A method for simultaneous detection of 16 components in Camellia chrysantha by LC-MS

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for simultaneously detecting the contents of 16 components in golden camellia by liquid chromatography-mass spectrometry, comprising the following steps: (1) preparation of a standard solution; (2) preparation of a standard curve; (3) preparation of a test solution; (4) determination method: taking the test solution of step (3), using a liquid chromatography-mass spectrometry detection method for detection, and calculating the concentrations of the 16 components in the golden camellia sample to be tested by the standard curve of step (2). The present invention can analyze the quality of golden camellia, and the established liquid chromatography-mass spectrometry method has high sensitivity, strong specificity, and good reproducibility, which provides a basis for improving the quality control standard of golden camellia.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for simultaneously detecting the contents of 16 components in golden camellia by liquid chromatography-mass spectrometry. Background Art

[0002] Camellia nitidissima is a rare plant with both medicinal and edible properties. It is mainly distributed in southwest my country and northern Vietnam. It is a first-class protected plant in my country and enjoys the reputation of "Queen of Tea". At present, there are 42 species and 5 variants of Camellia nitidissima reported in the world, of which 29 species and 5 variants are produced in southern and southwestern Guangxi. As a folk herbal medicine in Guangxi, Camellia nitidissima contains many active ingredients, including flavonoids, saponins, volatile oils, polysaccharides, amino acids and trace elements. It has high medicinal value and is being paid attention to by more and more people. According to the "Guangxi Traditional Chinese Medicine Standard Volume 2", Camellia nitidissima has the functions of clearing away heat and detoxifying, diuresis and swelling, and can be used for pharyngitis, dysentery, nephritis, edema, urinary tract infection, icteric hepatitis, liver cirrhosis ascites, hypertension, sores, and tumor prevention. In recent years, pharmacological studies on Camellia nitidissima have also shown that it has multiple activities such as antioxidant, hypoglycemic, anti-depressant, anti-anxiety, anti-obesity, antibacterial, and anti-cancer. Therefore, a rapid and simple analytical method is needed to evaluate the quality of multiple active ingredients in Camellia chrysantha.

[0003] So far, the quantification of chemical components in Camellia chrysantha is mostly done by high performance liquid chromatography-ultraviolet detection (HPLC-UV), but not all detected components have good ultraviolet absorption and are also interfered by the matrix. The sensitivity of this determination method is not enough to be suitable for the detection of extracts. In view of this, it is urgent to establish a highly sensitive and rapid method for determining the content of compounds in Camellia chrysantha alcohol extracts to provide more basis for the construction of internal control quality standards for Camellia chrysantha extracts. Summary of the invention

[0004] The present invention aims to solve the above technical problems and provides a method for simultaneously detecting the contents of 16 components in Camellia chrysantha by liquid chromatography-mass spectrometry. The determination method is simple and can realize the simultaneous detection of multiple effective components in Camellia chrysantha.

[0005] The technical solution of the present invention is:

[0006] A method for simultaneously detecting the contents of 16 components in golden camellia by liquid chromatography-mass spectrometry, comprising the following steps:

[0007] (1) Preparation of standard solution: Gallic acid, catechin, epicatechin, ferulic acid, rutin, isoquercetin, quercetin, apigenin-7-glucoside, myricetin, phloridzin, quercetin, kaempferol, hyperoside, luteolin, schaftoside, and astilbin were dissolved in methanol respectively, then mixed and diluted to obtain standard solutions containing 16 compounds;

[0008] (2) preparing a standard curve: taking the standard solution of step (1), detecting it by liquid chromatography-mass spectrometry, recording the peak area corresponding to each concentration of the compound, and drawing a linear standard curve with the peak area as the ordinate and the concentration as the abscissa;

[0009] (3) Preparation of test solution: Take the Camellia chrysantha sample to be tested, add 0.1 mol / L hydrochloric acid-ethanol standard solution, heat and extract, filter, cool the filtrate, adjust the pH to 3.5-4, evaporate to dryness at 45°C, add methanol to dissolve, filter, and dilute to obtain the test solution;

[0010] (4) Determination method: Take the test solution of step (3) and detect it by liquid chromatography-mass spectrometry. Calculate the concentrations of the 16 components in the Camellia chrysantha sample using the standard curve of step (2).

[0011] As a preferred solution, in step (3), the solid-liquid ratio of the Camellia chrysantha sample to the 0.1 mol / L hydrochloric acid-ethanol standard solution is 1:(30-50).

[0012] As a preferred embodiment, the liquid chromatography-mass spectrometry conditions are as follows:

[0013] Liquid chromatography conditions: chromatographic column: ZORBAX RRHD Eclipse plus C18; mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile, elution mode is gradient elution, flow rate: 0.2mL / min; column temperature: 30℃; injection volume: 10μL;

[0014] Mass spectrometry conditions: electrospray ionization source ESI, spray voltage: -4500V; ion source temperature: 450°C, nitrogen as heating gas and nebulizer gas, nebulizer gas: 45psi, heating gas: 45psi, curtain gas: 30psi; scanning mode is multiple reaction monitoring mode MRM, and the retention time of each ion pair is 50ms.

[0015] As a preferred scheme, the elution program of the gradient elution is: 0-5min, 13-13% mobile phase B; 5-6min, 13-15% mobile phase B; 6-15min, 15-18% mobile phase B; 15-17min, 18-23% mobile phase B; 17-19min, 23-25% mobile phase B; 19-23min, 25-25% mobile phase B; 23-24min, 25-30% mobile phase B; 24-27min, 30-60% mobile phase B; 27-28min, 60-13% mobile phase B; 28-30min, 13-13% mobile phase B.

[0016] Due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention adopts liquid chromatography-mass spectrometry technology to establish a method for determining the contents of 16 compounds in Camellia chrysantha, which can be used to analyze the quality of Camellia chrysantha. The established liquid chromatography-mass spectrometry method has high sensitivity, strong specificity and good reproducibility, and provides a basis for improving the quality control standards of Camellia chrysantha. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the MRM chromatogram of the standard solution of the present invention;

[0018] Figure 2 It is the MRM chromatogram of the sample solution of the golden camellia tea leaves in the present invention;

[0019] Figure 3 It is the MRM chromatogram of the sample solution of the four-season golden flower tea leaves in the present invention;

[0020] Figure 4 It is the MRM chromatogram of the sample solution of common golden camellia tea in the present invention;

[0021] Figure 5 It is the MRM chromatogram of the Dongxing golden flower tea sample solution in the present invention;

[0022] Figure 6 It is the MRM chromatogram of the sample Camellia japonica test solution in the present invention;

[0023] Figure 7 It is the MRM chromatogram of the sample Camellia chrysantha test solution in the present invention;

[0024] Figure 8 It is the MRM chromatogram of the sample Dongxing golden camellia test solution in the present invention;

[0025] Fig. 9 It is the MRM chromatogram of the sample solution of Camellia odorata in the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Instruments and reagents used in the examples of the present invention: Triple Quad TM 5500+ Ready mass spectrometer, equipped with ESI source, Analyst 1.7.1 software, SCIEX OS 1.6.1 data acquisition software, PeakView1.2 data processing software (AB Sciex, USA); HPLC system LC-30A (Shimadzu, Japan); S60H ultrasonic cleaner (Elma, Germany); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); AE240 electronic balance (METTLER TOLEDO); HH-8 digital display constant temperature water bath (Changzhou Huaguan Instrument Manufacturing Co., Ltd.); low-temperature coolant circulation pump (Shanghai Xiande Experimental Instrument Co., Ltd.); DFT-200A200g portable high-speed pulverizer (Wenling Linda Machinery Co., Ltd.).

[0028] Samples: Ordinary golden flower tea (picked in Nawan, Fangchenggang City, Guangxi in August 2019), Visible Vein Golden Flower Tea (picked in Shangyue, Fangchenggang, Guangxi in August 2019), Four Seasons Golden Flower Tea (picked in Shangxin, Chongzuo City, Guangxi in August 2019), Dongxing Golden Flower Tea (picked in Shangyue, Fangchenggang, Guangxi in March 2022), Ordinary Golden Flower Camellia (picked in Fangchenggang, Guangxi in March 2021), Four Seasons Golden Flower Camellia (picked in Jingxi City, Guangxi in March 2021), Dongxing Golden Flower Camellia (picked in Dongxing, Guangxi in March 2021), Concave Vein Golden Flower Camellia (picked in Fangchenggang, Guangxi in March 2021).

[0029] Reference substances: gallic acid, catechin, epicatechin, rutin, isoquercetin (≥98.48%), quercetin, apigenin-7-glucoside, myricetin, phloridzin, quercetin, kaempferol, and myricetin were purchased from Shanghai Anpu Experimental Technology Co., Ltd., ferulic acid, hyperoside (≥94.3%), and luteolin were purchased from China Food and Drug Inspection Institutes, schaftoside (≥98%) was purchased from Chengdu Munster Biotechnology Co., Ltd., and astilbin (≥98%) was purchased from Beijing Zhongke Yiyou Chemical Technology Research Institute.

[0030] Reagents: anhydrous ethanol (analytical grade, Guangdong Guanghua Technology Co., Ltd.), acetonitrile, methanol (chromatographic grade, Shanghai Anpu Experiment Technology Co., Ltd.), formic acid (chromatographic grade, Guangdong Guanghua Technology Co., Ltd.), 0.1 mol / L hydrochloric acid ethanol standard solution (analytical grade, Guangdong Wengjiang Chemical Reagent Co., Ltd.), ultrapure water prepared by Milli-Q pure water machine (≥18.25 MΩ*cm).

[0031] Example 1

[0032] 1. Solution Preparation

[0033] 1.1. Preparation of standard curve solution

[0034] Diluent: acetonitrile-0.1% formic acid water (volume ratio of 13:87).

[0035] Standard curve solution: Take 16 compounds (gallic acid, catechin, epicatechin, ferulic acid, rutin, isoquercetin, quercetin, apigenin-7-glucoside, myricetin, phloridzin, quercetin, kaempferol, hyperoside, luteolin, schaftoside, astilbin), accurately weigh, place in a 10mL volumetric flask, add chromatographic methanol to dissolve and dilute to the mark to obtain each reference stock solution. Accurately pipette each reference stock solution into the same volumetric flask, gradually dilute the stock solution with a diluent to obtain the standard solution of 16 compounds.

[0036] 1.2. Preparation of test solution

[0037] Samples of Xianmai Jinhua tea leaves (picked in Shangyue, Fangchenggang, Guangxi in August 2019), Siji Jinhua tea leaves (picked in Shangxin, Chongzuo, Guangxi in August 2019), Ordinary Jinhua tea leaves (picked in Shangyue, Fangchenggang, Guangxi in August 2019), Dongxing Jinhua tea leaves (picked in Shangyue, Fangchenggang, Guangxi in March 2022), Concave-veined Jinhua camellia (picked in Fangchenggang, Guangxi in March 2021), Siji Jinhua camellia (picked in Jingxi, Guangxi in March 2021), Dongxing Jinhua camellia (picked in Dongxing, Guangxi in March 2021), and Ordinary Jinhua camellia (picked in Fangchenggang, Guangxi in March 2021) were crushed by a Chinese herbal medicine grinder, passed through a 40-mesh sieve, 5g of each was weighed, and 250ml 0.1 mol / L hydrochloric acid-ethanol standard solution (solid-liquid ratio 1:50) was Soxhlet extracted at 90°C for 8 h, filtered, the filtrate was cooled to room temperature, the pH was adjusted to 3.5, evaporated to dryness using a rotary evaporator at 45°C, 5 ml of methanol was added for ultrasonic dissolution, filtered through a 0.22 μm filter membrane, and then diluted 100 times with a diluent to obtain the test solution.

[0038] 2. Liquid chromatography-mass spectrometry conditions:

[0039] Liquid chromatography conditions: Chromatographic column: ZORBAX RRHD Eclipse plus C18 (RRHD) (1.8 μm, 100 mm×2.1 mm, Agilent). Mobile phase: 0.1% formic acid water (phase A)-acetonitrile (phase B); linear gradient elution as follows: 0-5 min, 13-13% B; 5-6 min, 13-15% B; 6-15 min, 15-18% B; 15-17 min, 18-23% B; 17-19 min, 23-25% B; 19-23 min, 25-25% B; 23-24 min, 25-30% B; 24-27 min, 30-60% B; 27-28 min, 60-13% B; 28-30 min, 13-13% B. Flow rate: 0.2mL / min; column temperature: 30℃; injection volume: 10μL. Equilibration time: 10 minutes. Mass spectrometry conditions: Triple Quad TM 5500+ Ready mass spectrometer, electrospray ion source (ESI), spray voltage: -4500V; ion source temperature (TEM): 450℃, nitrogen as heating gas and nebulizer gas, nebulizer gas (Gas1): 45psi, heating gas (Gas2): 45psi, curtain gas (CUR): 30psi; scanning mode is multiple reaction monitoring mode (MRM), and the retention time of each ion pair is 50ms. The Manual Tuning function in the Analyst 1.7.1Software workstation was used to optimize the collision energy (CE) and declustering voltage (DP) parameters of 16 compounds. The mass spectrometry parameters of each compound after optimization are shown in Table 1.

[0040] Table 1 MS / MS parameters of analytes in MRM mode

[0041]

[0042] 3. Methodological investigation

[0043] 3.1. Linear equation, linear range, detection limit and quantification limit

[0044] Take the standard solution prepared under 1.1 and inject it into the LC-MS instrument. The MS / MS parameters of the analyte are shown in Table 1. With the concentration (ng / mL) as the horizontal axis (X) and the peak area of ​​the quantitative ion of each compound as the vertical axis (Y), draw the standard curve of each compound and perform a linear regression equation. The standard solution was gradually diluted and injected for analysis. The concentration corresponding to the signal-to-noise ratio of 3 was used as the detection limit (LOD), and the concentration corresponding to the signal-to-noise ratio of 10 was used as the quantification limit (LOQ). The results showed that the linear relationship was good, as shown in Table 2. The chromatogram of the standard solution is shown in Figure 1 shown.

[0045] Table 2 Linear equations, linear ranges, detection limits and quantification limits of 16 reference substances

[0046]

[0047] 3.2. Precision

[0048] The same sample solution of Camellia chrysantha was taken and injected 6 times continuously according to the conditions of liquid chromatography-mass spectrometry to examine the intra-day precision; the inter-day precision was measured for 3 consecutive days. The RSD of the intra-day precision of each compound was less than 2.76%, and the RSD of the inter-day precision of each compound was less than 2.51%, indicating that the method has good precision. See Table 3.

[0049] 3.3. Repeatability

[0050] Take the same sample of Camellia chrysantha and prepare 6 test solutions in parallel according to the method in 1.2. In accordance with the conditions of liquid chromatography-mass spectrometry, inject the samples separately to examine the reproducibility of the method. The RSD values ​​of the repeatability tests of 16 compounds are all less than 8.80%, indicating that the method has good reproducibility. See Table 3.

[0051] 3.4. Stability

[0052] The same sample solution was taken and tested at 0h, 6h, 12h, 18h, 24h and 48h according to the conditions of liquid chromatography-mass spectrometry. The RSD values ​​of the stability test of each compound were all less than 9.74%, indicating that the test sample had good stability. See Table 3.

[0053] Recovery rate

[0054] Weigh 5g of Camellia chrysantha with known compound contents, add low, medium and high concentrations of mixed reference solution, prepare test solution according to 1.2 method, and prepare three copies of each concentration in parallel. The recovery test shows that at three different concentrations of addition, the average recovery of 16 compounds is between 95.58% and 103.41%, and the RSD values ​​are all less than 6.40%, indicating that the recovery of 16 compounds is qualified. See Table 3.

[0055] Table 3 Precision, reproducibility, recovery and 48h stability of 16 compounds

[0056]

[0057] Example 2

[0058] The preparation method and determination of the standard solution are the same as those in Example 1.

[0059] The samples to be tested, namely, Camellia sinensis tea leaves, Four Seasons Camellia sinensis tea leaves, Common Camellia sinensis tea leaves, Dongxing Camellia sinensis tea leaves, Concave Vein Camellia sinensis tea leaves, Four Seasons Camellia sinensis tea leaves, Dongxing Camellia sinensis tea leaves, and Common Camellia sinensis tea leaves, were crushed by a Chinese herbal medicine grinder, passed through a 40-mesh sieve, 5 g of each was weighed, 250 ml of 0.1 mol / L hydrochloric acid-ethanol standard solution (solid-liquid ratio 1:30) was added, and Soxhlet extraction was performed at 90°C for 8 h, filtered, and the filtrate was cooled to room temperature, the pH was adjusted to 4, and evaporated to dryness using a rotary evaporator at 45°C, 5 ml of methanol was added for ultrasonic dissolution, filtered through a 0.22 μm filter membrane, and then diluted 100 times with a diluent to obtain a test solution. The test solution was injected into the LC-MS instrument, and the results of the determination and calculation of the contents of 16 compounds in Camellia sinensis are shown in Table 4. The MRM chromatogram of the test solution of Camellia sinensis tea leaves is shown in Table 4. Figure 2 As shown, the MRM chromatogram of the test solution of Four Seasons Golden Flower Tea is as follows Figure 3 As shown, the MRM chromatogram of the test solution of common golden tea leaves is as follows Figure 4 As shown, the MRM chromatogram of Dongxing Jinhua tea sample solution is as follows Figure 5 As shown, the MRM chromatogram of the four-season golden camellia sample solution is as follows Figure 6 As shown, the MRM chromatogram of the common Camellia chrysantha test solution is as follows Figure 7 As shown, the MRM chromatogram of the Dongxing Camellia chrysantha test solution is as follows Figure 8 As shown, the MRM chromatogram of the test solution of Camellia ovata is as follows Fig. 9 shown.

[0060] Table 4 Contents of 16 compounds in 8 samples of Camellia chrysantha (ug / g)

[0061]

[0062] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for simultaneously detecting the contents of 16 components in Camellia chrysantha by liquid chromatography-mass spectrometry, characterized in that: The following steps are involved: (1) Preparation of standard solution: Gallic acid, catechin, epicatechin, ferulic acid, rutin, isoquercetin, quercetin, apigenin-7-glucoside, myricetin, phloridzin, quercetin, kaempferol, hyperoside, luteolin, schaftoside, and astilbin were dissolved in methanol respectively, then mixed and diluted to obtain standard solutions containing 16 compounds; (2) preparing a standard curve: taking the standard solution of step (1), detecting it by liquid chromatography-mass spectrometry, recording the peak area corresponding to each concentration of the compound, and drawing a linear standard curve with the peak area as the ordinate and the concentration as the abscissa; (3) Preparation of test solution: Take the Camellia chrysantha sample to be tested, add 0.1 mol / L hydrochloric acid-ethanol standard solution, heat and extract, filter, cool the filtrate, adjust the pH to 3.5-4, evaporate to dryness at 45°C, add methanol to dissolve, filter, and dilute to obtain the test solution; (4) Determination method: Take the test solution of step (3), detect it by liquid chromatography-mass spectrometry, and calculate the concentration of 16 components in the Camellia chrysantha sample to be tested by the standard curve of step (2); The liquid chromatography-mass spectrometry conditions are as follows: Liquid chromatography conditions: chromatographic column: ZORBAX RRHD Eclipse plus C18; mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile, elution mode is gradient elution, flow rate: 0.2mL / min; column temperature: 30℃; injection volume: 10μL; Mass spectrometry conditions: electrospray ionization source ESI, spray voltage: -4500V; ion source temperature: 450°C, nitrogen as heating gas and nebulizer gas, nebulizer gas: 45psi, heating gas: 45psi, curtain gas: 30psi; scanning mode: multiple reaction monitoring mode MRM, retention time of each ion pair is 50ms; The elution program of the gradient elution is: 0-5min, 13-13% mobile phase B; 5-6min, 13-15% mobile phase B; 6-15min, 15-18% mobile phase B; 15-17min, 18-23% mobile phase B; 17-19min, 23-25% mobile phase B; 19-23min, 25-25% mobile phase B; 23-24min, 25-30% mobile phase B; 24-27min, 30-60% mobile phase B; 27-28min, 60-13% mobile phase B; 28-30min, 13-13% mobile phase B.

2. The method for simultaneously detecting the contents of 16 components in Camellia chrysantha by liquid chromatography-mass spectrometry according to claim 1, characterized in that: In step (3), the solid-liquid ratio of the Camellia chrysantha sample to the 0.1 mol / L hydrochloric acid-ethanol standard solution is 1:(30-50).

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