Multi-component determination method for sterilizing and itching-relieving lotion

Abstract

The invention belongs to the technical field of medicine detection, and discloses a multi-component determination method of a sterilizing and itching-relieving lotion, which can accurately determine the contents of astilbin, berberine hydrochloride and osthole in the sterilizing and itching-relieving lotion. The method comprises the following steps: preparation of a reference substance solution: precisely weighing astilbin, berberine hydrochloride and osthole respectively, and dissolving in a methanol solvent to prepare the reference substance solution; preparing a test solution, namely precisely measuring the sterilizing and itching-relieving lotion, adding a methanol solution, diluting and filtering to obtain the test solution; and determining the content of multiple components: respectively sucking the test solution and the reference solution, injecting the test solution and the reference solution into a high performance liquid chromatograph for determination to obtain a chromatogram, and calculating the contents of astilbin, berberine hydrochloride and osthole according to a standard curve regression equation. The determination method is simple and convenient to operate, good in reproducibility and free of negative control interference, and can be efficiently used for quality control of the sterilizing and itching-relieving lotion.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a multi-component determination method for a bactericidal and antipruritic lotion. Background Art

[0002] The bactericidal and antipruritic lotion is composed of six medicinal materials, including Sophora flavescens, Phellodendron chinense, Cnidium monnieri, Smilax glabra, Atractylodes macrocephala, and Borneol. It has the effects of clearing away heat and detoxifying, killing insects and relieving itching. It is mainly used to improve symptoms such as excessive vaginal discharge and vulvar itching caused by trichomoniasis, fungal vaginitis, nonspecific vaginitis and vulvar pruritus.

[0003] The premise of ensuring the stability of the efficacy of antiseptic and antipruritic lotion is to conduct scientific testing on its quality, among which the determination of the content of multiple important active ingredients is particularly important. Since the compound preparation of Chinese patent medicine has many material bases, most of which are the result of synergistic effects, it is difficult for a single ingredient to effectively control the quality of the compound Chinese medicine preparation, and the determination of the content of multiple ingredients has become necessary.

[0004] Although the content determination method of antiseptic and antipruritic lotion has been made public, such as Meng Xiaoxia et al. established an HPLC fingerprint inspection method for antiseptic and antipruritic lotion [1-3] However, it fails to clearly identify the medicinal flavor and indicator ingredients, making it difficult to effectively control the quality of the bactericidal and antipruritic lotion, and the operation is relatively complicated.

[0005] Another example is the patent with announcement number CN 109633055B, which also discloses a method for determining the content of multiple components of a bactericidal and antipruritic lotion. The quality of the lotion can be controlled by determining the contents of matrine, atractylodesin, berberine hydrochloride, osthole and astilbene, but the method is complicated to operate. For example, the method for preparing the sample solution is relatively cumbersome, which is as follows: take 5 mL of bactericidal and antipruritic lotion and dilute it 1 to 3 times with water, ultrasonically treat it for 10 min, make up to volume and shake well, transfer it to a separatory funnel, add 2 mL of ammonia water and shake well; then extract it with chloroform for 3 times, combine the chloroform solutions, evaporate to dryness in a water bath, dissolve the residue with methanol and make up to 10 mL, and shake well.

[0006] Therefore, the prior art needs to be further improved. Summary of the invention

[0007] In view of the problems existing in the above-mentioned prior art, the present invention provides a multi-component determination method for a bactericidal and antipruritic lotion. The determination method is simple to operate, has good reproducibility, has no interference from the negative control, and can be effectively used for the quality control of the bactericidal and antipruritic lotion.

[0008] To achieve the above objectives, this application provides the following technical solutions: The present application provides a multi-component determination method for a bactericidal and antipruritic lotion, which includes a method for determining the content of astilbin, berberine hydrochloride and osthole. The method for determining the content of astilbin, berberine hydrochloride and osthole includes the following steps: Preparation of reference solution: accurately weigh astilbin, berberine hydrochloride and osthole respectively, dissolve them in methanol solvent to prepare reference solution; Preparation of the test solution: Accurately measure the antiseptic and antipruritic lotion, add methanol solution to dilute and filter, and obtain the test solution.

[0009] Determination of multiple components: The test solution and the reference solution were respectively drawn and injected into a high performance liquid chromatograph for determination to obtain a chromatogram, and the contents of astilbin, berberine hydrochloride and osthole were calculated according to the standard curve regression equation.

[0010] The specific method is: calculate the content of the component in the test solution corresponding to the reference solution based on the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the component in the test solution corresponding to the reference solution in the chromatogram.

[0011] In terms of the selection of index ingredients, modern pharmacological studies have shown that antiseptic and antipruritic lotions have the effects of clearing away heat and detoxifying, as well as killing bacteria and relieving itching. Among them, osthole, the main coumarin active ingredient in Cnidium monnieri, has significant effects in antibacterial, anti-inflammatory and insecticidal effects; berberine, jatrorrhizine, phellodendronine and bamipine, the main alkaloid active ingredients in Phellodendron chinense, have obvious pharmacological effects such as anti-inflammatory, antibacterial and analgesic effects; atractylodesin, the main volatile oil active ingredient in Atractylodes lancea, has significant effects in antibacterial and anti-inflammatory effects; astilbin and smilax glabra, the main flavonoid active ingredients in Smilax glabra, have obvious pharmacological effects such as antibacterial, anti-inflammatory and analgesic effects. Therefore, in the selection of test ingredients, berberine hydrochloride is the index ingredient of Phellodendron chinense; astilbin is the index ingredient of Smilax glabra; osthole is the index ingredient of Cnidium monnieri; and atractylodesin is the index ingredient of Atractylodes lancea.

[0012] Preferably, in the method for determining the contents of astilbin, berberine hydrochloride and osthole, the volume fraction of methanol in the methanol solvent is 100%.

[0013] Preferably, in the method for determining the content of astilbin, berberine hydrochloride and osthole, the preparation method of the test solution is: take the bactericidal and antipruritic lotion, dilute it 5 times with 100% methanol, shake well, and filter.

[0014] Preferably, in the method for determining the content of astilbin, berberine hydrochloride and osthole, the preparation method of the reference solution is: accurately weigh appropriate amounts of astilbin, berberine hydrochloride and osthole reference substances, respectively, and add methanol to prepare reference solutions with mass concentrations of 186.08, 453.44 and 124.23 μg▪mL-1, respectively.

[0015] Preferably, in the method for determining the content of astilbin, berberine hydrochloride and osthole, the elution program in the high performance liquid chromatography is: 0-20 min, 5% B→22% B; 20-35 min, 22% B→35% B; 35-50 min, 35% B→90% B; 50-55 min, 90% B.

[0016] Preferably, in the method for determining the contents of astilbin, berberine hydrochloride and osthole, the conditions of high performance liquid chromatography are: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution-acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30℃.

[0017] Preferably, in the method for determining the contents of astilbin, berberine hydrochloride and osthole, the detection wavelength used in the high performance liquid chromatography is: 1-44 min, 280 nm; 45-55 min, switched to 322 nm.

[0018] Experiments have shown that based on the above chromatographic method, 280nm was selected as the detection wavelength, and astilbin and berberine hydrochloride could be accurately determined with good reproducibility; and when the wavelength was switched to 322nm at 45-55min, osthole was accurately determined, and the results showed good wavelength reproducibility.

[0019] Atractylodes lancea is an important raw material for the bactericidal and antipruritic lotion and is an important indicator for evaluating the quality of the drug. The effective ingredients of Atractylodes lancea are mostly concentrated in volatile components, among which the compound with the highest content is atractylodesin. Therefore, the identification of atractylodesin components is helpful for more comprehensive quality control of the bactericidal and antipruritic lotion.

[0020] Therefore, on the basis of the above, further, the multi-component determination method of the bactericidal and antipruritic lotion also includes a method for identifying atractylodesin, and the method for identifying atractylodesin includes the following steps: Preparation of reference solution: weigh atractylodesin and dissolve it in methanol solvent to prepare a reference solution; Preparation of the test solution: Accurately measure the antiseptic and antipruritic lotion, add methanol solution to dilute and filter, and obtain the test solution.

[0021] Determination of atractylodesin: aspirate 1 % of the test solution and the reference solution respectively, inject into the high performance liquid chromatograph, and record the chromatogram; the elution procedure of the liquid chromatography is gradient elution.

[0022] Optionally, the preparation method of the atractylodesin reference solution is: accurately weigh appropriate amounts of atractylodesin reference substances, and add methanol to prepare reference solutions with a mass concentration of 0.26 μg▪mL-1.

[0023] Preferably, in the method for identifying atractylodesin in the bactericidal and antipruritic lotion, the conditions of high performance liquid chromatography are: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution-acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30°C; The detection wavelength is: 1~44min, 280nm; 45~57min, switch to 340nm.

[0024] Preferably, in the method for identifying atractylodesin in the bactericidal and antipruritic lotion, the elution procedure in the high performance liquid chromatography is: 0-20 min, 5% B→22% B; 20-35 min, 22% B→35% B; 35-50 min, 35% B→90% B; 50-57 min, 90% B.

[0025] The present invention has the following beneficial effects: The multi-component determination method of the bactericidal and antipruritic lotion provided by the present invention is simple to operate, and not only simplifies the processing methods of the reference solution and the test solution, but also can accurately determine the contents of the three components of astilbin, berberine hydrochloride and osthole in the bactericidal and antipruritic lotion, and the determination results are accurate, repeatable and stable, and there is no interference from the negative control; on this basis, a method for identifying atractylodesin in the bactericidal and antipruritic lotion is further provided, based on the precise analysis of the above four components, thereby ensuring a more comprehensive quality control of the bactericidal and antipruritic lotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the high performance liquid chromatogram of astilbin reference substance solution; Figure 2 is the HPLC chromatogram of berberine hydrochloride reference solution; Figure 3 is the HPLC chromatogram of osthole reference solution; Figure 4 This is the HPLC chromatogram of the test solution of the antiseptic and antipruritic lotion; Peak 1 is astilbin; Peak 2 is berberine hydrochloride; Peak 3 is osthole; Figure 5 The HPLC chromatogram of the negative sample solution lacking Smilax glabra (astilbin); Figure 6 The HPLC chromatogram of the negative sample solution lacking Phellodendron chinense (berberine hydrochloride); Figure 7 The HPLC chromatogram of the negative sample solution lacking Cnidium monnieri (osthole); Figure 8 is the HPLC chromatogram of the atractylodesin reference substance solution; Fig. 9 is the HPLC chromatogram of the negative sample solution lacking atractylodesin; Fig.10 It is the HPLC chromatogram of atractylodesin in the test solution of the antiseptic and antipruritic lotion under the detection conditions; 1 is atractylodesin; Fig.11 The HPLC chromatograms of three groups under different conditions in Comparative Example 1 are shown; Fig.12 The chromatogram results of comparative example 2 detected at wavelengths of 220 nm, 240 nm and 260 nm; Fig.13 The chromatogram results of comparative example 2 detected at wavelengths of 280 nm, 300 nm and 320 nm; Fig.14 The chromatogram results of Comparative Example 3 in which the extraction solvents were set to water, 25% methanol, and 50% methanol respectively; Fig.15 The chromatogram results of Ratio 3 when the extraction solvent was set to 75% methanol and 100% methanol respectively. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0028] 1. Instruments and reagents Instruments: LC-20AT liquid chromatograph (Shimadzu Corporation, Japan), XS205DU electronic balance (1:100,000, Mettler-Toledo Instrument Co., Ltd.); AB204-S electronic balance (1:10,000, Mettler-Toledo Instrument Co., Ltd.).

[0029] Reference substances: astilbin (purchased from China Food and Drug Inspection Institutes, batch number: 111798-201805, content is 93.6%), berberine hydrochloride (purchased from China Food and Drug Inspection Institutes, batch number: 110713-202015, content is 85.9%), osthole (purchased from China Food and Drug Inspection Institutes, batch number: 110822-201710, content is 99.5%); atractylodesin (purchased from China Food and Drug Inspection Institutes, batch number: 111924-201806, content is 99.5%).

[0030] Reagents: Methanol, formic acid, and triethylamine were of analytical grade, acetonitrile was of chromatographic grade, and water was ultrapure water.

[0031] Test sample: Antibacterial and antipruritic lotion was provided by Guizhou Changsheng Pharmaceutical Co., Ltd.

[0032] Example 1 Determination of the Contents of Astilbin, Berberine Hydrochloride and Osthole in Antibacterial and Antipruritic Lotion In order to better control the quality of the antiseptic and antipruritic lotion, ensure the efficacy and simplify the operation, based on the study of four medicinal materials, namely, Phellodendron chinense, Fructus Cnidium monnieri, Smilax glabra and Atractylodes lancea, this embodiment provides an HPLC content determination method for three components, namely, astilbin, berberine hydrochloride and osthole, in the antiseptic and antipruritic lotion, so as to ensure a more comprehensive quality control of the antiseptic and antipruritic lotion.

[0033] 1. The method for determining the contents of astilbin, berberine hydrochloride and osthole in the bactericidal and antipruritic lotion provided in this embodiment comprises the following steps: (1) Preparation of reference solution: Astilbin, berberine hydrochloride and osthole were accurately weighed and dissolved in 100% methanol solvent to obtain a mass concentration of 86.08 μg / mL -1 , 453.44μg▪mL -1 , 124.23μg▪mL -1 of reference solution; (2) Preparation of test solution: Take 2 ml of antiseptic and antipruritic lotion and place it in a 10-ml volumetric flask. Add methanol to dilute to the mark, shake well, and filter.

[0034] (3) Preparation of negative sample solution According to the prescription and preparation process of the antiseptic and antipruritic lotion, negative samples lacking Phellodendron amurense, Cnidium monnieri or Smilax glabra were prepared respectively, and then negative control solutions lacking Phellodendron amurense, Sophora flavescens, Cnidium monnieri and Smilax glabra were prepared according to the preparation method of the test sample solution in (2).

[0035] (4) Determination of content of multiple components: 20 μl of the test solution and the reference solution were respectively taken and injected into the high performance liquid chromatograph for determination to obtain a chromatogram, and the contents of astilbin, berberine hydrochloride and osthole were calculated according to the standard curve regression equation. Specifically, the contents of the components corresponding to the reference solution in the test solution were calculated according to the concentration of the reference solution, the peak area of ​​the reference solution in the chromatogram, and the peak area of ​​the components corresponding to the reference solution in the test solution in the chromatogram.

[0036] The method of described high performance liquid chromatography is as follows: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution (A)-acetonitrile (B); Flow rate: 1.0 mL / min; Column temperature: 30℃.

[0037] The elution procedure was: 0~20min, 5%B→22%B; 20~35min, 22%B→35%B; 35~50min, 35%B→90%B; 50~55min, 90%B.

[0038] The detection wavelength is: 1~44min, 280nm; 45~55min, switch to 322nm.

[0039] Ten batches of antiseptic and antipruritic lotion samples were taken and assayed according to the above method. The contents of astilbin, berberine hydrochloride and osthole were calculated according to the standard curve regression equation.

[0040] The experimental results are shown in Table 1, which show that the method provided in this embodiment can accurately determine the content of astilbin, berberine hydrochloride and osthole in the bactericidal and antipruritic lotion, and the method established in this embodiment is simple to operate, has good reproducibility, has no interference from the negative control, and has high feasibility.

[0041] Table 1 Results of multiple batches of assays 2. Methodological investigation 2.1 Exclusivity Investigation (1) Methods: The test solution and the reference solution were prepared according to the above method, and 20 μl of each solution was taken and injected into a high performance liquid chromatograph for measurement to obtain a chromatogram.

[0042] (2) Results and analysis The HPLC chromatogram results of astilbin reference substance, berberine hydrochloride reference substance and osthole reference substance are shown in Figure 1~Figure 3, while the chromatogram of the antiseptic and antipruritic lotion sample is Figure 4 The chromatogram results of negative samples lacking Phellodendron chinense, Cnidium monnieri or Smilax glabra are shown in the table below. Figure 5~Figure 7 From the above chromatogram analysis, it can be seen that peak 1 is astilbin, peak 2 is berberine hydrochloride, and peak 3 is osthole.

[0043] From the above results, it can be seen that in the negative sample spectrum, no chromatographic peak is shown at the same retention time as that of astilbin, berberine hydrochloride and osthole reference substances, so the negative sample has no effect on the determination and there is no negative interference.

[0044] 2.2 Analysis of linear relationships (1) Methods Accurately pipette 0.2 mL, 0.5 mL, 1.0 mL, 2 mL, and 5 mL of the astilbin reference substance stock solution (i.e., the aforementioned reference substance solution) prepared in this example into a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare a series of concentrations of astilbin reference substance solutions.

[0045] Accurately pipette 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2 mL of the berberine hydrochloride reference substance stock solution (i.e., the aforementioned reference substance solution) prepared by the aforementioned method into a 5 mL volumetric flask, add methanol to dilute to the scale, and shake well to prepare a series of berberine hydrochloride reference substance solutions.

[0046] Accurately pipette 0.1 mL, 0.25 mL, 0.5 mL, 1.0 mL, and 2.5 mL of the osthole reference substance stock solution (i.e., the aforementioned reference substance solution) prepared by the aforementioned method into a 10 mL volumetric flask, add methanol to dilute to the scale, and shake well to prepare a series of osthole reference substance solutions.

[0047] Take 20 µL of the above series of concentration reference solution for injection respectively, and perform HPLC analysis according to the above method.

[0048] (2) Results and analysis Linear regression was performed with the peak area integral value as the ordinate (Y) and the injection volume (μg) as the abscissa (X), and the regression equations of astilbin, berberine hydrochloride and osthole were obtained. The results are shown in Table 2. It can be seen that the linear range of this determination method is wide and the minimum limit of detection is smaller.

[0049] Table 2 Regression equation and linear range 2.3 Instrument precision test (1) Methods Take the mixed reference solution (astilbin 9.3μg / mL -1 , berberine hydrochloride 45.3μg▪mL-1 and osthole 3.1μg▪mL -1 The mixed reference solution was injected 6 times in succession, 20 μL each time, and the peak area values ​​of astilbin, berberine hydrochloride and osthole were determined according to the aforementioned method of this example, and the RSD values ​​of the contents were calculated.

[0050] (2) Experimental results: The results showed that the RSD values ​​of astilbin, berberine hydrochloride and osthole were 0.42%, 0.5% and 0.46%, respectively, which indicated that the determination method of this example had good precision.

[0051] 2.4 Repeatability test (1) Methods Take the same batch of samples (batch number: 15230509), and prepare 6 test solutions in parallel according to the preparation method of the test solution described above. Perform HPLC determination according to the method described above in this example to calculate the contents of astilbin, berberine hydrochloride and osthole and the corresponding RSD values.

[0052] (2) Experimental results The results showed that the RSD values ​​of the contents of the three components, astilbin, berberine hydrochloride and osthole, were 0.97%, 0.6% and 1.42%, respectively. This result shows that the repeatability of the determination method in the embodiment is good.

[0053] 2.5 Sample recovery test (1) Methods Take the sample with measured content (batch number: 15230509), take 6 portions, accurately measure 2.5 ml of each portion, place in a 25 mL volumetric bottle, and add the reference substance concentrated solution (astilbin 66.96 μg / mL -1 , berberine hydrochloride 144.0μg▪mL -1 and osthole 9.92μg▪mL -1 2.5 mL of the mixed reference solution) was diluted to the mark with methanol, shaken and filtered to obtain the test solution. The determination was carried out according to the above method, and the average recovery rate and RSD of astilbin, berberine hydrochloride and osthole were calculated.

[0054] (2) Experimental results The results are shown in Table 3, which shows that the average recoveries of the three components, astilbin, berberine hydrochloride and osthole, are 97.88%, 98.79% and 98.21%, respectively, which indicates that the method of this embodiment has good sample recovery.

[0055] Table 3 Results of sample recovery test 2.6 Solution stability test (1) Experimental methods Take the same test solution and operate according to the method of this example to determine the peak areas of the three components, astilbin, berberine hydrochloride and osthole, at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h, respectively, and calculate the RSD values ​​of the contents of the three components.

[0056] (2) Experimental results and analysis The results showed that the RSD values ​​of the contents of astilbin, berberine hydrochloride and osthole were 1.39%, 1.41% and 1.15% respectively. The results showed that the three components of astilbin, berberine hydrochloride and osthole were basically stable within 24 hours, and the solution prepared by this method had good stability and could meet the detection needs.

[0057] Example 2 Identification method of atractylodesin in antiseptic and antipruritic lotion 1. This embodiment further provides a method for qualitative identification of atractylodesin in a bactericidal and antipruritic lotion based on Example 1, the method comprising the following steps: (1) Preparation of reference solution: Accurately weigh appropriate amounts of atractylodesin reference substance and add methanol to prepare reference substance solutions with a mass concentration of 0.26 μg▪mL-1.

[0058] (2) Preparation of test solution: Take an appropriate amount of this product, mix well, accurately measure 2 mL, place in a 10 mL volumetric flask, add methanol to dilute to the scale, shake well, filter, and obtain.

[0059] (3) Preparation of negative sample solution According to the prescription and preparation process of the antiseptic and antipruritic lotion, a negative sample lacking Atractylodes lancea was prepared, and then a negative control solution lacking Atractylodes lancea was prepared according to the preparation method of the test solution in (2).

[0060] (4) Detection of atractylodesin Accurately pipette 20µl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, perform the measurement, and obtain the chromatogram.

[0061] The method of described high performance liquid chromatography is as follows: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution (A)-acetonitrile (B); Flow rate: 1.0 mL / min; Column temperature: 30℃.

[0062] The elution procedure was: 0~20min, 5%B→22%B; 20~35min, 22%B→35%B; 35~50min, 35%B→90%B; 50~57min, 90%B.

[0063] The detection wavelength is: 1~44min, 280nm; 45~57min, switch to 340nm.

[0064] 2. Methodological investigation 2.1 Specificity Investigation (1) Methods Accurately pipette appropriate amounts of the test solution and reference solution prepared according to the above method of this embodiment, and perform the measurement according to the above method.

[0065] (2) Results and analysis The chromatogram of atractylodesin reference substance is shown in Figure 8 The chromatogram of the negative sample solution lacking Atractylodes lancea is shown in Fig. 9 , the chromatogram of the antiseptic and antipruritic lotion sample is shown in Fig.10 , the result shows that peak 1 is atractylodesin. In the negative sample spectrum, the chromatographic peak is not displayed at the same retention time as the atractylodesin reference substance. Therefore, the negative sample has no effect on the determination, and the negative has no interference with the determination results of the present invention.

[0066] 2.2 Determination of Identification of Atractylodesin (1) Methods Ten batches of antiseptic and antipruritic lotion samples were taken, and test solutions were prepared according to the aforementioned method of this example, and atractylodesin was identified under the aforementioned chromatographic conditions.

[0067] (2) Results and analysis The identification results of atractylodesin are shown in Table 4, which shows that the above method provided in this embodiment can accurately detect atractylodesin in the bactericidal and antipruritic lotion.

[0068] Table 4 Content determination results Comparative Example 1 1. Experimental methods: In this embodiment, test groups 1 to 3 are set. The detection steps of test groups 1 to 3 are the same as those of embodiment 1, except that the mobile phase "0.1% formic acid-0.2% triethylamine solution-acetonitrile" in embodiment 1 is replaced by methanol-water and acetonitrile-water solution, respectively, to conduct experiments and record chromatograms.

[0069] Methanol water: water (A)-methanol (B) was the mobile phase, gradient elution (0~15min, 5%B→18%B; 15~35min, 18%B→30%B; 35~50min, 30%B→90%B; 50~55min, 90%B), column temperature 30℃, flow rate 1mL•min-1, detection wavelength: 280nm; 45~60min, switch to 322nm.

[0070] Acetonitrile water: water (A)-acetonitrile (B) was the mobile phase, gradient elution (0~20min, 5%B→22%B; 20~40min, 22%B→35%B; 40~55min, 35%B→80%B; 55~58min, 80%B), column temperature 30℃, flow rate 1mL•min-1, detection wavelength: 280nm; 45~58min, switch to 322nm.

[0071] 2. Experimental results and analysis The chromatogram results of test groups 1 and 2 correspond to Fig.11 A~B, the above results of this comparative example 1~2 are compared with those of Example 1 Figure 4 After comparison, it can be found that when using methanol-water and acetonitrile-water systems, some chromatographic peaks have serious tailing. After adding 0.1% formic acid to the aqueous phase, the peak shapes of astilbin and osthole are significantly improved, and the separation effect of acetonitrile is better than that of methanol. When 0.2% triethylamine is added on the basis of formic acid, the peak shape of berberine hydrochloride in the chromatogram is better.

[0072] Comparative Example 2 1. Experimental methods The detection steps of this embodiment are the same as those of Example 1, except that the detection wavelength of the chromatographic conditions in Example 1, "1-44 min, 280 nm; 45-55 min, switched to 322 nm" is replaced with "220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm1 (1-55 min unified wavelength), the experiment is carried out, and the chromatogram is recorded.

[0073] 2. Experimental results and analysis Figure 12~Figure 13 A~F are the color spectra detected under the conditions of 220nm, 240nm, 260nm, 280nm, 300nm and 320nm, respectively. It can be seen that when the detection wavelength is above 300nm, the number of chromatographic peaks decreases. After comparison, at 280nm, the peaks are not only numerous, but also symmetrical in height and have good separation, so 280nm is selected as the detection wavelength. After further optimization, it is found that the chromatographic peaks detected by the method of Example 1 are best separated and have better reproducibility.

[0074] Comparative Example 3 1. Experimental methods: In this embodiment, multiple test groups are set up, and the detection steps of each test group are the same as those in Example 2, except that the extraction solvents used to dissolve the reference substance and the test substance are set to water, 25% methanol, 50% methanol, 75% methanol and 100% methanol, respectively, to conduct experiments and record chromatograms.

[0075] 2. Experimental results and analysis The results are as follows Figure 14~Figure 15 A~E are the chromatograms detected using water, 25% methanol, 50% methanol, 75% methanol and 100% methanol as the extraction solvents, respectively. From the comparison of the chromatogram results, it can be seen that the peaks obtained when 100% methanol is used as the extraction solvent are higher and the peak separation is large, so 100% methanol is used as the extraction solvent.

[0076] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for determining multiple components of a bactericidal and antipruritic lotion, characterized in that: The invention relates to a method for determining the content of astilbin, berberine hydrochloride and osthole, and the method for determining the content of astilbin, berberine hydrochloride and osthole comprises the following steps: Preparation of reference solution: accurately weigh astilbin, berberine hydrochloride and osthole respectively, dissolve them in methanol solvent to prepare reference solution; Preparation of the test solution: accurately measure the antiseptic and antipruritic lotion, add methanol solution for dilution and filtration to obtain the test solution; Determination of multi-component content: The test solution and the reference solution were respectively aspirated and injected into a high performance liquid chromatograph for determination to obtain a chromatogram. The contents of astilbin, berberine hydrochloride and osthole were calculated according to the standard curve regression equation.

2. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 1, characterized in that: In the content determination method of astilbin, berberine hydrochloride and osthole, the volume fraction of methanol solvent is 100%.

3. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 2, characterized in that: In the content determination method of astilbin, berberine hydrochloride and osthole, the preparation method of the test solution is: take the bactericidal and antipruritic lotion, dilute it 5 times with methanol, shake well, and filter.

4. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 3, characterized in that: In the method for determining the content of astilbin, berberine hydrochloride and osthole, the preparation method of the reference solution is as follows: accurately weigh appropriate amounts of astilbin, berberine hydrochloride and osthole reference substances, add methanol to obtain a mass concentration of 186.08 μg / mL -1 , 453.44μg▪mL -1 , 124.23μg▪mL -1 of the reference solution.

5. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 4, characterized in that: In the method for determining the contents of astilbin, berberine hydrochloride and osthole, the elution procedure in the high performance liquid chromatography is: 0-20 min, 5% B→22% B; 20-35 min, 22% B→35% B; 35-50 min, 35% B→90% B; 50-55 min, 90% B.

6. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 5, characterized in that: In the method for determining the contents of astilbin, berberine hydrochloride and osthole, the conditions of high performance liquid chromatography are: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution-acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30℃.

7. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 1, characterized in that: In the method for determining the contents of astilbin, berberine hydrochloride and osthole, the detection wavelengths used in the high performance liquid chromatography are: 1-44 min, 280 nm, to detect astilbin and berberine hydrochloride; 45-55 min, switched to 322 nm, to detect osthole.

8. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 7, characterized in that: Also included is a method for identifying atractylodesin, which comprises the following steps: Preparation of reference solution: weigh atractylodesin and dissolve it in methanol solvent to prepare a reference solution; Preparation of the test solution: accurately measure the antiseptic and antipruritic lotion, add methanol solution for dilution and filtration to obtain the test solution; Determination of atractylodesin: aspirate 1 % of the test solution and the reference solution respectively, inject into the high performance liquid chromatograph, and record the chromatogram; the elution procedure of the liquid chromatography is gradient elution.

9. The multi-component determination method of the bactericidal and antipruritic lotion according to claim 8, characterized in that: In the identification method of atractylodesin, the conditions of high performance liquid chromatography are: Chromatographic column: Shimadzu ODS-3 C18 column (250×4.6mm, 5μm); Mobile phase: 0.1% formic acid-0.2% triethylamine solution-acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30°C; The detection wavelength is: 1~44min, 280nm; 45~57min, switch to 340nm.

10. The method for determining multiple components of the bactericidal and antipruritic lotion according to claim 9, characterized in that: The identification method of atractylodesin, the elution procedure in the high performance liquid chromatography is: 0-20min, 5%B→22%B; 20-35min, 22%B→35%B; 35-50min, 35%B→90%B; 50-57min, 90%B.

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