sample lacking eucalyptol, the negative sample lacking menthol, the negative sample lacking blumen oil, and the negative sample lacking eucalyptol, menthol and blumen oil, and prepare each negative sample solution according to the preparation method of the test solution.

[0051] 5. Chromatographic conditions: Agilent DB-WAX capillary column (30 m×0.32 mm, 0.25 μm); carrier gas: high-purity nitrogen (purity>99.999%); constant pressure: 4.73 psi (1 psi=6.895 kPa); inlet temperature: 240°C; detector temperature: 240°C; split ratio: 10:1; programmed temperature (initial temperature: 40°C, maintained for 2 min, increased to 230°C at 10°C / min, maintained for 10 min); injection volume: 0.3 μL.

[0052] Accurately pipette the reference solution, donor solution and each negative sample solution, inject them into the gas chromatograph and measure to obtain the result.

[0053] The results are attached Figure 1-6 shown.

[0054] The results showed that the chromatographic peaks were well separated and there was no interference with the negative. The chromatographic peak of the internal standard naphthalene was located between borneol and dianthin, and the chromatographic peak separation effect of naphthalene was good, which could prevent the occurrence of systematic errors. In addition, the price of naphthalene was relatively low, so naphthalene was selected as the internal standard.

[0055] Example 2

[0056] This embodiment discloses the investigation of the linear relationship of each index component, specifically:

[0057] Take the reference substance stock solution of Example 1, accurately pipette 0.30, 0.40, 0.50, 1.00, and 2.00 mL into different containers, then accurately add 0.35 mL of the internal standard solution to each container, and dilute to 5 mL with methanol to obtain reference substance solutions of different concentrations.

[0058] The chromatographic conditions of Example 1 were used for determination, and regression was performed with the mass concentration of each reference substance as the abscissa (X) and the ratio of the peak area of ​​each reference substance to the internal standard as the ordinate (Y). Menthol had a good linear relationship at 0.0170-0.1020 mg / mL, camphor had a good linear relationship at 0.0022-0.0138 mg / mL, linalool had a good linear relationship at 0.0456-0.2904 mg / mL, and menthol had a good linear relationship at 0.0027-0.0140 mg / mL.

[0059] The linear relationship was good at 0.0169 mg / mL, borneol had a good linear relationship at 0.0080-0.0498 mg / mL, and oleanol had a good linear relationship at 0.0016-0.0976 mg / mL.

[0060] The results show that the six index components of the present invention have good linear relationships within their respective ranges.

[0061] Table 2 Linear relationship of each component

[0062] Element Regression equation r Linear range, mg / mL Eucalyptus Y=930.13X+0.7509 0.9997 0.0170～0.1020 Camphor Y=1851.9X+0.2952 0.9999 0.0022～0.0138 Linalool Y=628.94X+0.3135 0.9998 0.0456～0.2904 Menthol Y=2075.5X+3.6112 1.0000 0.0027～0.0169 Borneol Y=1497.7X+0.5412 1.0000 0.0080～0.0498 Caryophyllene Y=183.36X+1.4695 0.9992 0.0016～0.0976

[0063] Example 3

[0064] This embodiment discloses the precision study of the method of the present invention, specifically:

[0065] The reference solution of Example 1 was accurately aspirated, and the sample was measured 6 times according to the chromatographic conditions of Example 1. The RSDs of the areas of menthol, camphor, eucalyptol, linalool, borneol and dianthin were 0.94%, 1.75%, 2.25%, 1.65%, 1.93% and 1.76%, respectively, indicating that the instrument had good precision.

[0066] Example 4

[0067] This embodiment discloses the stability test of the method of the present invention, specifically:

[0068] Take the test solution of Example 1 (room temperature 20°C, relative humidity 80%), and according to the chromatographic conditions of Example 1, sample injection and measurement were performed at 0, 3, 6, 12, and 24 h, respectively. The RSDs of menthol, camphor, eucalyptol, linalool, borneol, and dianthin areas were 1.69%, 2.87%, 2.51%, 1.87%, 2.39%, and 1.06%, respectively, indicating that the solution had good stability within 24 h.

[0069] Example 5

[0070] This embodiment discloses a repeatability test of the method of the present invention, specifically:

[0071] From the same batch of condensate, 6 test solutions were prepared according to the method of Example 1. The samples were injected and measured according to the chromatographic conditions of Example 1. The RSDs of menthol, camphor, eucalyptol, linalool, borneol and dianthin were 1.89%, 2.25%, 2.43%, 1.56%, 2.49% and 3.00%, respectively, indicating that the method had good repeatability.

[0072] Example 6

[0073] This embodiment discloses a sample recovery test of the method of the present invention, specifically:

[0074] Take 9 portions of the same batch of condensate with known contents of each component, each portion is about 0.1g, accurately weighed, and add reference solution at 0.8, 1, and 1.2 times the content of each component, respectively, and dilute to 10mL with methanol. Prepare the test solution according to the method of Example 1, and inject and measure according to the chromatographic conditions of Example 1 to calculate the recovery rate. The results are shown in Table 3. The recovery rate of the method of the present invention is good.

[0075] Table 3 Sample recovery determination results

[0076]

[0077]

[0078] Example 7 This example discloses the use of the method of the present invention to determine the sample content, specifically:

[0079] Three batches of condensate were taken and 9 test solutions were prepared according to the method of Example 1, 3 in each batch. The samples were injected and measured under the chromatographic conditions of Example 1, the chromatograms were recorded, and the contents of the six components were calculated according to the standard curve method. The results are shown in Table 4.

[0080] Table 4 Determination results of the content of each component (mg·g -1 )

[0081] Sample No. Menthol Camphor Eucalyptus Linalool Borneol Caryophyllene S1 1.4620 0.0994 0.5256 0.0874 0.3910 0.0839 S2 1.4665 0.0998 0.5260 0.0862 0.3925 0.0847 S3 1.5485 0.1049 0.5316 0.0929 0.4180 0.0875 S4 1.4690 0.0969 0.5365 0.0874 0.4039 0.0858 S5 1.4743 0.0974 0.5362 0.0863 0.4054 0.0843 S6 1.4728 0.0975 0.5297 0.0877 0.4046 0.0840 S7 1.5430 0.1043 0.5310 0.0939 0.4163 0.0869 S8 1.4633 0.0986 0.5259 0.0869 0.3916 0.0841 S9 1.5398 0.1033 0.5311 0.0934 0.4173 0.0876

[0082] Comparative Example 1

[0083] Compared with Example 1, the heating rate of this comparative example is different, and the other conditions are the same.

[0084] In this comparative example, the initial temperature was 40°C, maintained for 2 min, then raised to 210°C at 20°C / min and maintained for 8 min.

[0085] The results are attached Fig.15 As shown, the chromatographic peaks of the test solution are effectively separated.

[0086] Comparative Example 2

[0087] Compared with Example 1, the split ratio of this comparative example is different, and the other conditions are the same.

[0088] The split ratio of this comparative example is 20:1. The results are shown in the attached Figure 7 As shown, the chromatographic peaks of the test solution are effectively separated.

[0089] Comparative Example 3

[0090] Compared with Example 1, the chromatographic column used in this comparative example is different, and the other conditions are the same.

[0091] This comparative example uses INNOWAX, HP-5, and DB-1701 chromatographic columns for testing.

[0092] The results are attached Figure 8-10 As shown, these three chromatographic columns cannot effectively separate the components in the sample.

[0093] Comparative Example 4

[0094] This comparative example uses diethyl malonate, m-xylene, n-propanol, and n-butanol as internal standards. The internal standard is added to the test sample according to the method of Example 1 to prepare the test solution. The chromatographic conditions of Example 1 are used for determination. The results are shown in the attached figure. Figure 11-14 When selecting an internal standard, the position of the internal standard's chromatographic peak should be close to that of the component being measured, or between the chromatographic peaks of several components being measured. The retention times of m-xylene, n-propanol, and n-butanol are too short, and they are far away from the positions of the chromatographic peaks of the components being measured. Therefore, they are not suitable as internal standards.

[0095] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining six components in Wan Jin Xiang refreshing gel, characterized in that: The gas chromatography method was used, with naphthalene as the internal standard, and the chromatographic conditions were: a polyethylene glycol capillary column, nitrogen as the carrier gas, an injection port temperature of 230-260°C, a detector temperature of 230-260°C; the detector type was a hydrogen flame ionization detector; Program temperature rise: initial temperature 40℃, hold for 2min, increase to 230℃ at 10℃ / min, hold for 10min; The polyethylene glycol capillary column is an Agilent DB-WAX capillary column; The six ingredients are menthol, camphor, eucalyptol, linalool, borneol and dianthin.

2. The method according to claim 1, characterized in that The capillary column has specifications of 30m×0.32mm and 0.25μm.

3. The method according to claim 1 or 2, characterized in that: During measurement, the system constant pressure is 4.65-4.80psi.

4. The method according to claim 1 or 2, characterized in that: During measurement, the system constant pressure was 4.73 psi.

5. The method according to claim 1 or 2, characterized in that: The injection port temperature was 240°C and the detector temperature was 240°C.

6. The method according to claim 1 or 2, characterized in that: The split ratio is 10:

1.

7. The method according to claim 1, characterized in that The following steps are involved: Step 1. Prepare an internal standard solution: weigh an appropriate amount of naphthalene, add methanol to dissolve it, and prepare an internal standard solution with a concentration of 50-200 μg / mL; Step 2. Prepare reference solution: weigh appropriate amount of menthol reference, camphor reference, eucalyptol reference, linalool reference, borneol reference and dianthin reference respectively, dissolve them in methanol to prepare stock solution of each reference, then take the stock solution and internal standard solution of each reference respectively, put them in the same container, mix them evenly, and prepare reference solution; the content of each component in the reference solution is: menthol 10-30 μg / mL, camphor 1-3 μg / mL, eucalyptol 2-4 μg / mL, linalool 20-80 μg / mL, borneol 4-12 μg / mL, dianthin 1-3 μg / mL, naphthalene 8-30 μg / mL; Step 3. Preparation of the test solution: Pipette 0.1-0.3 g of Wan Jin Xiang refreshing gel and 0.2-0.4 mL of internal standard solution into the same container, dilute with methanol and make up to 10 mL, shake well, and obtain the solution; Accurately pipette 0.1-0.5uL of internal standard solution, reference solution and test solution respectively, inject into gas chromatograph and measure to obtain the result.

8. The method according to claim 7, characterized in that The concentration of the internal standard solution was 100 μg / mL.

9. The method according to claim 7, characterized in that: The contents of the components in the reference solution are as follows: 15-20 μg / mL of menthol, 2.0-2.5 μg / mL of camphor, 2.5-3 μg / mL of eucalyptol, 45-48 μg / mL of linalool, 7-9 μg / mL of borneol, 1.2-2 μg / mL of oleanolic acid, and 14.5-16.5 μg / mL of naphthalene.

10. The method according to claim 7, characterized in that In the step 3, 0.2 g of Wan Jin Xiang refreshing gel and 0.35 mL of internal standard solution were respectively taken into the same container, and the volume was diluted with methanol to 10 mL.

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