Method for determining content of saponin components in pseudo-ginseng

Abstract

The invention relates to a method for determining the content of saponin components in panax notoginseng, the determination method comprises the step of determining the content of the saponin components in the panax notoginseng by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the conditions of ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) comprise a C18 chromatographic column and a C18 guard column. The UHPLC-MS / MS method for simultaneously determining the contents of the 22 saponin components in the pseudo-ginseng medicinal material is established, the 22 saponin components in the pseudo-ginseng medicinal material can be simultaneously, rapidly and quantitatively analyzed within 27 min, and the determination of the contents of three saponins such as 20-O-glucose ginsenoside Rf, 5, 6-dehydroginsenoside Rd and ginsenoside Ro in the pseudo-ginseng medicinal material is reported for the first time.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and relates to a method for determining the content of saponin components in Panax notoginseng, and particularly to a UHPLC-MS / MS method for determining the content of saponin components in Panax notoginseng. Background Art

[0002] Panax notoginseng, derived from the dried roots and rhizomes of Panax notoginseng (Burk.) FHChen, a plant of the Araliaceae family, has the effects of dispersing blood stasis and stopping bleeding, reducing swelling and relieving pain, and has a long history of medicinal use and extremely high medicinal value. Its authentic production area is Wenshan, Yunnan, but due to reasons such as continuous cropping obstacles, in recent years, the production area of ​​Panax notoginseng has gradually radiated to surrounding areas, such as Kunming, Yuxi, Qujing, Yunnan Province, and even in Sichuan, Chongqing, Guizhou and other provinces and cities.

[0003] Saponins are the main chemical components of Panax notoginseng and are also recognized as the main active ingredients. There are many types of saponins. The content of saponins in Panax notoginseng from different origins is different, and the efficacy of saponins of different contents is different. Generally speaking, the higher the content of saponins, the better the quality of Panax notoginseng. Therefore, the detection of saponin content can identify the origin of Panax notoginseng and its quality.

[0004] The 2020 edition of the Chinese Pharmacopoeia states that the detection of Panax notoginseng content is carried out by high performance liquid chromatography, with octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, water as mobile phase B, elution according to the gradient elution conditions in the table below, and the detection wavelength is 203nm. The theoretical plate number calculated based on the Panax notoginseng saponin R1 peak should be no less than 4000. Flow rate: 1ml / min; column temperature: 25℃.

[0005] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～12 19 81 12～60 19→36 81→64

[0006] Preparation of reference solution: Accurately weigh appropriate amounts of ginsenoside Rg1 reference, ginsenoside Rb1 reference and notoginsenoside R1 reference, add methanol to make a mixed solution containing ginsenoside Rg10.4 mg, ginsenoside Rb10.4 mg and notoginsenoside R10.1 mg per 1 ml.

[0007] Preparation of test solution: Take 0.6 g of the powder of the product (passed through a No. 4 sieve), weigh it accurately, add 50 ml of methanol accurately, weigh it, leave it overnight, keep it at a slight boil for 2 hours in a water bath at 80°C, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0008] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0009] The standard stipulates that this product contains ginsenoside Rg1(C 42 H 72 O 14 )、ginsenoside Rb1(C 54 H 92 O 23 ) and notoginsenoside R1(C 47 H 80 O 18 ) shall not be less than 5.0%.

[0010] However, the above detection method has a long analysis time, requiring more than 60 minutes, and a relatively high flow rate. Therefore, there are problems such as large consumption of mobile phase, long time consumption, high pressure of the liquid phase system, and unsatisfactory peak shape. In addition, the above detection method detects few types of saponins.

[0011] Compared with high performance liquid chromatography, mass spectrometry quantitative analysis technology has greatly increased the number of components that can be measured simultaneously, and has higher sensitivity and accuracy, so it has become an important means of determining the content of chemical components in traditional Chinese medicine. However, the establishment of mass spectrometry quantitative analysis methods usually requires complex exploratory experiments and methodological verification experiments, and the more types of components that are measured simultaneously, the more difficult it is to establish the method. Currently, the number of chemical components in Panax notoginseng determined using mass spectrometry quantitative methods is reported in the literature to be less than 20. [1-3] , for example, there are 18 [1] , 8 types [2] and 19 kinds [3] , and the content determination of three saponins, including 20-O-glucose ginsenoside Rf, 5,6-dehydroginsenoside Rd, and ginsenoside Ro, has not been reported. Summary of the invention

[0012] In order to identify Panax notoginseng from different origins, it is necessary to increase the number of components for content determination as much as possible. The present application provides a new method for determining the content of saponin components in Panax notoginseng, in particular, a new UHPLC-MS / MS method for determining the content of saponin components in Panax notoginseng. The method of the present invention can simultaneously and rapidly quantitatively analyze 22 saponin components in Panax notoginseng within 27 minutes. The flow rate of the method of the present invention is 0.4 mL / min, and the mobile phase A used is 0.1% formic acid water, and the mobile phase B is 0.1% formic acid acetonitrile, and the mobile phase consumption is low. According to methodological verification, the method of the present invention has good repeatability, good precision, good stability, and good accuracy.

[0013] The present invention adopts the following technical scheme to achieve the above-mentioned purpose.

[0014] A method for determining the content of saponins in Panax notoginseng, the method comprising using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to determine the content of saponins in Panax notoginseng, wherein the conditions of ultra high performance liquid chromatography in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) include: C 18 Chromatographic columns and C 18 Guard column.

[0015] Preferably, the C 18 Chromatographic column: Waters ACQUITY UPLC BEH C 18 (2.1mm×100mm, 1.7μm); the C 18 Guard column: Waters ACQUITY UPLC BEH C 18 (2.1 mm × 5 mm, 1.7 μm);

[0016] Preferably, the conditions of ultra high performance liquid chromatography in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) further include:

[0017] Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; the gradient elution program was as follows:

[0018]

[0019] Preferably, the conditions of ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) also include: flow rate: 0.4 mL / min; column temperature: 40° C.; injection volume: 1 μL.

[0020] Preferably, the mass spectrometry conditions in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) include: ESI ion source, negative ion mode; drying gas (N2) flow rate: 10.0 L / min; nebulizing gas flow rate: 3.0 L / min; heating gas flow rate: 10.0 L / min.

[0021] Preferably, the mass spectrometry conditions in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) also include: heating module temperature: 400°C; interface voltage: 3.0 kV; interface temperature: 300°C, detector voltage: 2.00 kV; DL temperature: 250°C.

[0022] Preferably, the saponin component is selected from 20-O-glucosylginsenoside Rf (20-O-glucoginsenoside Rf), notoginsenoside R1 (Notoginsenoside R1), ginsenoside Rg1 (GinsenosideRg1), ginsenoside Re (Ginsenoside Re), Vietnamese ginsenoside R4 (Vinaginsenoside R4), ginsenoside Rf (Ginsenoside Rf), notoginsenoside R2 (Notoginsenoside R2), notoginsenoside R4 (NotoginsenosideR4), ginsenoside Rh1 (Ginsenoside Rh1), notoginsenoside Fa (Notoginsenoside Fa), ginsenoside Rg2 (Ginsenoside Rg2), ginsenoside F3 (Ginsenoside F3), ginsenoside Ra3 (Ginsenoside Ra3), ginsenoside Rb1 (Ginsenoside Rb1), ginsenoside Ro (Ginsenoside Ro), ginsenoside Ra1, ginsenoside F1, 5,6-dehydroginsenoside Rd, ginsenoside Rd, gypenoside XVII, ginsenoside F2 and ginsenoside Rg3.

[0023] Preferably, the retention time (t R ), ion pair information (MRM transition) and fragmentation voltage (CE) are shown in the following table:

[0024]

[0025]

[0026] Preferably, the assay method includes the preparation of a reference solution, and the preparation of the reference solution is as follows:

[0027] Weigh 1.0 mg of each saponin reference substance, place them in 15 mL centrifuge tubes, add 10 mL of 70% methanol to dissolve, fully dissolve and mix, and filter through a 0.22 μm PTFE microporous filter to obtain the reference substance.

[0028] Preferably, the assay method comprises the preparation of a sample solution, and the sample solution is prepared as follows:

[0029] Grind the Panax notoginseng medicinal material, pass it through the pharmacopoeia No. 4 sieve, weigh 1.0 g of the sieved Panax notoginseng medicinal material powder, put it in a 100 mL conical flask, accurately add 20 mL of 70% methanol with a pipette, seal it with a sealing film, extract it with ultrasound (500W) at room temperature for 60 minutes, cool it to room temperature for 10 minutes, aspirate the supernatant and pass it through a 0.22 μm PTFE microporous filter, and dilute it 25 times with 70% methanol to obtain it.

[0030] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0031] The present invention establishes a UHPLC-MS / MS method for simultaneously determining the contents of 22 saponin components in Panax notoginseng medicinal materials, which can simultaneously and rapidly quantitatively analyze the 22 saponin components in Panax notoginseng within 27 minutes; among them, three saponins, namely 20-O-glucose ginsenoside Rf, 5,6-dehydroginsenoside Rd, and ginsenoside Ro, are the first to be established in Panax notoginseng medicinal materials for content determination, which has not been reported before; and the method for simultaneously determining the contents of the remaining 19 components in Panax notoginseng medicinal materials is also established for the first time in the present invention.

[0032] The flow rate of the method of the invention is 0.4 mL / min, the mobile phase A used is 0.1% formic acid water, the mobile phase B is 0.1% formic acid acetonitrile, and the mobile phase consumption is low.

[0033] The method of the present invention has good repeatability, precision, stability and accuracy through methodology verification.

[0034] The present invention establishes a UHPLC-MS / MS method for simultaneously determining the contents of 22 saponin components in Panax notoginseng medicinal materials, and determines the mass spectrometry parameters such as ion pairs (parent ion-daughter ion) and CE values ​​for the quantitative determination of 22 saponins. A simple, fast and efficient sample extraction method is determined. According to the peak shape and elution time, the mobile phase system (methanol-water, acetonitrile-water) and the proportion of formic acid added (0.05%, 0.1%), as well as the gradient elution program, flow rate (0.2mL / min, 0.3mL / min, 0.4mL / min) and column temperature (30°C, 35°C, 40°C) are explored. Under the premise of simultaneously quantifying 22 saponins with large differences in content, the sample dilution multiple and injection volume are determined, and finally an LC-MS method for simultaneously determining the contents of 22 saponin components in Panax notoginseng medicinal materials is established. The established content determination method is methodologically validated (including linearity, repeatability, precision, stability and accuracy). The results showed that the method had good repeatability, good instrument precision, and the sample solution was stable within 48 hours. This method can be used to determine the contents of 22 saponin components in Panax notoginseng. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The MRM pattern spectra of 22 saponin components (compound numbers are shown above) show the retention time (t R ), ion pair information (MRM transition) and fragmentation voltage (CE) table);

[0036] Figure 2 In the process of method optimization, the initial sample solution was diluted 100 times, 5 μL was injected, and the results were obtained by the MRM method;

[0037] Figure 3 In the process of method optimization, the initial sample solution was diluted 100 times, 5 μL was injected, and the cycle time was adjusted to 1.428 sec. The results were obtained by measuring according to the MRM method;

[0038] Figure 4 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, 1 μL was injected, and the results were determined according to the MRM method;

[0039] Figure 5 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, 2 μL was injected, and the results were determined according to the MRM method;

[0040] Figure 6 for Figure 5 Enlarged view of ginsenoside Rb1 peak;

[0041] Figure 7 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 11;

[0042] Figure 8 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 12;

[0043] Fig. 9 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0044] Fig.10 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, the dwell time was set to 7 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0045] Fig.11In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, the dwell time was set to 10 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0046] Fig.12 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, the dwell time was set to 18 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0047] Fig.13 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, the dwell time was set to 12 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0048] Fig.14 In the process of method optimization, the sample solution was not diluted, the cycle time was set to 1.428 sec, the dwell time was set to 16 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0049] Fig.15 After replacing the DL tube for routine instrument maintenance, the method was optimized in this case without diluting the sample solution, setting the cycle time to 1.428 sec, setting the dwell time to 14 msec, injecting 1 μL, and measuring the results according to the gradient elution program in Table 13;

[0050] Fig.16 To dilute the sample solution 100 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the results were measured according to the gradient elution program in Table 13;

[0051] Fig.17 To dilute the sample solution 50 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the results were measured according to the gradient elution program in Table 13;

[0052] Fig.18 To dilute the sample solution 50 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 2 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0053] Fig.19 To dilute the sample solution 50 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 5 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0054] Fig. 20 To dilute the sample solution 25 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0055] Fig.21 To dilute the sample solution 20 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0056] Fig. 22 To dilute the sample solution 25 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the dwell time of the 22 components to be tested was optimized at about 10 msec, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0057] Fig.23 To dilute the sample solution 25 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the dwell time of the 22 components to be tested was optimized at about 10 msec, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0058] Fig.24 To dilute the sample solution 25 times, the cycle time was set to 1.428 sec, the dwell time was set to 14 msec, 1 μL was injected, and the dwell time of the 22 components to be tested was optimized at about 10 msec, and the results were obtained by measuring according to the gradient elution program in Table 13;

[0059] Fig.25 In order to dilute the sample solution 25 times, the cycle time was set to 1.428 sec, the residence time was set to 14 msec, 1 μL was injected, and the residence time of the 22 components to be tested was optimized at around 10 msec. The results were measured according to the gradient elution program in Table 13. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0061] Example 1 :A UHPLC-MS / MS method for determining the content of saponins in Panax notoginseng 1. Experimental materials

[0062] (I) Sample information

[0063] The three-year-old Panax notoginseng sample WS-21 (collection location: Dongjiao Village, Shuguang Township, Guangnan County, Wenshan Prefecture, Yunnan Province, collection time: 2020.09.18) was identified as the dried root of Panax notoginseng (Burk.) FHChen of the Araliaceae family. The voucher specimen is stored in the Pharmacognosy Laboratory, School of Pharmacy, Peking University.

[0064] (II) Reference substances

[0065] Notoginsenoside R1 (lot No: MUST-21011910); Ginsenoside Rg1 (lot No: MUST-20110810); Ginsenoside Rb1 (lot No: MUST-21063015); 5,6-didehydroginsenoside Rd (lot No: MUST-20082805); Notoginsenoside R2 (lot No: MUST-21042812); Ginsenoside F2 (lot No: MUST-21042502); Ginsenoside Rd (lot No: MUST-21071208); Ginsenoside Re (lot No: MUST-21063015); 5,6-dehydroginsenoside Rd (lot No: MUST-20082805); No: MUST-21070810) was purchased from Chengdu Munster Biotechnology Co., Ltd.

[0066] 20-O-glucoginsenoside Rf (lot No: PS210406-10); Ginsenoside F3 (lot No: PS020410); Ginsenoside F1 (lot No: PS020389); Ginsenoside Rh1 (lot No: PS010067); Ginsenoside Rg3 (lot No: PS020427); Ginsenoside Rg2 (lot No: PS020018); Ginsenoside Ra3 (lot No: PS210406-11); Ginsenoside Ra ... Notoginsenoside Fa (lot No: PS010258); Ginsenoside Rf (lot No: PS010816); Ginsenoside Ro (lot No: PS010731); and Vinaginsenoside R4 (lot No: PS210406-15) were purchased from Chengdu Pusi Biotechnology Co., Ltd.

[0067] Notoginsenoside R4 (lot No: J23GB152507) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Gypenoside XVII (lot No: BBP04020) was purchased from Yunnan Xili Biotechnology Co., Ltd.

[0068] There are 22 reference substances in total, and the purity of all of them measured by HPLC area normalization method is ≥98%.

[0069] (III) Instruments and equipment

[0070] SHIMADZU 8050UHPLC-MS / MS liquid spectrometer (SHIMADZU, Japan): including controller (CBM-20A), binary pump (LC-30AD), autosampler (SIL-30AC), online degasser (DGC-20A3), column oven (CTO-20AC); chromatographic column: Waters ACQUITY UPLC BEH C 18(2.1 mm × 100 mm, 1.7 μm); Guard column: Waters ACQUITY UPLC BEH C 18 (2.1mm×5mm, 1.7μm); one-hundred-thousandth balance (METTLER TOLEDO, XS205 DU, Mettler-Toledo Instrument (Shanghai) Co., Ltd.); one-tenth-thousandth balance (OHAUS, AR1140, USA); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water preparation instrument (Millipore, USA); grinder (Xiaoxiong Electric Appliance Co., Ltd., FSJ-A05N6); Pharmacopoeia No. 4 sieve (R40 / 3 series, Shaoxing Shangyu Huafeng Hardware Instrument Co., Ltd.).

[0071] (IV) Reagents and consumables

[0072] Acetonitrile (LC-MS grade, Fisher Scientific, lot No: 207296); methanol (LC-MS grade, Fisher Scientific, lot No: 206409); formic acid (LC-MS grade, Fisher Scientific, lot No: 205178); water: Milli-Q ultrapure water; 0.22 μm PTFE microporous filter (Beijing Dima Technology Co., Ltd., lot No: F201201).

[0073] 2. Experimental Methods

[0074] (I) Preparation of reference solution

[0075] Weigh 1.0 mg of each of the 22 reference substances, place them in 15 mL centrifuge tubes, add 10 mL of 70% methanol to dissolve, and after sufficient dissolution and mixing, filter through a 0.22 μm PTFE microporous filter to obtain a solution of each reference substance with a concentration of 0.1 mg / mL.

[0076] (II) Experimental methods

[0077] The MRM method was optimized in the negative ion mode using the reference solution with a concentration of 0.1 mg / mL to determine the ion pairs (parent ion-daughter ion) and CE values ​​suitable for quantification, as shown in Table 2. The sample extraction method, extraction solvent, solid-liquid ratio and mobile phase type (methanol, acetonitrile), the proportion of formic acid added, the gradient elution program, the sample dilution multiple, the column temperature, the flow rate, the injection volume, etc. were explored, and the sample preparation method and the chromatographic and mass spectrometric conditions were finally determined.

[0078] (III) Sample preparation

[0079] Take Panax notoginseng medicinal material and grind it, pass it through the pharmacopoeia No. 4 sieve, weigh 1.0g of Panax notoginseng medicinal material powder (WS-21), accurately weigh it, put it in a 100mL conical bottle, accurately add 20mL of 70% methanol with a pipette, and seal it with a sealing film. Ultrasonic extraction (500W) at room temperature for 60min (the actual ultrasonic water temperature varies between 18 and 23℃), cool it to room temperature for 10min, draw the supernatant through a 0.22μm PTFE microporous filter, and dilute it 25 times with 70% methanol to obtain the sample solution to be tested.

[0080] (IV) Chromatographic and mass spectrometry conditions

[0081] Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 1 μL; gradient elution program is shown in Table 1; ESI ion source, negative ion mode; drying gas (N2) flow rate: 10.0 L / min, nebulizing gas flow rate: 3.0 L / min, heating gas flow rate: 10.0 L / min, heating module temperature: 400°C, interface voltage: 3.0 kV, interface temperature: 300°C, detector voltage: 2.00 kV, DL temperature: 250°C.

[0082] Table 1: Gradient elution program for UHPLC-MS / MS content determination method

[0083]

[0084] Table 2: Names and retention times (t R ), ion pair information (MRM transition) and fragmentation voltage (CE)

[0085]

[0086] The MRM mode spectra of the 22 components to be tested in the negative ion mode of the obtained Panax notoginseng sample solution are as follows: Figure 1 The compound numbers are shown in Table 2.

[0087] 3. Methodological Validation

[0088] (I) Linear

[0089] Accurately weigh 22 reference substances in a volumetric bottle, add 70% methanol to dissolve, dilute to scale, shake well, and obtain a mixed reference substance stock solution. Dilute 2, 4, 5, 10, 20, 40, 100, 200, 500, 800 times to obtain a series of mixed reference substance solutions. According to the above chromatographic and mass spectrometric conditions, inject 1 μL and measure them respectively. Perform linear regression on the peak area (y) and concentration (x, ng / mL) to obtain the regression equation, linear range, correlation coefficient (r2 The concentration when the signal-to-noise ratio (S / N) was 3 was taken as the limit of detection (LOD), and the concentration when the signal-to-noise ratio (S / N) was 10 was taken as the limit of quantification (LOQ). The results are shown in Table 3.

[0090] Table 3 Linear regression data, limit of detection (LOD) and limit of quantification (LOQ) of 22 components to be tested

[0091]

[0092]

[0093] (II) Repeatability

[0094] Grind the Panax notoginseng medicinal material and pass it through the pharmacopoeia No. 4 sieve. Weigh 6 portions of the Panax notoginseng medicinal material powder (WS-21) obtained by grinding, 1.0 g each, accurately weigh, and prepare 6 sample solutions according to the sample preparation method. The solutions were measured according to the above-mentioned chromatographic and mass spectrometric conditions, and the peak areas of 22 saponin components were recorded. The results showed that the RSD% of the peak area values ​​met the requirements (Table 4), indicating that the method had good repeatability.

[0095] Table 4 Methodology validation - repeatability (n = 6, unit: peak area)

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] (IV) Stability

[0104] Grind the Panax notoginseng medicinal material, pass it through the pharmacopoeia No. 4 sieve, weigh 1.0 g of the Panax notoginseng medicinal material powder (WS-21), accurately weigh it, prepare 1 sample solution according to the sample preparation method, and measure it at 0, 4, 8, 24, and 48 h according to the above-mentioned chromatographic and mass spectrometric conditions, record the peak areas of 22 components to be measured, and the RSD% of the peak area values ​​meet the requirements (Table 7), indicating that the sample solution is stable within 48 h.

[0105] Table 7 Methodology Validation - Stability (n = 6, unit: peak area)

[0106]

[0107] (V) Accuracy (sample recovery rate)

[0108] Grind the Panax notoginseng medicinal material, pass it through the pharmacopoeia No. 4 sieve, weigh 9 portions of the Panax notoginseng medicinal material powder (WS-21) obtained by grinding, 1.0 g each, accurately weighed, divided into 3 groups, 3 portions in each group, accurately added a certain amount of 22 kinds of saponin mixed reference solution (the amount of reference added in each group is equivalent to 80%, 100%, and 120% of the content of each reference in the medicinal material, respectively), prepared each sample solution according to the sample preparation method, and measured them respectively according to the above-mentioned chromatographic and mass spectrometric conditions, calculated the sample recovery rates of 22 saponins, and the results are shown in Table 8. The sample recovery rates of 22 saponins all met the requirements.

[0109] Table 8 Methodology Verification - Accuracy (Sample Recovery)

[0110]

[0111] It should be noted that the sample recovery values ​​of 5 of the above 22 saponins (Ginsenoside F1, 20-O-glucoginsenosideRf, Ginsenoside Rg2, Ginsenoside Rf, Vinaginsenoside R4) are significantly deviated from 100%. According to the provisions of the guidelines for analytical method validation in the Chinese Pharmacopoeia (Volume 4 of the 2020 edition), the average sample recovery rates of these 5 saponins are all within the prescribed recovery limit range; the RSD% of the recoveries of 5 of the 22 saponins (20-O-glucoginsenosideRf, Vinaginsenoside R4, Ginsenoside Ra3, Ginsenoside Rb1, Notoginsenoside R4) slightly exceed the acceptable range specified in the Pharmacopoeia (Table 9), but the Pharmacopoeia also stipulates that the recovery limit can be appropriately relaxed in the analysis of complex matrices, component content less than 0.01%, and multiple components. In view of the fact that this method simultaneously determines a large number of components and the content of most components is less than 0.01%, it is judged that the sample recovery test results of the 22 saponins meet the requirements of the pharmacopoeia.

[0112] Table 9 Content magnitude, average recovery (%), RSD% of 8 saponins in Panax notoginseng with recovery values ​​significantly deviating from 100% or with large RSD% and acceptable range of recovery limit and RSD% specified in the pharmacopoeia

[0113]

[0114] Example 2 :Method exploration experiment

[0115] The applicant conducted a large number of exploratory experiments before finally determining the method of determining the content of saponins in Panax notoginseng of the present invention. The following describes part of the exploratory experiment process by which the applicant determined the method of the present invention.

[0116] 1. Initial sample preparation method and chromatographic and mass spectrometric conditions

[0117] The initial sample preparation method and chromatographic and mass spectrometric conditions used by the applicant are as follows:

[0118] Sample preparation: Weigh 1.0g of Panax notoginseng powder, accurately weigh it, place it in a 100mL conical flask, accurately add 20mL of 70% methanol with a pipette, and seal it with a sealing film. Ultrasonic extraction (500W) at room temperature for 60min (the actual ultrasonic water temperature varies between 18 and 23°C), cool it to room temperature for 10min, and draw the supernatant through a 0.22μm PTFE microporous filter to obtain the sample solution to be tested.

[0119] Chromatographic and mass spectrometry conditions: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; gradient elution program is shown in Table 10; ESI ion source, negative ion mode; drying gas (N2) flow rate: 10.0 L / min, nebulizing gas flow rate: 3.0 L / min, heating gas flow rate: 10.0 L / min, heating module temperature: 400°C, interface voltage: 3.0 kV, interface temperature: 300°C, detector voltage: 2.00 kV, DL temperature: 250°C.

[0120] Table 10 Initial gradient elution program

[0121]

[0122] 2. Method Optimization Process

[0123] 1. After diluting the initial sample solution 100 times, inject 5 μL and measure according to the MRM method. The results are ( Figure 2 ) showed a “flat-top peak”, but the signal response intensity was not high. It was speculated that the concentration of the sample solution was too high, or parameters such as the dwell time or loop time needed to be optimized.

[0124] 2. After diluting the initial sample solution 100 times, 2 μL was injected, and the measured result still showed a "flat peak", so it was speculated that parameters such as residence time or cycle time needed to be optimized.

[0125] 3. After diluting the initial sample solution 100 times, inject 5 μL again and adjust the cycle time to 1.428 sec. The measured results are ( Figure 3 )The peak shape is normal.

[0126] 4. Based on the above conditions, the sample solution was not diluted and 1 μL was injected. The separation effect and peak shape were acceptable ( Figure 4 ).

[0127] 5. Based on the above conditions, try to increase the sample injection volume to 2μL ( Figure 5 ) and found that the peak type of ginsenoside Rb1 was "forked" ( Figure 6 ), so it was determined that 1 μL should be injected if this concentration was used.

[0128] 6. Inject 1 μL of sample solution and adjust the gradient elution program as shown in Table 11. The results show that the peak separation effect is not good at 5 to 18 minutes ( Figure 7 ), the elution gradient needs to be further optimized.

[0129] Table 11 Gradient elution program

[0130]

[0131] 7. Inject 1 μL of sample solution and adjust the gradient elution program as shown in Table 12. The results show that the peak separation effect is not good at 0 to 5 min ( Figure 8 ), the elution gradient needs to be further optimized.

[0132] Table 12 Gradient elution program

[0133]

[0134] 8. 1 μL of sample solution was injected, and the gradient elution program was adjusted several times. Finally, the gradient elution program was adjusted to that shown in Table 13. The results were measured ( Fig. 9 ) showed that the separation effect and peak shape were good under this condition, and the elution gradient could be used basically.

[0135] Table 13 Gradient elution program

[0136]

[0137] 9. Next, optimize the dwell time parameter and set the dwell time to 7, 10, 18, 12, and 16 msec respectively. Inject 1 μL of sample solution respectively and get the results ( Fig.10 , 11 , 12, 13, 14) show that when the residence time is greater than 14 msec, the peak shape is "broken line-shaped", and too short a residence time will also affect the peak shape. Therefore, after comparing the peak shape and separation under various conditions, it is considered to set the residence time to 14 msec.

[0138] 10. After the DL tube of the instrument was replaced, based on the currently optimized conditions, a "flat peak" would appear in the sample solution ( Fig.15 ), therefore, it is necessary to reduce the concentration of the sample solution after replacing the DL tube.

[0139] 11. Dilute the sample solution 100 times, 50 times, 25 times and 20 times respectively, and at the same time explore the injection volume (1μL, 2μL, 5μL), and the results are as follows Fig.16 , 17 , 18, 19, 20, and 21. Based on the comprehensive judgment of the peak shape and signal intensity, the appropriate sample concentration was determined to be 25 times dilution of the sample extraction solution and 1 μL injection. Next, the residence time parameters were optimized again.

[0140] 12. The residence time of the compound was optimized at about 10msec, and the results were as follows Fig. 22 , 23 , 24, and 25.

[0141] Based on the results of the above optimization conditions, the retention time values ​​of the 22 saponins to be tested were finally determined, which can ensure that the peak shape of these 22 compounds is good, and the test results are stable and accurate. The sample preparation method was finally determined as follows: 1.0g of Panax notoginseng powder was weighed accurately and placed in a 100mL conical flask, 20mL of 70% methanol was accurately added with a pipette, and sealed with a sealing film. Ultrasonic extraction (500W) was performed at room temperature for 60min (the actual ultrasonic water temperature varied between 18 and 23°C), cooled to room temperature for 10min, the supernatant was drawn through a 0.22μm PTFE microporous filter, and diluted 25 times with 70% methanol to obtain the sample solution to be tested.

[0142] The chromatographic and mass spectrometric conditions are as follows: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile; flow rate: 0.4 mL / min; column temperature: 40°C; gradient elution program is shown in Table 1; ESI ion source, negative ion mode; drying gas (N2) flow rate: 10.0 L / min, nebulizing gas flow rate: 3.0 L / min, heating gas flow rate: 10.0 L / min, heating module temperature: 400°C, interface voltage: 3.0 kV, interface temperature: 300°C, detector voltage: 2.00 kV, DL temperature: 250°C.

[0143] The names and retention times (t R ), ion pair information (MRM transition), dwell time (dwell time) and fragmentation voltage (CE) are shown in Table 2.

Claims

1. A method for determining the content of saponins in Panax notoginseng, the method comprising using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to determine the content of saponins in Panax notoginseng, wherein: The conditions of ultra high performance liquid chromatography in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) include: C 18 Chromatographic columns and C 18 Guard column.

2. The assay method according to claim 1, wherein The C 18 Chromatographic column: Waters ACQUITY UPLC BEHC 18 (2.1mm×100mm, 1.7μm); the C 18 Guard column: Waters ACQUITY UPLC BEH C 18 (2.1mm×5mm, 1.7μm).

3. The assay method according to claim 1 or 2, wherein The conditions of ultra high performance liquid chromatography in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) also include: Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile; the gradient elution program was as follows:

4. The assay method according to any one of claims 1 to 3, wherein The ultra-high performance liquid chromatography conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) also include: flow rate: 0.4 mL / min; column temperature: 40° C.; injection volume: 1 μL.

5. The assay method according to any one of claims 1 to 4, wherein The mass spectrometry conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) include: ESI ion source, negative ion mode; drying gas (N2) flow rate: 10.0 L / min; nebulizing gas flow rate: 3.0 L / min; heating gas flow rate: 10.0 L / min.

6. The assay method according to any one of claims 1 to 5, wherein The mass spectrometry conditions in the ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) also include: heating module temperature: 400°C; interface voltage: 3.0 kV; interface temperature: 300°C, detector voltage: 2.00 kV; DL temperature: 250°C.

7. The assay method according to any one of claims 1 to 6, wherein The saponin components include 20-O-glucosylginsenoside Rf (20-O-glucoginsenoside Rf), notoginsenoside R1 (Notoginsenoside R1), ginsenoside Rg1 (Ginsenoside Rg1), ginsenoside Re (Ginsenoside Re), Vietnamese ginsenoside R4 (VinaginsenosideR4), ginsenoside Rf (Ginsenoside Rf), notoginsenoside R2 (Notoginsenoside R2), notoginsenoside R4 (Notoginsenoside R4), ginsenoside Rh1 (Ginsenoside Rh1), notoginsenosideFa (NotoginsenosideFa), ginsenoside Rg2 (Ginsenoside Rg2), ginsenoside F3 (Ginsenoside F3), ginsenoside Ra3 (Ginsenoside Ra3), ginsenoside Rb1 (Ginsenoside Rb1), ginsenoside Ro (Ginsenoside Ro), ginsenoside Ra1, ginsenoside F1, 5,6-dehydroginsenoside Rd, ginsenoside Rd, gypenoside XVII, ginsenoside F2 and ginsenoside Rg3.

8. The assay method according to any one of claims 1 to 7, wherein The retention time of the saponin components (t R ), ion pair information (MRM transition) and fragmentation voltage (CE) are shown in the following table:

9. The assay method according to any one of claims 1 to 8, wherein The determination method includes the preparation of a reference solution, and the preparation of the reference solution is as follows: Weigh 1.0 mg of each saponin reference substance, place them in 15 mL centrifuge tubes, add 10 mL of 70% methanol to dissolve, fully dissolve and mix, and filter through a 0.22 μm PTFE microporous filter to obtain the reference substance.

10. The assay method according to any one of claims 1 to 9, wherein The assay method includes the preparation of a sample solution, and the sample solution is prepared as follows: Grind the Panax notoginseng medicinal material, pass it through the pharmacopoeia No. 4 sieve, weigh 1.0 g of the sieved Panax notoginseng medicinal material powder, put it in a 100 mL conical flask, accurately add 20 mL of 70% methanol with a pipette, seal it with a sealing film, extract it with ultrasound (500W) at room temperature for 60 minutes, cool it to room temperature for 10 minutes, aspirate the supernatant and pass it through a 0.22 μm PTFE microporous filter, and dilute it 25 times with 70% methanol to obtain it.

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