Construction method and application of HPLC (High Performance Liquid Chromatography) specific chromatogram of sunshine prevention and treatment cream

Abstract

The invention discloses a construction method and application of an HPLC (High Performance Liquid Chromatography) specific chromatogram of sunburn prevention and treatment cream, and relates to the technical field of pharmaceutical analysis. The sunshine prevention and treatment cream is composed of honeysuckle, perfoliote knotweed herb, sedum sarmentosum, dayflower, radix polygonati officinalis, lithospermum erythrorhizon, aloe, asparagus fern, lucid ganoderma, coix seed and bee pollen, and is one of representative products of Guizhou Miao medicine brands. The specific chromatogram of the sunshine prevention and treatment cream is established by adopting a high performance liquid chromatography, and the five medicinal materials including honeysuckle, lithospermum erythrorhizon, aloe, sedum sarmentosum and perfoliote knotweed herb can be identified at the same time. The HPLC characteristic chromatogram of the sunshine prevention and treatment cream established by the method can effectively characterize the quality of the sunshine prevention and treatment cream, and is beneficial to comprehensively monitoring the quality of medicinal materials. Meanwhile, the method has the advantages of being simple, convenient, stable, high in precision, good in reproducibility and the like, and the authenticity of the product can be rapidly and accurately identified.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for constructing an HPLC characteristic spectrum of a sunscreen cream and an application thereof. Background Art

[0002] Sunburn prevention cream is one of the representative products of Guizhou Miao medicine brand (drug approval number: National Medicine Standard Z20025534), which has the effects of clearing away heat and detoxifying, cooling blood and removing spots, and is used to prevent and treat sunburn caused by heat and toxicity. The product has significant clinical efficacy and is favored by patients and doctors.

[0003] The sunscreen cream is made up of 11 herbs: honeysuckle, angelica sinensis, scutellaria baicalensis, coleus scabra, polygonatum odoratum, lithospermum officinale, aloe vera, asparagus cochinchinensis, ganoderma lucidum, coix seed, and bee pollen. The current quality standard only tests the content of chlorogenic acid in honeysuckle, which cannot fully reflect the quality of the sunscreen cream.

[0004] As a product, according to Article 5 of the "Administrative Measures for Post-Marketing Changes of Drugs (Trial)", "the holder shall take the initiative to conduct post-marketing research on drugs to achieve full life cycle management of drugs. Holders are encouraged to use new production technologies, new methods, new equipment, and new scientific and technological achievements to continuously improve and optimize production processes, continuously improve drug quality, and enhance drug safety, effectiveness, and quality controllability." With the continuous development of new technologies and methods in the pharmaceutical field, it is necessary to further improve the quality standards of sunscreen creams.

[0005] Based on this, the present invention provides a method for constructing an HPLC characteristic spectrum of a sunscreen cream and its application, aiming to effectively characterize the quality of the sunscreen cream and quickly identify the authenticity of the product. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an HPLC characteristic spectrum of a sunscreen cream and its application. To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The method for constructing the HPLC characteristic spectrum of the sunburn prevention cream of the present invention comprises the following steps:

[0008] S1 Preparation of test solution:

[0009] Take 5g of sun protection cream, weigh it accurately, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent until dry; wash the residue with acetonitrile-water mixed solution in batches, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column, elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain.

[0010] Preparation of S2 reference solution:

[0011] Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 0.15 mg per 1 mL.

[0012] Preparation of S3 reference solution:

[0013] Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh them accurately, and add methanol to make mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL.

[0014] S4 HPLC determination:

[0015] Accurately pipette the test solution, reference solution and control solution prepared in the above steps respectively, inject them into high performance liquid chromatography, and measure to obtain the result.

[0016] In the method for constructing the HPLC characteristic spectrum of the sunscreen cream of the present invention, the volume ratio of methanol to 25% hydrochloric acid in the methanol-25% hydrochloric acid mixed solution in step S1 is 4:1, and the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 1:9.

[0017] In the method for constructing the HPLC characteristic spectrum of the sunscreen cream of the present invention, the inner diameter of the polyamide column in step S1 is 1.0 cm, the column length is 15 cm, the polyamide particle size is 30-60 mesh, and it is prepared by wet column packing.

[0018] In the method for constructing the HPLC characteristic spectrum of the sunburn prevention cream of the present invention, the chromatographic conditions of the high performance liquid chromatography determination in step S4 are as follows:

[0019] Chromatographic column: C18 chromatographic column, with octadecylsilane bonded silica gel as filler; column temperature: 25-35°C; flow rate: 0.8-1.2 mL / min; detection wavelength: 250 nm; injection volume: 10 μL; mobile phase: methanol as mobile phase A, 0.05% formic acid solution as mobile phase B, and gradient elution.

[0020] Preferably, in the method for constructing the HPLC characteristic spectrum of the sunscreen cream of the present invention, the column temperature in the chromatographic conditions of the high performance liquid chromatography determination in step S4 is 30° C. and the flow rate is 1.0 mL / min.

[0021] The elution procedure of the gradient elution of the present invention is as follows:

[0022]

[0023] The specifications of the chromatographic column of the present invention are: column length is 250 mm, inner diameter is 4.6 mm, and particle size is 5 μm.

[0024] The HPLC characteristic spectrum of the sunburn prevention and treatment cream of the present invention comprises 6 characteristic peaks: peak 1 and peak S are characteristic components of honeysuckle, peak S is chlorogenic acid; peak 3 is aloin, which is a characteristic component of aloe; peak 4 is quercetin, which is a characteristic component of herba schizonepetae and herba schizonepetae; peak 5 is kaempferol, which is a characteristic component of herba schizonepetae; peak 6 is β,β'-dimethylacryloyl akanin, which is a characteristic component of lithospermum parkii.

[0025] The HPLC characteristic spectrum of the sun protection cream of the present invention has six characteristic peaks, with peak S as the reference peak, and the relative retention times of the remaining peaks are: peak 1 is 0.78, peak 3 is 1.08, peak 4 is 1.19, peak 5 is 1.34, and peak 6 is 1.60.

[0026] Application of the method for constructing the HPLC characteristic spectrum of the sunburn prevention cream of the present invention in the quality control of the sunburn prevention cream.

[0027] Beneficial effects of the present invention:

[0028] The method for constructing the HPLC characteristic spectrum of the sunscreen cream provided by the present invention simultaneously realizes the common control of the evaluation index components of chlorogenic acid, aloin, quercetin, kaempferol and β,β'-dimethylacryloyl akanin. The constructed characteristic spectrum contains 6 characteristic peaks, which can effectively characterize the quality of the sunscreen cream.

[0029] The method for constructing the HPLC characteristic spectrum of the sunburn prevention and treatment cream provided by the present invention further optimizes the chromatographic conditions of the high performance liquid chromatography analysis, and the chromatographic analysis is performed through a gradient elution program. The obtained HPLC characteristic spectrum has a good peak shape and a good separation effect.

[0030] The method for constructing the HPLC characteristic spectrum of the sunburn prevention cream provided by the present invention has the advantages of being simple, stable, highly precise and reproducible, and can achieve the purpose of quickly and accurately identifying the authenticity of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Mixed reference sample

[0032] Figure 2 Sun protection cream test sample chart

[0033] Figure 3 Control feature map

[0034] Figure 4 Chlorogenic acid reference substance spectrum

[0035] Figure 5 Aloin reference substance spectrum

[0036] Figure 6 Quercetin reference substance spectrum

[0037] Figure 7 Kaempferol reference substance spectrum

[0038] Figure 8 β,β'-dimethylacryloyl akanin reference substance spectrum

[0039] Fig. 9 Negative spectrum of honeysuckle

[0040] Fig.10 Aloe Negative Spectrum

[0041] Fig.11 Double negative spectrum of Radix Glehniae and Herba Lycopodii

[0042] Fig.12 Negative spectrum of spathiphyllum

[0043] Fig.13 Negative spectrum of Lithospermum officinale

[0044] Fig.14 Characteristic spectrum of precision test of sun protection cream

[0045] Fig.15 Repeatability test characteristic spectrum of sun protection cream

[0046] Fig.16 Characteristic spectrum of stability test of sun protection cream

[0047] Fig.17 Characteristic spectrum of different flow rates of sun protection cream

[0048] Fig.18 Characteristic spectrum of sun protection cream at different column temperatures

[0049] Fig.19 Characteristic spectrum of sun protection cream investigated by different chromatographic columns DETAILED DESCRIPTION

[0050] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only used for explanation and illustration, and do not constitute a limitation on the technical solution of the present invention.

[0051] Example 1

[0052] The method for constructing the HPLC characteristic spectrum of the sun protection cream comprises the following steps:

[0053] S1 Preparation of test solution

[0054] Take 5g of sunburn prevention cream, accurately weigh it, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid (4:1) mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent until dry; wash the residue with acetonitrile-water (1:9) mixed solution in batches, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column (with an inner diameter of 1.0cm, a column length of 15cm, a polyamide particle size of 30-60 mesh, and is prepared by wet column packing), elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain;

[0055] Preparation of S2 reference solution

[0056] Take an appropriate amount of chlorogenic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.15 mg per 1 mL.

[0057] Preparation of S3 reference solution

[0058] Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh accurately, and add methanol to prepare mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL;

[0059] S4 HPLC

[0060] Accurately pipette 10 μL of the test solution, reference solution and control solution prepared in the above steps respectively, inject into the high performance liquid chromatograph, and measure according to the following chromatographic conditions to obtain.

[0061] Chromatographic conditions:

[0062] Chromatographic column: C18 chromatographic column (4.6×250mm, 5μm), with octadecylsilane bonded silica as filler; column temperature: 30°C; flow rate: 1.0mL / min; detection wavelength: 250nm; mobile phase: methanol as mobile phase A, 0.05% formic acid solution as mobile phase B, gradient elution, the gradient elution program is as follows:

[0063]

[0064] Example 2

[0065] The method for constructing the HPLC characteristic spectrum of the sun protection cream comprises the following steps:

[0066] S1 Preparation of test solution

[0067] Take 5g of sunburn prevention cream, accurately weigh it, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid (4:1) mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent until dry; wash the residue with acetonitrile-water (1:9) mixed solution in batches, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column (with an inner diameter of 1.0cm, a column length of 15cm, a polyamide particle size of 30-60 mesh, and is prepared by wet column packing), elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain;

[0068] Preparation of S2 reference solution

[0069] Take an appropriate amount of chlorogenic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.15 mg per 1 mL.

[0070] Preparation of S3 reference solution

[0071] Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh accurately, and add methanol to prepare mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL;

[0072] S4 HPLC

[0073] Accurately pipette 10 μL of the test solution, reference solution and control solution prepared in the above steps respectively, inject into the high performance liquid chromatograph, and measure according to the following chromatographic conditions to obtain.

[0074] Chromatographic conditions:

[0075] Chromatographic column: C18 chromatographic column (4.6×250mm, 5μm), with octadecylsilane bonded silica as filler; column temperature: 25°C; flow rate: 0.8mL / min; detection wavelength: 250nm; mobile phase: methanol as mobile phase A, 0.05% formic acid solution as mobile phase B, gradient elution, the gradient elution program is as follows:

[0076]

[0077] Example 3

[0078] The method for constructing the HPLC characteristic spectrum of the sun protection cream comprises the following steps:

[0079] S1 Preparation of test solution

[0080] Take 5g of sunburn prevention cream, accurately weigh it, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid (4:1) mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent until dry; wash the residue with acetonitrile-water (1:9) mixed solution in batches, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column (with an inner diameter of 1.0cm, a column length of 15cm, a polyamide particle size of 30-60 mesh, and is prepared by wet column packing), elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain;

[0081] Preparation of S2 reference solution

[0082] Take an appropriate amount of chlorogenic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.15 mg per 1 mL.

[0083] Preparation of S3 reference solution

[0084] Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh accurately, and add methanol to prepare mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL;

[0085] S4 HPLC

[0086] Accurately pipette 10 μL of the test solution, reference solution and control solution prepared in the above steps respectively, inject into the high performance liquid chromatograph, and measure according to the following chromatographic conditions to obtain.

[0087] Chromatographic conditions:

[0088] Chromatographic column: C18 chromatographic column (4.6×250mm, 5μm), with octadecylsilane bonded silica as filler; column temperature: 35°C; flow rate: 1.2mL / min; detection wavelength: 250nm; mobile phase: methanol as mobile phase A, 0.05% formic acid solution as mobile phase B, gradient elution, the gradient elution program is as follows:

[0089]

[0090] Example 4

[0091] Application of the method for constructing HPLC characteristic spectrum of sunscreen cream in quality control of sunscreen cream:

[0092] According to the method for constructing the HPLC characteristic spectrum of the sunburn prevention and treatment cream provided by the present invention, the content of each index component of the sunburn prevention and treatment cream product is determined.

[0093] Example 5

[0094] Application of the method for constructing HPLC characteristic spectrum of sunscreen cream in quality control of sunscreen cream:

[0095] According to the method for constructing the HPLC characteristic spectrum of the sunburn prevention cream provided by the present invention, HPLC analysis is performed on the sunburn prevention cream to identify the authenticity of the product.

[0096] In order to further verify the reliability of the present invention, the inventor conducted a series of experiments, the excerpts of which are as follows: 1. Medicinal materials and reagents

[0097] Chlorogenic acid reference substance (China Food and Drug Inspection Institute; batch number: 110753-202119); quercetin reference substance (China Food and Drug Inspection Institute; batch number: 100081-201610); kaempferol reference substance (China Food and Drug Inspection Institute; batch number: 110861-202214); aloin reference substance (China Food and Drug Inspection Institute; batch number: 110787-202109); β,β'-dimethylacryloyl akanin reference substance (China Food and Drug Inspection Institute; batch number: 111689-201805); sunscreen cream (Guizhou Green Sun Pharmaceutical Co., Ltd.). Water was Wahaha purified water, methanol and acetonitrile were chromatographically pure (Tian Di Co., Ltd., USA), acetonitrile, hydrochloric acid, and methanol were analytically pure (Chongqing Wansheng Chuandong Chemical Co., Ltd.).

[0098] 2. Instruments

[0099] Electronic balance (Japan Shimadzu Instrument Co., Ltd., AUW-220D); CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Jiangsu Province, KQ-500DE); electronic balance (Sartorius, Shimadzu Libror AEL 40SM); high performance liquid chromatograph (Japan Shimadzu Instrument Co., Ltd., LC-2010AHT); chromatographic columns: ZORBAX Eclipse XDB-C18 (4.6×250mm, 5μm); Agilent 5TC-C18 (4.6×250mm, 5μm); XTERRA MS C18 (4.6×250mm, 5μm).

[0100] 3. Construction of HPLC characteristic spectrum of sun protection cream

[0101] 3.1 Preparation of test solution

[0102] Take 5g of sun protection cream, accurately weigh it, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid (4:1) mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent to dryness; add acetonitrile-water (1:9) mixed solution to wash the residue in batches, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column (with an inner diameter of 1.0cm, a column length of 15cm, a polyamide particle size of 30-60 mesh, and is prepared by wet column packing), elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain.

[0103] 3.2 Preparation of reference solution

[0104] Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add methanol to make a solution containing 0.15 mg per 1 mL.

[0105] 3.3 Preparation of reference solution

[0106] Take appropriate amount of quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance, accurately weigh them, and add methanol to prepare quercetin reference substance solution, aloin reference substance solution, kaempferol reference substance solution and β,β'-dimethylacryloyl akanin reference substance solution, respectively.

[0107] Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh them accurately, and add methanol to make mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL.

[0108] 3.4 Chromatographic analysis conditions

[0109] Chromatographic column: C 18 Chromatographic column (4.6×250 mm, 5 μm) with octadecylsilane bonded silica as filler; column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 250 nm; mobile phase: methanol as mobile phase A, 0.05% formic acid solution as mobile phase B, gradient elution program see Table 1.

[0110] Table 1 Gradient elution program

[0111] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5 95 20～30 5～10 95～90 30～40 10～25 90～75 40～50 25～40 75～60 50～65 40～70 60～30 65～72 70 30

[0112] 3.5 Determination method

[0113] Accurately pipette 10 μL of the test solution, reference solution, and control solution respectively, inject into the high performance liquid chromatograph, and measure to obtain the result.

[0114] 3.6 Results and Analysis

[0115] The results show that the HPLC characteristic spectrum of the sunscreen cream shows 6 characteristic peaks, of which peak 2 is chlorogenic acid, peak 3 is aloin, peak 4 is quercetin, peak 5 is kaempferol, and peak 6 is β,β'-dimethylacryloyl akanin. Figure 1 As shown; the sample spectrum of the sun protection cream is as follows Figure 2 As shown; the control characteristic spectrum is as Figure 3 As shown in the figure: S1 is the characteristic spectrum of the sun protection cream, S2 is the characteristic spectrum of the mixed reference substance; the chlorogenic acid reference substance spectrum, the aloin reference substance spectrum, the quercetin reference substance spectrum, the kaempferol reference substance spectrum, and the β,β'-dimethylacryloyl akanin reference substance spectrum are shown in Figures 4 to 8 shown.

[0116] 4. Chromatographic peak identification

[0117] 4.1 Preparation of negative solution

[0118] Take 5g of each of the negative samples of honeysuckle, lithospermum, aloe and creeping vine, and the double negative samples of angelica sinensis and creeping vine, accurately weigh them, place them in a stoppered conical flask, add 50mL of a mixed solution of methanol-25% hydrochloric acid (4:1), heat and reflux for 1h, cool, shake well, filter, and recover the solvent to dryness; wash the residue with a mixed solution of acetonitrile-water (1:9) in several times, transfer the washing solution to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to a polyamide column (with an inner diameter of 1.0cm, a column length of 15cm, a polyamide particle size of 30-60 meshes, and is prepared by wet column packing), elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain.

[0119] 4.2 Determination method

[0120] According to the chromatographic analysis conditions under "3. Construction of HPLC characteristic spectrum of sun protection cream", HPLC determination was carried out.

[0121] 4.3 Results and Analysis

[0122] The results show that in the HPLC characteristic spectrum of the sunscreen cream test sample: Peak 1 and Peak 2 are characteristic components of honeysuckle, of which Peak 2 is chlorogenic acid, and there is no interference in the negative control; Peak 3 is a characteristic component of aloe, and there is no interference in the negative control; Peak 4 is a characteristic component of Herba Lycoris and Herba Glechomae, and there is no interference in the negative control; Peak 5 is a characteristic component of Herba Lycoris, and there is no interference in the negative control; Peak 6 is a characteristic component of Lithospermum officinale, and there is no interference in the negative control. The negative spectrum of honeysuckle, the negative spectrum of aloe, the double negative spectrum of Herba Lycoris and Herba Glechomae, the negative spectrum of Herba Lycoris, and the negative spectrum of Lithospermum officinale are as follows: Figures 9 to 13 shown.

[0123] 5. Selection of chromatographic conditions

[0124] 5.1 Selection of mobile phase and elution gradient

[0125] Take 5 g of sunscreen cream, weigh accurately, and prepare the test solution according to the method under "3.1 Preparation of test solution", using methanol and acetonitrile as mobile phase A, respectively, and 0.05% formic acid solution as mobile phase B, and perform gradient elution according to the elution program set in Tables 2 to 5.

[0126] Table 2 Gradient elution program 1

[0127] Time (minutes) Methanol A(%) 0.05% Formic acid (%) 0～20 5 95 20～30 5～10 95～90 30～40 10～25 90～75 40～50 25～40 75～60 50～65 40～70 60～30 65～72 70 30

[0128] Table 3 Gradient elution program 2

[0129]

[0130]

[0131] Table 4 Gradient elution program 3

[0132] Time (minutes) Methanol A(%) 0.05% Formic acid (%) 0～20 5～10 95～90 20～35 10～20 90～80 35～50 20～40 80～60 50～60 40～70 60～30 60～72 70 30

[0133] Table 5 Gradient elution program 4

[0134] Time (minutes) Acetonitrile A(%) 0.05% Formic acid (%) 0～20 5～10 95～90 20～35 10～20 90～80 35～50 20～40 80～60 50～60 40～70 60～30 60～72 70 30

[0135] 5.2 Selection of detection wavelength

[0136] A DAD detector was used to perform full wavelength scanning to collect chromatographic information at wavelengths of 230 nm, 250 nm, 270 nm, 290 nm, 310 nm, 330 nm, and 350 nm, respectively.

[0137] 5.3 Results and Analysis

[0138] 5.3.1 Selection of mobile phase and elution gradient

[0139] The results showed that the chromatographic peak shape was good and the separation was good when gradient elution was performed under the conditions of gradient elution program 1; the peak shape of quercetin was poor and the tailing was serious when gradient elution was performed under the conditions of gradient elution program 2, and the separation degree of chlorogenic acid peak and impurity peak was poor and the peak shape was bifurcated; the aloin peak and impurity peak overlapped and could not be separated when gradient elution was performed under the conditions of gradient elution program 3; the peak shape of quercetin was poor and the tailing was serious when gradient elution was performed under the conditions of gradient elution program 4. Therefore, gradient elution program 1 was selected as the elution program for this study.

[0140] 5.3.2 Selection of detection wavelength

[0141] The results showed that the chromatographic peak separation and peak shape were better and the baseline was more stable at 250nm wavelength, which was better than other wavelengths. Therefore, 250nm was selected as the detection wavelength in this study.

[0142] 6. Methodological Validation

[0143] 6.1 Precision investigation

[0144] Take 5g of sunscreen cream, weigh accurately, prepare the test solution according to the method in "3.1 Preparation of test solution", and measure according to the conditions in "3.4 Chromatographic analysis conditions", inject 6 times continuously, and measure the precision of the instrument. Take the characteristic peak of chlorogenic acid (peak 2) as the reference peak, calculate the relative retention time of other peaks, and calculate RSD.

[0145] 6.2 Repeatability study

[0146] Weigh 5 g of each of 6 samples of sunscreen cream, accurately weigh them, prepare the test solution according to the method in "3.1 Preparation of test solution", and perform the determination according to the conditions in "3.4 Chromatographic analysis conditions". Take the characteristic peak of chlorogenic acid (peak 2) as the reference peak, calculate the relative retention time of other peaks, and calculate the RSD.

[0147] 6.3 Stability investigation

[0148] Take 5g of sunburn prevention cream, weigh accurately, prepare the test solution according to the method under "3.1 Preparation of test solution", accurately draw 10μL of the test solution, inject at 0, 2, 4, 6, 8, and 12h, and measure according to the conditions under "3.4 Chromatographic analysis conditions". Take the characteristic peak of chlorogenic acid (peak 2) as the reference peak, calculate the relative retention time of other peaks, and calculate RSD.

[0149] 6.4 Durability test

[0150] 6.4.1 Investigation of different flow rates

[0151] Take 5 g of sunscreen cream, weigh accurately, prepare the test solution according to the method under "3.1 Preparation of test solution", and measure according to the conditions under "3.4 Chromatographic analysis conditions". The flow rates are 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min respectively, to investigate the durability of the method for different mobile phase flow rates.

[0152] 6.4.2 Investigation of different column temperatures

[0153] Take 5 g of sunscreen cream, weigh accurately, prepare the test solution according to the method in "3.1 Preparation of test solution", and measure according to the conditions in "3.4 Chromatographic analysis conditions". The column temperatures are 25℃, 30℃, and 35℃ respectively, and the durability of the method at different column temperatures is examined.

[0154] 6.4.3 Investigation of different chromatographic columns

[0155] Take 5 g of sunscreen cream, weigh accurately, prepare the test solution according to the method in "3.1 Preparation of test solution", select ZORBAX Eclipse XDB-C18, Agilent 5TC-C18, and XTERRA MS C18 chromatographic columns respectively for determination according to the conditions in "3.4 Chromatographic analysis conditions" to examine the durability of the method on different chromatographic columns.

[0156] 6.5 Results and Analysis

[0157] 6.5.1 Precision investigation

[0158] The results show that the relative retention time RSD of the five identified peaks is in the range of 0.00 to 0.24%, indicating that the precision of the instrument is good. The results are shown in Table 6 and the precision spectrum is shown in Fig.14 .

[0159] Table 6 Retention time and relative retention time table (precision study)

[0160]

[0161] 6.5.2 Repeatability study

[0162] The results show that the relative retention time RSD of the five identification peaks of the six repeatability experimental samples is in the range of 0.00-0.52%, indicating that the repeatability of the characteristic spectrum is good. The results are shown in Table 7, and the repeatability spectrum is shown in Fig.15 .

[0163] Table 7 Retention time and relative retention time table (repeatability study)

[0164]

[0165]

[0166] 6.5.3 Stability Study

[0167] The results showed that the relative retention time RSD of the five identified peaks of the sample was in the range of 0.00 to 0.23%, indicating that the test solution had good stability within 12 hours. The results are shown in Table 8, and the stability test spectrum is shown in Fig.16 .

[0168] Table 8 Retention time and relative retention time table (stability study)

[0169]

[0170] 6.5.4 Durability test

[0171] 6.5.4.1 Investigation of different flow rates

[0172] The results show that the separation of each chromatographic peak is good at different flow rates, and the relative retention time RSD of the five identification peaks is in the range of 0.00-0.16%. This shows that the method has good durability for flow rate. The results are shown in Table 9. Fig.17 .

[0173] Table 9 Retention time and relative retention time table at different flow rates

[0174]

[0175]

[0176] 6.5.4.2 Investigation of different column temperatures

[0177] The results show that the relative retention time RSDs of the five peaks are all within the range of 0.00 to 0.17%, indicating that the method has good durability to column temperature. The results are shown in Table 10. Fig.18 .

[0178] Table 10 Retention time and relative retention time table at different column temperatures

[0179]

[0180] 6.5.4.3 Investigation of different chromatographic columns

[0181] The results show that this method has low specificity for the stationary phase selection, and the peak shape and separation of the chromatographic column are excellent. Therefore, this method has good durability for the selection of chromatographic columns. The results are shown in Table 11. Fig.19 .

[0182] Table 11 Retention time and relative retention time table of different chromatographic columns

[0183]

[0184]

[0185] In summary, the present invention strengthens the preparation specificity identification and overall quality control by constructing the HPLC characteristic spectrum of the sun protection cream, further improves the preparation quality standard, and thus ensures the safety, stability and quality controllability of the preparation.

[0186] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing an HPLC characteristic spectrum of a sunscreen cream, characterized in that: The steps include: S1 Preparation of test solution: Take 5g of sunburn prevention cream, weigh it accurately, put it in a stoppered conical flask, add 50mL of methanol-25% hydrochloric acid mixed solution, heat and reflux for 1h, cool, shake well, filter, and recover the solvent until dry; wash the residue with acetonitrile-water mixed solution in batches, transfer the washing liquid to a 10mL volumetric flask, and make up to the scale with the above acetonitrile-water mixed solution; accurately measure 5mL, add it to the polyamide column, elute with 50mL of water, discard the water, and then elute with 100mL of methanol, collect the eluate, and evaporate to dryness; dissolve the residue with methanol and transfer it to a 10mL volumetric flask, add methanol to the scale, shake well, and obtain; Preparation of S2 reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.15 mg per 1 mL. Preparation of S3 reference solution: Take appropriate amount of chlorogenic acid reference substance, quercetin reference substance, aloin reference substance, kaempferol reference substance and β,β'-dimethylacryloyl akanin reference substance respectively, weigh accurately, and add methanol to prepare mixed reference substance solutions containing 0.18 mg, 0.08 mg, 0.2 mg, 0.01 mg and 0.01 mg of each reference substance per 1 mL; S4 HPLC determination: Accurately pipette the test solution, reference solution and control solution prepared in the above steps respectively, inject them into high performance liquid chromatography, and measure to obtain the result.

2. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 1, characterized in that: In step S1, the volume ratio of methanol to 25% hydrochloric acid in the methanol-25% hydrochloric acid mixed solution is 4:1, and the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 1:

9.

3. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 1, characterized in that: The inner diameter of the polyamide column in step S1 is 1.0 cm, the column length is 15 cm, the polyamide particle size is 30-60 mesh, and it is prepared by wet column packing.

4. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography determination in step S4 are as follows: Chromatographic column: C 18 Chromatographic column, using octadecylsilane bonded silica gel as filler; column temperature: 25-35°C; flow rate: 0.8-1.2 mL / min; detection wavelength: 250 nm; injection volume: 10 μL; mobile phase: using methanol as mobile phase A and 0.05% formic acid solution as mobile phase B, for gradient elution.

5. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 4, characterized in that: The column temperature was 30°C and the flow rate was 1.0 mL / min.

6. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 4, characterized in that: The elution procedure of the gradient elution is as follows:

7. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 4, characterized in that: The specifications of the chromatographic column are: column length of 250 mm, inner diameter of 4.6 mm, and particle size of 5 μm.

8. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 1, characterized in that: The HPLC characteristic spectrum of the obtained sunscreen prevention and treatment ointment contains 6 characteristic peaks: peak 1 and peak S are characteristic components of honeysuckle, and peak S is chlorogenic acid; peak 3 is aloin, which is a characteristic component of aloe; peak 4 is quercetin, which is a characteristic component of herba schizonepetae and herba schizonepetae; peak 5 is kaempferol, which is a characteristic component of herba schizonepetae; peak 6 is β,β'-dimethylacryloyl akanin, which is a characteristic component of lithospermum parkii.

9. The method for constructing the HPLC characteristic spectrum of the sunscreen cream according to claim 8, characterized in that: The relative retention times of the six characteristic peaks are as follows: peak 1 is 0.78, peak 3 is 1.08, peak 4 is 1.19, peak 5 is 1.34, and peak 6 is 1.60, taking peak S as the reference peak.

10. Use of the method for constructing the HPLC characteristic spectrum of the sunscreen cream according to any one of claims 1 to 9 in the quality control of the sunscreen cream.

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