A method for simultaneously determining the contents of multiple psoraleae substances in Bufei Huoxue capsules by using a one-test-multiple-evaluation method

Abstract

The present invention relates to the field of traditional Chinese medicine, and specifically to a method for simultaneously determining the content of multiple psoralen substances in Bufei Huoxue Capsules by a one-test-multiple-evaluation method. Bufei Huoxue Capsules include Astragalus, Red Peony Root and Psoralen. The method uses psoralen as an internal reference substance to respectively determine the relative correction factors of psoralen, isopsoralen, isopsoralen and bakuchiol compared to psoralen; obtain a test solution, wherein the test solution contains the contents of the Bufei Huoxue Capsules; perform high performance liquid chromatography on the test solution, and determine the content of psoralen and at least one substance selected from psoralen, isopsoralen, isopsoralen and bakuchiol in Bufei Huoxue Capsules based on the relative correction factors of psoralen, isopsoralen, isopsoralen or bakuchiol compared to psoralen. The method is stable and reliable and can be used for quality control of Bufei Huoxue Capsules.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to a method for simultaneously determining the contents of multiple psoralea corylifolia substances in Bufei Huoxue capsules by adopting a one-test-multiple-evaluation method. Background Art

[0002] Bufei Huoxue Capsule is composed of three Chinese medicinal herbs: Astragalus, Red Peony Root, and Psoralea corylifolia. It has the effects of invigorating qi and promoting blood circulation, tonifying the lungs and strengthening the kidneys. It is included in the 2020 edition of the Chinese Pharmacopoeia. The Psoralea corylifolia in the prescription has anti-inflammatory, kidney-tonifying and yang-tonifying, and anti-aging effects, and the furanocoumarins it contains are the main pharmacodynamic material basis for the biological activity of the whole prescription. At present, the quantitative requirements of the pharmacopoeia for Bufei Huoxue Capsule are concentrated on astragaloside I and paeoniflorin, and Psoralea corylifolia is not involved. According to the theory of traditional Chinese medicine, the efficacy of traditional Chinese medicine is the result of the combined effect of multiple components. The quality control and evaluation of traditional Chinese medicine is one of the key issues restricting its modernization. Its multi-component and multi-functional characteristics determine that it is difficult to comprehensively evaluate the quality of a single component. Therefore, multi-component and multi-index content determination has become a quality evaluation model widely recognized internationally. However, traditional methods have defects such as large consumption of reference materials, high cost, and complex operation. The existing technical methods either use psoralen as an internal standard to determine the content of ligustilide in Chuanxiong medicinal materials by one test and multiple evaluations, or use one test and multiple evaluations to detect psoralea medicinal materials. These methods are not suitable for the content determination of multiple psoralea substances in Bufei Huoxue Capsules. The accuracy of the methods is poor, which is not conducive to the quality and efficacy control of Bufei Huoxue Capsules. Further improvements are needed on how to quantitatively determine multiple components in Bufei Huoxue Capsules. This method is more accurate, takes less time, and is more efficient when simultaneously determining the content of multiple psoralea substances in Bufei Huoxue Capsules. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. Wang Zhimin et al. proposed a multi-index quality control method of one test and multiple evaluations, and their research ideas have been widely used in the quality control of some Chinese medicinal materials and Chinese patent medicine preparations. In the course of research, the inventor found that for Bufei Huoxue Capsules, one test and multiple evaluations can be used for research, and creatively found that psoralen is used as an internal reference substance, and the contents of these five components in the finished preparation are determined by establishing the relative correction factors between the components of the other four kinds of psoralens, and the feasibility and applicability of the one test and multiple evaluations method are confirmed. Through the method provided by the present invention, the contents of psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol in Bufei Huoxue Capsules can be determined simultaneously. The method provided is simple, fast and accurate, and the relative correction factor has good credibility and accuracy. It can be used for content determination when the reference substance is in short supply, and can reduce solvent consumption, shorten analysis time, and be more conducive to efficiency improvement and cost compression.

[0004] Specifically, the present invention provides the following technical solutions:

[0005] In one aspect of the present invention, the present invention provides a method for simultaneously determining the content of psoralea corylifolia in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method, wherein the Bufei Huoxue Capsule comprises astragalus, red peony root and psoralea corylifolia, and the method comprises:

[0006] Using psoralen as the internal reference substance, the relative correction factors of psoralen, isopsoralen, isopsoralen or bakuchiol compared with psoralen were determined respectively;

[0007] preparing a test solution, wherein the test solution contains the contents of the Bufei Huoxue Capsule;

[0008] The test solution is subjected to high performance liquid chromatography detection, and the content of psoralen and at least one substance selected from psoralen, isopsoralen, isopsoralen, and bakuchiol in Bufei Huoxue Capsule is determined based on the relative correction factor of psoralen, isopsoralen, isopsoralen or bakuchiol compared to psoralen.

[0009] According to an embodiment of the present invention, the method for simultaneously determining the content of Psoralea corylifolia in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method described above may further include the following technical features:

[0010] According to an embodiment of the present invention, the method further comprises: obtaining the determination result of psoralen, isopsoralen, psoralen or isopsoralen at a wavelength of 300 to 320 nanometers, and obtaining the determination result of bakuchiol at a wavelength of 250 to 270 nanometers.

[0011] According to an embodiment of the present invention, the measurement results of psoralen, isopsoralen, psoralen or isopsoralen are obtained at a wavelength of 305 to 315 nanometers, and the measurement results of bakuchiol are obtained at a wavelength of 255 to 265 nanometers. According to an embodiment of the present invention, the measurement results of psoralen, isopsoralen, psoralen or isopsoralen are obtained at a wavelength of 308, 310 or 312 nanometers, and the measurement results of bakuchiol are obtained at a wavelength of 258, 260 or 262 nanometers.

[0012] According to an embodiment of the present invention, the relative correction factor of the psoralen to the psoralen is 3.8 to 4.5, preferably 4.1 to 4.3.

[0013] According to an embodiment of the present invention, the relative correction factor of the isopsoralen to the psoralen is 3.7 to 4.5, preferably 3.9 to 4.2.

[0014] According to an embodiment of the present invention, the relative correction factor of the isopsoralen relative to the psoralen is 0.85 to 1.3, preferably 0.95 to 1.1.

[0015] According to an embodiment of the present invention, the relative correction factor of the bakuchiol relative to the psoralen is 1 to 1.5, preferably 1.1 to 1.4.

[0016] According to an embodiment of the present invention, the test solution is obtained by dissolving the contents of Bufei Huoxue Capsule in methanol. According to a preferred embodiment of the present invention, the methanol is 40% methanol. According to a preferred embodiment of the present invention, after dissolving in 40% methanol, ultrasonic treatment is performed for 20 to 50 minutes to obtain the test solution.

[0017] According to an embodiment of the present invention, the HPLC detection conditions are as follows:

[0018] The chromatographic column is a C18 chromatographic column, preferably any one of Angilent Eclipse Plus C18, Angilent Proshell HPHC18, and Ekzo Nobel Kromasil C18 chromatographic columns;

[0019] The mobile phase includes methanol and an aqueous phase containing phosphoric acid, wherein the volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05% to 0.5%, preferably 0.05% to 0.2%;

[0020] The volume flow rate is 0.3-1.0 mL / min, preferably 0.5-0.7 mL / min;

[0021] The column temperature is 20 to 40 degrees Celsius, preferably 25 to 35 degrees Celsius;

[0022] The detection wavelength is 300-320 nm for 0-50 minutes and 250-270 nm for 50-80 minutes.

[0023] According to a preferred embodiment of the present invention, the HPLC detection conditions are as follows:

[0024] Angilent Eclipse Plus C18 column, 150 mm × 4.6 mm, 3.5 μm;

[0025] The mobile phase was methanol and water containing 0.1% by volume phosphoric acid;

[0026] The gradient elution conditions were:

[0027] Time (min) Methanol(%) 0.1% Phosphoric acid water (%) 0～10 25～25 75～75 10～50 25～50 75～50 50～55 50～100 50～0 55～65 100～100 0～0

[0028] Column temperature: 30°C;

[0029] Volume flow rate 0.6ml / min;

[0030] The detection wavelength for 0 to 50 min was 310 nm, and the detection wavelength for 50 to 65 min was 260 nm.

[0031] In another aspect of the present invention, the present invention provides a method for simultaneously determining the contents of psoralen, isopsoralen, isopsoralen, isobakuchiol and psoralen in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method, wherein the Bufei Huoxue Capsule comprises astragalus, red peony root and psoralea corylifolia, and the method comprises:

[0032] (1) preparing reference substance stock solutions of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol respectively using methanol, and mixing them to obtain reference substance solutions containing psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol;

[0033] (2) taking the contents of the Bufei Huoxue capsule and dissolving it in 40% methanol to obtain a test solution;

[0034] (3) taking the reference solution of step (1), performing high performance liquid chromatography under different injection volume conditions, using psoralen as the internal reference substance, and determining the relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared to psoralen;

[0035] (4) taking the test solution in step (2), performing high performance liquid chromatography detection, and obtaining the determination results of psoralen, isopsoralen, psoralen and isopsoralen at a wavelength of 300 to 320 nanometers, and obtaining the determination results of bakuchiol at a wavelength of 250 to 270 nanometers.

[0036] The invention provides a method for determining the content of psoralea corylifolia in Bufei Huoxue capsules by using a one-measurement multiple-evaluation method. The provided method is not interfered by components in astragalus and red peony root, and the results obtained by the one-measurement multiple-evaluation method are close to those obtained by the external standard method, and the difference is not statistically significant. The provided method is stable and reliable, and can be used for quality control of Bufei Huoxue capsules.

[0037] According to an embodiment of the present invention, the method provided above for simultaneously determining the contents of psoralen, isopsoralen, isopsoralen, isobakuchiol and psoralen in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method may further include the following technical features:

[0038] By scanning the absorption wavelength of each psoralea corylifolia component under different wavelength conditions, it is found that psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol in psoralea corylifolia all have good absorption at 250-270 nanometers; however, the retention time of psoralen and isopsoralen is close to that of the chromatographic peak of peony in this wavelength, and the requirement of separation greater than 1.5 cannot be met; therefore, the inventor creatively discovered that: the content of psoralen, isopsoralen, psoralen and isopsoralen is detected at a wavelength of 300-320 nanometers, and the content of bakuchiol is detected at 250-270 nanometers, which can achieve accurate detection of the content of psoralen in Bufei Huoxue Capsule. According to a preferred embodiment of the present invention, in step (4), the measurement results of psoralen, isopsoralen, psoralen and isopsoralen are obtained at a wavelength of 305-315 nanometers, and the measurement results of bakuchiol are obtained at a wavelength of 255-265 nanometers.

[0039] According to a preferred embodiment of the present invention, the determination results of psoralen, isopsoralen, psoralen and isopsoralen are obtained under the conditions of 308-312 nanometers, for example, at 308, 310 or 312 nanometers; and the determination results of bakuchiol are obtained under the conditions of 258-262 nanometers, for example, at 258, 260 or 262 nanometers. Thus, accurate detection of the content of psoralen in Bufei Huoxue Capsule can be achieved.

[0040] According to an embodiment of the present invention, the methanol in step (2) is 40% methanol. When 40% methanol is used for dissolution, the obtained chromatogram has rich chromatographic peak information, strong signals of each peak to be measured, and a relatively stable baseline, so the result is more accurate.

[0041] According to an embodiment of the present invention, after dissolving in methanol, ultrasonic treatment is performed for 20 to 50 minutes to obtain a test solution. Thus, the obtained chromatogram has rich chromatographic peak information, strong signals of each peak to be tested, and a relatively stable baseline, and the result is more accurate.

[0042] According to an embodiment of the present invention, step (2) further comprises: (2-1) weighing the contents of the Bufei Huoxue Capsule, placing it in a stoppered container, adding 40% methanol, weighing the mass, and obtaining a first weighing solution; (2-2) subjecting the first weighing solution to ultrasonic treatment for 25 to 40 minutes, weighing the mass of the solution after ultrasonic treatment, and obtaining a second weighing solution; (2-3) determining the mass lost by the second weighing solution compared to the first weighing solution, supplementing it with 40% methanol, and filtering it with a 0.45 micron filter membrane to obtain the test solution. Thus, the chromatogram obtained has rich chromatographic peak information, strong signals of each peak to be measured, and a relatively stable baseline, and the result is more accurate.

[0043] According to an embodiment of the present invention, step (3) further comprises: (3-1) taking the reference solution of step (1), injecting 1, 2, 4, 6, 8, and 10 microliters respectively, and performing high performance liquid chromatography detection to obtain corresponding test results; (3-2) based on the test results, taking psoralen as the internal reference substance, respectively calculating the relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared to psoralen under different injection conditions, and taking the average value to obtain the average relative correction factor. The result obtained thereby is more accurate.

[0044] According to an embodiment of the present invention, the relative correction factor of the psoralen to the psoralen is 3.8-4.5, preferably 4.1-4.3; the relative correction factor of the isopsoralen to the psoralen is 3.7-4.5, preferably 3.9-4.2; the relative correction factor of the isopsoralen to the psoralen is 0.85-1.3, preferably 0.95-1.1; the relative correction factor of the bakuchiol to the psoralen is 1-1.5, preferably 1.1-1.4.

[0045] According to an embodiment of the present invention, the high performance liquid chromatography detection conditions in step (3) and step (4) are as follows: the chromatographic column is a C18 chromatographic column, preferably any one of Angilent Eclipse Plus C18, Angilent Proshell HPH C18, and EkzoNobel Kromasil C18 chromatographic columns; the mobile phase comprises methanol and an aqueous phase containing phosphoric acid, and the volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05% to 0.5%, preferably 0.05% to 0.2%; the volume flow rate is 0.3 to 1.0 mL / min, preferably 0.5 to 0.7 mL / min; the column temperature is 20 to 40 degrees Celsius, preferably 25 to 35 degrees Celsius; the detection wavelength is 300 to 320 nanometers for 0 to 50 minutes and 250 to 270 nanometers for 50 to 80 minutes.

[0046] According to a preferred embodiment of the present invention, the HPLC detection conditions are as follows:

[0047] Angilent Eclipse Plus C18 column, 150 mm × 4.6 mm, 3.5 μm;

[0048] The mobile phase was methanol and water containing 0.1% by volume phosphoric acid;

[0049] The gradient elution conditions were:

[0050] Time (min) Methanol(%) 0.1% Phosphoric acid water (%) 0～10 25～25 75～75 10～50 25～50 75～50 50～55 50～100 50～0 55～65 100～100 0～0

[0051] Column temperature: 30°C;

[0052] Volume flow rate 0.6ml / min;

[0053] The detection wavelength for 0 to 50 min was 310 nm, and the detection wavelength for 50 to 65 min was 260 nm.

[0054] The beneficial effects achieved by the present invention are as follows: the method provided by the present invention is simple, fast and accurate, and the relative correction factor is less affected by the chromatographic column, column temperature, mobile phase, volume flow rate, etc., has good credibility and accuracy, can be used for content determination when the reference substance is in short supply, and can reduce solvent consumption and shorten analysis time, thereby improving efficiency and reducing components; it can be used for quality control of Bufei Huoxue Capsules. Moreover, the provided method is indifferent to the external standard method, and is accurate and controllable. For example, the psoralen, the isopsoralen, the psoralen, the isopsoralen and the bakuchiol have good linear relationships in the ranges of 2.102-84.113 μg / ml, 2.049-81.977 μg / ml, 1.920-76.811 μg / ml, 1.766-70.652 μg / ml, and 1.976-79.042 μg / ml, respectively. For example, the average sample recovery rates of the psoralen, the isopsoralen, the psoralen, the isopsoralen and the bakuchiol are 99% to 105%, 98% to 102%, 98% to 103%, 95% to 105%, and 98% to 103%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a graph of the high performance liquid chromatography results of the reference solution provided in Example 1 of the present invention.

[0056] Figure 2 It is a graph of the high performance liquid chromatography results of the test solution provided in Example 1 of the present invention.

[0057] Figure 3 It is a graph of the HPLC results of the negative test solution provided in Example 1 of the present invention.

[0058] Figure 4 This is a full-wavelength scanning 3D result diagram of the Bufei Huoxue Capsule test solution provided in Example 3 of the present invention, wherein the 3D scanning result at a wavelength of 190 to 800 nm is shown.

[0059] Figure 5 It is a graph of the high performance liquid chromatography result of the test solution measured by the prior art provided in Comparative Example 1 of the present invention.

[0060] in Figure 1 and Figure 2Label number 1 is psoralen, label number 2 is isopsoralen, label number 3 is psoralen, label number 4 is isopsoralen, and label number 5 is bakuchiol. DETAILED DESCRIPTION

[0061] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0062] The one-test-multiple-evaluation method is to measure a readily available, inexpensive and effective component (as an internal reference) to achieve simultaneous determination of the contents of multiple components. This method can reduce the cost of testing while solving the problem of insufficient reference materials.

[0063] The present invention provides a method for simultaneously determining the content of psoralea corylifolia in Bufei Huoxue capsule by using a one-measurement-multiple-evaluation method. The Bufei Huoxue capsule comprises astragalus, red peony root and psoralea corylifolia. The method comprises:

[0064] Using psoralen as the internal reference substance, the relative correction factors of psoralen, isopsoralen, isopsoralen or bakuchiol compared with psoralen were determined respectively;

[0065] preparing a test solution, wherein the test solution contains the contents of the Bufei Huoxue Capsule;

[0066] The test solution is subjected to high performance liquid chromatography detection, and the contents of psoralen and at least two substances selected from psoralen, isopsoralen, isopsoralen, and bakuchiol in Bufei Huoxue Capsule are determined based on the relative correction factors of psoralen, isopsoralen, isopsoralen or bakuchiol compared to psoralen.

[0067] The method provided by the present invention uses psoralen as an internal reference substance and can simultaneously determine the contents of at least two psoralen components in Bufei Huoxue Capsule. For example, the contents of psoralen and at least one substance selected from psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol can be simultaneously determined.

[0068] The present invention provides a method for simultaneously determining the content of psoralea corylifolia in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method. The Bufei Huoxue Capsule comprises astragalus, red peony root and psoralea corylifolia. The method comprises:

[0069] (1) preparing reference substance stock solutions of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol respectively using methanol, and mixing them to obtain reference substance solutions containing psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol;

[0070] (2) taking the contents of the Bufei Huoxue capsule and dissolving it in 40% methanol to obtain a test solution;

[0071] (3) taking the reference solution of step (1), performing high performance liquid chromatography under different injection volume conditions, using psoralen as the internal reference substance, and determining the average relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared to psoralen;

[0072] (4) taking the test solution in step (2), performing high performance liquid chromatography detection, and obtaining the determination results of psoralen, isopsoralen, psoralen and isopsoralen at a wavelength of 300 to 320 nanometers, and obtaining the determination results of bakuchiol at a wavelength of 250 to 270 nanometers.

[0073] In at least some embodiments of the present invention, the present invention provides a method for determining the content of psoralea corylifolia in Bufei Huoxue Capsule using a one-measurement-multiple-evaluation method, wherein the Bufei Huoxue Capsule comprises astragalus, red peony root and psoralea corylifolia, and the method comprises:

[0074] (1) preparing reference substance stock solutions of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol respectively using methanol, and mixing them to obtain reference substance solutions containing psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol;

[0075] (2) taking the contents of the Bufei Huoxue capsule, dissolving it in 40% methanol, ultrasonically treating it for 30 to 50 minutes, and filtering it with a microporous filter membrane to obtain a test solution;

[0076] (3) taking the reference solution of step (1), performing high performance liquid chromatography under different injection volume conditions, using psoralen as the internal reference substance, and determining the average relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared to psoralen;

[0077] (4) taking the test solution in step (2), performing high performance liquid chromatography detection, and obtaining the determination results of psoralen, isopsoralen, psoralen and isopsoralen at a wavelength of 300 to 320 nanometers, and obtaining the determination results of bakuchiol at a wavelength of 250 to 270 nanometers.

[0078] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications are used. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0079] Example 1

[0080] Example 1 provides a method for simultaneously determining the contents of five psoralea corylifolia components in Bufei Huoxue Capsules using a one-measurement-multiple-evaluation method.

[0081] The instruments and samples used are as follows:

[0082] 1. Instrument

[0083] LC-20AD high performance liquid chromatograph (Shimadzu International Trading Co., Ltd., Japan);

[0084] LC-2030C 3D Plus (Shimadzu International Trading Co., Ltd., Japan);

[0085] Angilent Proshell HPH C18 column (150 mm × 4.6 mm, 4 μm);

[0086] Angilent Eclipse Plus C18 column (150 mm × 4.6 mm, 3.5 μm);

[0087] Ekzo Nobel Kromasil Classic C18 column (150 mm × 4.6 mm, 3.5 μm);

[0088] Labsolutions workstation (Shimadzu International Trading Co., Ltd., Japan);

[0089] Ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0090] AL104 (1 / 10,000), XS105 (1 / 100,000) electronic balances (Mettler-Toledo Instruments (Shanghai) Co., Ltd.);

[0091] DHG-9145A electric heating blast drying oven (Shanghai Huitai Instrument Manufacturing Co., Ltd.);

[0092] Disposable 2 ml sterile syringe (Jiangsu Kangyou Medical Instrument Co., Ltd.);

[0093] Nylon 66 microporous filter membrane (13 mm × 0.45 μm, Tianjin Jinteng Experimental Equipment Co., Ltd.).

[0094] 2. Samples

[0095] Psoralen (batch number Y07A7S12664, purity 98.3%),

[0096] Isopsoralen (batch number Y27A9S60238, purity 99.6%),

[0097] Isopsoralen (batch number C05M10Q82078, purity 99.5%),

[0098] Bakuchiol (batch number YM0320SA14, purity 99.2%);

[0099] The above four reference substances were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0100] Psoralen (batch number 110739-201918, purity 99.6%), this reference substance was purchased from China Food and Drug Inspection Institutes.

[0101] Phosphoric acid (lot number 10015418);

[0102] Methanol for pretreatment (batch number 10014118, analytical grade) (Sinopharm Chemical Reagent Co., Ltd.);

[0103] Methanol for liquid phase analysis (Batch No. 40064292, chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.);

[0104] The water is pure water;

[0105] There are 4 batches of Bufei Huoxue Capsules (Guangdong Leiyunshang Pharmaceutical Co., Ltd.), with batch numbers 012007, 011953, 011824, and 012001 respectively.

[0106] The specific measurement methods and results are as follows:

[0107] 3. Methods

[0108] (1) Chromatographic conditions: Angilent Eclipse Plus C18 column (150 mm × 4.6 mm, 3.5 μm); mobile phase consisting of water and methanol containing 0.1% by volume phosphoric acid, gradient elution (program see Table 1); column temperature 30°C; volume flow rate 0.6 mL / min; detection wavelength 310 nm (0-50 min), 260 nm (50-65 min); injection volume 5 μL.

[0109] Table 1 Gradient elution program

[0110] Time (min) Methanol(%) 0.1% Phosphoric acid water (%) 0～10 25～25 75～75 10～50 25～50 75～50 50～55 50～100 50～0 55～65 100～100 0～0

[0111] (2) Reference solution:

[0112] Accurately weigh appropriate amounts of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol reference substances, place them in 10 ml volumetric bottles, dissolve in methanol and dilute to scale, shake well, prepare 262.85, 256.18, 240.04, 220.79, 247.01 μg / ml stock solutions, and store at 4°C for later use. Accurately pipette 1 mL of each of the above stock solutions, place them in the same 10 ml volumetric bottle, dilute to scale with methanol, shake well, and obtain a mixed reference solution, which is stored at 4°C for later use.

[0113] (3) Test solution:

[0114] Take 0.35 g of the content of Bufei Huoxue Capsule, weigh it accurately, put it in a stoppered conical flask, add 50 mL of 40% methanol accurately, stopper it tightly, weigh it, ultrasonicate it for 30 min (250 W, 40 kHz), take it out, cool it, weigh it again, make up for the lost mass with 40% methanol, shake it well, take the supernatant and filter it through a 0.45 μm microporous filter membrane to obtain the product.

[0115] (4) Negative test solution:

[0116] A negative sample lacking Psoralea corylifolia and containing only Radix Astragali and Radix Paeoniae Rubra was prepared according to the process, and a negative test solution was prepared in the same manner as described in “(3) Test solution”.

[0117] (5) Methodological investigation

[0118] (5-1) System adaptability and specificity inspection:

[0119] Take the mixed reference solution, test solution and negative test solution respectively, and perform the analysis according to the chromatographic conditions under “(1) Chromatographic conditions”. The results are as follows: Figure 1 shown.

[0120] As can be seen from the figure, the separation degrees of the five components and their adjacent chromatographic peaks are all greater than 1.5, the theoretical plate numbers of the internal reference component psoralen are all above 90,000, and there is no negative interference, indicating that the method has good specificity.

[0121] (5-2) Linear relationship investigation: Accurately pipette the stock solution and mixed reference solution under "(2) Reference solution" and dilute them with methanol 3.125, 6.25, 12.5, 25, 50, and 125 times, respectively. Accurately pipette 5 μL of mixed reference solution of different concentrations and inject and measure under the chromatographic conditions under "(1) Chromatographic conditions". Regression was performed with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The results are shown in Table 2. It can be seen that each component has a good linear relationship within its respective range.

[0122] Table 2 Linear relationship of each component

[0123] Element Linear regression equation r Linear range (μg / ml) Psoralen y=5687.5x-438.71 1 2.102-84.113 Isopsoralen y=6022.3x-528.6 1 2.049-81.977 Psoralen y=23950x-1942.8 1 1.920-76.811 Isopsoralen y=24761x-1074.5 1 1.766-70.652 Bakuchiol y=18655x-1530.4 1 1.976-79.042

[0124] (5-3) Precision test:

[0125] Take the mixed reference solution under “(2) Reference solution” and inject it six times under the chromatographic conditions under “(1) Chromatographic conditions”. The peak areas of the components are shown in Table 3.

[0126] Table 3 Peak areas of six different injection batches of each component

[0127] Element Psoralen Isopsoralen Psoralen Isopsoralen Bakuchiol 1 191751 148351 841422 808965 741105 2 190502 147891 847717 812703 740835 3 190058 146958 848820 814400 738783 4 187194 144842 841523 807415 730084 5 187567 145134 845707 812061 731386 6 187056 144736 847886 813863 730625 RSD 1.06％ 1.11％ 0.39％ 0.34％ 0.72％

[0128] It is not difficult to see from the results given in Table 3 that the RSDs of the contents of psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol measured in the experiment are 1.06%, 1.11%, 0.39%, 0.34% and 0.72%, respectively, indicating that the instrument has good precision.

[0129] (5-4) Stability test:

[0130] 0.35 g of the contents of Bufei Huoxue Capsule (batch number: 021007) was accurately weighed and, according to the test sample preparation method under "(3) Test sample solution", 5 μL of the solution was accurately drawn at 0, 2, 4, 8, 12, and 24 h under the chromatographic conditions under "(1) Chromatographic conditions" and injected into the liquid chromatograph. The peak areas of the components are shown in Table 4.

[0131] Table 4 Peak areas of each component under different time conditions

[0132] Time (hr) Psoralen Isopsoralen Psoralen Isopsoralen Bakuchiol 0 686357 446548 847061 626991 392308 2 680134 442439 833282 619316 388821 4 673554 438459 829916 615533 384488 6 675229 439443 833226 617937 386526 12 667669 433802 821299 609861 380875 24 659992 433716 824087 611254 382269 RSD 1.37％ 1.14％ 1.09％ 1.00％ 1.10％

[0133] It is not difficult to see from the results given in Table 4 that the RSDs of the contents of psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol measured in the experiment are 1.37%, 1.14%, 1.09%, 1.00% and 1.10%, respectively, indicating that the test solution has good stability within 24 hours.

[0134] (5-5) Repeatability test:

[0135] Take several capsules of Bufei Huoxue Capsules (Batch No.: 021007), mix the contents, accurately weigh 6 portions, and inject 5 μl of each portion according to the test sample preparation method under "(3) Test sample solution" and the chromatographic conditions under "(1) Chromatographic conditions". The peak area and content of each component were analyzed and recorded. The results are shown in Table 5.

[0136] Table 5 Content and RSD of each component in the test sample

[0137]

[0138] It is not difficult to see from the results given in Table 5 that the RSD values ​​of psoralen, isopsoralen, psoralen, isopsoralen and bakuchiol contents were 1.39%, 0.87%, 1.13%, 1.06% and 0.73%, respectively, indicating that the method has good repeatability.

[0139] (5-6) Sample recovery test:

[0140] Accurately weigh 6 portions of Bufei Huoxue Capsules with known contents from the same batch (batch number: 021007), prepare the test solution according to the method under “(3) Test solution”, add equal amounts of reference solution in a 1:1 ratio, and perform the analysis under the chromatographic conditions under “(1) Chromatographic conditions”. Calculate the recovery rate. The results are shown in Table 6.

[0141] Table 6 Experimental results of recovery rate of each component

[0142]

[0143]

[0144] (6) Calculation of relative correction factor:

[0145] Using psoralen as the internal reference substance, the relative correction factors (RCF) of psoralen, isopsoralen, isopsoralen and bakuchiol to psoralen were calculated. The results are shown in Table 7.

[0146] Table 7 Relative correction factors of each component

[0147]

[0148] (7) Determination of test sample content

[0149] Four batches of Bu Fei Huo Xue Capsules were taken, and about 0.35 g of each batch was accurately weighed. The test solution was prepared according to the "(3) Test solution". 5 μL of the reference solution and the test solution were accurately pipetted. The samples were injected and measured under the chromatographic conditions in "(1) Chromatographic conditions". The results were compared with the external standard method (using the standard of each substance for detection). The results are shown in Table 8. It can be seen that the results obtained by the two methods are close and the difference is not statistically significant.

[0150] Table 8 Comparison of the results obtained by one-test-multiple-evaluation method and external standard method

[0151]

[0152] It is not difficult to see from the above method and the results obtained that the method provided by the present invention is accurate and reliable for determining the content of Psoralea corylifolia in Bu Fei Huo Xue Capsules, and the method is simple and can be used for quality control of Bu Fei Huo Xue Capsules.

[0153] Example 2

[0154] Example 2 The preparation method of the test solution was studied and investigated. The experiment found that after ultrasonic treatment with 40% methanol for 30 minutes and filtration with a microporous membrane, the test results of the test solution obtained were more accurate and reliable. The details are as follows:

[0155] This example investigates the effects of 100% methanol, 40% methanol by volume, water ultrasonically filtered through a microporous membrane, methanol, water Soxhlet reflux extraction, and methanol dissolution and injection on the test results. The experiment found that when 40% methanol was injected after ultrasonic injection, the chromatogram obtained had rich information on the chromatogram peaks, strong signals of each peak to be measured, and a relatively stable baseline. The ultrasonic time was also investigated, and it was found that there was no significant difference in the signal intensity of the peak to be measured when ultrasonically 30 minutes was compared with ultrasonically 60 minutes and Soxhlet extraction for 1 hour. Therefore, 40% methanol ultrasonically 30 minutes and then filtered through a microporous membrane was used as the test solution preparation method.

[0156] Example 3

[0157] Example 3 The detection wavelength of high performance liquid chromatography was studied. The experiment found that although all components of Psoralea corylifolia have good absorption under the condition of 260 nanometers, some components of Psoralea corylifolia will be affected by other components in Bufei Huoxue Capsule. Therefore, the experiment selected 310nm as the detection wavelength of psoralen, isopsoralen, psoralen, and isopsoralen, and 260nm as the detection wavelength of bakuchiol, which can obtain more accurate results. The details are as follows:

[0158] In this embodiment, when the reference substance solutions of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol were scanned at full wavelength, it was found that all five had good absorption at a wavelength of 260 nm, but the retention times of the chromatographic peaks of psoralen and isopsoralen were similar to those of paeoniflorin at a wavelength of 260 nm, and the requirement of separation degree>1.5 could not be met. Therefore, 310 nm was selected as the detection wavelength of the first four components, and 260 nm was selected as the detection wavelength of bakuchiol.

[0159] Example 4

[0160] Example 4 considers the influence of different substances as internal reference substances on the determination results. It is found that psoralen is the main active ingredient of Bufei Huoxue Capsule, which is easy to obtain, cheap, easy to obtain, has a moderate retention time and good separation, so it is selected as the internal reference substance.

[0161] Example 5

[0162] Example 5 investigated the durability of the relative correction factor, and investigated the repeatability of the relative correction factor of each component under different chromatographic columns, volume flow rates, column temperatures, detection wavelengths, and mobile phases, to verify the feasibility and technical adaptability of the one-measurement-multiple-evaluation method in the quality control of Bufei Huoxue Capsules. The results showed that the durability of the relative correction factor under different conditions was good, with RSD < 5%, indicating that the method was stable and feasible and could be used for the content determination of the above five components. The details are as follows:

[0163] 1. Chromatographic column:

[0164] 5 μL of the mixed reference solution under the item "Reference solution of Example 1 (2)" was accurately drawn, and the sample was injected and measured using Angilent Proshell HPH C18, Angilent Eclipse Plus C18, and Ekzo Nobel Kromasil C18 chromatographic columns respectively under the chromatographic conditions of item "2.1" to investigate the influence of different chromatographic columns on the relative correction factor. The results are shown in Table 9. It can be seen from the results given in Table 9 that the influence of different chromatographic columns is not obvious, and the system durability is good.

[0165] Table 9 Effect of different chromatographic columns on relative correction factors

[0166] Chromatographic columns Psoralen Isopsoralen Isopsoralen Bakuchiol Proshell HPH C18 4.246 4.013 0.969 1.314 Eclipse C18Plus 4.208 3.975 0.965 1.278 Kromasil Classic C18 4.296 4.060 0.964 1.266 average value 4.250 4.016 0.966 1.286 RSD 1.04％ 1.06％ 0.28％ 1.92％

[0167] 2. Volume flow rate:

[0168] 5 μl of the mixed reference solution under the item "reference solution (2) in Example 1" was accurately drawn, and the sample was injected and measured under the chromatographic conditions under the item "chromatographic conditions (1) in Example 1", and the effect of the volume flow rate of 0.5, 0.6, and 0.7 ml / min on the relative correction factor was investigated. The results are shown in Table 10. It can be seen from the results given in Table 10 that different volume flow rates have little effect on the relative correction factor, and the system has good durability.

[0169] Table 10 Effect of different volume flow rates on relative correction factors

[0170]

[0171] 3. Column temperature:

[0172] 5 μL of the mixed reference solution under the item "reference solution (2) in Example 1" was taken and injected under the chromatographic conditions under the item "chromatographic conditions (1) in Example 1" to investigate the effects of different column temperatures of 25, 30, and 35°C on the relative correction factor. The results are shown in Table 11. It can be seen from the results given in Table 11 that different column temperatures have little effect on the relative correction factor and the durability is good.

[0173] Table 11 Effect of different column temperatures on relative correction factors

[0174]

[0175]

[0176] 4. Mobile phase:

[0177] 5 μL of the mixed reference solution under the item "reference solution (2) in Example 1" was taken and injected under the chromatographic conditions under the item "chromatographic conditions (1) in Example 1" to investigate the effects of 0.05%, 0.1%, and 0.2% phosphoric acid / water phase (v / v, volume ratio) on the relative correction factor. The results are shown in Table 12. As can be seen from the results in Table 12, the effects of different ratios of phosphoric acid-water phase are not obvious, and the system has good durability.

[0178] Table 12 Effect of different ratios of phosphoric acid to water on relative correction factors

[0179]

[0180] 5. Detection wavelength:

[0181] 5 μL of the mixed reference solution under the item "reference solution (2) in Example 1" was accurately drawn, and the sample was injected and measured under the chromatographic conditions under the item "chromatographic conditions (1) in Example 1", and the effects of 308, 310, 312 nm on the relative correction factors of psoralen, isopsoralen and isopsoralen and 258, 260, 262 nm on bakuchiol were investigated respectively. The results are shown in Table 13. From the results given in Table 13, it can be seen that the detection wavelength has no obvious effect on the relative correction factor, and the system durability is good.

[0182] Table 13 Effect of different detection wavelengths on relative correction factors

[0183] RCF Psoralen Isopsoralen Isopsoralen Bakuchiol 308nm / 258nm 4.272 3.856 0.974 1.317 310nm / 260nm 4.233 4.002 0.959 1.352 312nm / 262nm 4.192 4.175 0.952 1.346 average value 4.233 4.011 0.962 1.338 RSD 0.95％ 3.99％ 1.13％ 1.38％

[0184] Example 6

[0185] In order to correctly locate the chromatographic peaks of the other four components in Bufei Huoxue Capsule when only a single reference substance is used, this example selects the relative retention time of each component to the internal reference substance as the positioning standard, and conducts a repeatability test on it under different instruments and chromatographic columns. The results show that the relative retention value can locate the chromatographic peak more accurately. The details are as follows:

[0186] The repeatability of the relative retention value and the retention time difference in different instruments and chromatographic columns was investigated, and the results are shown in Table 14. As shown in Table 14, the relative retention value fluctuates less and is relatively stable in different instrument chromatographic columns. The method provided by the present invention is more tolerant to instruments.

[0187] Table 14 Effects of different instruments and columns on relative retention values

[0188]

[0189] Combined with the results given in the above embodiments, this experiment established a one-measurement-multiple-evaluation method to simultaneously determine the contents of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol in Bufei Huoxue Capsule. The method is simple, fast, and accurate, and the relative correction factor has good credibility and accuracy. It can be used for content determination when the reference substance is in short supply, and can reduce solvent consumption, shorten analysis time, and is more conducive to efficiency improvement and cost compression.

[0190] Comparative Example 1

[0191] The contents of the five psoralea corylifolia components in Bufei Huoxue Capsule were determined according to the method of Example 1, except that the chromatographic conditions were as follows:

[0192] Chromatographic column: AkzoNobel Kromasil 100-5-C18 (4.6×250 mm, 5 μm);

[0193] Mobile phase: acetonitrile (A)-0.1% phosphoric acid solution (B);

[0194] The mobile phase gradient elution program is shown in Table 15.

[0195] Table 15 Mobile phase gradient elution time program

[0196]

[0197]

[0198] Detection wavelength: 245nm;

[0199] Column temperature: 30°C;

[0200] Flow rate: 1.0 mL / min;

[0201] Injection volume: 5 μL.

[0202] The results are shown in Table 16 and Figure 5 As shown, the method of this comparative example may cause other substances to interfere with the target component to be measured, and good separation cannot be achieved, and the content measured by the method has a large error. The content measured by the method of the present invention is more accurate.

[0203] Table 16 Differences in the content of ingredients in Bufei Huoxue Capsules measured by different methods

[0204]

[0205] In this document, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0206] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0207] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for simultaneously determining the content of psoralea corylifolia in Bufei Huoxue Capsule using a one-measurement-multiple-evaluation method, wherein the Bufei Huoxue Capsule comprises astragalus, red peony root, paeoniflorin and psoralea corylifolia, characterized in that: The method comprises: Using psoralen as the internal reference substance, the relative correction factors of psoralen, isopsoralen, isopsoralen and bakuchiol compared with psoralen were determined respectively; Obtaining a test solution, wherein the test solution contains the contents of the Bufei Huoxue Capsule; The test solution is subjected to high performance liquid chromatography detection, and the determination results of psoralen, isopsoralen, psoralen and isopsoralen are obtained at a wavelength of 300 to 320 nanometers for 0 to 50 minutes, and the determination results of bakuchiol are obtained at a wavelength of 250 to 270 nanometers for 50 to 80 minutes; Determine the content of psoralen and psoralen, isopsoralen, isopsoralen and bakuchiol in Bufei Huoxue Capsule based on the relative correction factor of psoralen, isopsoralen, isopsoralen or bakuchiol compared to psoralen; The relative correction factor of the psoralen to the psoralen is 3.8 to 4.5; The relative correction factor of the isopsoralen to the psoralen is 3.7 to 4.5; The relative correction factor of the isopsoralen relative to the psoralen is 0.85 to 1.3; The relative correction factor of the bakuchiol relative to the psoralen is 1 to 1.5; The HPLC detection conditions are as follows: The chromatographic column is a C18 column; The mobile phase includes methanol and an aqueous phase containing phosphoric acid, and the volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05-0.5%; The volume flow rate is 0.3-1.0 mL / min; The column temperature is 20-40℃.

2. The method according to claim 1, characterized in that The determination results of psoralen, isopsoralen, psoralen or isopsoralen are obtained at a wavelength of 305 to 315 nanometers, and the determination results of bakuchiol are obtained at a wavelength of 255 to 265 nanometers.

3. The method according to claim 1, characterized in that The measurement results of psoralen, isopsoralen, psoralen or isopsoralen are obtained at a wavelength of 308, 310 or 312 nanometers, and the measurement results of bakuchiol are obtained at a wavelength of 258, 260 or 262 nanometers.

4. The method according to claim 1, characterized in that: The relative correction factor of the psoralen to the psoralen is 4.1 to 4.3; The relative correction factor of the isopsoralen to the psoralen is 3.9 to 4.2; The relative correction factor of the isopsoralen relative to the psoralen is 0.95 to 1.1; The relative correction factor of the bakuchiol relative to the psoralen is 1.1 to 1.

4.

5. The method according to claim 1, characterized in that The test solution is obtained by dissolving the contents of Bufei Huoxue Capsule in methanol.

6. The method according to claim 5, characterized in that The methanol is 40% methanol.

7. The method according to claim 5, characterized in that After dissolving with methanol, ultrasonic treatment is performed for 20 to 50 minutes to obtain a test solution.

8. The method according to claim 1, characterized in that The HPLC detection conditions are as follows: The chromatographic column is any one of Angilent Eclipse Plus C18, Angilent Proshell HPH C18, and Ekzo NobelKromasil C18; The volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05% to 0.2%; The volume flow rate is 0.5-0.7 mL / min; The column temperature is 25-35 degrees Celsius.

9. The method according to claim 1, characterized in that: The HPLC detection conditions are as follows: Angilent Eclipse Plus C18 column, 150 mm × 4.6 mm, 3.5 μm; The mobile phase was methanol and water containing 0.1% by volume phosphoric acid; The gradient elution conditions were: Column temperature: 30°C; Volume flow rate 0.6ml / min; The detection wavelength for 0 to 50 min was 310 nm, and the detection wavelength for 50 to 65 min was 260 nm.

10. A method for simultaneously determining the contents of psoralen, isopsoralen, isopsoralen, isobakuchiol and psoralen in Bufei Huoxue Capsule by using a one-measurement-multiple-evaluation method, wherein the Bufei Huoxue Capsule comprises astragalus, red peony root, paeoniflorin and psoralen, characterized in that: The method comprises: (1) preparing reference substance stock solutions of psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol respectively using methanol, and mixing them to obtain reference substance solutions containing psoralen, isopsoralen, psoralen, isopsoralen, and bakuchiol; (2) taking the contents of the Bufei Huoxue Capsule and dissolving it with methanol to obtain a test solution; (3) taking the reference solution of step (1), performing high performance liquid chromatography under different injection volume conditions, using psoralen as the internal reference substance, and determining the relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared to psoralen; (4) taking the test solution in step (2) and performing high performance liquid chromatography detection, obtaining the determination results of psoralen, isopsoralen, psoralen and isopsoralen at a wavelength of 300 to 320 nanometers for 0 to 50 minutes, and obtaining the determination results of bakuchiol at a wavelength of 250 to 270 nanometers for 50 to 80 minutes; The mobile phase for the high performance liquid chromatography detection comprises methanol and an aqueous phase containing phosphoric acid, wherein the volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05 to 0.5%; The gradient elution conditions were: 。 11. The method according to claim 10, characterized in that In step (4), the determination results of psoralen, isopsoralen, psoralen and isopsoralen are obtained at a wavelength of 305 to 315 nanometers, and the determination results of bakuchiol are obtained at a wavelength of 255 to 265 nanometers.

12. The method according to claim 10, characterized in that The assay results for psoralen, isopsoralen, psoralen and isopsoralen were obtained at a wavelength of 308, 310 or 312 nanometers, and the assay results for bakuchiol were obtained at a wavelength of 258, 260 or 262 nanometers.

13. The method according to claim 10, characterized in that The methanol in step (2) is 40% methanol.

14. The method according to claim 10, characterized in that After dissolving with methanol, ultrasonic treatment is performed for 20 to 50 minutes to obtain a test solution.

15. The method according to claim 10, characterized in that Step (2) further comprises: (2-1) Weigh the contents of the Bufei Huoxue Capsule, place it in a stoppered container, add 40% methanol, weigh it, and obtain a first weighing solution; (2-2) subjecting the first weighing solution to ultrasonic treatment for 25 to 40 minutes, weighing the solution after the ultrasonic treatment to obtain a second weighing solution; (2-3) Determine the mass lost by the second weighing solution compared to the first weighing solution, make up with 40% methanol, and filter using a 0.45 μm filter membrane to obtain the test solution.

16. The method according to claim 10, characterized in that Step (3) further comprises: (3-1) taking the reference solution of step (1), injecting 1, 2, 4, 6, 8, and 10 μl of the reference solution, respectively, and performing high performance liquid chromatography to obtain corresponding test results; (3-2) Based on the detection results, taking psoralen as the internal reference substance, the relative correction factors of psoralen, isopsoralen, isopsoralen, and bakuchiol compared with psoralen under different injection conditions were calculated respectively, and the average value was taken to obtain the average relative correction factor.

17. The method according to claim 10, characterized in that The high performance liquid chromatography detection conditions in step (3) and step (4) are as follows: The chromatographic column is a C18 column; The mobile phase includes methanol and an aqueous phase containing phosphoric acid, and the volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05-0.5%; The volume flow rate is 0.3-1.0 mL / min; The column temperature is 20-40 degrees Celsius.

18. The method according to claim 10, characterized in that The high performance liquid chromatography detection conditions in step (3) and step (4) are as follows: The chromatographic column is any one of Angilent Eclipse Plus C18, Angilent Proshell HPH C18, and Ekzo NobelKromasil C18; The volume ratio of phosphoric acid in the aqueous phase containing phosphoric acid is 0.05% to 0.2%; The volume flow rate is 0.5-0.7 mL / min; The column temperature is 25-35 degrees Celsius.

19. The method according to claim 10, characterized in that The HPLC detection conditions are as follows: Angilent Eclipse Plus C18 column, 150 mm × 4.6 mm, 3.5 μm; The mobile phase was methanol and water containing 0.1% by volume phosphoric acid; The gradient elution conditions were: Column temperature: 30°C; Volume flow rate 0.6ml / min; The detection wavelength for 0 to 50 min was 310 nm, and the detection wavelength for 50 to 65 min was 260 nm.

Images