HPLC-QAMS (High Performance Liquid Chromatography-Quantitative Analysis Mass Spectrometry) synchronous quantitative detection method for four rare ginsenosides in cooked pseudo-ginseng

Abstract

The invention provides an HPLC-QAMS (High Performance Liquid Chromatography-Quantitative Analysis Mass Spectrometry) synchronous quantitative detection method for four rare ginsenosides in cooked pseudo-ginseng, and belongs to the technical field of traditional Chinese medicine detection. According to the method, ginsenoside 20 (R)-Rg3 is taken as an internal reference, an HPLC-QAMS (High Performance Liquid Chromatography-Quantitative Amplified Mass Spectrometry) method is adopted, and simultaneous quantification of four ginsenosides is realized through verified relative correction factors; rSDlt; the method has the advantages of high accuracy (the recovery rate is 91.95-101.34%, RSDlt, 1.8%), high linearity (Rgt, 0.9997) and high stability (RSDlt, 3.5%), and the reference standard cost can be reduced by 75%. The QAMS method is excellent in green analysis score and blue applicability grade index. By analyzing 15 batches of samples, the consistency (relative to ESM, RE% lt, 5%) of the samples is confirmed, and the transfer rate of 20 (R) G-Rg3 is 85.82% by optimizing the extraction condition.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, and in particular relates to a simultaneous quantitative HPLC-QAMS method for four rare ginsenosides in processed Panax notoginseng. Background Technology

[0002] Panax notoginseng, a precious medicinal herb of the Araliaceae family, has a long history of clinical application in Traditional Chinese Medicine (TCM), with its medicinal value dating back to the Ming Dynasty's *Compendium of Materia Medica*. It is renowned for its "raw toxin-reducing and cooked tonic" properties—the raw form disperses blood stasis and stops bleeding, while the cooked form tonifies and strengthens the body, a classic example of the TCM theory of "different treatments for raw and cooked herbs." Modern pharmacological studies have confirmed that the bioactive substances in Panax notoginseng are mainly saponins, numbering over 150, primarily including protopanaxadiol (PPD type, such as Rb1 and Rd) and protopanaxtriol (PPT type, such as Rg1 and Re) saponins. These components exert hemostatic, anti-inflammatory, and cardiovascular protective effects by regulating key signaling pathways such as PI3K / AKT and NF-κB.

[0003] Although these saponins are crucial, their analytical standards, especially epimer 20 ( S G-Rg3 and 20 ( R G-Rg3 is expensive and often in short supply. 20 ( S The market price of Rg3 can reach approximately $2,500 per milligram. This has become a major bottleneck for routine industrial quality control (QC) using the traditional external standard method (ESM), as ESM requires a separate reference standard for each analyte. The high cost severely limits the frequency and scalability of batch monitoring. Quality Assay Multiple Analysis (QAMS), which quantifies multiple components using a relative correction factor (RCF) established with a single reference standard, offers a promising solution for quality control, but its application in processed Panax notoginseng is currently lacking. Furthermore, a core challenge of QAMS is ensuring the robustness and transferability of RCF values. RCF values ​​are susceptible to changes in chromatographic conditions (such as column type, HPLC type, detection wavelength, and flow rate), leading to significant drift (10%) and affecting the reliability of the method. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a simultaneous quantitative detection method for four rare ginsenosides in processed Panax notoginseng by HPLC-QAMS, so as to solve the problem of high quality control costs.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a simultaneous quantitative HPLC-QAMS method for the detection of four rare ginsenosides in processed Panax notoginseng, comprising the following steps: 1) Mix cooked Panax notoginseng with an ethanol solution, reflux and extract to obtain cooked Panax notoginseng extract; 2) Ginsenosides Rk3, Rh4, and 20 ( S -Rg3 and 20 ( R Rg3 standard was dissolved in methanol to prepare mixed standard solutions of different concentrations. The mixed standard solutions were detected by HPLC, and a mixed standard curve was plotted with mass concentration as the abscissa and peak area as the ordinate. 3) Based on the standard curve in step 2), the mixed standard solution was analyzed and recorded by HPLC to detect ginsenosides Rk3, Rh4, and 20 (…). S -Rg3 and 20 ( R The peak area and concentration of ginsenoside 20 (Rg3) were determined by ginsenoside 20. R )-Rg3 is an internal reference. Ginsenosides Rk3, Rh4, and 20 are calculated according to formula (1). S RCF of -Rg3 (1) In formula (1), F 20(R)Rg3 / mi The target object mi = Rk3 / Rh4 / 20 ( S )-Rg3 relative to internal reference 20 ( R The correction factor for -Rg3; A 20(R)Rg3 C 20(R)Rg3 20 (of the mixed reference standards) R Peak area and concentration of )-Rg3; 4) Add ginsenoside 20 ( R Ginsenoside 20 at different concentrations was prepared by dissolving Rg3 standard in methanol. R )-Rg3 standard solution, with ginsenoside 20 ( R Ginsenoside 20 (Rg3 standard solution) was detected by HPLC. A plot of ginsenoside 20 was constructed with mass concentration on the x-axis and peak area on the y-axis. R )-Rg3 standard curve; Ginsenosides Rk3, Rh4, and 20 were obtained from processed Panax notoginseng extract by HPLC. S -Rg3, 20 ( R The peak area of ​​ginsenoside 20 (Rg3) is based on ginsenoside 20. R )-Rg3 standard curve determination of 20 ( ) of processed Panax notoginseng R The content of ginsenosides Rk3, Rh4, and 20 was calculated according to formula (2). S The concentration of )-Rg3; (2) In formula (2), A s and C s (mg / mL) is the internal reference substance ginsenoside 20 (R Peak area and concentration of )-Rg3, A i and C i (mg / mL) represents the analyte components ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng sample. S Peak area and concentration of )-Rg3; 5) Calculate the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract according to formula (3). S The content of )-Rg3; (3) In formula (3), R i (mg / g) represents the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract. S The content of )-Rg3, m(g) is the weight of cooked Panax notoginseng weighed when preparing the sample solution, and V is the constant volume of the sample solution.

[0006] Preferably, the volume fraction of the ethanol solution in step 1) is 50-70%.

[0007] Preferably, the mass ratio of the cooked Panax notoginseng and the ethanol solution in step 1) is 1:5~10.

[0008] Preferably, the reflux extraction temperature in step 1) is 70~90℃, and the reflux extraction time is 1~2h.

[0009] Preferably, the reflux extraction in step 1) is performed 2 to 6 times.

[0010] Preferably, the instrument conditions for the HPLC detection are as follows: analysis is performed on an Agilent 1260 Infinity IILC system, with a quaternary pump, G7131C, pressure range up to 600 bar, an autosampler with a temperature-controlled sample tray, column chamber with temperature accuracy of ±0.5℃, diode array detector, and high-sensitivity flow cell.

[0011] Preferably, the chromatographic conditions for the HPLC detection are as follows: Agilent ZORBAX SB-C18 column, 250 mm × 4.6 mm, 5 μm particle size; mobile phase acetonitrile-water, volume ratio 40:60, isobaric elution; flow rate 1.0 mL / min; column temperature 25 °C; detection wavelength 203 nm; injection volume 10 μL.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention uses ginsenoside 20 ( R G-Rk3 was used as an internal control, and the relative correction factors (G-Rk3: 0.5768, G-Rh4: 0.4690, 20) were validated using HPLC-QAMS.S (G-Rg3: 1.0924), achieving simultaneous quantification of four ginsenosides; RSD < 2.0%, demonstrating high accuracy (recovery rate: 91.95-101.34%, RSD < 1.8%) and linearity (R). 2 >0.9997) and stability (RSD <3.5%), reducing reference standard costs by 75%. The QAMS method performed excellently in green analysis (AGREE) (score: 0.76 vs. ESM 0.63) and blue applicability grade index (BAGI) (score: 77.5 vs. ESM 65.0). Analysis of 15 batches of samples confirmed sample consistency (RE% <5% relative to ESM), and optimized extraction (60% ethanol, 5 cycles × 1.5 h) for 20 ( R The transfer rate of G-Rg3 was 85.82%. This work solved the bottleneck of SPN industrialization, ensured the consistency of bioactivity, and established a cost-effective and environmentally friendly quality control model. Attached Figure Description

[0013] Figure 1 This is an extraction experiment of processed Panax notoginseng (where A is a pie chart of the contribution rate of the comprehensive score of the four-factor orthogonal experiment; B is a bar chart of the comprehensive score of the four-factor orthogonal experiment; C is the effect of the number of extractions on the ginsenoside 20 in the processed Panax notoginseng extract). R (-Rg3 average transfer rate influence trend chart). Figure 2 It consists of four ginsenosides: Rk3, Rh4, and 20. S Rg3 and 20 ( R The standard curve of Rg3; Figure 3 This is an HPLC chromatogram of the mixed reference solution and the processed Panax notoginseng extract (where A is the mixed reference solution, B is the processed Panax notoginseng extract, 1 is ginsenoside Rk3, 2 is ginsenoside Rh4, 3 is ginsenoside 20-). S )-Rg3,4 is ginsenoside 20-( R )-Rg3); Figure 4 It analyzes the greenness (AGREE) and blue applicability rating index (BAGI) scores (where A1 is the QAMS greenness score, A2 is the ESM greenness score, B1 is the QAMS blue applicability score, and B2 is the ESM blue applicability score). Detailed Implementation

[0014] This invention provides a simultaneous quantitative HPLC-QAMS method for the detection of four rare ginsenosides in processed Panax notoginseng, comprising the following steps: 1) Mix cooked Panax notoginseng with an ethanol solution, reflux and extract to obtain cooked Panax notoginseng extract; 2) Ginsenosides Rk3, Rh4, and 20 ( S -Rg3 and 20 ( R Rg3 standard was dissolved in methanol to prepare mixed standard solutions of different concentrations. The mixed standard solutions were detected by HPLC, and a mixed standard curve was plotted with mass concentration as the abscissa and peak area as the ordinate. 3) Based on the standard curve in step 2), the mixed standard solution was analyzed and recorded by HPLC to detect ginsenosides Rk3, Rh4, and 20 (…). S -Rg3 and 20 ( R The peak area and concentration of ginsenoside 20 (Rg3) were determined by ginsenoside 20. R )-Rg3 is an internal reference. Ginsenosides Rk3, Rh4, and 20 are calculated according to formula (1). S RCF of -Rg3 (1) In formula (1), F 20(R)Rg3 / mi The target object mi = Rk3 / Rh4 / 20 ( S )-Rg3 relative to internal reference 20 ( R The correction factor for -Rg3; A 20(R)Rg3 C 20(R)Rg3 20 (of the mixed reference standards) R Peak area and concentration of )-Rg3; 4) Add ginsenoside 20 ( R Ginsenoside 20 at different concentrations was prepared by dissolving Rg3 standard in methanol. R )-Rg3 standard solution, with ginsenoside 20 ( R Ginsenoside 20 (Rg3 standard solution) was detected by HPLC. A plot of ginsenoside 20 was constructed with mass concentration on the x-axis and peak area on the y-axis. R )-Rg3 standard curve; Ginsenosides Rk3, Rh4, and 20 were obtained from processed Panax notoginseng extract by HPLC. S -Rg3, 20 ( R The peak area of ​​ginsenoside 20 (Rg3) is based on ginsenoside 20. R )-Rg3 standard curve determination of 20 ( ) of processed Panax notoginseng R The content of ginsenosides Rk3, Rh4, and 20 was calculated according to formula (2). S The concentration of )-Rg3; (2) In formula (2), A s and C s (mg / mL) is the internal reference substance ginsenoside 20 (R Peak area and concentration of )-Rg3, A i and C i (mg / mL) represents the analyte components ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng sample. S Peak area and concentration of )-Rg3; 5) Calculate the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract according to formula (3). S The content of )-Rg3; (3) In formula (3), R i (mg / g) represents the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract. S The content of )-Rg3, m(g) is the weight of cooked Panax notoginseng weighed when preparing the sample solution, and V is the constant volume of the sample solution.

[0015] In this invention, the volume fraction of the ethanol solution is preferably 50-70%, more preferably 60%; the mass ratio of the cooked Panax notoginseng to the ethanol solution is preferably 1:5-10, more preferably 1:7.5; the reflux extraction temperature is preferably 70-90℃, more preferably 80℃; the reflux extraction time is preferably 1-2 h, more preferably 1.5 h; the number of reflux extractions is preferably 2-6 times, more preferably 5 times; the HPLC detection instrument conditions are: analysis on an Agilent 1260 Infinity II LC system, quaternary pump, G7131C, pressure range up to 600 bar, autosampler with a temperature-controlled sample tray, column chamber: temperature accuracy ±0.5℃, diode array detector and high-sensitivity flow cell; the HPLC detection chromatographic conditions are: Agilent column. ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm particle size; mobile phase acetonitrile-water, volume ratio 40:60, isobaric elution; flow rate 1.0 mL / min; column temperature 25 ℃; detection wavelength 203 nm; injection volume 10 μL.

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] The reagents, standards, and samples involved in this invention: chromatographic solvents (methanol and acetonitrile, HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared using the Millipore system (Millipore, Bedford, MA, USA), and the purified water was sourced from Wahaha Group (Hangzhou, China). Ginsenoside standards: G-Rk3 and G-Rh4, were from Stanford Analytical Chemistry (Stanford, CA, USA); 20 ( S G-Rg3 was provided by Chengdu GLP Biotechnology Co., Ltd. (Chengdu, China); 20 ( R G-Rg3 was purchased from the National Institute for Food and Drug Control (NIFDC, Beijing, China). Purity verification: All standards were verified by HPLC peak area normalization, and the purity was greater than 98% (see Table 1 for details).

[0018] Table 1 Standard Product Information

[0019] Purity values ​​were obtained from the supplier's Certificate of Analysis (COA). General chemicals: Analytical grade reagents were purchased from Beijing Chemical Plant (Beijing, China). To verify the reliability of the QAMS method, this invention used QAMS and external standard method (ESM) to perform parallel quantification of four saponins in 15 batches of processed Panax notoginseng. Information on the 15 batches of processed Panax notoginseng is shown in Table 2. All Panax notoginseng was purchased from Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province, China. Sample identification was performed according to the Chinese Pharmacopoeia (2025 Edition) using macroscopic and microscopic examinations.

[0020] Table 2. Sample Information of Prepared Panax notoginseng Powder Slices

[0021] Example 1

[0022] 1. Single-factor optimization of the extraction process of ginsenosides from processed Panax notoginseng powder

[0023] The optimal extraction conditions for ginsenosides from processed Panax notoginseng powder were determined using a single-factor optimization method. Sample preparation: 0.6 g of processed Panax notoginseng powder (prepared by selecting 60 Panax notoginseng roots according to agricultural grading standards to ensure uniform maturity and structural integrity. Fresh roots were air-dried at 53℃ for 50 h to achieve a moisture content ≤8%, and then mechanically pulverized into coarse powder using a hammer mill. The powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm) to separate particles with a diameter of 2-4 mm; fragments that were too small or too large were discarded. The sieved particles were soaked in pure water (100% w / w) at 25℃ for 2 h to achieve uniform moisture distribution. Then, the material was steamed at a controlled temperature of 120℃ for 5 h in a layered tray (≤3 cm high, covered with bamboo mat). After steaming, the material was dried at 60℃ for 4.5 h until the moisture content dropped below 9%). Evaluation of Extraction Variables: Methods: Reflux and ultrasonic-assisted extraction (UAE; 40 kHz, 250 W); Reflux time: 0.5, 1.0, or 1.5 h (reflux extraction only); Solvent volume: 50 mL vs 100 mL (60% ethanol reflux for 1 h); Solvent concentration: methanol (40–100% v / v) or ethanol (40–100% v / v), both by reflux for 1 h. Under each condition, the mixture was cooled to 25 ± 2 °C, solvent loss was replenished to constant weight, homogenized, and filtered through a 0.45 μm nylon membrane. Key trends (e.g., 1.0 h reflux is superior to short / long reflux; 60% ethanol yields comparable efficiency to 80% methanol with lower toxicity) guided the selection of the final protocol. Ginsenoside Rk3, ginsenoside Rh4, ginsenoside 20- ( S Rg3 and ginsenoside 20- ( R The method for determining the Rg3 content is the same as in Example 2.

[0024] Experimental results are shown in Table 3.

[0025] Table 3. Results of Single-Factor Experiments on Extraction Process of Panax notoginseng Powder

[0026] Table 3 shows that ethanol reflux extraction (ERE) enriched G-Rk3, G-Rh4, and 20 (G-Rk3, G-Rh4, and G-Rh4) in processed Panax notoginseng powder. S G-Rg3 and 20 ( RThe extraction efficiency of the four target ginsenosides G-Rg3 was significantly better than that of ultrasound-assisted extraction (p<0.05). Optimization of key parameters showed that: Extraction time: The total ginsenoside content reached its peak at 1 h (an increase of 32.7% compared to 0.5 h, p=0.003), but decreased by 18.4% at 1.5 h (p=0.017), indicating thermal degradation / isomerization of rare saponins; Solvent selection: Ethanol was chosen based on its equilibrium polarity, as it effectively dissolves target ginsenosides (such as PPD- and ppt-type) while minimizing co-extraction of lipophilic impurities. Comparative studies showed that the extraction efficiency of 60% ethanol (17.8018 mg / g) was comparable to that of 80% methanol (17.8474 mg / g), but with higher safety and lower residual toxicity, meeting the requirements for industrial-scale production; Solvent volume: There was no statistically significant difference between 50 mL and 100 mL (p=0.236), supporting flexible scale-adjusted production.

[0027] 2. Orthogonal experiment on the extraction process of ginsenosides from processed Panax notoginseng powder

[0028] The extraction parameters (ethanol concentration, solid-liquid ratio, extraction time, and extraction frequency) were systematically optimized using an L9 (3^4) orthogonal array design (Table 4).

[0029] Table 4. Orthogonal Experiment for Extraction of Total Saponins from Panax notoginseng (L9(3)) 4 Experimental plan

[0030] In all experiments, experimental matrices were constructed to maintain a constant total solvent volume (300 mL) to ensure comparability of extraction efficiencies. Each experiment was performed in triplicate (n=3) to assess variability, and the average value was reported.

[0031] The basic principle of parameter selection: (1) Solid-liquid ratio: Experiment 1 (1:10) vs. Experiment 2 (1:5). The higher ratio (1:10) reduces solvent consumption but may limit mass transfer efficiency. A balance ratio (1:5) was included to verify scalability. The total solvent volume was kept constant (300 mL) to isolate the effect of solid loading.

[0032] (2) Extraction cycle: Optimize the number of cycles (2 vs 4) to balance extraction integrity and energy efficiency. Extending the extraction cycle (4×) effectively improves the heat resistance of ginsenosides (e.g., 20 ( R The recovery rate of G-Rg3 is achieved without causing measurable thermal degradation.

[0033] The transfer rate was 20 ( R G-Rg3: (4) Total extraction yield: (5) Experimental results are shown in Tables 5 and 6.

[0034] Table 5 L9 (3) 4 Orthogonal experimental table

[0035] Table 6. Orthogonal Experiment Analysis Table (Comprehensive Score)

[0036] Tables 5 and 6 show that the orthogonal array design systematically evaluated the effects of ethanol concentration (A: 50%, 60%, 100%), solid-liquid ratio (B: 1:5, 1:7.5, 1:10), extraction time (C: 1.0, 1.5, 2.0 h), and extraction times (D: 3, 4, 5) on extraction efficiency. A comprehensive scoring model was used to evaluate the 20 (… R The G-Rg3 transfer rate (weight: 80%) and total extraction rate (weight: 20%) were integrated. The results showed that the number of extractions (D) and extraction time (C) significantly affected the overall score (FD=45.231, p<0.05; FC=31.462, p<0.05), contributing 55.4% and 38.5% respectively. In contrast, ethanol concentration (A) and solid-liquid ratio (B) had no statistically significant difference (p>0.05). The histogram of the overall score showed a linear positive correlation between the extraction cycle (D) and the score, with 4 cycles increasing the score by 42.3% compared to 3 cycles; the extraction time (C) peaked at 1.5 h (score: 7.8) but decreased at 2.0 h, possibly due to the heat-resistant degradation of the bioactive compounds. Considering industrial feasibility, the optimal parameters were determined to be: 60% ethanol, solid-liquid ratio 1:7.5, and 4 extractions (1.5 h each).

[0037] 3. Optimization study of key parameters of extraction process

[0038] Based on the results of the orthogonal experiments, single-factor experiments were conducted to optimize the extraction frequency. Three batches of processed Panax notoginseng granules were extracted by reflux with 7.5 volumes of 60% ethanol (preparation method: 60 Panax notoginseng roots were selected according to agricultural grading standards to ensure uniform maturity and structural integrity. Fresh roots were air-dried at 53℃ for 50 h to achieve a moisture content ≤8%, and then mechanically pulverized into coarse powder using a hammer mill. The powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm) to separate particles with a diameter of 2-4 mm; fragments that were too small or too large were discarded. The sieved particles were soaked in pure water (100% w / w) at 25℃ for 2 h to achieve uniform moisture distribution. Then, they were steamed at a controlled temperature of 120℃ for 5 h in a layered tray (≤3 cm high, covered with bamboo mat). After steaming, the material was dried at 60℃ for 4.5 h until the moisture content dropped below 9%) (30 g). Each cycle was 1.5 h, with extraction frequencies of 4, 5, and 6 times (one extraction per condition). The combined extract was filtered, concentrated under reduced pressure to recover ethanol, and then lyophilized into powder. The total extract yield was calculated as the ratio of the mass of the dried extract to the mass of the initial sample × 100%. The 20 ( R The content of G-Rg3 was determined by the concentration of 20 g / L in the extract. R )G-Rg3 quality and 20 (in raw materials) R The transfer rate was determined by multiplying the mass ratio of G-Rg3 by 100%.

[0039] Experimental results: as shown in Tables 7 and 8. Figure 1 As shown.

[0040] Table 7. Drying mass, yield, and 20 (%) of each experimental extract. R Results of Rg3 content and transfer rate

[0041] Table 8. Extracts of Panax notoginseng from different extraction times, 20 (… R Comparison of average transfer rates of Rg3

[0042] From Table 7, Table 8 and Figure 1 It can be seen that the single-factor experiment under orthogonal optimization conditions shows that the extraction cycle is a factor affecting 20 ( R The most significant variable in the transfer efficiency of G-Rg3. Comparative analysis of extraction cycles 4, 5, and 6 showed that: 20 ( RThe transfer rate of G-Rg3 increased with increasing extraction cycles, but with diminishing marginal returns: (a) the transfer rate after 5 cycles was 85.82%, an improvement of 5.29% compared to 4 cycles; (b) the further improvement after 6 cycles was only 1.96% (total 87.78%, p<0.05, 5 cycles). The total extraction rate decreased by 8.3% from 4 to 6 cycles (p=0.012), due to thermal degradation during the long processing time. Crucially, the 1.96% gain from 5 to 6 cycles resulted in disproportionate energy and time costs, while 5 cycles balanced efficiency and cost-effectiveness. The results indicate that 5 cycles maximized the transfer rate by 20% (…). R G-Rg3 recovery rate. The final extraction protocol used 7.5 volumes of 60% ethanol for 5 cycles (1.5 h each).

[0043] Example 2

[0044] Simultaneous quantitative determination of four rare ginsenosides in processed Panax notoginseng by HPLC-QAMS

[0045] 1. Preparation of standard solutions

[0046] G-Rk3, Rh4 and 20 ( S Accurately weigh 40 mg of each G-Rg3 standard and dissolve them in methanol in 25 mL flasks to prepare concentrations of 1.5570 mg / mL (G-Rk3), 1.5605 mg / mL (G-Rh4), and 1.4719 mg / mL (20 mg / mL). S The stock solution of G-Rg3. Additionally, 20 ( R G-Rg3 (20 mg) was dissolved in methanol in a 100 mL volumetric flask to obtain a stock solution of 0.17114 mg / mL. Equal portions of G-Rk3, G-Rh4, and 20 mg / mL were then dissolved in methanol. S Transfer 1 mL of each of the G-Rg3 stock solution to a 10 mL volumetric flask, and mix with 5 mL of 20 ( R The G-Rg3 stock solution was mixed and diluted with 60% ethanol to the desired volume, yielding final concentrations of 0.1557 mg / mL (G-Rk3), 0.1561 mg / mL (G-Rh4), and 0.1472 mg / mL (20 mg / mL). S A mixed working solution of G-Rg3. 0.0856 mg / mL (20 ( R (G-Rg3). Before injection, all diluted solutions were filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) to remove particulate contaminants. Calibration curves were constructed by plotting peak areas based on their respective concentrations. Figure 2 This ensures robust quantification within the validated linear dynamic range (Table 9).

[0047] Table 9. Concentration Table for Standard Solution Preparation (mg / ml)

[0048] Depend on Figure 2 It can be seen that, based on the HPLC results of six standards with different concentrations, the functional relationship between the injection volume (X) and the peak area (Y) was obtained. The results show that Rk3, Rh4, and 20 ( S -Rg3 and 20 ( R )-Rg3 of R 2 All values ​​are greater than 0.9997, indicating a good linear relationship.

[0049] It is worth noting that 20 ( R G-Rg3 was intentionally kept at a low concentration in the stock solution because its solubility in methanol is relatively poor compared to the other three ginsenosides, as determined by the initial solubility assessment. However, all four analytes exhibited sufficient solubility in methanol, facilitating efficient serial dilutions to produce working solutions compatible with HPLC analysis.

[0050] 2. Sample preparation

[0051] Accurately weigh 0.6 g of processed Panax notoginseng powder (accuracy: ±0.1 mg) and place it in a round-bottom flask. Reflux extraction: Add 50 mL of 60% (v / v) ethanol and record the total weight. Reflux the mixture in an 80℃ water bath for 1 h to obtain the extract. Purify the extract using macroporous resin and dry to obtain processed Panax notoginseng saponins. Specific conditions and steps are as follows: Column: Φ50×300 mm glass column, packed with D101 resin (loading: 0.2 g / mL, flow rate: 0.5 BV / h). Elution: Washing: Remove water-soluble impurities with 4 BV deionized water (pH 6.0); Elution: Recover saponins with 4 BV 100% ethanol (pH 7.0). Anion exchange resin decolorization: Column: Φ30×200 mm column (height-to-diameter ratio 10:1), packed with D941 resin. Conditions: Mobile phase: pH 4.0 phosphate buffer (0.1 M), flow rate 1 BV / h; Selective elution: 3 BV 68% ethanol (pH 4.0) to remove acidic impurities, yielding a fraction with >98% purity of the target compound. Post-purification treatment: The combined eluent was spray-dried (inlet 265±10℃, outlet 80±5℃, pressure >0.4MPa) to obtain a uniform powder, which was then sieved through a 120-mesh sieve and stored at <30% humidity. The obtained powder was sieved through a 120-mesh sieve, homogenized for 30 min, and stored in a light-proof container at <30% humidity. An appropriate amount of cooked Panax notoginseng saponin powder was accurately weighed and dissolved in methanol to a concentration of 1 mg / ml. Filtration: The solution was filtered through a 0.22 μm PVDF membrane to remove particulate matter, followed by HPLC injection.

[0052] 3. HPLC analysis

[0053] Chromatography system

[0054] Instrumentation: Analysis was performed on an Agilent 1260 Infinity IILC system equipped with: a quaternary pump (G7131C, pressure range up to 600 bar), an autosampler with a temperature-controlled sample tray, a column chamber (temperature accuracy: ±0.5°C), a diode array detector (DAD), and a high-sensitivity flow cell (G7117B). Chromatographic conditions: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm particle size); mobile phase acetonitrile-water (40:60, v / v), isobaric elution; flow rate 1.0 mL / min; column temperature 25°C; detection wavelength 203 nm; injection volume 10 μL. System suitability: 20 ( R (G-Rg3>6000 theoretical plate, sample analysis: chromatograms of reference standard and processed Panax notoginseng extract are shown in the figure) Figure 3 .

[0055] 4. Validation of HPLC analytical method

[0056] The rationality of the HPLC analytical method was verified through evaluations of specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity and range, precision, stability, repeatability, and accuracy. The verification procedure was designed as follows: LOD: signal-to-noise ratio method (S / N≥3, N=3); LOQ: determined by three times the LOD concentration, ensuring a signal-to-noise ratio (S / N) >10; linearity and range: accurate extraction of ginsenosides G-Rk3, G-Rh4, and 20 ( S G-Rg3 and 20 ( R Aliquots (2, 4, 6, 8, 10, and 12 μL) of the mixed reference standard solution G-Rg3 were injected into the HPLC system under the specified chromatographic conditions of the profile chromatography system (volume was directly adjusted to avoid analyte loss due to dilution). Precision (intra-day): The same test solution was analyzed six times consecutively within one day to evaluate intra-day precision. Precision (inter-day): The same sample was measured six times daily over three consecutive days, and inter-day precision was calculated using comprehensive multi-day data. Repeatability: Six independent sample formulations from the same batch were analyzed in parallel to assess repeatability. Accuracy: Accuracy was determined by standard spiking recovery experiments. A known amount of reference standard (in a 1:1 ratio with the target analyte) was added to the test solution, and the recovery rate (%) was calculated. Solution stability: The stability of the sample solution was evaluated at 0, 2, 4, 8, 12, 16, 20, 24, and 36 hours after preparation under controlled storage conditions.

[0057] Experimental results are shown in Table 10. The LOD and LOQ ranged from 1.50 to 5.28 µg / mL and 4.49 to 15.9 µg / mL, respectively.

[0058] Table 10 Methodological Validation of HPLC Analytical Methods

[0059] RSD: Relative Standard Deviation.

[0060] Table 10 shows that the validation results of the method are expressed as the relative standard deviation (RSD) of the contents of the four ginsenosides, which has high reliability. The lower the RSD value, the higher the precision. For repeatability assessment, the RSD values ​​of all target peaks in the six replicate analyses of the same sample were all below 0.38%, and the precision (daytime) RSD values ​​of all target peaks were all below 1.8%. The RSD values ​​of the contents of the four ginsenosides measured at multiple time points within 36 hours at room temperature were <1.2%, confirming the stability of the sample. The repeatability of the method was validated using six independently prepared samples from the same batch, and the RSD values ​​of all peaks were <1.0%. Under known standard concentrations, the spiked recoveries of the four major saponins were evaluated, and the spiked recoveries ranged from 91.95% to 101.34%, with RSDs ranging from 0.93% to 1.8%.

[0061] 5. Establishment of QAMS analysis method

[0062] ① Establishment of a multi-component quantitative analysis and evaluation model using the single-label method (QAMS)

[0063] In this invention, ginsenoside 20 ( R 20-Rg3 is the most stable and readily available of the saponins in processed Panax notoginseng. It is easily separated under conventional chromatographic conditions, the separation technology is mature, and its acquisition cost is relatively low. It also exhibits significant activity in various aspects, including anticancer, immune enhancement, antioxidant, anti-inflammatory, and cardiovascular protection. Therefore, 20-Rg3... R Rg3 is the most suitable internal standard for the determination of Panax notoginseng content.

[0064] ② Relative correction factor

[0065] The core step in establishing the QAMS method is to achieve multi-component quantification by calculating the relative correction factor (RCF) between the internal reference and the target component. The specific steps are as follows: Preparation of standard solution: Accurately pipette the mixed standard solution containing ginsenosides Rk3, Rh4, and 20 ( S )-Rg3 and 20-( R 2, 4, 6, 8, 10, and 12 μL of Rg3 were injected and analyzed under the chromatographic conditions described above.

[0066] RCF calculation: with 20 (R Rg3 is an internal reference. The RCF values ​​of each component are calculated according to formula (1): (1) In formula (1), F 20(R)Rg3 / mi The target object (mi=Rk3 / Rh4 / 20) S )-Rg3) relative to internal reference 20 ( R The correction factor for -Rg3; A 20(R)Rg3 C 20(R)Rg3 20 (of the mixed reference standards) R Peak area and concentration of )-Rg3.

[0067] Experimental results are shown in Table 11.

[0068] Table 11 Relative Correction Factors for Saponins

[0069] Table 11 shows that within the validated linear range, saponin content is directly proportional to the chromatographic peak area. Changes in injection volume alter both saponin content and detector response; however, the relative correction factor (RCF) theoretically remains constant. The average RCF value calculated for different injection volumes is used as the final result, and its relative standard deviation (RSD) reflects the stability of the calculation. To ensure accuracy, all RCF values ​​are uniformly retained to four decimal places. Ginsenosides G-Rk3, G-Rh4, and 20 ( S The average RCFs of G-Rg3 were 0.5768, 0.4690 and 1.0924, respectively, with a maximum RSD of 1.1%, which is significantly lower than the threshold of 2.0%, confirming that the calculated RCFs have robust stability.

[0070] ③QAMS calculation of Panax notoginseng saponin content

[0071] First, according to ginsenoside 20 ( R The standard curve of 20-Rg3 was used to determine the content of 20-Rg3 in different batches of processed Panax notoginseng. R The content of ginsenoside 20 (Rg3) in 15 batches of processed Panax notoginseng was determined by QAMS. R The content of )-Rg3.

[0072] According to formula (2), the ginsenosides Rk3, Rh4 and 20 in processed Panax notoginseng were calculated. S The concentration of )-Rg3.

[0073] (2)

[0074] In formula (2), A s and C s(mg / mL) is the internal reference substance ginsenoside 20 ( R Peak area and concentration of )-Rg3, A i and C i (mg / mL) represents the analyte components (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample. S The peak area and concentration of )-Rg3).

[0075] Finally, the analytes (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample were calculated according to formula (3). S The content of )-Rg3).

[0076] (3)

[0077] In formula (3), R i (mg / g) represents the analyte components (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample. S The content of )-Rg3), m (g) is the weight of cooked Panax notoginseng weighed when preparing the sample solution, which is 0.6000g in this invention, and V is the constant volume of the sample solution, which is 50mL in this invention.

[0078] 6. Durability check

[0079] Flow rate variations (0.9, 1.0, and 1.1 mL / min), detection wavelengths (202, 203, and 204 nm), HPLC systems (ThermoFisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity), and columns [Agilent ZORBAX-C18 (4.6 × 250 mm, 5 μm), Shimadzu VP-ODS (4.6 × 250 mm, 5 μm), Inertsil ODS-3C18 (4.6 × 250 mm, 5 μm)] were used to evaluate the efficacy of different concentrations of ginsenosides G-Rk3, G-Rh4, and 20 (G-Rh4) in ginsenosides detected by QAMS. S The effect of the relative correction factor (RCF) of G-Rg3 was systematically reviewed.

[0080] Experimental results are shown in Table 12.

[0081] Table 12 Relative correction factors of saponins under different conditions

[0082] As shown in Table 12, the RCF exhibits good stability under all conditions, with RSDs of flow rate variation <1.0%, wavelength adjustment <1.3%, and system / column variation <2.0%. All deviations are within acceptable limits (5.0%), confirming the robustness of this method for routine application in different laboratory environments.

[0083] 7. Consistency assessment between QAMS and external standard methods

[0084] To verify the reliability of the QAMS method, this invention, in accordance with the General Principles of the Chinese Pharmacopoeia 9101, employed both QAMS and external standard method (ESM) to perform parallel quantification of four saponins in 15 batches of SPN. The consistency between the methods was evaluated using relative error (RE%), calculated as: RE% = [(QAMS - ESM) / EMS] × 100%.

[0085] Experimental results are shown in Table 13.

[0086] Table 13 Contents of four saponins in 15 batches of processed Panax notoginseng determined by ESM and QAMS methods

[0087] Table 13 shows that G-Rk3, G-Rh4 and 20 ( S The average RE% values ​​for G-Rg3 were -0.18%, 0.83%, and 0.87%, respectively. All RE% values ​​fell within ±0.87% of the target, confirming the accuracy and stability of the established QAMS method. Crucially, QAMS utilizes expensive reference standards (especially 20... S The consumption of G-Rg3 was reduced by 75%, while demonstrating excellent consistency (RSD < 3.5%) across three HPLC systems (ThermoFisher Ultimate 3000, Shimadzu LC-20AD, and Agilent 1260Infinity) and multiple C18 columns. This consistency ensures method transferability between laboratory and instrument platforms, resulting in highly comparable and reliable data. By significantly reducing barriers to industrial-scale multi-component monitoring (due to scarce / expensive standards) and lowering analytical costs, QAMS represents a key step towards establishing a unified, standardized quality assessment system for SPNs and their formulations, enabling intelligent manufacturing and end-to-end quality control of traditional Chinese medicine.

[0088] 8. Evaluate the greenness of the proposed method.

[0089] The environmental sustainability of the analytical workflow is assessed using the AGREE index, which quantifies compliance with the 12 principles of Green Analytical Chemistry (GAC). The assessment will employ a single-label (QAMS) method for quantitative analysis of multiple components and compare it with conventional external analytical methods.

[0090] Key parameters of the Standard Method (ESM) include: reduced reference standard consumption: QAMS eliminates 20 ( R The QAMS method requires 500 μg / mL stock solution for G-Rg3 standards, while ESM requires 60% (v / v) ethanol for sample preparation, whereas ESM uses 100% methanol. Energy efficiency is also a factor; QAMS avoids repeated chromatographic calibration, reducing instrument run time by 40%. The AGREE score (range: 0-1) reflects eco-friendliness, with higher values ​​indicating better compliance with GAC principles. Furthermore, the BAGI score assesses the method's industrial applicability across three dimensions: ease of operation: QAMS requires minimal operator training, avoiding complex sample derivation; throughput: analysis time per batch is reduced to <30 minutes (compared to 120 minutes for ESM); cost-effectiveness: solvent and standard costs are reduced by 35% and 60%, respectively. A BAGI score >0.7 indicates high practical value for routine analysis.

[0091] Experimental results: such as Figure 4 As shown, the QAMS method scored significantly higher (0.76) than the external standard method (ESM, 0.63), indicating that the proposed QAMS method has superior environmental friendliness and reduced environmental impact. Furthermore, in terms of functionality and usability assessed by the BAGI (Blue Applicability Rank Index) index, QAMS and ESM scored 77.5 and 65.0 respectively, confirming that both methods have high usability, with QAMS improving sustainability and operational efficiency by 19.2%.

[0092] As can be seen from the above embodiments, the present invention uses ginsenoside 20 ( RUsing G-Rg3 as an internal control, the HPLC-QAMS method was employed. With validated relative correction factors (G-Rk3: 0.5768, G-Rh4: 0.4690, 20(S)G-Rg3: 1.0924), simultaneous quantification of four ginsenosides was achieved. The RSD was <2.0%, demonstrating high accuracy (recovery: 91.95-101.34%, RSD <1.8%), linearity (R²>0.9997), and stability (RSD <3.5%), reducing the cost of the reference standard by 75%. The QAMS method showed excellent performance in AGREE (0.76 vs. ESM 0.63) and BAGI (77.5 vs. ESM 65.0). Analysis of 15 batches of samples confirmed sample consistency (RE% < 5% relative to ESM), and optimized extraction (60% ethanol, 5 cycles × 1.5 h) was performed on 20 ( R The transfer rate of G-Rg3 was 85.82%.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A simultaneous quantitative HPLC-QAMS method for the determination of four rare ginsenosides in processed Panax notoginseng, characterized in that, Includes the following steps: 1) Mix cooked Panax notoginseng with an ethanol solution, reflux and extract to obtain cooked Panax notoginseng extract; 2) Ginsenosides Rk3, Rh4, and 20 ( S -Rg3 and 20 ( R Rg3 standard was dissolved in methanol to prepare mixed standard solutions of different concentrations. The mixed standard solutions were detected by HPLC, and a mixed standard curve was plotted with mass concentration as the abscissa and peak area as the ordinate. 3) Based on the standard curve in step 2), the mixed standard solution was analyzed and recorded by HPLC to detect ginsenosides Rk3, Rh4, and 20 (…). S -Rg3 and 20 ( R The peak area and concentration of ginsenoside 20 (Rg3) were determined by ginsenoside 20. R )-Rg3 is an internal reference. Ginsenosides Rk3, Rh4, and 20 are calculated according to formula (1). S RCF of -Rg3 （1） In formula (1), F 20(R)Rg3 / mi The target object mi = Rk3 / Rh4 / 20 ( S )-Rg3 relative to internal reference 20 ( R The correction factor for -Rg3; A 20(R)Rg3 C 20(R)Rg3 20 (of the mixed reference standards) R Peak area and concentration of )-Rg3; 4) Add ginsenoside 20 ( R Ginsenoside 20 at different concentrations was prepared by dissolving Rg3 standard in methanol. R )-Rg3 standard solution, with ginsenoside 20 ( R Ginsenoside 20 (Rg3 standard solution) was detected by HPLC. A plot of ginsenoside 20 was constructed with mass concentration on the x-axis and peak area on the y-axis. R )-Rg3 standard curve; Ginsenosides Rk3, Rh4, and 20 were obtained from processed Panax notoginseng extract by HPLC. S -Rg3, 20 ( R The peak area of ​​ginsenoside 20 (Rg3) is based on ginsenoside 20. R )-Rg3 standard curve determination of 20 ( ) of processed Panax notoginseng R The content of ginsenosides Rk3, Rh4, and 20 was calculated according to formula (2). S The concentration of )-Rg3; （2） In formula (2), A s and C s (mg / mL) is the internal reference substance ginsenoside 20 ( R Peak area and concentration of )-Rg3, A i and C i (mg / mL) represents the analyte components ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng sample. S Peak area and concentration of )-Rg3; 5) Calculate the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract according to formula (3). S The content of )-Rg3; （3） In formula (3), R i (mg / g) represents the ginsenosides Rk3, Rh4, and 20 in the processed Panax notoginseng extract. S The content of )-Rg3, m(g) is the weight of cooked Panax notoginseng weighed when preparing the sample solution, and V is the constant volume of the sample solution.

2. The HPLC-QAMS simultaneous quantitative detection method according to claim 1, characterized in that, Step 1) The volume fraction of the ethanol solution is 50-70%.

3. The HPLC-QAMS simultaneous quantitative detection method according to claim 1, characterized in that, In step 1), the mass ratio of the cooked Panax notoginseng to the ethanol solution is 1:5~10.

4. The HPLC-QAMS simultaneous quantitative detection method according to claim 1, characterized in that, Step 1) The reflux extraction temperature is 70~90℃, and the reflux extraction time is 1~2h.

5. The HPLC-QAMS simultaneous quantitative detection method according to claim 1, characterized in that, Step 1) involves 2 to 6 reflux extractions.

6. The HPLC-QAMS simultaneous quantitative method according to claim 1, characterized in that, The instrument conditions for the HPLC detection were as follows: analysis was performed on an Agilent 1260 Infinity IILC system with a quaternary pump, G7131C, pressure range up to 600 bar, an autosampler with a temperature-controlled sample tray, column chamber with temperature accuracy of ±0.5℃, diode array detector, and high-sensitivity flow cell.

7. The HPLC-QAMS simultaneous quantitative method according to claim 1, characterized in that, The chromatographic conditions for the HPLC detection were as follows: Agilent ZORBAX SB-C18 column, 250 mm × 4.6 mm, 5 μm particle size; mobile phase acetonitrile-water, volume ratio 40:60, isobaric elution; flow rate 1.0 mL / min; column temperature 25 ℃; detection wavelength 203 nm; injection volume 10 μL.

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