Method for simultaneous determination of multiple effective components in callicarpa nudiflora by UPLC-UV

Abstract

The invention discloses a method for simultaneously determining various effective components in callicarpa nudiflora through UPLC-UV (Ultra Performance Liquid Chromatography-Ultraviolet). The method comprises the following steps: (1) preparing a reference substance solution; (2) preparing a test solution; and (3) carrying out UPLC-UV detection. The method is easy and convenient to operate, high in sensitivity, good in precision and good in reproducibility, and the quality stability and the clinical curative effect of the callicarpa nudiflora and the product can be guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, and specifically relates to a method for simultaneously determining multiple active ingredients in Callicarpa nudiflora. Background Technology

[0002] Callicarpa nudiflora Hook. et Arn., a plant in the Verbenaceae family, is a dried leaf of the plant. It is mainly produced in Hainan, Guangdong, and Guangxi provinces. Other names include Zei Zi Ye, Bu Fa, Gan Feng Chai, Da Ban Jiu Mi, Jie Jie Hong, and Yin Tang Ye. It is one of the representative medicinal materials of the Li ethnic group and a native medicinal material of Hainan Province. Callicarpa nudiflora has anti-inflammatory, detoxifying, dampness-resolving, and hemostatic effects. It is often used for inflammatory swellings caused by bacterial infections, acute infectious hepatitis, and internal and external bleeding. Callicarpa nudiflora mainly contains flavonoids, phenylethyl glycosides, terpenes, and phenolic acids. The 2020 edition of the Chinese Pharmacopoeia uses luteolin and verbascoside as content indicators. However, there is currently no good method for simultaneously determining the content of multiple active ingredients in Callicarpa nudiflora. Summary of the Invention

[0003] The purpose of this invention is to address the current quality control situation of Callicarpa nudiflora by providing a method for simultaneously determining multiple active ingredients in Callicarpa nudiflora, which is beneficial for effective quality control of Callicarpa nudiflora medicinal materials and their products, and ensures their clinical efficacy.

[0004] A method for simultaneous determination of multiple active ingredients in *Callicarpa nudiflora* by UPLC-UV includes the following steps:

[0005] (1) Preparation of reference solution:

[0006] Accurately weigh appropriate amounts of verbascoside, isoruascoside, piracetamin III, forsythoside B, luteolin, 6-OH-luteolin, caffeic acid, and decaffeoyl cimicifugoside reference standards, dissolve them in methanol, and prepare reference standard solutions.

[0007] (2) Preparation of the test solution:

[0008] Weigh out the naked flower purple artichoke, add methanol, centrifuge by sonication, take the supernatant, filter it through a microporous membrane, and take the filtrate to obtain the naked flower purple artichoke test solution.

[0009] (3) UPLC-UV detection:

[0010] The reference solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) were respectively injected into the ultra-high performance liquid chromatograph for determination;

[0011] The chromatographic conditions were as follows: mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water; gradient elution program: 0–0.2 min, 5% A; 0.2–2.0 min, 5%–5.5% A; 2.0–4.5 min, 5.5% A; 4.5–5.0 min, 5.5%–12% A; 5.0–15.0 min, 12%–12.5% ​​A; 15.0–18.5 min, 12.5%–13% A; 18.5–20.0 min, 13%–17% A; 20.0–21.0 min, 17%–98% A; 21.0–24.0 min, 98% A; 24.0–25.0 min, 98%–5% A; PDA detector, detection wavelength: 270–290 nm.

[0012] Preferably, steps (1) and (2) are as follows:

[0013] (1) Preparation of reference solution:

[0014] Accurately weigh appropriate amounts of verbascoside, isoruascoside, piracetamin III, forsythoside B, luteolin, 6-OH-luteolin, caffeic acid, and decaffeoyl cimicifugoside reference standards, place them in centrifuge tubes, add 60%-80% methanol to dissolve them, and prepare reference standard solutions.

[0015] (2) Preparation of the test solution:

[0016] Weigh out the naked-flowered purple artichoke, add 60%-80% methanol, centrifuge by sonication, take the supernatant, filter it through a microporous membrane, and take the filtrate to obtain the naked-flowered purple artichoke test solution.

[0017] Preferably, the preparation of the test solution in step (2) is as follows: weigh 50mg-500mg of Callicarpa nudiflora, crush it into fine powder, add 5mL-50mL of 65%-75% methanol, centrifuge by sonication, take the supernatant, filter it through a microporous membrane, and take the filtrate to obtain the test solution of Callicarpa nudiflora.

[0018] Preferably, the chromatographic column used in the chromatographic conditions is an ACQUITYUPLC CORTECS T3 ultra-high performance liquid chromatography column.

[0019] Preferably, step (3) is as follows:

[0020] (3) UPLC-UV detection:

[0021] The reference solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) were respectively injected into the ultra-high performance liquid chromatograph for determination;

[0022] The chromatographic conditions were as follows: Column: ACQUITYUPLC CORTECS T3 ultra-high performance liquid chromatography column; Column temperature: 40℃; Mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water; Gradient elution program: 0–0.2 min, 5% A; 0.2–2.0 min, 5%–5.5% A; 2.0–4.5 min, 5.5% A; 4.5–5.0 min, 5.5%–12% A; 5.0–15.0 min, 12%–12.5% ​​A; 15.0–18.5 min, 1 2.5%-13% A; 18.5-20.0 min, 13%-17% A; 20.0-21.0 min, 17%-98% A; 21.0-24.0 min, 98% A; 24.0-25.0 min, 98%-5% A; flow rate 0.5 mL / min-1 mL / min; PDA detector; detection wavelength: 270-290 nm; column temperature 35℃-40℃; injection volume 0.8 μL-1 μL.

[0023] More preferably, a method for simultaneous determination of multiple active ingredients in *Callicarpa nudiflora* by UPLC-UV includes the following steps:

[0024] (1) Preparation of reference solution:

[0025] Accurately weigh appropriate amounts of verbascoside, isoruascoside, piracetamidine III, forsythoside B, luteolin, 6-OH-luteolin, caffeic acid, and decaffeoyl cimicifugoside reference standards, place them in centrifuge tubes, add 70% methanol to dissolve them, and prepare reference standard solutions.

[0026] (2) Preparation of the test solution:

[0027] Weigh 50 mg of Callicarpa nudiflora, add 5 mL of 70% methanol, centrifuge by sonication, take the supernatant, filter it through a microporous membrane, and take the filtrate to obtain the Callicarpa nudiflora test solution.

[0028] (3) UPLC-UV detection:

[0029] The reference solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) were respectively injected into the ultra-high performance liquid chromatograph for determination;

[0030] The chromatographic conditions were as follows: Column: ACQUITYUPLC CORTECS T3 ultra-high performance liquid chromatography column; Column temperature: 40℃; Mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water; Gradient elution program: 0–0.2 min, 5% A; 0.2–2.0 min, 5%–5.5% A; 2.0–4.5 min, 5.5% A; 4.5–5.0 min, 5.5%–12% A; 5.0–15.0 min, 12%–12.5% ​​A; 15… 0–18.5 min, 12.5%–13% A; 18.5–20.0 min, 13%–17% A; 20.0–21.0 min, 17%–98% A; 21.0–24.0 min, 98% A; 24.0–25.0 min, 98%–5% A; flow rate 0.5 mL / min; PDA detector; detection wavelength: 280 nm; column temperature 40 °C; injection volume 1 μL.

[0031] Preferably, the preparation of the reference solution in step (1) is as follows:

[0032] Accurately weigh appropriate amounts of verbascoside, isoverascoside, berberine glycoside III, forsythoside B, luteolin, 6-OH-luteolin, caffeic acid, and decaffeoyl cimicifugoside reference standards, place them in centrifuge tubes, dissolve them in 70% methanol, and mix the reference standards in a certain proportion to prepare a solution containing 1147.5 μg·mL verbascoside. -1 Verbascoside 199 μg·mL -1 Berberine III 97 μg·mL -1 Forsythoside B 180 μg·mL -1 Luteolin glycoside 226.5 μg·mL -1 Caffeic acid 37.5 μg·mL -1 Decaffeoyl cimicifugain 85 μg·mL -1 6-OH-luteolin 157 μg·mL -1 The mixed solution is the reference solution.

[0033] Preferably, the preparation of the test solution in step (2) is as follows:

[0034] Take the naked flower purple artichoke and grind it separately using a ball mill. Accurately weigh 50 mg, add 5 mL of 70% methanol, extract by ultrasonication for 40 min, centrifuge at 2000 r for 10 min, take the supernatant, filter it through a 0.22 μm filter membrane, and take the filtrate to obtain the naked flower purple artichoke test solution.

[0035] To better understand this method, the technical solution of this invention application will be further described as follows:

[0036] 1. Materials

[0037] 1.1 Instruments and Reagents

[0038] GZX-GF101-3-BS-Ⅱ and GZX-GF101-3-BS-H electric thermostatic drying ovens (Shanghai Yuejin Medical Instrument Co., Ltd.); M1-L213C Midea microwave oven (Guangdong Midea Kitchen Appliances Manufacturing Co., Ltd.); Waters AcquityUPLC-I-Class ultra-high performance liquid chromatography-tandem Synapt HD-XS high-resolution mass spectrometer, Masslynx 4.2 mass spectrometry workstation (Waters Corporation, USA); Waters Acquity UPLC-I-Class liquid chromatography system (including binary high-pressure solvent manager, autosampler, PDA detector, Empower 3 workstation, Waters Corporation, USA), ACQUITYUPLC CORTECS T3 column (2.1 x 100 mm, 1.6 μm), ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 μm), ACQUITYUPLC CORTECS C18 + Chromatographic columns (2.1 x 100 mm, 1.6 μm) and Kinetex XB-C18 columns (2.1 x 100 mm, 1.7 μm) (Phenomenex, USA). Analytical balance (Mettler-Toledo, Switzerland); Retsch MM400 ball mill (Leitsch GmbH, Germany); centrifuge (Eppendorf GmbH, Germany); KQ-100DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 0.22 μm syringe filter (Pall, USA).

[0039] Reference standards verbascoside (batch number: 20050607), isoverascoside (batch number: 20050611), berberine III (batch number: 19083046), forsythoside B (batch number: 20050718), luteolin (batch number: 20010608), luteolin (batch number: 20032606), caffeic acid (batch number: 20090806), decaffeoyl luteolin (batch number: 19061324), and 6-OH-luteolin (batch number: 22040302) were all purchased from Beijing Rongcheng Xinde Technology Co., Ltd., with a purity ≥98%. Chromatographic grade acetonitrile and methanol (Merck, Germany); ultrapure water was prepared using a Milli-Q pure water system (resistivity ≥18.2 MΩ, Millipore, USA).

[0040] 1.2 Sample Collection

[0041] The sample of *Callicarpa nudiflora* was collected at the Fushan Base in Hainan Province at the end of February 2022. It was identified by Researcher Zhang Xiaobo of the Center for Medicinal Herbs Resources, China Academy of Chinese Medical Sciences, as the dried leaves of *Callicarpa nudiflora* Hook. et Arn., a plant of the Verbenaceae family, and is stored at the Center for Medicinal Herbs Resources, China Academy of Chinese Medical Sciences.

[0042] 2 methods

[0043] 2.1 Preparation of reference solution

[0044] Accurately weigh appropriate amounts of verbascoside, isoverascoside, berberine III, forsythoside B, luteolin, luteolin, caffeic acid, decaffeoyl cimicifugoside, and 6-OH-luteolin reference standards, place them in 2 mL centrifuge tubes, dissolve them in 70% methanol, and mix the reference standards in a certain proportion to prepare a solution containing 1147.5 μg / mL verbascoside. -1 Verbascoside 199 μg·mL -1 Berberine III 97 μg·mL -1 Forsythoside B 180 μg·mL -1 Luteolin glycoside 226.5 μg·mL -1 Luteolin 40 μg·mL -1 Caffeic acid 37.5 μg·mL -1 Decaffeoyl cimicifugain 85 μg·mL -1 6-OH-luteolin 157 μg·mL -1 The mixed solution was used as a mother liquor for later use, and then gradually diluted to different concentrations for later use.

[0045] 2.2 Preparation of the test solution

[0046] The samples of *Callicarpa nudiflora* were pulverized separately using a ball mill. 50 mg of each sample was accurately weighed and added to 5 mL of 70% methanol. The mixture was then ultrasonically extracted for 40 min. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the sample solutions for each *Callicarpa nudiflora*. 100 μL of each *Callicarpa nudiflora* sample solution was taken and mixed thoroughly for quality control.

[0047] 2.3 Optimization of chromatographic conditions

[0048] 2.3.1 UPLC-Q-TOF-MS Qualitative Analysis Conditions

[0049] Chromatographic conditions: Waters CORTECS T3 column (2.1 x 100 mm, 1.6 μm); mobile phase: 0.1% formic acid acetonitrile (A) - 0.1% formic acid water (B); gradient elution: 0–0.2 min, 5% A; 0.2–2 min, 5%–10% A; 2–3.5 min, 10%–11% A; 3.5–10 min, 11%–13% A; 10–18 min, 13%–26% A; 18–33 min, 26%–40% A; 33–36 min, 40%–48% A; 36–40 min, 48%–98% A; 40–43 min, 98% A; flow rate: 0.5 mL / min; column temperature: 40 °C; injection volume: 1 μL.

[0050] Mass spectrometry conditions: ESI negative ion, MSE acquisition mode. Capillary voltage 2.5 kV (-); cone voltage 40 V; nitrogen as solvent gas, 1000 L·h. -1 The solvent temperature was 450℃, the ion source temperature was 120℃, the scan range was 50~1200Da m / z, the collision gas was argon, the trap collision energy was 6eV for low-energy scans and 20-60eV for high-energy scans. Leucine enkephalin was used as the accurate mass number correction solution [554.2615(-) and 556.2771(+)]. MassLynx was used as the operating system.

[0051] Data Processing: A database of chemical constituents of the *Callicarpa* genus (415 compounds) was compiled through literature review and supplementation, including compound names, molecular formulas, and corresponding mol files (.mol). Based on UPLC-Q-TOF-MS technology, the raw mass spectrometry data acquired in MSE Continuum mode were imported into UNIFI 1.9 analysis software (Waters Inc., USA), and the program was set up to perform analytical processes including peak extraction, ion merging, and custom library search and identification.

[0052] 2.3.2 Qualitative Analysis using UPLC-Q-TOF-MS

[0053] To comprehensively characterize the overall composition of *Callicarpa nudiflora*, identify the structures of its main compounds, and determine the indicative components for its content determination, this study first characterized *Callicarpa nudiflora* using baseline ion current chromatograms (BPI) in positive and negative ion modes and ultraviolet chromatograms at 280 nm using UPLC-UV-Q-TOF MS (see [link to study]). Figure 1 By verifying and analyzing UNIFY identification data, especially the precise molecular weight, fragment ions, possible fragmentation pathways, and retention times of compounds under negative ion conditions, and referring to information on previously identified compounds in *Callicarpa nudiflora* and the *Callicarpa* genus, the structures of 35 compounds in *Callicarpa nudiflora* were identified (see [link to UNIFY identification data]). Figure 1 (and Table 1), mainly phenylethanol glycosides, flavonoids, iridoid glycosides and triterpenoids.

[0054] The phenylethanol glycosides in *Callicarpa nudiflora* are mostly composed of phenylethanol aglycone and 2-3 different sugar groups and caffeoyl groups. Except for caffeoyl-based cimicifuga glycosides (which lack a caffeoyl group) and cistanche glycoside D (which contains a ferulic acyl group), these compounds all produce strong [MH]- and [MH-C9H6O3]- ions, and have strong caffeoyl characteristic ion signals at m / z 179.03, 161.02, and 133.04. The signal of phenylethanol at m / z 153.05 is very weak or almost invisible, but its dehydration signal at m / z 135.04 is consistent with the fragment ions of caffeoyl groups. Ferulic acyl substitution produces obvious ions at m / z 193.05, 175.04, and 160.01. The mass spectrometry fragments and abundance of isomers of these compounds are relatively consistent, making it difficult to identify the structure of isomers by mass spectrometry. However, the position of the substituent in the central sugar affects the chromatographic retention time. The further the substituent is in the central sugar, the longer the retention time, as seen in compounds 18 and 20.

[0055] Flavonoids typically form a quasi-molecular ion peak of [MH]-. Flavonoid glycosides readily form the deglycosylated aglycone ion [Yo]-. The aglycone readily forms fragment ions of de-CO and CO2. Aglycones with methoxy substitutions also have characteristic de-CH3 ions, such as in compound 32. Ions such as m / z 151.00 and 133.03 generated by RDA cleavage of the C ring can help determine the position of substituents on the aglycone, as in compounds 11 and 15.

[0056] Iridoid glycosides are generally glycosides with iridoid alcohols as the parent nucleus and sugar linkages, and often have caffeoyl or ferulic acyl substitutions. They readily generate strong [MH]- and [MH-C9H12O5]- ions, which lose 200 Da of neutral (C9H) ions. 12 The O5 signal is key information for determining whether a compound is an iridoid glycoside. In addition, there will be strong characteristic ion signals of caffeoyl or ferulic oxytocin.

[0057] Triterpenes produce a strong [MH]- signal, while triterpenoid glycosides produce a strong [M+HCOO]- signal, which is significantly different from other types of compounds. Triterpenoid glycosides readily undergo deglycosylation to produce triterpenoid aglycones, which in turn readily form a series of dehydration signals. The signals of fragments under positive ions are more pronounced, as seen in compounds 28 and 33.

[0058] Table 1. UPLC-Q-TOF-MS data of identified compounds in *Callicarpa nudiflora*.

[0059]

[0060]

[0061] Note: *Identified by comparison with reference standard.

[0062] Note.*Comparedandidentifiedbythereferencestandards.

[0063] 2.3.3 Multivariate statistical analysis of UPLC-Q-TOF-MS data from *Callicarpa nudiflora* samples

[0064] Comparison of UPLC-MS spectra of different batches of Callicarpa nudiflora can be found in [link to UPLC-MS spectra]. Figure 2 Significant differences were observed in the responses of the main compounds among different batches of samples, particularly in the peak responses of decaffeoyl compounds such as cimicifugoside, forsythoside B, verbascoside, and isorhizoside. To further and more accurately evaluate the changes in the main chemical components, we performed quantitative analysis on the compounds with higher content.

[0065] 2.3.4 Selection of Quantitative Compounds

[0066] The identification results of the identified compounds on the LC-UV-MS spectrum of *Callicarpa nudiflora* indicate that ( Figure 2 The decaffeoyl compounds cimicifugoside (3), 6-OH-luteolin (11), berberine III (12), nudifloside (14), luteolin (15), forsythoside B (16), verbascoside (18), luteolin-4'-O-glucosinolate (19), and isoratoscoside (20) showed high responses in mass spectrometry and ultraviolet light. These compounds are also active compounds and potential Q-markers in Callicarpa nudiflora. Therefore, this study, considering factors such as mass spectrometry response, ultraviolet absorption, pharmacological activity, and ease of use, determined eight compounds—decaffeoyl cimicifugoside (3), caffeic acid (6), 6-OH-luteolin (11), berberine III (12), luteolin (15), forsythoside B (16), verbascoside (18), and isoratoscoside (20)—as indicators for the content determination of Callicarpa nudiflora.

[0067] 2.3.5 Optimization of chromatographic conditions

[0068] In reversed-phase chromatography of *Callicarpa nudiflora*, baseline separation is difficult to achieve because some phenylethanoid glycosides and flavonoids have similar chromatographic retention capabilities. For example... Figure 3As shown in the UV chromatogram and BPI chromatogram, a single peak is observed at 8.65 min. However, in the extracted ion chromatogram, peaks of m / z 523 and m / z 447 are observed around 8.65 min, indicating co-elution. Similarly, co-elution of m / z 755 and m / z 477 is observed at 10.50 min. Therefore, to further optimize chromatographic conditions to improve compound resolution and achieve accurate quantification, this study optimized the chromatographic conditions and compared the separation effects of ACQUITY UPLC CORTECS T3, HSS T3, CORTECSC18+, and Kinetex XB-C18 columns. Figure 4 As shown in the figure, berberine III (12), nudifloside (14), luteolin (15), forsythoside B (16), and the unknown compound m / z 653 were difficult to separate at baseline on chromatography. Comparison revealed that the CORTECS T3 column showed the best separation effect for the target components in quantitative analysis. Further optimization of the chromatographic conditions on the CORTECS T3 column resulted in good separation of all analytes in the standard and *Callicarpa nudiflora* samples. Figure 5 The compound to be quantified did not co-elute during mass spectrometry detection.

[0069] 2.4 Quantitative Analysis Chromatographic Conditions

[0070] Chromatographic column: ACQUITY UPLC CORTECS T3 ultra-high performance liquid chromatography column, column temperature: 40℃, mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, gradient elution program: 0~0.2min, 5% A; 0.2~2.0min, 5%-5.5% A; 2.0~4.5min, 5.5% A; 4.5~5.0min, 5.5%-12% A; 5.0~15.0min, 12%-12.5% ​​A; 15.0~18.5min, 12.5%-13% A; 18.5~20.0min, 13%-17% A; 20.0~21.0min, 17%-98% A; 21.0~24.0min, 98% A; 24.0~25.0min, 98%-5% A. Flow rate: 0.5 mL / min. PDA detector, detection wavelength: 280 nm. Column temperature: 40 °C; injection volume: 1 μL.

[0071] 2.5 Methodological Examination

[0072] 2.5.1 Examination of Linear Relationships

[0073] Mixed reference solutions of different concentrations were analyzed under the conditions described in section 2.4. A standard curve was established with peak area as the ordinate and the mass concentration of each reference standard as the abscissa. The linear regression equations, correlation coefficients, linear ranges, limits of detection (LODs), and limits of quantitation (LOQs) for the eight reference standards are shown in Table 2. The results indicate that the eight compounds exhibit good linear range and linear relationship when analyzed using this method, and the detection sensitivity is high.

[0074] Table 2. Linear equations, correlation coefficients, and linear ranges for the eight compounds.

[0075]

[0076] 2.5.2 Precision Test

[0077] The mixed reference solution was injected six times consecutively under the conditions described in section 2.4. The RSDs of the peak areas of 6-OH-luteolin, berberine III, caffeic acid, forsythoside B, verbascoside, luteolin, decaffeoyl cimicifugoside, and isoratascoside were 0.71%, 1.26%, 2.51%, 1.89%, 0.55%, 0.96%, 1.82%, and 0.95%, respectively, indicating that the quantitative analysis method had good precision.

[0078] 2.5.3 Repeatability Test

[0079] Six portions of the same batch of *Callicarpa nudiflora* samples were accurately weighed and prepared into test solutions using the method described in section 2.2. The solutions were analyzed under the chromatographic conditions described in section 2.4. The peak area RSDs of 6-OH-luteolin, berberine III, caffeic acid, forsythoside B, verbascoside, luteolin, decaffeoyl cimicifugoside, and isoratascoside were 0.55%, 2.17%, 1.16%, 1.03%, 0.45%, 1.02%, 2.01%, and 1.26%, respectively. The results indicate that the quantitative analysis method has good repeatability.

[0080] 2.5.4 Stability Assessment

[0081] 50 mg of *Callicarpa nudiflora* sample was weighed and a test solution was prepared using the method described in section 2.2. The solution was analyzed at 0, 2, 4, 8, 12, 24, and 48 h under the conditions described in section 2.4. The peak areas RSD of 6-OH-luteolin, berberine III, caffeic acid, forsythoside B, verbascoside, luteolin, decaffeoyl cimicifugoside, and isoratascoside in the sample were 0.93%, 2.86%, 1.40%, 0.91%, 0.70%, 0.78%, 1.52%, and 0.93%, respectively. The results indicate that the sample has good stability within 48 h.

[0082] 2.5.5 Recovery rate assessment

[0083] Nine 50 mg portions of a known concentration of *Callicarpa nudiflora* sample powder were accurately weighed. Reference solutions were added in precise ratios of 1:0.8, 1:1, and 1:1.2 according to the content of each reference standard in the sample, with three portions of each ratio. The test solutions were prepared according to the method described in section 2.2, and the compounds were injected separately to determine their content. The recoveries and RSDs were calculated. The average recoveries of 6-OH-luteolin, berberine III, caffeic acid, forsythoside B, verbascoside, luteolin, decaffeoyl cimicifugoside, and isoratascoside were 1.01, 1.00, 1.00, 1.02, 1.03, 0.97, 0.98, and 0.98, respectively, and the average RSDs were 1.34%, 1.83%, 3.24%, 1.06%, 0.75%, 1.57%, 3.20%, and 1.37%, respectively. The recovery rates of all eight reference standards met the requirements.

[0084] The beneficial effects of this invention are as follows: This invention establishes a method for the simultaneous determination of eight compounds with high content, which is simple to operate, highly sensitive, highly precise, and has good reproducibility, thus helping to ensure the quality stability and clinical efficacy of Callicarpa nudiflora medicinal materials and products. Attached Figure Description

[0085] Figure 1 UPLC-UV-MS spectra of *Callicarpa nudiflora* samples: (A) 280 nm chromatogram, (B) positive ion chromatogram, (C) negative ion chromatogram.

[0086] Figure 2 Comparative UPLC-MS spectra of different batches of Callicarpa nudiflora samples

[0087] Figure 3 BPI spectrum, UV chromatogram at 280 nm, and extracted ion chromatogram of UPLC-Q-TOF MS(-) for samples of *Callicarpa nudiflora*.

[0088] Figure 4 Comparison of UPLC-MS separation effects of different types of chromatographic columns: 3. Decaffeoyl citric acid; 11. 6-OH-luteolin; 12. berberine III; 14. nudifloside; 15. luteolin; 16. forsythoside B; 18. verbascoside; 20. isorhabditis oleracea glycoside

[0089] Figure 5Comparison of UPLC-UV chromatograms and base peak ion chromatograms of mixed reference solution and *Callicarpa nudiflora* test solution: (A) UV spectrum of reference (280 nm); (B) UV spectrum of *Callicarpa nudiflora* sample (280 nm); (C) BPI spectrum of reference; (D) BPI spectrum of *Callicarpa nudiflora* sample. 3. Decaffeoyl leucosin; 6. Caffeic acid; 11. 6-OH-luteolin; 12. Pestoside III; 15. Luteolin; 16. Forsythoside B; 18. Verbascoside; 20. Isorbascoside Detailed Implementation

[0090] Example 1

[0091] A method for simultaneous determination of multiple active ingredients in *Callicarpa nudiflora* by UPLC-UV includes the following steps:

[0092] (1) Preparation of reference solution:

[0093] Accurately weigh appropriate amounts of verbascoside, isoverascoside, berberine glycoside III, forsythoside B, luteolin, 6-OH-luteolin, caffeic acid, and decaffeoyl cimicifugoside reference standards, place them in 2 mL centrifuge tubes, dissolve them in 70% methanol, and mix the reference standards in a certain proportion to prepare a solution containing 1147.5 μg / mL verbascoside. -1 Verbascoside 199 μg·mL -1 Berberine III 97 μg·mL -1 Forsythoside B 180 μg·mL -1 Luteolin glycoside 226.5 μg·mL -1 Caffeic acid 37.5 μg·mL -1 Decaffeoyl cimicifugain 85 μg·mL -1 6-OH-luteolin 157 μg·mL -1 The mixed solution is the reference solution.

[0094] (2) Preparation of the test solution:

[0095] Take the naked flower purple artichoke and grind it separately using a ball mill. Accurately weigh 50 mg, add 5 mL of 70% methanol, extract by ultrasonication for 40 min, centrifuge at 2000 r for 10 min, take the supernatant, filter it through a 0.22 μm filter membrane, and take the filtrate to obtain the naked flower purple artichoke test solution.

[0096] (3) UPLC-UV detection:

[0097] The reference solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) were respectively injected into the ultra-high performance liquid chromatograph for determination;

[0098] The chromatographic conditions were as follows: column: CORTECS T3 column; column temperature: 40℃; mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water; gradient elution program: 0–0.2 min, 5% A; 0.2–2.0 min, 5%–5.5% A; 2.0–4.5 min, 5.5% A; 4.5–5.0 min, 5.5%–12% A; 5.0–15.0 min, 12%–12.5% ​​A; 15.0–18.5 min, 12.5%–13% A; 18.5–20.0 min, 13%–17% A; 20.0–21.0 min, 17%–98% A; 21.0–24.0 min, 98% A; 24.0–25 min, 98%–5% A; flow rate: 0.5 mL / min. PDA detector, detection wavelength: 280nm. Column temperature: 40℃; injection volume: 1μL.

[0099] (4) Sample content determination

[0100] Content determination data for 11 batches of samples. The results are shown in Table 3.

[0101] Table 3. Content of various components in 11 batches of Callicarpa nudiflora (mg·g) -1 (n=6)

[0102]

Claims

1. A method for simultaneously determining a plurality of effective components in a naked flower of a purple gem by UPLC-UV, characterized in that, It comprises the following steps: (1) preparation of a control solution: Accurately weigh a proper amount of verbascoside, isoverbascoside, picroside Ⅲ, forsythoside B, jacoboside, 6-OH-jacoboside, caffeic acid, and decaffeoyl jatamansin, respectively, add methanol to dissolve them, and prepare a control solution; (2) preparation of a test solution: Weigh a proper amount of Callicarpa nudiflora, add methanol, and ultrasonically centrifuge it, take the supernatant, filter it through a microporous filter, and take the filtrate, thus obtaining a test solution of Callicarpa nudiflora; (3) UPLC-UV detection: Take the control solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) respectively, and inject them into an ultra-high performance liquid chromatograph for determination; The chromatographic conditions are as follows: mobile phase: A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, gradient elution program: 0-0.2 min, 5% A; 0.2-2.0 min, 5%-5.5% A; 2.0-4.5 min, 5.5% A; 4.5-5.0 min, 5.5%-12% A; 5.0-15.0 min, 12%-12.5% A; 15.0-18.5 min, 12.5%-13% A; 18.5-20.0 min, 13%-17% A; 20.0-21.0 min, 17%-98% A; 21.0-24.0 min, 98% A; 24.0-25.0 min, 98%-5% A, PDA detector, detection wavelength: 270-290 nm. 2.The method for simultaneously determining the plurality of effective components in the Callicarpa nudiflora by UPLC-UV according to claim 1, characterized in that, The steps (1) and (2) are as follows: (1) preparation of a control solution: Accurately weigh a proper amount of verbascoside, isoverbascoside, picroside Ⅲ, forsythoside B, jacoboside, 6-OH-jacoboside, caffeic acid, and decaffeoyl jatamansin, respectively, add 60%-80% methanol to dissolve them in a centrifuge tube, and prepare a control solution; (2) preparation of a test solution: Weigh a proper amount of Callicarpa nudiflora, add 60%-80% methanol, ultrasonically centrifuge it, take the supernatant, filter it through a microporous filter, and take the filtrate, thus obtaining a test solution of Callicarpa nudiflora.

3. The method for simultaneously determining the plurality of effective components in the naked flower of Lonicera similis by UPLC-UV according to claim 1 or 2, characterized in that, The chromatographic conditions further comprise that the chromatographic column is an ACQUITY UPLC CORTECS T3 ultra-high performance liquid chromatographic column. 4.The method for simultaneously determining the plurality of effective components in Ligustrum robustum according to claim 1, characterized in that, The step (3) is as follows: (3) UPLC-UV detection: Take the control solution and the test solution of Callicarpa nudiflora obtained in steps (1) and (2) respectively, and inject them into an ultra-high performance liquid chromatograph for determination; The chromatographic conditions are as follows: chromatographic column is ACQUITY UPLC CORTECS T3 ultra-high performance liquid chromatographic column, column temperature is 40 DEG C, mobile phase is A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, gradient elution program is 0-0.2min, 5% A; 0.2-2.0min, 5%-5.5% A; 2.0-4.5min, 5.5% A; 4.5-5.0min, 5.5%-12% A; 5.0-15.0min, 12%-12.5% A; 15.0-18.5min, 12.5%-13% A; 18.5-20.0min, 13%-17% A; 20.0-21.0min, 17%-98% A; 21.0-24.0min, 98% A; 24.0-25.0min, 98%-5% A, flow rate is 0.5mL / min-1mL / min, PDA detector, detection wavelength is 270-290nm, column temperature is 35 DEG C-40 DEG C; injection amount is 0.8 μL-1 μL.

5. The method for simultaneously determining the plurality of effective components in Ligustrum robustum by UPLC-UV according to claim 1, characterized in that, Comprise the following steps: (1) preparation of control solution: Respectively, a certain amount of vitexin, isovitexin, picroside Ⅲ, forsythoside B, galuteolin, 6-OH-galuteolin, caffeic acid, and decaffeoyl-epimediumin reference substances are weighed accurately, placed in a centrifuge tube, dissolved by adding 70% methanol, and prepared into a reference solution; (2) preparation of test solution: 50mg of naked flower purple pearl is weighed, 5mL of 70% methanol is added, ultrasonic centrifugation is carried out, the supernatant is taken, filtered by microporous filter membrane, and the filtrate is taken, to obtain the naked flower purple pearl test solution; (3) UPLC-UV detection: The reference solution and the naked flower purple pearl test solution obtained in steps (1) and (2) are respectively taken, and injected into the ultra-high performance liquid chromatograph for determination; The chromatographic conditions are as follows: chromatographic column is ACQUITY UPLC CORTECS T3 ultra-high performance liquid chromatographic column, column temperature is 40 DEG C, mobile phase is A: 0.1% formic acid acetonitrile, B: 0.1% formic acid water, gradient elution program is 0-0.2min, 5% A; 0.2-2.0min, 5%-5.5% A; 2.0-4.5min, 5.5% A; 4.5-5.0min, 5.5%-12% A; 5.0-15.0min, 12%-12.5% A; 15.0-18.5min, 12.5%-13% A; 18.5-20.0min, 13%-17% A; 20.0-21.0min, 17%-98% A; 21.0-24.0min, 98% A; 24.0-25.0min, 98%-5% A, flow rate is 0.5mL / min, PDA detector, detection wavelength is 280nm, column temperature is 40 DEG C; injection amount is 1 μL.

6. The method for simultaneously determining the plurality of effective components in Ligustrum robustum by UPLC-UV according to claim 1 or 2 or 5, characterized in that, The preparation of the reference solution in step (1) is: Take a certain amount of verbascoside, isoverbascoside, picroside Ⅲ, forsythoside B, jacoboside, 6-OH-jacoboside, caffeic acid, and 7-O-β-D-glucopyranosyl-epicatechin-3-O-β-D-glucopyranoside reference substance respectively, and place them in centrifuge tubes. Dissolve them in 70% methanol. Mix the reference substances in a certain proportion to prepare a mixed solution containing verbascoside 1147.5 μg·mL -1 , isoverbascoside 199 μg·mL -1 , picroside Ⅲ 97 μg·mL -1 , forsythoside B 180 μg·mL -1 , jacoboside 226.5 μg·mL -1 , caffeic acid 37.5 μg·mL -1 , and 7-O-β-D-glucopyranosyl-epicatechin-3-O-β-D-glucopyranoside 85 μg·mL -1 , 6-OH-jacoboside 157 μg·mL -1 , which is the reference solution.

7. The method for simultaneously determining the plurality of effective components in the naked flower of Lonicera similis by UPLC-UV according to claim 1 or 2 or 5, characterized in that, The preparation of the test solution in step (2) is: Take the naked flower of purple gem by ball mill respectively, accurately take 50 mg, add 70% methanol 5 mL, ultrasonic extraction 40 min, 2000 r centrifugal 10 min, take supernatant, through 0.22 μm filter membrane, take the filter solution, namely naked flower of purple gem test solution.

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