Method for detecting contents of four fat-soluble components in Tibetan radix salviae miltiorrhizae by quantitative analysis of multi-components by single marker

Abstract

The invention relates to the field of medicine detection, in particular to a quantitative analysis of multi-components by single marker (QAM) detection method for the content of four fat-soluble components in Tibetan salvia miltiorrhiza, which comprises the following steps: step 1, preparing a Tibetan salvia miltiorrhiza reference substance and a Tibetan salvia miltiorrhiza test sample from a Tibetan salvia miltiorrhiza raw material; step 2, eluting the prepared Tibetan radix salviae miltiorrhizae reference substance, and drawing a standard curve according to the contents of the four fat-soluble components to obtain a regression equation; and step 3, carrying out sample introduction on a Tibetan radix salviae miltiorrhizae test sample to obtain peak areas of the four fat-soluble components, and substituting the peak areas into the regression equation to obtain the contents of the four fat-soluble components in the Tibetan radix salviae miltiorrhizae test sample. According to the method, 20 batches of Tibetan radix salviae miltiorrhizae are measured through the established QAMS method, an external standard method is used for verifying that the content of the components measured through the two methods has no significant difference, and it is indicated that the QAMS method is accurate and feasible in content measurement of the four fat-soluble components in the Tibetan radix salviae miltiorrhizae.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a one-test-multiple-evaluation detection method for the contents of four fat-soluble components in Tibetan salvia miltiorrhiza. Background Art

[0002] Tibetan salvia, scientifically known as Salvia castanea Dielsf. tomentosa Stib, is a variant of Salvia castanea Dielsf. tomentosa Stib, a plant of the genus Salvia in the Lamiaceae family. It is native to the Nyingchi region of Tibet. In December 2005, Salvia castanea was included in the local medicinal material standard by the Tibet Autonomous Region Food and Drug Administration and named "Tibetan salvia." Research has shown that Tibetan salvia contains both fat-soluble and water-soluble components. The fat-soluble components include tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone, which have pharmacological activities such as anti-myocardial ischemia, improving myocardial infarction, anti-thrombosis, preventing and delaying Alzheimer's disease, and alleviating hyperammonemia and hepatic encephalopathy.

[0003] Quantitative Analysis of Multi-components by Single Marker (QAMS) is an innovative multi-index quality control method proposed by Wang Zhimin and others at the China Academy of Chinese Medical Sciences in 2006. By establishing relative correction factors between target components, this method enables simultaneous quantitative analysis of multiple components using a single reference standard, providing a new approach to addressing the bottleneck of "reference standard dependence" in the quality control of traditional Chinese medicines. Its core approach is to utilize a readily available, inexpensive, and stable reference standard as an internal standard. By optimizing chromatographic conditions and methodological validation, metrics such as relative retention time, UV spectral similarity, and relative correction factors are calculated to achieve qualitative and quantitative analysis of multiple components. Specific steps include selecting an internal standard, establishing correction factors, and performing simultaneous quantitative analysis. QAMS offers advantages such as cost-effectiveness, convenience, accuracy, and environmental friendliness, and has been widely used in the quality control of traditional Chinese medicines, decoction pieces, and preparations. As an emerging analytical technology, QAMS holds promising prospects for development and application. In the future, this method is expected to be promoted and applied in more fields, further promoting the improvement of the quality control system for traditional Chinese medicines.

[0004] Literature: Zuo Lan, Meng Shengnan. Application progress of one-test-multiple-evaluation method in traditional Chinese medicine analysis[J]. Chinese Pharmacy, 2016, 27(18): 2589-2592.

[0005] Wang Xin, Qin Yao, Wang Dejiang, et al. Application progress of one-test-multiple-evaluation method in quality control of traditional Chinese medicine[J]. Chinese Traditional Patent Medicine, 2016, 38(02): 395-402.

[0006] Liu Changxiao, Chen Shilin, Xiao Xiaohe, et al. Quality Marker (Q-Marker) of traditional Chinese medicine: a new concept for quality control of traditional Chinese medicine products [J]. Chinese Traditional and Herbal Drugs, 2016, 47(09): 1443-1457.

[0007] When using the external standard method to detect the contents of 4 liposoluble components in Salvia przewalskii Maxim., there are defects such as large consumption of reference substances, many types of reference substances, and complex and cumbersome operations. Moreover, the preparation methods of some reference substances are complex and the supply is short, which limits the application of this method in scientific research, actual production and market supervision. Therefore, there is a need to provide a simple, efficient, accurate and low-cost detection method to determine the contents of 4 liposoluble components in Salvia przewalskii Maxim. Summary of the Invention

[0008] The purpose of the present invention is to provide a multi-component simultaneous determination method for detecting the contents of 4 liposoluble components in Salvia przewalskii Maxim., which can quickly detect the contents of 4 liposoluble components in Salvia przewalskii Maxim.

[0009] To achieve the above purpose, the technical solution of the present application is as follows:

[0010] A multi-component simultaneous determination method for detecting the contents of 4 liposoluble components in Salvia przewalskii Maxim. includes the following steps:

[0011] Step 1: Take the raw materials of Salvia przewalskii Maxim. to prepare reference substances and test samples of Salvia przewalskii Maxim.;

[0012] Step 2: Elute the prepared reference substances of Salvia przewalskii Maxim., and draw a standard curve based on the contents of 4 liposoluble components to obtain a regression equation;

[0013] Among them, the regression equation is:

[0014] Tanshinone ⅡA: Y = 4832.87X, R 2 = 0.9996;

[0015] Tanshinone Ⅰ: Y = 22319.4X, R 2 = 0.9996;

[0016] Cryptotanshinone: Y = 43162.1X, R 2 = 0.9995;

[0017] Dihydrotanshinone: Y = 27185.7X, R 2 = 0.9996;

[0018] Among them: R 2 represents the goodness of fit of the regression model to the data;

[0019] Step 3: Inject the tested sample of *Salvia przewalskii* var. *mandarinorum* to obtain the peak areas of 4 liposoluble components, substitute the peak areas into the regression equation, and obtain the contents of 4 liposoluble components in the tested sample of *Salvia przewalskii* var. *mandarinorum*.

[0020] Specifically, in step 1, 5 batches of *Salvia przewalskii* var. *mandarinorum* from Milin, Bomi, Bayi, and Langxian are selected as the raw materials of the tested sample of *Salvia przewalskii* var. *mandarinorum*, with a total of 20 batches, numbered S1, S2... S20.

[0021] Furthermore, step 1 includes:

[0022] Step 1.1: Weigh tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone, mix them, add methanol to dissolve, and prepare the reference substance of *Salvia przewalskii* var. *mandarinorum*.

[0023] Step 1.2: Take the tested samples of *Salvia przewalskii* var. *mandarinorum* of batches S1, S2... S20, add methanol respectively, weigh them, then perform ultrasonic treatment, cool them, weigh them again for the second time, and make up the weight reduced in the second weighing with methanol to prepare the tested sample 1 of *Salvia przewalskii* var. *mandarinorum*.

[0024] Step 1.3: Take 6 portions of the tested sample of *Salvia przewalskii* var. *mandarinorum* of batch S1, add methanol respectively, weigh them, then perform ultrasonic treatment, cool them, weigh them again for the second time, and make up the weight reduced in the second weighing with methanol to prepare the tested sample 2 of *Salvia przewalskii* var. *mandarinorum*.

[0025] Among them, the ratio of the tested sample of *Salvia przewalskii* var. *mandarinorum* to methanol in step 1.2 and step 1.3 is 3 g: 500 mL.

[0026] Even further, step 2 includes:

[0027] Step 2.1: Dilute the reference substance of *Salvia przewalskii* var. *mandarinorum* in step 1 to obtain the diluted solution of the reference substance of *Salvia przewalskii* var. *mandarinorum*.

[0028] Step 2.2: Elute the diluted solution of the reference substance of *Salvia przewalskii* var. *mandarinorum* in step 1.1 with an acetonitrile - 0.02% phosphoric acid mixed solution as the mobile phase under chromatographic conditions.

[0029] Step 2.3: Based on step 2.2, plot a standard curve with the peak area as the ordinate Y and the mass concentration of the diluted solution of the reference substance of *Salvia przewalskii* var. *mandarinorum* as the abscissa X, and calculate the regression equation.

[0030] Step 2.4: Based on step 2.3, obtain the regression equations of 4 liposoluble components in the diluted solution of the reference substance of *Salvia przewalskii* var. *mandarinorum*.

[0031] In the above technical solution, step 3 includes:

[0032] Step 3.1: Inject the tested sample 1 of *Salvia przewalskii* var. *mandarinorum* of batch S1 prepared in step 1.2 under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively.

[0033] Step 3.2: Place the Tibetan Salvia miltiorrhiza test sample 1 of Batch S1 prepared in Step 1.2 in the dark at room temperature for different times, inject samples under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively;

[0034] Step 3.3: Inject the Tibetan Salvia miltiorrhiza test sample 2 prepared in Step 1.3 under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. Significantly reduce detection costs and resource dependence

[0037] The traditional external standard method requires separate provision of high-purity reference substances for each target component, while the reference substances for some lipophilic components in Tibetan Salvia miltiorrhiza (such as tanshinone IIA, cryptotanshinone, etc.) are expensive or in limited supply. By establishing a relative correction factor (RCF) and retention time correlation model, the present invention only requires 1 easily obtainable main component reference substance to simultaneously determine 4 components, reducing the reference substance cost by more than 70%, and avoiding the limitation of scarce reference substances on detection.

[0038] 2. Improve detection efficiency and throughput

[0039] By optimizing chromatographic conditions (such as using a gradient elution program) and combining the method of multi-component determination with a single standard, simultaneous separation and quantification of 4 lipophilic components are achieved. The research by Fu Haitan, Zhang Qianrui, and Wu Fangjian. Research progress of modern analytical techniques in the quality control of traditional Chinese medicines [J]. China Pharmaceuticals, 2019, 28(22): 96-99 shows that the single analysis time is shortened by 60% compared with the traditional separate detections, and there is no need to repeatedly replace the reference substance, which is especially suitable for high-throughput detection of a large number of samples (such as enterprise quality control or drug regulatory sampling).

[0040] 3. The methodology has strong robustness and the results are accurate and reliable

[0041] After systematic verification, the RSDs of the relative correction factors of the 4 lipophilic components are all <3% under different concentrations, instruments, and laboratory conditions, and the retention time drift correction model R 2 >0.999. Compared with the classical external standard method, the relative errors of the content determination results are all <5%, proving that the repeatability and accuracy of the method meet the requirements of the Chinese Pharmacopoeia and can replace the traditional detection scheme.

[0042] 4. Promote the standardization of the quality control of Tibetan Salvia miltiorrhiza

[0043] In view of the current situation of the single quality control index for the fat-soluble active components (such as tanshinones) of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib., the present invention realizes the synchronous monitoring of multiple components for the first time, which can more comprehensively reflect the internal quality of the medicinal materials, provide key technical support for establishing a specific quality evaluation system for Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib., and contribute to the modernization and internationalization process of Tibetan medicine.

[0044] 5. Improvement of environmental friendliness and operation safety

[0045] Reduce the consumption of organic solvents (such as methanol and acetonitrile) by about 40%, reduce the pressure of waste liquid treatment; at the same time, avoid the frequent contact of experimental personnel with highly toxic reference substances (such as some tanshinone derivatives), which is in line with the development trend of green analytical chemistry. Description of the drawings

[0046] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention.

[0047] Figure 1 It is the chromatogram obtained by eluting the reference substance solution of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. with different mass concentrations after dilution of the present invention under the chromatographic conditions with a gradient of acetonitrile - 0.02% phosphoric acid mixed solution as the mobile phase;

[0048] Figure 2 It is the curve graph of the peak area of tanshinone IIA and the mass concentration of the reference substance solution of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of the present invention;

[0049] Figure 3 It is the curve graph of the peak area of tanshinone I and the mass concentration of the reference substance solution of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of the present invention;

[0050] Figure 4 It is the curve graph of the peak area of cryptotanshinone and the mass concentration of the reference substance solution of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of the present invention;

[0051] Figure 5 It is the curve graph of the peak area of dihydrotanshinone and the mass concentration of the reference substance solution of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of the present invention. Detailed implementation manners

[0052] The following is a description of the preferred embodiments of the present invention with reference to the drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0053] Embodiment

[0054] A method for multi-component determination of four liposoluble components in Salvia castanea Diels, in which the instruments used in the experimental determination process are Shimadzu LC-2040 high performance liquid chromatograph (Shimadzu (China) Co., Ltd.); PL-S100T ultrasonic cleaner (Dongguan Kangshijie Ultrasonic Technology Co., Ltd.); Chinese herbal medicine grinder (Tianjin Test Instrument Co., Ltd.); UPT-11-10T ultra-pure water machine (Shenzhen Youpute Co., Ltd.).

[0055] The S1, S2... S20 batches of Salvia castanea Diels used in the experiment were entrusted to local farmers for collection and procurement, mainly produced in Milin, Bomi, Bayi and Langxian areas of Tibet. Submitted by Associate Professor Jiang Zhao of Tibet Minzu University, and identified as the roots of Salvia castanea f. tomentosa E. Peter. of Labiatae by first-generation sequencing. Refer to Table 1:

[0056] Table 1: Sample numbers and origin information are shown in the table

[0057] Medicinal material number Place of origin Source area S1 Mainling Commercially available S2 Mainling Commercially available S3 Mainling Commercially available S4 Mainling Commercially available S5 Mainling Commercially available S6 Bomi Commercially available S7 Bomi Commercially available S8 Bomi Commercially available S9 Bomi Commercially available S10 Bomi Commercially available S11 Lhunze Commercially available S12 Lhunze Commercially available S13 Lhunze Commercially available S14 Lhunze Commercially available S15 Lhunze Commercially available S16 Bayi Commercially available S17 Bayi Commercially available S18 Bayi Commercially available S19 Bayi Commercially available S20 Bayi Commercially available

[0058] A method for multi-component determination of four liposoluble components in Salvia castanea Diels, comprising the following steps:

[0059] Step 1: Select 5 batches of Salvia castanea Diels from Milin, Bomi, Bayi and Langxian respectively, a total of 20 batches, numbered S1, S2... S20;

[0060] It should be noted that the cryptotanshinone reference substance (batch number: J07HB184109), tanshinone I reference substance (batch number: A14IB222878), tanshinone IIA reference substance (batch number: F18IB207456), and dihydrotanshinone I reference substance (batch number: J06HB178293) in Step 1 were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0061] Step 2: Prepare the reference substance of Salvia castanea Diels, and prepare the test samples of Salvia castanea Diels based on S1, S2... S20 batches;

[0062] Step 2.1: Weigh tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone, mix them and dissolve them in methanol to prepare the reference substance of Salvia castanea Diels.

[0063] Among them, the specific amounts of the 4 liposoluble components weighed in Step 2.1 are 0.95 g of tanshinone IIA, 0.85 g of tanshinone I, 0.95 g of cryptotanshinone, and 0.6 g of dihydrotanshinone; the amount of methanol added is 10 mL.

[0064] Step 2.2: Take 0.3 g of the Tibetan Salvia miltiorrhiza Bunge samples from batches S1, S2... S20 respectively, add 50 mL of methanol to each, weigh, then perform ultrasonic treatment, let it cool and weigh again, and make up the weight reduced during the second weighing with methanol to obtain the Tibetan Salvia miltiorrhiza Bunge sample 1.

[0065] Step 2.3: Take 6 portions of 0.3 g each of the Tibetan Salvia miltiorrhiza Bunge sample from batch S1, add 50 mL of methanol to each, weigh, then perform ultrasonic treatment, let it cool and weigh again, and make up the weight reduced during the second weighing with methanol to obtain the Tibetan Salvia miltiorrhiza Bunge sample 2.

[0066] It should be noted that before weighing the Tibetan Salvia miltiorrhiza Bunge from batches S1, S2... S20, it is necessary to filter through a No. 3 sieve.

[0067] Step 2.4: Take 24 portions of 0.15 g each of the Tibetan Salvia miltiorrhiza Bunge sample from batch S1, add the corresponding Tibetan Salvia miltiorrhiza Bunge reference substance with an equivalent content of liposoluble components to each portion of the Tibetan Salvia miltiorrhiza Bunge sample from batch S1, and prepare the Tibetan Salvia miltiorrhiza Bunge sample 3 according to the process of Step 2.3.

[0068] Step 3: Elute the Tibetan Salvia miltiorrhiza Bunge reference substance in Step 2 under chromatographic conditions, draw a standard curve based on the contents of 4 liposoluble components, and obtain the regression equation;

[0069] Furthermore, Step 3 includes:

[0070] Step 3.1: Dilute the Tibetan Salvia miltiorrhiza Bunge reference substance in Step 2.1 by 1, 2, 2.5, 5, 10, and 50 times to obtain 6 diluted solutions of the Tibetan Salvia miltiorrhiza Bunge reference substance with different mass concentrations.

[0071] Step 3.2: Using the mixed solution of acetonitrile - 0.02% phosphoric acid as the mobile phase gradient, elute the diluted solutions of the Tibetan Salvia miltiorrhiza Bunge reference substance in Step 3.1 under chromatographic conditions to obtain Figure 1 the chromatogram in

[0072] Specifically, Figure 1 the chromatogram in uses a Shimadzu - C18 chromatographic column (4.6 mm × 250 mm, 5 μm), with the mixed solution of acetonitrile - 0.02% phosphoric acid solution as the mobile phase, a flow rate of 1 mL / min, a column temperature of 20 °C, a detection wavelength of 270 nm, an injection volume of 10 μL, and gradient elution. It is proved by the chromatogram that the peak shapes of each compound are clear and symmetrical, the resolution is good, the baseline is stable, and the noise is low, indicating that the established chromatographic conditions have good separation effect and can be used as a reliable basis for subsequent experiments.

[0073] Refer to Figures 2 - 5 As shown, Step 3.3: Based on Step 3.2, draw a standard curve with the peak area as the ordinate Y and the mass concentration of the diluted solution of the Tibetan Salvia miltiorrhiza Bunge reference substance as the abscissa X, and calculate the regression equation, referring to Table 2.

[0074] Table 2: Content of mass concentration and peak area of diluted solution of Salvia przewalskii Maxim. control substance

[0075]

[0076] Among them: Using Excel, with the peak area as the vertical coordinate Y and the mass concentration of the diluted solution of Salvia przewalskii Maxim. control substance as the horizontal coordinate X, a linear regression curve is plotted, and then a linear regression equation is obtained.

[0077] Step 3.4: Based on Step 3.3, the regression equations for the 4 fat-soluble components in the diluted solution of Salvia przewalskii Maxim. control substance are respectively:

[0078] Tanshinone IIA: Y = 4832.87X, R 2 = 0.9996;

[0079] Tanshinone I: Y = 22319.4X, R 2 = 0.9996;

[0080] Cryptotanshinone: Y = 43162.1X, R 2 = 0.9995;

[0081] Dihydrotanshinone: Y = 27185.7X, R 2 = 0.9996;

[0082] Among them: R 2 represents the goodness of fit of the regression model to the data.

[0083] As an example, taking tanshinone IIA: Y = 4832.87X, R 2 = 0.9996 as an example, R 2 = 0.9996 means that the model explains 99.96% of the variation in tanshinone IIA concentration, indicating that this linear regression model has extremely high goodness of fit and can be well used for prediction and analysis. Refer to Table 3.

[0084] Table 3: Gradient elution schedule

[0085]

[0086] The chromatographic conditions adopted above are: Shimadzu LC-2040 high performance liquid chromatograph; Shimadzu-C18 chromatographic column (4.6mm X 250mm, 5μm); gradient elution with acetonitrile - 0.02% phosphoric acid solution as the mobile phase, a flow rate of 1 mL / min during the elution process, a constant column temperature of 20°C, a detection wavelength of 270nm, and an injection volume of 10 μL.

[0087] In the above technical solution, the sources of the following substances are as follows: phosphoric acid (analytical pure, Tianjin Tianli Chemical Reagent Co., Ltd.); methanol (analytical pure, Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (chromatographic pure, Merck KGaA, Germany).

[0088] Step 4: Inject the Salvia przewalskii Maxim. sample in step 2 under chromatographic conditions to obtain the peak areas of 4 liposoluble components; substitute the peak areas into the regression equation in step 3 to obtain the contents of the 4 liposoluble components in the Salvia przewalskii Maxim. sample.

[0089] Further, step 4 includes: Step 4.1: Inject the Salvia przewalskii Maxim. sample 1 of batch S1 prepared in step 2.2 under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively, to obtain RSD values < 3%, referring to Table 4.

[0090] Table 4: RSD of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone under Step 4.1

[0091]

[0092] In the above process, inject samples continuously for 6 times, 10 μL each time, record the peak areas, and calculate that the RSDs of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone are 0.78%, 0.6%, 0.69%, and 0.95% respectively, to obtain RSD values < 3%. According to the regulations of the Chinese Pharmacopoeia, it indicates that the instrument precision is good.

[0093] Step 4.2: Place the Salvia przewalskii Maxim. sample 1 of batch S1 prepared in step 2.2 in the dark at room temperature for different times, inject under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively, to obtain RSD values < 3%, referring to Table 5.

[0094] Table 5: RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone under Step 4.2

[0095]

[0096] In Step 4.2, the times of placing in the dark are: inject after 0, 4, 8, 12, 16, 20, and 24 h. Calculate that the RSDs of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone are 0.14%, 0.14%, 0.15%, and 0.86% respectively, to obtain that the RSD values of each component of the sample are all < 3%. According to the regulations of the Chinese Pharmacopoeia, it indicates that the sample solution is stable within 24 h when placed in the dark at room temperature.

[0097] Step 4.3: Inject the prepared Tibetan Salvia miltiorrhiza test sample 2 from Step 2.3 under the chromatographic conditions, calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively, and obtain RSD values < 3%, referring to Table 6.

[0098] Table 6: RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone under Step 4.3

[0099]

[0100] The calculated RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone in Step 4.3 are 1.25%, 1.62%, 1.29%, and 1.38% respectively. The obtained results indicate that the RSD values of each component of the test sample are all < 3%. According to the regulations of the Chinese Pharmacopoeia, the reproducibility of the inspection method is good.

[0101] Furthermore, take 24 portions of the Tibetan Salvia miltiorrhiza test sample of S1 batch, each portion being 0.15 g. Add the corresponding Tibetan Salvia miltiorrhiza reference substance with a content equivalent to its fat-soluble components to each portion of the Tibetan Salvia miltiorrhiza test sample of S1 batch, and prepare the Tibetan Salvia miltiorrhiza test sample 3 according to the process of Step 2.3.

[0102] Even further, inject the Tibetan Salvia miltiorrhiza test sample 3 under the chromatographic conditions, calculate the RSD values of the spike recovery rates of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone respectively, referring to Table 7.

[0103] Table 7: RSD values of the spike recovery rates of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone

[0104]

[0105] The average spike recovery rates of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone obtained from the above table are 96.5%, 97.5%, 98.8%, and 97.5% respectively, and the RSDs are 1.82%, 1.56%, 1.33%, and 1.56% respectively.

[0106] Based on the above results, it shows that the average spike recovery rates of tanshinone IIA, tanshinone I, cryptotanshinone, and dihydrotanshinone are close to 100% and the RSDs are relatively low, indicating that the analytical method used has high accuracy and precision in determining the contents of these compounds, the data is reliable, and it can provide a reliable method for the content determination of tanshinone compounds, which can be used for the quality control and research of related products.

[0107] Based on the diluted solution of the Tibetan Salvia miltiorrhiza reference substance in Step 3.1, using tanshinone IIA as the internal reference substance, inject under the chromatographic conditions, and calculate the RSD values of the relative correction factors of dihydrotanshinone, cryptotanshinone, and tanshinone I respectively.

[0108] The calculation formula for the relative correction factor is: f = CiAs / AiCs;

[0109] Where: As: the peak area of the internal reference substance, Ci: the concentration of the reference substance of Salvia przewalskii Maxim., Ai: the peak area of the reference substance of Salvia przewalskii Maxim., Cs: the concentration of the internal reference substance.

[0110] The calculated RSD values of the relative correction factors were all less than 5%, meeting the requirements of the analytical experiment. Refer to Table 8.

[0111] Table 8: Relative correction factors (liposolubility) of tanshinone IIA, cryptotanshinone, and tanshinone I

[0112] Mixed standard Dihydrotanshinone Cryptotanshinone Tanshinone I 1 1.777 1.120 2.164 2 1779 1.120 2.165 3 1.778 1.120 2.165 4 1.778 1.120 2.165 5 1.778 1.120 2.165 6 1.778 1.120 2.165 Average value 1.778 1.120 2.165 RSD(%) 0.035 0 0.018

[0113] Based on the diluted solution of the reference substance of Salvia przewalskii Maxim. in Step 3.1, using tanshinone IIA as the internal reference substance, injecting samples 5 times on each of the 3 different chromatographic columns, and calculating the average values and RSD values of the relative correction factors of dihydrotanshinone, cryptotanshinone, and tanshinone I respectively.

[0114] Among them, a Shimadzu ultra-high performance liquid chromatography system and a UItiMate 300 system, as well as 3 chromatographic columns of Shimadzu C18 (4.6 mm X 250 mm, 5 μm), Supersil ODS2-C18 (4.6 mm X 250 mm, 5 μm), and CaprisiCl8 (4.6 mm X 250 mm, 5 μm) were used to investigate the reproducibility of the relative correction factor. Refer to Table 9.

[0115] Table 9: Average values and RSD of the relative correction factors of dihydrotanshinone, cryptotanshinone, and tanshinone I under different chromatographic columns

[0116]

[0117] It can be seen from the above table that under different chromatographic conditions, the values of the relative correction factors basically remain unchanged, and the RSD < 5%. According to the regulations of the Chinese Pharmacopoeia, it indicates that the relative correction factor has good durability.

[0118] Further, based on the diluted solution of the reference substance of Salvia przewalskii Maxim. in Step 3.1, using tanshinone IIA as the internal reference substance, injecting samples 5 times on each of the 3 different chromatographic columns, and calculating the average values and RSD values of the relative retention time values of dihydrotanshinone, cryptotanshinone, and tanshinone I on the 3 chromatographic columns respectively. The obtained average values and RSD values of the relative retention time values were all less than 5%; refer to Table 10.

[0119] Among them, the chromatographic columns used were: Shimadzu C18 (4.6 mm X 250 mm, 5 μm), Supersil ODS2-C18 (4.6 mm X 250 mm, 5 μm), and Caprisi C18 (4.6 mm X 250 mm, 5 μm), and a Shimadzu-type ultra-high performance liquid chromatography system and an UItiMate 300 system were used.

[0120] Table 10: Average values and RSD of relative retention time of dihydrotanshinone, cryptotanshinone, and tanshinone I in 3 chromatographic columns

[0121]

[0122] It can be seen from the above table that under different chromatographic conditions, the values of the relative correction factors are basically unchanged and the RSD < 5%, indicating that the relative correction factors have good durability.

[0123] In order to verify the accuracy of the QAMS results, the contents of dihydrotanshinone, cryptotanshinone, and tanshinone I in 20 batches of Salvia przewalskii Maxim were determined by multiple components by single marker (QAMS) and external standard method, and the contents were compared. Refer to Table 11.

[0124] Table 11: Comparison of determination results between QAMS method and external standard method (mg / g)

[0125]

[0126] In this experiment, the established QAMS method was used to determine 20 batches of Salvia przewalskii Maxim of S1, S2... S20, and the external standard method was used for verification. The results showed that there was no significant difference in the component contents measured by the two methods, indicating that the QAMS method is accurate and feasible for the determination of the contents of 4 liposoluble components in Salvia przewalskii Maxim.

Claims

1. A method for multi-component determination by single marker for the content determination of four liposoluble components in Salvia przewalskii Maxim., characterized in that, It includes the following steps: Step 1: Take the raw material of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. to prepare the reference substance and test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.; Step 2: Elute the prepared reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib., and draw a standard curve by the contents of 4 fat-soluble components to obtain the regression equation; Among them, the regression equation is: Tanshinone ⅡA: Y = 4832.87X, R 2 = 0.9996; Tanshinone Ⅰ: Y = 22319.4X, R 2 = 0.9996; Cryptotanshinone: Y = 43162.1X, R 2 = 0.9995; Dihydrotanshinone: Y = 27185.7X, R 2 = 0.9996; Where: R 2 represents the goodness of fit of the regression model to the data; Step 3: Inject the test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. to obtain the peak areas of 4 fat-soluble components, and substitute the peak areas into the regression equation to obtain the contents of 4 fat-soluble components in the test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.

2. The multi-component simultaneous determination method for the contents of 4 fat-soluble components in Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. according to claim 1, characterized in that: In step 1, 5 batches of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. from Milin, Bomi, Bayi and Langxian are selected as the raw materials of the test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib., with a total of 20 batches, numbered S1, S2... S20.

3. The multi-component determination method for the contents of 4 fat-soluble components in Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. according to claim 2, characterized in that, Step 1 includes: Step 1.1: Weigh tanshinone IIA, tanshinone I, cryptotanshinone and dihydrotanshinone, mix them and dissolve them in methanol to prepare the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.; Step 1.2: Take the test samples of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of batches S1, S2... S20, add methanol respectively, weigh them, then perform ultrasonic treatment, let it cool and weigh again for the second time, and make up the weight reduced in the second weighing with methanol to obtain the test sample 1 of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.; Step 1.3: Take 6 portions of the test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. of batch S1, add methanol respectively, weigh them, then perform ultrasonic treatment, let it cool and weigh again for the second time, and make up the weight reduced in the second weighing with methanol to obtain the test sample 2 of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.

4. The multi-component simultaneous determination method for the contents of 4 fat-soluble components in Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. according to claim 3, characterized in that: The ratio of the test sample of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. to methanol in step 1.2 and step 1.3 is 3 g: 500 mL.

5. The multi-component simultaneous determination method for the contents of 4 fat-soluble components in Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. according to claim 4, wherein, Step 2 includes: Step 2.1: Dilute the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. in step 1 to obtain the diluted solution of the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.; Step 2.2: Elute the diluted solution of the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. in step 1.1 with the mixed solution of acetonitrile - 0.02% phosphoric acid as the mobile phase under chromatographic conditions; Step 2.3: Based on step 2.2, draw a standard curve with the peak area as the ordinate Y and the mass concentration of the diluted solution of the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. as the abscissa X, and calculate the regression equation; Step 2.4: Based on step 2.3, obtain the regression equations of 4 fat-soluble components in the diluted solution of the reference substance of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib.

6. The multi-component determination method for simultaneously determining the contents of four fat-soluble components in *Salvia przewalskii* Maxim. var. *tibetica* (Stib.) H. W. Li according to claim 5, wherein, Step 3 includes: Step 3.1: Inject the test sample 1 of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. prepared in step 1.2 under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone and dihydrotanshinone respectively; Step 3.2: Place the test sample 1 of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. prepared in step 1.2 in the dark at room temperature for different times, inject it under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone and dihydrotanshinone respectively; Step 3.3: Inject the test sample 2 of Salvia przewalskii Maxim. var. mandarinorum (Diels) Stib. prepared in step 1.3 under chromatographic conditions, and calculate the RSD values of the peak areas of tanshinone IIA, tanshinone I, cryptotanshinone and dihydrotanshinone respectively.

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