Fingerprint spectrum detection method of heart-stabilizing traditional Chinese medicine composition

Abstract

The invention provides a fingerprint spectrum detection method of a heart-stabilizing traditional Chinese medicine composition, the detection method comprises a fingerprint spectrum and an effective index component determination method, a characteristic spectrum is established by adopting a high performance liquid chromatography-mass spectrometry method, and meanwhile, the effective index component determination method is used for determining the effective index component of the heart-stabilizing traditional Chinese medicine composition. Chemical characteristic index components in rhizoma nardostachyos, pseudo-ginseng, codonopsis pilosula and rhizoma polygonati medicinal materials in the heart-stabilizing granules are determined. According to the present invention, 22 batches of the heart stabilizing particles are determined, 8 index components such as polygonatum sibiricum alkali A, lobetyolin, desoxynardostinol A, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd are identified, and the detection method has characteristics of rapid detection (detection time is less than 18 min), relatively comprehensive qualitative detection index relative components, high sensitivity, high accuracy, and the like. The internal quality of the compound traditional Chinese medicine composition can be objectively represented.

Description

Technical Field

[0001] This invention belongs to the field of quality testing of effective components of traditional Chinese medicine, and in particular relates to a fingerprint spectrum detection method for a stable traditional Chinese medicine composition. Background Technology

[0002] The traditional Chinese medicine composition for calming the heart described in this invention is Wenxin Granules, a core product of Shandong Buchang Pharmaceutical Co., Ltd. It is prepared from the extraction and processing of traditional Chinese medicinal herbs including Codonopsis pilosula, Polygonatum sibiricum, Panax notoginseng, amber, and Nardostachys jatamansi. Its indications are: tonifying Qi and nourishing Yin, calming palpitations and restoring pulse, promoting blood circulation and removing blood stasis; used for symptoms such as palpitations, shortness of breath, and fatigue caused by deficiency of both Qi and Yin and obstruction of the heart vessels. The applicant of this invention has prior patent applications for this product, with application numbers: 01131734.5 (formula), 200410081398.2 (preparation method), 200530090041.6 (packaging box), 200610042976.0 (quality control method), 200610042974.1 (quality testing standard method), 200810232536.0 (prevention of allergic diseases), and 20081018393. 0.X (fracture application), 201510145198.7 (volatile component detection method), 201610013406.2 (high performance liquid chromatography detection method), 201811308826.9 (codonopsis glycoside analogs), 201910483321.4 (isopinene-type diterpenoids), 202210084515.9 (multi-index component detection method), 202210355751.X (Nardostachys jatamansi volatile component fingerprint spectrum). Currently, the existing standard for Wenxin Granules is the 2020 edition of the Chinese Pharmacopoeia, Part I, and the test items include appearance; TLC identification of Codonopsis pilosula, Panax notoginseng, and Polygonatum sibiricum; routine test items and HPLC content determination of Panax notoginseng. Since the traditional Chinese medicine composition of this invention is a compound preparation, relying solely on a single index-based effective component is insufficient to truly reflect the overall quality control level of the traditional Chinese medicine composition of this invention.

[0003] A systematic review of existing technical literature on this product by the applicant of this invention reveals that Li Qiyan and Wang Rongmei, in their paper "Determination of Saponin Components in Wenxin Granules by Rapid Solvent Extraction-Ultra-High Performance Liquid Chromatography," published in the *Journal of Pharmaceutical Analysis*, 2015-03-31, used this method to determine the content of Panax notoginseng saponin R1, ginsenoside Rg1, and ginsenoside Rb1 in Wenxin Granules. Acetonitrile (A)-water (B) was used as the mobile phase, with gradient elution (0–10 min, 81% B; 10–17 min).

[0004] The extraction time was reduced from 14 h to 25 min (17-18 min, 45% B → 81% B; 18-25 min, 81% B), with a column temperature of 25℃ and a detection wavelength of 203 nm. Compared with the conventional extraction method, the extraction time was shortened from 14 h to 25 min, significantly improving the sample processing efficiency. Li Xiaofeng, Zhang Shaoqiang, et al., Analysis and Identification of Main Components of Wenxin Granules Based on UPLC / Q-TOF MS / MS. Tianjin Journal of Traditional Chinese Medicine Impurities, 2020-08-12. The article states that using acetonitrile-water (containing 0.1% formic acid by volume) as the mobile phase gradient elution at a flow rate of 0.3 mL / min, with an electrospray ionization source, the main components of Wenxin Granules were sugars, saponins, and flavonoids, with high abundances of notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1. The problem with the methods in the existing technical literature is that they cannot simultaneously determine the chemical components in Nardostachys jatamansi, Panax notoginseng, Codonopsis pilosula, and Polygonatum sibiricum under the same chromatographic conditions. In order to detect the characteristic components of the four Chinese herbal medicines Nardostachys jatamansi, Panax notoginseng, Codonopsis pilosula, and Polygonatum sibiricum as much as possible, a high performance liquid chromatography-mass spectrometry method is adopted to establish the characteristic chromatographic detection method of the stable Chinese herbal medicine composition of the present invention. Summary of the Invention

[0005] This invention provides a fingerprint spectrum detection method for a stable traditional Chinese medicine composition. The detection method includes fingerprint spectrum and determination method of effective indicator components. The detection method has the advantages of short detection time, comprehensive detection of indicator components, and good operation.

[0006] This invention employs high-performance liquid chromatography-mass spectrometry (HPLC-MS) to establish characteristic spectra and to determine the chemical characteristic components of Nardostachys jatamansi, Panax notoginseng, Codonopsis pilosula, and Polygonatum sibiricum in Wenxin granules. Twenty-two batches of Wenxin granules were analyzed, and eight indicator components were identified: Polygonatine A, Codonopsis pilosula glycoside, deoxyninthol A, Panax notoginsenoside R1, Ginsenoside Rg1, Ginsenoside Re, Ginsenoside Rb1, and Ginsenoside Rd. This detection method features rapid detection speed (detection time less than 18 minutes), relatively comprehensive qualitative detection indicators, and can objectively characterize the quality of the Wenxin traditional Chinese medicine composition of this invention.

[0007] The technical solution of this invention patent application is as follows:

[0008] A fingerprint spectrum detection method for a traditional Chinese medicine composition for stabilizing the body, characterized in that the detection method includes the following steps:

[0009] (1) Preparation of reference solution: Weigh out Polygonatum sibiricum A, Codonopsis pilosula glycoside, deoxynaphthol A, Panax notoginsenoside R1, Ginsenoside Rg1, Ginsenoside Re, Ginsenoside Rb1, and Ginsenoside Rd, add 20-50% methanol to prepare a mixed reference solution.

[0010] (2) Preparation of test solution: Take the Wenxin traditional Chinese medicine composition, add 20-50% methanol, sonicate for 30 minutes, replenish the weight loss with 20-50% methanol, shake well, dilute to the mark with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0011] (3) Chromatographic and mass spectrometry conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.01% formic acid solution containing 2 mmol / L ammonium formate was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the flow rate was 0.2-0.5 ml per minute.

[0012]

[0013]

[0014] (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatography-mass spectrometry instrument, and determine the result.

[0015] Preferably, the fingerprint spectrum detection method is characterized in that the mass spectrometry conditions in step (3) of the detection method are as follows: a triple quadrupole mass spectrometer is used, and multiple reaction monitoring (MRM) is performed in electrospray ionization (ESI) positive ion mode. The monitored ion pairs are shown in the table below:

[0016]

[0017] Preferably, in the preparation of the reference solution (1) and the test solution (2) of the detection method, the concentration of methanol is 30%.

[0018] Preferably, the mass spectrometry conditions for step (1) of the detection method are as follows: the concentration of the mixed reference solution is: a mixed solution containing 0.03 μg of scutellarin A, 0.1 μg of codonopsis pilosula glycoside, 0.5 μg of deoxynaphthol A, 0.2 μg of notoginsenoside R1, 11 μg of ginsenoside Rg, 0.2 μg of ginsenoside Re, 11 μg of ginsenoside Rb, and 1 μg of ginsenoside Rd per 1 ml is prepared by adding 30% methanol.

[0019] Preferably, the specifications of the octadecylsilane-bonded silica gel filler are: Shim-pack Velox C18, column length of 10cm, inner diameter of 2.1mm, and particle size of 1.8μm.

[0020] The fingerprint spectrum detection method for the stable traditional Chinese medicine composition described in this invention can be used for the quality detection method of the internal control intermediates and finished products of the stable traditional Chinese medicine of this invention.

[0021] The beneficial effects of the technical solution of this invention are as follows:

[0022] (1) This invention uses high-performance liquid chromatography-mass spectrometry to establish a characteristic fingerprint spectrum. Simultaneously, it determines the chemical characteristic components of *Nardostachys chinensis*, *Panax notoginseng*, *Codonopsis pilosula*, and *Polygonatum sibiricum* in the *Wenxin* traditional Chinese medicine composition. Twenty-two batches of *Wenxin* granules were tested. The results showed that the ion chromatography of the test samples extracted with the specified mass-to-charge ratio ion pairs in all 22 batches exhibited eight characteristic peaks corresponding to the reference standard chromatogram. Among these, eight indicator components were identified: Polygonatine A, Codonopsis pilosula glycoside, deoxyninthol A, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd. This detection method has the advantages of fast detection speed (detection time less than 18 minutes), relatively comprehensive qualitative detection indicators, and can objectively characterize the intrinsic quality of the traditional Chinese medicine composition of this invention.

[0023] (2) This invention also establishes eight methods for detecting the content of various indicators. The methodological content determination results show that: ① The retention time (min) precision test results for each peak area are: RSD value 0.02%–0.29%; the peak area precision test results are: 1.32%–5.73%; the peak retention time repeatability test results are: 0.02%–0.43%; the peak area repeatability test results are: 2.65%–6.14%; the peak retention time (min) stability test results are: 0.02%–0.49%; and the peak area stability test results are: 1.89%–11.36%. Therefore, the eight methods established in this invention for detecting the content of Polygonatum sibiricum A, Codonopsis pilosula glycoside, deoxynaphthol A, Panax notoginseng saponin R1, Ginsenoside Rg1, Ginsenoside Re, Ginsenoside Rb1, and Ginsenoside Rd have good precision, stability, and reliability, and can be used in the quality detection standards of the stable traditional Chinese medicine composition of this invention. Attached Figure Description

[0024] Figure 1 - TIC ion chromatogram of the reference solution;

[0025] Figure 2 - Fingerprint chromatogram of the test solution of Wenxin Granules (TIC ion chromatogram);

[0026] Figure 3 - Fingerprint chromatogram (TIC ion chromatogram) of Wenxin Granules test solution (sucrose-free); among which, chromatographic peak 1: Polygonatine A, chromatographic peak 2: Codonopsis pilosula glycoside, chromatographic peak 3: Deoxynardinol A, chromatographic peak 4: Panax notoginsenoside R1, chromatographic peak 5: Ginsenoside Rg1, chromatographic peak 6: Ginsenoside Re, chromatographic peak 7: Ginsenoside Rb1, chromatographic peak 8: Ginsenoside Rd;

[0027] Figure 4 -TIC ion chromatogram of Polygonatum rhizome solution;

[0028] Figure 5 -TIC ion chromatogram of Codonopsis pilosula medicinal material solution;

[0029] Figure 6 -TIC ion chromatogram of Nardostachys jatamansi herb solution;

[0030] Figure 7-3 TIC ion chromatogram of the seven medicinal materials solution;

[0031] Figure 8 - Comparative characteristic chromatogram of the traditional Chinese medicine composition of this invention (TIC ion current chromatogram). Detailed Implementation

[0032] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0033] Example 1: Fingerprint Spectrum Detection Method for the Stable Traditional Chinese Medicine Composition of the Present Invention

[0034] 1. Instruments and reagents

[0035] Instruments: Shimadzu LC-MS8050 ultra-high performance liquid chromatography-mass spectrometry system (Shimadzu Corporation, Japan); Triple Quad LC-MS / MS system TM 6500+ (Thermo Fisher Scientific); Mettler Toledo XSE205 electronic balance (Mettler, Switzerland);

[0036] Reference standards: Panax notoginseng saponin R1, National Institutes for Food and Drug Control (NIFDC), batch number 110745-201921, content calculated as 90.4%; Ginsenoside Rg1, NIFDC, batch number 110703-201933, content calculated as 93.4%; Ginsenoside Re, NIFDC, batch number 110754-202129, content calculated as 96.03%; Ginsenoside Rb1, NIFDC, batch number 110704-20223 0, content calculated as 95.1%; Ginsenoside Rd, National Institutes for Food and Drug Control (NIFDC), batch number 111818-202104, content calculated as 97.3%; Codonopsis pilosula glycoside, NIFDC, batch number 111732-201908; Deoxynardol A, batch number PS013043, purchased from Chengdu Pusi Biotechnology Co., Ltd.; Polygonatum odoratum alkaloid A, batch number B28511-1504, purchased from Shanghai Yuanye Biotechnology Co., Ltd. Samples: Detailed information on 22 batches of samples is shown in Table 1.

[0037] Table 1 Information on Stable Particle Samples

[0038]

[0039]

[0040]

[0041] 2. The fingerprint spectrum determination method for the stable traditional Chinese medicine composition of the present invention

[0042] 2.1 Determination of chromatographic and mass spectrometric conditions

[0043] The chromatographic and mass spectrometric conditions were determined based on the characteristic chromatograms of the intermediate 3-Nardostachys chinensis, Panax notoginseng, Codonopsis pilosula, and Polygonatum sibiricum water extract mixture. A Shimadzu Shim-pack Velox C18 column (10 cm column length, 2.1 mm inner diameter, 1.8 μm particle size) was used; acetonitrile was used as mobile phase A, and 0.01% formic acid solution containing 2 mmol / L ammonium formate was used as mobile phase B, with gradient elution performed according to the specifications in Table 2; the flow rate was 0.3 mL per minute.

[0044] Table 2 Gradient elution program

[0045]

[0046] A triple quadrupole mass spectrometer was used with multiple reaction monitoring (MRM) in positive ion mode via electrospray ionization (ESI). Interface temperature: 300℃, desolvation temperature: 526℃, heating gas flow rate: 20 L / min. Monitored ion pairs are shown in Table 3.

[0047] Table 3 Information on monitored ion pairs

[0048]

[0049]

[0050] 2.2 Investigation on the preparation of the test solution

[0051] 2.2.1 Investigation of extraction solvent

[0052] Take 0.5g or 0.25g of Wenxin Granules (batch number 2307009) (sugar-free batch number 2005017), accurately weigh them, and accurately add 25ml of 10% methanol, 30% methanol, 60% methanol, and 80% methanol respectively. Weigh them, sonicate for 30 minutes, weigh them again, make up the lost weight with the corresponding solvent, shake well, accurately measure 1ml, put it in a 10ml volumetric flask, add 30% methanol to dilute to the mark, shake well, filter, and take the filtrate to obtain the product.

[0053] Accurately pipette 2 μl of each of the above solutions and inject them into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Perform the determination under the determined chromatographic conditions, and compare the peak area of ​​ion pair ① with the sample amount (g). The results are shown in Tables 4 and 5.

[0054] Table 4. Results of Extraction Solvent Investigation (Peak Area / Sample Amount)

[0055]

[0056] Table 5. Results of Extraction Solvent Evaluation (Peak Area / Sample Amount) - Sugar-Free

[0057]

[0058] The results show that there is no significant difference in extraction efficiency when using 30% methanol, 60% methanol and 80% methanol for extraction, so 30% methanol was selected as the extraction solvent.

[0059] 2.2.2 Sampling Quantity Examination

[0060] Take 0.2g, 0.5g, 0.7g, 1.0g or 0.1g, 0.25g, 0.5g of Wenxin Granules (batch number 2307009) (sugar-free batch number 2005017), accurately weigh them, add 25ml of 30% methanol to each, weigh them, sonicate for 30 minutes, weigh them again, make up the weight loss with 30% methanol, shake well, accurately measure 1ml, put it in a 10ml volumetric flask, add 30% methanol to dilute to the mark, shake well, filter, and take the filtrate to obtain the product.

[0061] Accurately pipette 2 μl of each of the above solutions and inject them into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Perform the determination under the determined chromatographic conditions, and compare the peak area of ​​ion pair ① with the sample amount (g). The results are shown in Tables 6 and 7.

[0062] Table 6. Results of Sampling Quantity Investigation (Peak Area / Sampling Quantity)

[0063]

[0064]

[0065] Table 7. Results of Sampling Quantity Investigation (Peak Area / Sampling Quantity) - Sugar-Free

[0066]

[0067] The test results show that there is no significant difference in the test results when the sample amount is 0.2g and 0.5g, and when the sample amount for the sugar-free specification is 0.1g and 0.25g. Considering the operability of sampling, the sample amount is determined to be 0.5g and the sample amount for the sugar-free specification is 0.25g.

[0068] 2.2.3 Examination of Ultrasound Time

[0069] Take 0.5g or 0.25g of Wenxin Granules (batch number 2307009) (sugar-free batch number 2005017), accurately weigh them, add 25ml of 30% methanol to each, weigh them, and sonicate them for 15 minutes, 30 minutes, and 45 minutes respectively, weigh them again, make up the weight loss with 30% methanol, shake well, accurately measure 1ml, put it in a 10ml volumetric flask, add 30% methanol to dilute to the mark, shake well, filter, and take the filtrate to obtain the product.

[0070] Accurately pipette 2 μl of each of the above solutions and inject them into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Perform the determination under the determined chromatographic conditions, and compare the peak area of ​​ion pair ① with the sample amount (g). The results are shown in Tables 8-9.

[0071] Table 8 Results of Ultrasonic Time Study (Peak Area / Sampling Amount)

[0072]

[0073]

[0074] Table 9. Results of Ultrasonic Time Study (Peak Area / Sampling Amount) - Sugar-Free

[0075]

[0076] According to the test results, the extraction efficiency is basically the same when the ultrasonic time is 30 or 45 minutes, so the ultrasonic time of 30 minutes was selected.

[0077] In summary, the preparation method for the test sample is determined as follows: Take 0.5g or 0.25g of stable granules (sugar-free), weigh accurately, add 25ml of 30% methanol accurately, weigh accurately, sonicate for 30 minutes, weigh again, replenish the lost weight with 30% methanol, shake well, accurately measure 1ml, place in a 10ml volumetric flask, dilute to the mark with 30% methanol, shake well, filter, and collect the filtrate to obtain the test sample.

[0078] 2.3 Preparation of reference solution

[0079] Weigh appropriate amounts of the following reference standards: Polygonatum odoratum A, Codonopsis pilosula glycoside, deoxynardol A, Panax notoginseng saponin R1, Ginsenoside Rg1, Ginsenoside Re, Ginsenoside Rb1, and Ginsenoside Rd. Add 30% methanol to prepare a mixed solution containing 0.03 μg of Polygonatum odoratum A, 0.1 μg of Codonopsis pilosula glycoside, 0.5 μg of deoxynardol A, 0.2 μg of Panax notoginseng saponin R1, 11 μg of Ginsenoside Rg1, 0.2 μg of Ginsenoside Re, 11 μg of Ginsenoside Rb1, and 1 μg of Ginsenoside Rd per 1 ml.

[0080] 2.4 Preparation of the test solution

[0081] Take 0.5g or 0.25g of Wenxin granules (sugar-free), weigh accurately, add 25ml of 30% methanol accurately, weigh again, sonicate for 30 minutes, weigh again, replenish the lost weight with 30% methanol, shake well, accurately measure 1ml, place in a 10ml volumetric flask, dilute to the mark with 30% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0082] The assay involves precisely pipetting 2 μl each of the reference solution and the test solution, injecting them into a liquid chromatography-mass spectrometry (LC-MS) instrument, and measuring the results.

[0083] The ion chromatography of the test sample extracted with ion pairs of specified mass-to-charge ratio should show 8 characteristic peaks corresponding to the reference standard chromatogram.

[0084] 2.5 Identification and Assignment of Characteristic Peaks

[0085] Preparation of herbal solution: Take about 1g of crude powder of Panax notoginseng, Nardostachys chinensis, Codonopsis pilosula and Polygonatum sibiricum respectively, add appropriate amount of water and decoct twice, filter, combine the decoctions, concentrate to about 5ml, add 10ml of ethanol, let stand for 24 hours, take the supernatant, filter, and take the filtrate to obtain the solution.

[0086] Preparation of the test solution: Take 0.5g or 0.25g of Wenxin granules (sugar-free), weigh accurately, add 25ml of 30% methanol accurately, weigh accurately, sonicate for 30 minutes, weigh again, replenish the lost weight with 30% methanol, shake well, accurately measure 1ml, place in a 10ml volumetric flask, dilute to the mark with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0087] Accurately pipette 2 μl each of the reference solution (2.2) and the above solution into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument, and perform the determination under the determined chromatographic and mass spectrometric conditions to obtain the final result.

[0088] The results showed that peak 1 belonged to Polygonatum sibiricum, peak 2 to Codonopsis pilosula, peak 3 to Nardostachys jatamansi, and peaks 4, 5, 6, 7, and 8 all belonged to Panax notoginseng. The TIC ion chromatogram is shown below. Figures 1-7 .

[0089] 2.6 Selection of Judgment Method

[0090] The characteristic chromatograms of traditional Chinese medicine can generally be evaluated by retention time, relative retention time / relative peak area, etc. In this method, the 8 characteristic peaks are known components. Based on the results of the column and instrument robustness test, the characteristic peak retention time method is selected for evaluation. The relevant chromatographic peaks of the test sample characteristic chromatogram should have the same retention time as the corresponding reference chromatographic peak.

[0091] 2.7 Establishment of Feature Maps

[0092] The ion chromatography of the test sample extracted with ion pairs of specified mass-to-charge ratios should exhibit eight characteristic peaks corresponding to those of the reference standard. (See chromatogram.) Figure 8 .

[0093] 2.8 Methodological Validation

[0094] 2.8.1 Specificity Test

[0095] Preparation of the test solution: Take 0.5g or 0.25g of Wenxin granules (sugar-free), weigh accurately, add 25ml of 30% methanol accurately, weigh accurately, sonicate for 30 minutes, weigh again, replenish the lost weight with 30% methanol, shake well, accurately measure 1ml, place in a 10ml volumetric flask, dilute to the mark with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0096] Preparation of negative sample solution for Codonopsis pilosula: Take 4g of Polygonatum sibiricum, 0.6g of Panax notoginseng, and 2g of Nardostachys chinensis, add appropriate amount of water and decoct twice, filter, combine the decoctions, concentrate to about 10ml, add 20ml of ethanol, let stand for 24 hours, take the supernatant, filter, and take the filtrate to obtain the solution.

[0097] Preparation of negative sample solution for Polygonatum deficiency: Take 3g of Codonopsis pilosula, 0.6g of Panax notoginseng, and 2g of Nardostachys chinensis, add appropriate amount of water and decoct twice, filter, combine the decoctions, concentrate to about 10ml, add 20ml of ethanol, let stand for 24 hours, take the supernatant, filter, and take the filtrate to obtain the solution.

[0098] Preparation of negative sample solution for Panax notoginseng deficiency: Take 3g of Codonopsis pilosula, 4g of Polygonatum sibiricum and 2g of Nardostachys jatamansi, add appropriate amount of water and decoct twice, filter, combine the decoctions, concentrate to about 10ml, add 20ml of ethanol, let stand for 24 hours, take the supernatant, filter, and take the filtrate to obtain the solution.

[0099] Preparation of Nardostachys jatamansi negative sample solution: Take 3g of Codonopsis pilosula, 4g of Polygonatum sibiricum and 0.6g of Panax notoginseng, add appropriate amount of water and decoct twice, filter, combine the decoctions, concentrate to about 10ml, add 20ml of ethanol, let stand for 24 hours, take the supernatant, filter, take the filtrate, and that is the solution.

[0100] Accurately pipette 2 μl of each of the above solutions, namely the reference solution under "2.3" and the test solution under "2.4", and inject them into the high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Measure the solutions according to the determined chromatographic and mass spectrometric conditions to obtain the final result.

[0101] The results showed that no chromatographic peaks corresponding to the extraction ion chromatography of scutellarin A reference standard were observed in the negative sample solution lacking Polygonatum sibiricum, Codonopsis pilosula, desoxynaphthol A reference standard, or Panax notoginseng reference standards. These results indicated that the negative sample solutions lacking Polygonatum sibiricum did not exhibit any chromatographic peaks corresponding to the extraction ion chromatography of Panax notoginseng saponins R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, and ginsenoside Rd reference standards. Furthermore, no chromatographic peaks corresponding to the extraction ion chromatography of Panax notoginseng saponins R1, Rg1, Re, Rb1, and Rd reference standards were observed in the negative sample solutions lacking Polygonatum sibiricum, indicating good method specificity.

[0102] 2.8.2 Instrument precision test

[0103] Preparation of the test solution: Take 0.5g or 0.25g of Wenxin granules (sugar-free), weigh accurately, add 25ml of 30% methanol accurately, weigh accurately, sonicate for 30 minutes, weigh again, replenish the lost weight with 30% methanol, shake well, accurately measure 1ml, place in a 10ml volumetric flask, dilute to the mark with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0104] Accurately pipette 2 μl of each of the above solutions and inject them into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Perform the determination under the determined chromatographic conditions, and compare the retention time and peak area of ​​ion pair ①. The results are shown in Tables 10-13.

[0105] Table 10 Retention Time (min) Precision Test Results

[0106]

[0107] Table 11 Results of Peak Area Precision Test

[0108]

[0109]

[0110] Table 12 Retention Time (min) Precision Test Results - Sugar-Free

[0111]

[0112] Table 13 Peak Area Precision Test Results - Sugar-Free

[0113]

[0114]

[0115] The test results show that the instrument has good precision.

[0116] 2.8.3 Repeatability Test

[0117] Following the test solution preparation method described in section 2.8.2, six test solutions were prepared in parallel and injected into a high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. The solutions were then analyzed under the determined chromatographic conditions, and the retention time and peak area of ​​ion pair ① were compared using the sample size as indicators. The results are shown in Tables 14–17.

[0118] Table 14 Retention Time (min) Repeatability Test Results

[0119]

[0120] Table 15 Results of Peak Area Repeatability Test

[0121]

[0122] Table 16 Retention Time (min) Repeatability Test Results - Sugar-Free

[0123]

[0124]

[0125] Table 17 Peak Area Repeatability Test Results - Sugar-Free

[0126]

[0127] The experimental results show that the method has good repeatability.

[0128] 2.8.4 Stability Test

[0129] The test solution under the repeatability test was injected at 0, 4, 8, 12, 16, and 24 hours, 2 μl each time, for a total of 24 hours. The stability of the analyte in the test solution during the detection process was observed using the retention time and peak area of ​​ion pair ① as indicators. The results are shown in Tables 18-21.

[0130] Table 18 Results of Retention Time (min) Stability Test

[0131]

[0132]

[0133] Table 19 Peak area stability test results

[0134]

[0135] Table 20 Retention Time (min) Stability Test Results - Sugar-Free

[0136]

[0137]

[0138] Table 21 Peak Area Stability Test Results - Sugar-Free

[0139]

[0140] The experimental results show that the retention time stability of the analyte is good within 24 hours, which can meet the determination requirements; peaks 1 to 3 are stable within 24 hours with RSD% within 5%; peaks 4 to 8 are slightly less stable within 24 hours with RSD% within 11.36%, which can basically meet the determination requirements.

[0141] 2.8.5 Durability Study of Chromatographic Columns and Instruments

[0142] The selected chromatographic columns were: ① Shimadzu Shim-pack Velox C18 (10cm length, 2.1mm inner diameter, 1.8μm particle size) and ② Thermo Acclaim. TM RSLC 120C18 (column length 10cm, inner diameter 2.1mm, particle size 2.2μm), chromatographic column ③ Waters ACQUITY HSS T3 (column length 10cm, inner diameter 2.1mm, particle size 1.8μm) and Agilent SB C18 (column length 10cm, inner diameter 2.1mm, particle size 1.8μm) columns were used for column robustness testing. A Shimadzu LC-MS8050 ultra-high performance liquid chromatography-mass spectrometry system and an AB liquid chromatography-mass spectrometry system (TRIPLE QUAD) were selected. TM Thermo Fisher Scientific conducted a durability test on the 6500+ instrument.

[0143] The results showed that chromatographic columns from different brands exhibited significant differences in chromatographic behavior and poor durability, making relative retention time an unsuitable evaluation method. The same chromatographic column showed essentially consistent chromatographic behavior on both instruments, with good separation performance and reproducibility.

[0144] Table 22 Results of Durability Test Based on Retention Time (min)

[0145]

[0146] 3. Summary

[0147] Characteristic chromatograms were established using high performance liquid chromatography-mass spectrometry. The chemical components of Nardostachys jatamansi, Panax notoginseng, Codonopsis pilosula, and Polygonatum sibiricum in Wenxin granules were determined. The results showed that the ion chromatograms of the test samples extracted with the specified mass-to-charge ratio ion pairs of the 22 batches of samples all showed 8 characteristic peaks corresponding to the reference standard chromatograms.

Claims

1. A fingerprint spectrum detection method for a traditional Chinese medicine composition for stabilizing the body, characterized in that, The detection method includes the following steps: (1) Preparation of reference solution: Weigh out Polygonatum sibiricum A, Codonopsis pilosula glycoside, deoxynaphthol A, Panax notoginsenoside R1, Ginsenoside Rg1, Ginsenoside Re, Ginsenoside Rb1, and Ginsenoside Rd, add 20-50% methanol to prepare a mixed reference solution. (2) Preparation of test solution: Take the Wenxin traditional Chinese medicine composition, add 20-50% methanol, sonicate for 30 minutes, replenish the weight loss with 20-50% methanol, shake well, dilute to the mark with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution. (3) Chromatographic and mass spectrometry conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; Using acetonitrile as mobile phase A and a 0.01% formic acid solution containing 2 mmol / L ammonium formate as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.2–0.5 ml per minute. (4) Determination method: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatography-mass spectrometry instrument, and determine the result.

2. The fingerprint pattern detection method as described in claim 1, characterized in that, The detection method step (3) mass spectrometry conditions are as follows: a triple quadrupole mass spectrometer detector is used, and multiple reaction monitoring (MRM) is performed in electrospray ionization (ESI) positive ion mode. The monitored ion pairs are shown in the table below:

3. The fingerprint pattern detection method as described in claim 1, characterized in that, In the detection method, step (1) preparation of the reference solution and step (2) preparation of the test solution, the concentration of methanol is 30%.

4. The fingerprint pattern detection method as described in claim 1, characterized in that, The detection method step (1) mass spectrometry conditions are as follows: The concentration of the mixed reference solution is: a mixed solution containing 0.03 μg of scutellarin A, 0.1 μg of codonopsis pilosula glycoside, 0.5 μg of deoxynaphthol A, 0.2 μg of notoginsenoside R1, 11 μg of ginsenoside Rg, 0.2 μg of ginsenoside Re, 11 μg of ginsenoside Rb, and 1 μg of ginsenoside Rd per 1 ml is prepared by adding 30% methanol.

5. The fingerprint pattern detection method as described in claim 1, characterized in that, The specifications of the octadecylsilane-bonded silica gel filler in step (3) of the detection method are: Shim-pack Velox C18, column length 10cm, inner diameter 2.1mm, and particle size 1.8μm.

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