A method for establishing the GC-MSMS fingerprint of Guben Yanling Pills

Abstract

The present invention relates to the technical field of traditional Chinese medicine detection, and discloses a method for establishing a GC-MS / MS fingerprint of Guben Yanling Pills, including the following steps: preparation of instruments and reagents, preparation of solutions, GC-MS analysis conditions, experimental method and condition optimization, selection of temperature programming, analysis results of volatile components of Guben Yanling Pills, establishment of GC-MS / MS fingerprint of Guben Yanling Pills, establishment of fingerprint of Guben Yanling Pills, and similarity evaluation of fingerprint of Guben Yanling Pills. The method for establishing the GC-MS / MS fingerprint of Guben Yanling Pills can effectively detect the quality of Guben Yanling Pills accurately through the establishment of the fingerprint of Guben Yanling Pills, thereby effectively helping users quickly and accurately detect the quality and efficacy of Guben Yanling Pills, and thus helping enterprises quickly conduct batch detection of drugs, fundamentally solving the problem of quality standardization of traditional Chinese medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and particularly to a method for establishing a GC-MSMS fingerprint of Guben Yanling Pills. Background Art

[0002] Guben Yanling Pills strengthen the primordial qi, nourish yin, replenish marrow and essence, and strengthen muscles and bones. They are used for consumptive impairment, low back pain and general fatigue, palpitation and insomnia, emaciated skin, premature whitening of hair and beard, irregular menstruation, and loss of appetite, and are commonly applied in the field of traditional Chinese medicine.

[0003] In the prior art, in the process of measuring the corresponding data parameters of Guben Yanling Pills, only the corresponding product data is obtained through a single data detection method. During the process of product determination, no corresponding detection method is set for effective and rapid detection. As a result, when a large number of Guben Yanling Pills are batch-tested, relevant data cannot be effectively and rapidly detected, and the problem of the quality standardization of traditional Chinese medicine cannot be fundamentally solved. In view of this, we propose a method for the GC-MSMS fingerprint of Guben Yanling Pills. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing a GC-MSMS fingerprint of Guben Yanling Pills to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A method for establishing a GC-MSMS fingerprint of Guben Yanling Pills, the method for the GC-MSMS fingerprint of Guben Yanling Pills includes the following steps:

[0006] S1. Preparation of instruments and reagents;

[0007] S2. Preparation of solutions, the preparation of solutions includes: preparation of mixed alkanes, preparation of test solution;

[0008] S3. GC-MS analysis conditions, the GC-MS analysis conditions include:

[0009] The chromatographic column is SH-Rxi-5SilMS chromatographic column;

[0010] Temperature programming: The initial temperature is 50°C. After 2 minutes, the temperature is raised to 125°C at a rate of 6°C / min and held for 1 minute. Then the temperature is raised to 175°C at a rate of 8°C / min and held for 2 minutes. Then the temperature is raised to 240°C at a rate of 6°C / min and held for 3 minutes. Then the temperature is raised to 280°C at a rate of 3°C / min and held for 14 minutes; the injection volume is 1 μL; without splitting;

[0011] The carrier gas is high-purity He, with a total flow rate of 14 mL / min, a column flow rate of 1 mL / min, and a pressure of 43.6 kPa; the injection temperature is 260 °C;

[0012] EI ionization; the electron bombardment energy is 70 eV; the ion source temperature is 230 °C; the interface temperature is 280 °C; the solvent delay time is 4 min; the mass scanning range is m / z: 25 - 550;

[0013] S4. Experimental methods and condition optimization;

[0014] S5. Selection of the temperature programming, and the temperature programming includes Program 1 and Program 2;

[0015] S6. Analysis results of the volatile components of Guben Yanling Pills;

[0016] S7. Establishment of the GC-MS fingerprint of Guben Yanling Pills;

[0017] S8. Establishment of the fingerprint of Guben Yanling Pills;

[0018] S9. Similarity evaluation of the fingerprint of Guben Yanling Pills.

[0019] Optionally, the preparation of the instrument includes a GCMS-TQ8040 gas chromatography-mass spectrometry instrument, a SH-Rxi-5SilMS capillary column for chromatography, an electronic analytical balance, and a KQ-250B ultrasonic cleaner.

[0020] Optionally, the preparation of the reagents includes n-hexane, ethyl acetate, n-butanol, a mixed reference substance of C7-C40 n-alkanes, and distilled water, and the n-hexane, ethyl acetate, and n-butanol are all of analytical grade.

[0021] Optionally, the preparation of the mixed alkanes includes: taking 10 μL of the mixed reference substance of C7-C40 n-alkanes, adding ethyl acetate to 1 mL, to obtain a 1% mixed reference substance solution of n-alkanes.

[0022] Optionally, the preparation of the test solution includes: precisely weighing 1 g of Guben Yanling Pills, placing them in a conical flask, adding 3 mL of water, soaking and stirring until it becomes a suspension, adding 10 mL of ethyl acetate, ultrasonic extracting for 30 min, then cooling to room temperature, filtering with filter paper, separating the filtrate through a separatory funnel, taking the upper ethyl acetate extract, transferring it to a 10 mL volumetric flask, making up the volume to the mark with ethyl acetate, shaking well, and filtering through a 0.22 μm filter membrane to obtain the test solution.

[0023] Optionally, the data acquisition mode in the EI ionization is the full scan mode.

[0024] Optionally, step S4 further includes: investigating the extraction effects of different polar solvents such as n-hexane, n-butanol, and ethyl acetate on the volatile components of Guben Yanling Pills, and selecting the best extraction method according to the number of peaks and the magnitude of response values in the chromatogram.

[0025] Optionally, Program 1 includes:

[0026] The initial temperature is 50°C. After 2 minutes, the temperature is raised to 280°C at a rate of 5°C min-1 and held for 10 minutes.

[0027] The solvent delay time is 5 minutes.

[0028] The chromatographic peaks are relatively dispersed before 20 minutes.

[0029] The chromatographic peaks are poorly separated at 35 - 40 minutes, and a constant temperature platform is added; there are components that are not detected at the end of the program.

[0030] Optionally, Program 2 includes:

[0031] The initial temperature is 50°C. After 2 minutes, the temperature is raised to 125°C at a rate of 6°C min-1 and held for 1 minute.

[0032] The temperature is raised to 175°C at a rate of 8°C min-1 and held for 2 minutes.

[0033] The temperature is raised to 240°C at a rate of 6°C min-1 and held for 3 minutes.

[0034] The temperature is raised to 280°C at a rate of 3°C min-1 and held for 14 minutes, and the solvent delay time is 4 minutes.

[0035] Compared with the prior art, the present invention provides a method for establishing a GC-MSMS fingerprint of Guben Yanling Pills, having the following beneficial effects:

[0036] For the method for establishing the GC-MSMS fingerprint of Guben Yanling Pills, the similarity between 20 batches of samples and the reference chromatogram: the similarity between the fingerprint chromatograms of each batch of Guben Yanling Pills (big honey pills) and the reference fingerprint chromatogram is greater than 0.849, and the similarity between the fingerprint chromatograms of each batch of Guben Yanling Pills (small honey pills) and the reference fingerprint chromatogram is greater than 0.885, indicating that the chemical compositions of different batches of Guben Yanling Pills of the same dosage form are relatively consistent. By establishing the fingerprint of Guben Yanling Pills, the quality of Guben Yanling Pills can be effectively and accurately detected, thereby effectively helping users quickly and accurately detect the quality and efficacy of Guben Yanling Pills, and helping enterprises quickly conduct batch detection of drugs, fundamentally solving the problem of the quality standardization of traditional Chinese medicines. Description of the Drawings

[0037] Figure 1 Schematic diagram of the results of Method 2 of the present invention;

[0038] Figure 2 Schematic diagram of the results of Method 3 of the present invention with ethyl acetate as the solvent;

[0039] Figure 3 Schematic diagram of the results of Method 3 of the present invention with n-butanol as the solvent;

[0040] Figure 4 Schematic diagram of the results of Method 3 of the present invention with n-hexane as the solvent;

[0041] Figure 5 TLC diagram of the temperature-rising program 1 of the present invention;

[0042] Figure 6 TLC diagram of the temperature-rising program 2 of the present invention;

[0043] Figure 7 TIC diagram of the n-alkane mixed reference of the present invention;

[0044] Figure 8 Schematic diagram of the spectrum of X1 of the present invention;

[0045] Figure 9 Schematic diagram of the spectrum of X2 of the present invention;

[0046] Figure 10 Schematic diagram of the spectrum of X3 of the present invention;

[0047] Figure 11 Schematic diagram of the spectrum of X4 of the present invention;

[0048] Figure 12 Schematic diagram of the spectrum of X5 of the present invention;

[0049] Figure 13 Schematic diagram of the spectrum of X6 of the present invention;

[0050] Figure 14 Schematic diagram of the spectrum of X7 of the present invention;

[0051] Figure 15 Schematic diagram of the spectrum of X8 of the present invention;

[0052] Figure 16 Schematic diagram of the spectrum of X9 of the present invention;

[0053] Figure 17 Schematic diagram of the spectrum of X10 of the present invention;

[0054] Figure 18 Schematic diagram of the spectrum of D1 of the present invention;

[0055] Figure 19 Schematic diagram of the spectrum of D2 of the present invention;

[0056] Figure 20 Schematic diagram of the spectrum of D3 of the present invention;

[0057] Figure 21 Schematic diagram of the spectrum of D4 of the present invention;

[0058] Figure 22 Schematic diagram of the spectrum of D5 of the present invention;

[0059] Figure 23 Schematic diagram of the spectrum of D6 of the present invention;

[0060] Figure 24 Schematic diagram of the spectrum of D7 of the present invention;

[0061] Figure 25 Schematic diagram of the spectrum of D8 of the present invention;

[0062] Figure 26 Schematic diagram of the spectrum of D9 of the present invention;

[0063] Figure 27 Schematic diagram of the spectrum of D10 of the present invention;

[0064] Figure 28 Schematic diagram of the overlay map of the GC-MS fingerprint of the present invention;

[0065] Figure 29 Schematic diagram of the control fingerprint of the big honey pill of the present invention;

[0066] Figure 30 Schematic diagram of the control fingerprint of the small honey pill of the present invention;

[0067] Figure 31 Schematic diagram of the control fingerprint of the present invention. Detailed implementation manners

[0068] As Figures 1 - 31 shown, the present invention provides a technical solution: a method for establishing a GC-MSMS fingerprint of Guben Yanling Pills, comprising the following steps:

[0069] S1. Preparation of instruments and reagents.

[0070] The preparation of instruments includes a GCMS-TQ8040 gas chromatograph-mass spectrometer, a SH-Rxi-5SilMS capillary column, an electronic analytical balance, and a KQ-250B ultrasonic cleaner; the preparation of reagents includes n-hexane, ethyl acetate, n-butanol, a C7-C40 n-alkane mixed reference substance, and distilled water. N-hexane, ethyl acetate, and n-butanol are all of analytical grade;

[0071] Collect 20 groups of Guben Yanling Pills, and the sample information of the Guben Yanling Pills is shown in the following table:

[0072] Serial number Production batch number Dosage form Serial number Production batch number Dosage form X1 20220401 Small honeyed pills D1 20210901 Large honeyed pills X2 20211001 Small honeyed pills D2 20220302 Large honeyed pills X3 20210403 Small honeyed pills D3 20220301 Large honeyed pills X4 20221102 Small honeyed pills D4 20210804 Large honeyed pills X5 20211103 Small honeyed pills D5 20210405 Large honeyed pills X6 20220101 Small honeyed pills D6 20210703 Large honeyed pills X7 20210801 Small honeyed pills D7 20210502 Large honeyed pills X8 20211101 Small honeyed pills D8 20220603 Large honeyed pills X9 20220601 Small honeyed pills D9 20220201 Large honeyed pills X10 20220402 Small honeyed pills D10 20220501 Large honeyed pills

[0073] S2. Preparation of solutions, which includes: preparation of mixed alkanes and preparation of test solution.

[0074] The preparation of mixed alkanes includes: taking 10 μL of n-alkane C7-C40 mixed reference substance, adding ethyl acetate to 1 mL to obtain a 1% n-alkane mixed reference substance solution.

[0075] The preparation of test solution includes: accurately weighing 1 g of Guben Yanling Pills, placing them in a conical flask, adding 3 mL of water, soaking and stirring until a suspension is formed, adding 10 mL of ethyl acetate, ultrasonically extracting for 30 min, cooling to room temperature, filtering with filter paper, separating the filtrate through a separating funnel, taking the upper ethyl acetate extract, transferring it to a 10 mL volumetric flask, making up the volume to the mark with ethyl acetate, shaking well, and filtering through a 0.22 μm filter membrane to obtain the test solution.

[0076] S3. GC-MS analysis conditions.

[0077] The chromatographic column is SH-Rxi-5SilMS chromatographic column; temperature programming: initial temperature 50 °C, after 2 min, increasing the temperature to 125 °C at a rate of 6 °C min-1, holding for 1 min, increasing the temperature to 175 °C at a rate of 8 °C min-1, holding for 2 min, increasing the temperature to 240 °C at a rate of 6 °C min-1, holding for 3 min, increasing the temperature to 280 °C at a rate of 3 °C min-1, holding for 14 min; injection volume 1 μL; splitless; carrier gas is high-purity He, total flow rate 14 mL / min, column flow rate 1 mL / min, pressure 43.6 kPa; injection temperature 260 °C. EI ionization; electron bombardment energy 70 eV; ion source temperature 230 °C; interface temperature 280 °C; solvent delay time 4 min; mass scanning range m / z: 25 - 550, data acquisition mode is full scan mode.

[0078] S4. Experimental methods and condition optimization. The ultrasonic extraction method was used, and the extraction effects of different polar solvents such as n-hexane, n-butanol, and ethyl acetate on the volatile components of Guben Yanling Pills were investigated respectively, and the best extraction method was selected according to the number of peaks and the magnitude of response values in the chromatogram.

[0079] Method 1: Weigh accurately 1 g of Guben Yanling Pills, add 10 mL of water, soak and grind them. Transfer the suspension to a conical flask, perform ultrasonic extraction for 30 min, then transfer it to a separating funnel. Add another 10 mL of ethyl acetate, shake well and let it stand still. After layering, take the upper ethyl acetate extract and transfer it to a 10-mL volumetric flask. Dilute it to the mark with ethyl acetate, shake well, filter through a 0.22-μm filter membrane, and take the subsequent filtrate for analysis. In actual operation, since the sample is a honey pill with many components, it is very easy to emulsify during extraction and difficult to layer. Therefore, the method was optimized.

[0080] Method 2: Weigh accurately 1 g of Guben Yanling Pills, place them in a conical flask, add 10 mL of water, soak and stir until it becomes a suspension. Perform ultrasonic extraction for 30 min, then cool to room temperature, filter through filter paper. Transfer the filtrate to a separating funnel, add 10 mL of ethyl acetate, shake well and let it stand still. After layering, take the upper ethyl acetate extract and transfer it to a 10-mL volumetric flask. Dilute it to the mark with ethyl acetate, shake well, filter through a 0.22-μm filter membrane, and take the subsequent filtrate for analysis.

[0081] Method 3: Select three different polar solvents, ethyl acetate, n-butanol, and n-hexane, as extraction solvents. Weigh accurately 1 g of Guben Yanling Pills, place them in a conical flask, add 3 mL of water, soak and stir until it becomes a suspension. After adding 10 mL of the above extraction solvent, perform ultrasonic extraction for 30 min, then cool to room temperature, filter. Separate the filtrate through a separating funnel, take the upper organic phase, transfer it to a 10-mL volumetric flask, dilute it to the mark, shake well, filter through a 0.22-μm filter membrane, and take the subsequent filtrate for analysis.

[0082] When using Method 3 for sample treatment, both the number of chromatographic peaks and their response values are greater than those of Method 2. Therefore, Method 3 was selected. When using n-butanol as the extraction solvent, there are many impurities and the resolution of compounds is poor. Therefore, n-butanol is not considered as the extraction solvent. When using n-hexane as the extraction solvent, most of the compounds are saturated aliphatic hydrocarbons, and there are fewer other types of compounds with small peak areas. Therefore, n-hexane is not considered as the extraction solvent. When using ethyl acetate as the extraction solvent, the number of chromatographic peaks obtained is more than that obtained with n-hexane extraction, and the resolution is better. Therefore, ethyl acetate was selected as the extraction solvent. The analysis results are shown in Figures 1 - 4 。

[0083] S5. Selection of the heating program. In order to obtain better analysis efficiency and chromatographic peak resolution, the heating program was optimized in this experiment.

[0084] Program 1: The initial temperature is 50 °C, and after 2 min, the temperature is raised to 280 °C at a rate of 5 °C min-1 and maintained for 10 min. The solvent delay time is 5 min.

[0085] The chromatographic peaks were relatively dispersed 20 minutes ago, so the heating rate was increased; the chromatographic peaks were poorly separated between 35 - 40 minutes, so a constant temperature platform was added; there were components that were not detected at the end of the program, so the detection time was delayed.

[0086] Program 2: The initial temperature is 50°C. After 2 minutes, the temperature is raised to 125°C at a rate of 6°C min-1, held for 1 minute, then raised to 175°C at a rate of 8°C min-1, held for 2 minutes, then raised to 240°C at a rate of 6°C min-1, held for 3 minutes, and then raised to 280°C at a rate of 3°C min-1, held for 14 minutes. The solvent delay time is 4 minutes; the total ion current chromatogram is as Figures 5 - 6 .

[0087] S6. Analysis results of volatile components of Guben Yanling Pills.

[0088] In this section, the volatile components of Guben Yanling Pills were analyzed. To improve the accuracy of analysis and identification, the mass spectra corresponding to each chromatographic peak in the total ion current chromatogram obtained by GC-MS were retrieved through a standard spectral library, and qualitative comparison was carried out by assisting with the retention index method and comparison with reference substances. The relative percentage content of each component was calculated using the peak area normalization method. The retention index method is to detect and record the retention time of individual n-alkanes in the n-alkane mixed reference substance under the experimental analysis conditions, and calculate the retention value of the component to be measured using the two n-alkanes before and after the component to be measured, so as to improve the accuracy of qualitative identification. The linear programmed temperature RI calculation formula is:

[0089]

[0090] Where t x , t n and t n+1 (t n <t x <t n+1 ) are the retention times of the component to be analyzed and the n-alkane mixed standard product with carbon atoms between n and n + 1, respectively.

[0091] In this experiment, the mass spectra of each peak in the total ion current chromatogram were retrieved using the NIST17.L standard spectral library. The top several substances with the highest similarity were selected as the preferred ones and the similarity was not less than 80%. The C7 - C40 n-alkane mixed reference substance was used to calibrate each peak, and the component reference substance was used to confirm the reliability of the identification. Calculate the relative deviation of the measured retention index (RI exp ) and the reference retention index (RI lit ) (RI exp - RI lit ) / RI lit× 100%, and the matching structure with the closer values of the two having a smaller relative deviation is the best identified component. If the relative deviation is greater than 5%, it is considered that there is a large difference in the retention indices between the matched compound and the component to be measured. The n-alkane mixed reference standard was injected using the temperature programming "2.5.1" for the second time to obtain the total ion current chromatogram, as Figure 7 shown.

[0092] The analysis of the volatile components of Guben Yanling Pills is shown in the following table:

[0093] NO. Compound name CAS RT (min) <![CDATA[RI exp > <![CDATA[RI lit > SI Relative content % 1 2 - Furanmethanol 98-00-0 5.520 869 864 88 0.32 2 Benzene, 1,3 - dimethyl - 108-38-3 5.666 875 866 84 0.62 3 Hexanoic acid 142-62-1 8.391 984 974 85 0.07 4 2,5 - Furandicarboxaldehyde 823-82-5 11.020 1085 1079 95 0.19 5 Furylhydroxymethyl ketone 17678-19-2 11.181 1091 1086 92 0.26 6 Maltol 118-71-8 11.904 1119 1110 90 0.50 7 3 - Hydroxy - 2,3 - dihydromaltol 28564-83-2 12.689 1149 1154 81 6.37 8 Dehydromevalonic lactone 2381-87-5 13.235 1170 1169 82 0.20 9 3,5 - Dihydroxy - 2 - methyl - 4 - pyrone 1073-96-7 13.773 1191 1188 83 0.22 10 5 - Hydroxymethylfurfural 67-47-0 14.861 1232 1233 89 6.60 11 2 - Isopropyl - 5 - methylcyclohex - 3 - en - 1 - one - 15.643 1262 1251 84 0.04 12 n - Tridecane 629-50-5 16.659 1300 1300 85 0.13 13 Ylangene 14912-44-8 18.599 1382 1375 90 0.12 14 Vanillin 121-33-5 19.240 1410 1409 83 0.15 15 Caryophyllene 87-44-5 19.724 1434 1417 81 0.27 16 cis - Methylisoeugenol 6380-24-1 20.354 1465 1460 87 0.09 17 β - Chamigrene 18431-82-8 20.943 1494 1479 82 0.11 18 cis - γ - Bisabolene 13062-00-5 21.082 1501 1502 87 0.18 19 .alpha.-Cuprenene 29621-78-1 21.350 1515 1513 90 0.13 20 2,4 - Di - tert - butylphenol 96-76-4 21.411 1518 1513 90 0.73 21 Cadina - 1(10),4 - diene 16729-01-4 21.708 1534 1469 80 0.10 22 cis - γ - Bisabolene 13062-00-5 21.788 1538 1534 84 0.06 23 α - Nerolidol 40716-66-3 22.371 1568 1551 85 0.07 24 .gamma.-Asarone 5353-15-1 22.591 1580 1574 90 0.04 25 Cedrol 77-53-2 23.478 1621 1618 80 0.03 26 .beta.-Asarone 5273-86-9 23.609 1626 1617 88 1.69 27 Isospathulenol 88395-46-4 23.964 1641 1640 90 0.05 28 Ylangenol 41610-69-9 24.319 1656 1666 84 0.17 29 Aplotaxene 10482-53-8 24.618 1669 1675 95 0.38 30 Xanthoxylin 90-24-4 25.033 1686 1675 89 4.48 31 Acoramone 2020-90-8 26.969 1759 1751 90 0.27 32 tetradecanoic acid 544-63-8 27.098 1764 1761 85 0.14 33 Cuparenal 16982-01-7 27.436 1776 1775 88 0.08 34 β - Costol 515-20-8 27.680 1786 1774 88 0.20 35 ( + ) - Isovalencenol 22387-74-2 27.812 1791 1783 81 0.17 36 Thujopsenal 470-41-7 28.319 1811 1724 85 0.53 37 Dehydrosaussurealactone 28290-35-9 29.326 1853 1838 81 0.16 38 Caffeine 58-08-2 29.600 1864 1842 84 0.15 39 1 - Hexadecanol 36653-82-4 30.054 1884 1883 88 0.04 40 Hexadecanoic acid, methyl ester 112-39-0 31.075 1929 1925 91 0.52 41 Pentadecanoic acid 1002-84-2 31.850 1966 1878 89 1.36 42 Butylisobutyl phthalate 17851-53-5 31.998 1973 1973 84 0.21 43 .beta.-Cyclocostunolide 2221-82-1 32.382 1991 1983 84 0.20 44 Palmitic acid, ethyl ester 628-97-7 32.502 1996 1994 89 0.36 45 Dehydrocostuslactone 477-43-0 32.927 2018 2006 90 2.93 46 cis - 1 - Chloro - 9 - octadecene 16507-61-2 34.236 2086 2044 80 0.16 47 Linoleic acid, methyl ester 112-63-0 34.492 2100 2092 87 0.45 48 9 - Octadecenoic acid, methyl ester, (E)- 1937-62-8 34.588 2105 2109 90 0.59 49 Linoleyl acetate 5999-95-1 35.690 2168 2193 89 0.90 50 (E)-9 - Octadecenoic acid ethyl ester 6114-18-7 35.779 2173 2174 84 0.70 51 Octadecanamide 124-26-5 36.052 2188 2220 84 0.18 52 Ethyl 15 - methylheptadecanoate 57274-46-1 36.208 2197 2112 81 0.11 53 n - Eicosane 112-95-8 36.258 2200 2200 85 0.16 54 n - Tricosane 638-67-5 38.023 2300 2300 87 0.35 55 Ferruginol 514-62-5 39.097 2352 2335 85 0.44 56 13 - Docosenamide, (Z)- 112-84-5 39.480 2370 2427 87 0.74 57 n - Tetracosane 646-31-1 40.095 2400 2400 89 0.53 58 p - Cresol, 2,2'-methylenebis[6 - tert - butyl - 119-47-1 40.900 2436 2398 94 0.20 59 11 - Methylpentacosane 15689-71-1 42.339 2500 2529 81 1.73 60 β - Monopalmitin 23470-00-0 42.737 2517 2498 80 6.49 61 n - Hexacosane 630-01-3 44.615 2600 2600 92 1.06 62 13 - Docosenamide, (Z)- 112-84-5 48.945 2788 2625 84 0.83 63 n - Octacosane 630-02-4 49.197 2800 2800 90 1.32 64 n - Nonacosane 630-03-5 51.450 2900 2900 89 1.19 65 n - Triacontane 638-68-6 53.687 3000 3000 88 0.86 66 .gamma.-Tocopherol 7616-22-0 55.339 3065 3046 93 0.21 67 n - Hentriacontane 630-04-6 56.249 3101 3100 86 1.59 68 Vitamin E 59-02-9 57.616 3146 3149 92 0.28 69 .gamma.-Sitosterol 83-47-6 64.217 3330 3290 82 1.38

[0094] S7. Establishment of the GC-MS fingerprint of Guben Yanling Pills; The chemical components of traditional Chinese medicine are complex, and their types and contents are easily affected by the climate of the producing area, the harvesting time, and the processing method. In order to comprehensively reflect the common chemical characteristic components as a whole, the fingerprint of Guben Yanling Pills was established, and the characteristic peaks were shown in the form of a chromatogram like a fingerprint. As a method to control the internal quality of traditional Chinese medicine, the fingerprint of traditional Chinese medicine has been recognized by most countries, fundamentally solving the problem of the quality standardization of traditional Chinese medicine. In this study, the GC / MS fingerprint of Guben Yanling Pills was established to more comprehensively control the batch-to-batch quality of Guben Yanling Pills from an overall perspective;

[0095] S8. Establishment of the fingerprint of Guben Yanling Pills. Twenty batches of Guben Yanling Pills samples produced at different times were prepared for samples and detection, and the chromatogram is as Figures 8 - 17 shown. The total ion current chromatogram was exported in the format of a text file txt, including the retention time, peak area, and intensity information. Using the D9 chromatogram with better peak shape as the reference chromatogram, after multi-point calibration and Mark peak matching, 130 common chromatographic peaks were determined to generate a reference chromatogram. The superimposed chromatogram of the GC-MS fingerprints of twenty batches of Guben Yanling Pills is shown in the figure. The generated reference chromatogram is shown in Figures 28 - 31 .

[0096] S9. Similarity evaluation of the fingerprint of Guben Yanling Pills; The similarities of the 20 batches of samples and the reference chromatogram. The similarities of the fingerprints of each batch of Guben Yanling Pills (big honey pills) and the reference fingerprint chromatogram are all greater than 0.849, and the similarities of the fingerprints of each batch of Guben Yanling Pills (small honey pills) and the reference fingerprint chromatogram are all greater than 0.885, indicating that the chemical compositions of different batches of Guben Yanling Pills of the same dosage form are in good consistency. The similarity evaluation results of a batch of Guben Yanling Pills (big honey pills) are shown in the following table:

[0097] Serial number D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 R D1 1.000 0.914 0.931 0.959 0.869 0.884 0.727 0.872 0.957 0.933 0.960 D2 0.914 1.000 0.966 0.938 0.912 0.903 0.721 0.844 0.899 0.855 0.951 D3 0.931 0.966 1.000 0.922 0.853 0.850 0.725 0.807 0.915 0.905 0.941 D4 0.959 0.938 0.922 1.000 0.918 0.927 0.752 0.892 0.924 0.884 0.970 D5 0.869 0.912 0.853 0.918 1.000 0.941 0.728 0.817 0.822 0.784 0.923 D6 0.884 0.903 0.850 0.927 0.941 1.000 0.756 0.863 0.861 0.814 0.941 D7 0.727 0.721 0.725 0.752 0.728 0.756 1.000 0.814 0.767 0.709 0.849 D8 0.872 0.844 0.807 0.892 0.817 0.863 0.814 1.000 0.901 0.799 0.926 D9 0.957 0.899 0.915 0.924 0.822 0.861 0.767 0.901 1.000 0.954 0.959 D10 0.933 0.855 0.905 0.884 0.784 0.814 0.709 0.799 0.954 1.000 0.917 R 0.960 0.951 0.941 0.970 0.923 0.941 0.849 0.926 0.959 0.917 1.000

[0098] The similarity evaluation results of 10 batches of Guben Yanling Pills (small honey pills) are shown in the following table:

[0099] Serial number X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 R X1 1.000 0.956 0.907 0.780 0.889 0.924 0.957 0.988 0.910 0.874 0.968 X2 0.956 1.000 0.920 0.771 0.917 0.983 0.908 0.955 0.900 0.838 0.965 X3 0.907 0.920 1.000 0.921 0.982 0.916 0.882 0.913 0.979 0.960 0.978 X4 0.780 0.771 0.921 1.000 0.888 0.758 0.790 0.788 0.890 0.914 0.885 X5 0.889 0.917 0.982 0.888 1.000 0.917 0.877 0.904 0.973 0.959 0.969 X6 0.924 0.983 0.916 0.758 0.917 1.000 0.842 0.913 0.897 0.825 0.945 X7 0.957 0.908 0.882 0.790 0.877 0.842 1.000 0.976 0.871 0.867 0.944 X8 0.988 0.955 0.913 0.788 0.904 0.913 0.976 1.000 0.916 0.884 0.973 X9 0.910 0.900 0.979 0.890 0.973 0.897 0.871 0.916 1.000 0.983 0.970 X10 0.874 0.838 0.960 0.914 0.959 0.825 0.867 0.884 0.983 1.000 0.944 R 0.968 0.965 0.978 0.885 0.969 0.945 0.944 0.973 0.970 0.944 1.000

[0100] The above has generally described the present invention in detail. However, based on the present invention, some modifications or improvements can be made, which are obvious to those of ordinary skill in the art. Therefore, modifications or improvements that do not depart from the spirit and idea of the present invention are within the protection scope of the present invention.

Claims

1. A method for establishing the GC-MSMS fingerprint of Guben Yanling Pills, characterized in that: The method for the GC-MSMS fingerprint of Guben Yanling Pills comprises the following steps: S1. Preparation of the instrument and reagents; S2. Preparation of solutions, which includes preparation of mixed alkanes and preparation of the test solution; S3. GC-MS analysis conditions, which include: The chromatographic column is a SH-Rxi-5SilMS chromatographic column; Temperature rising program: Initial temperature is 50°C. After 2 min, the temperature is raised to 125°C at a rate of 6°C·min -1 and held for 1 min, then raised to 175°C at a rate of 8°C·min -1 and held for 2 min, then raised to 240°C at a rate of 6°C·min -1 and held for 3 min, then raised to 280°C at a rate of 3°C·min -1 and held for 14 min; Injection volume is 1 μL; Splitless; The carrier gas is high-purity He, with a total flow rate of 14 mL / min, a column flow rate of 1 mL / min, and a pressure of 43.6 kPa; the injection temperature is 260 °C; EI ionization; electron bombardment energy of 70 eV; ion source temperature of 230 °C; interface temperature of 280 °C; solvent delay time of 4 min; mass scanning range m / z: 25 - 550; The preparation of the mixed alkanes includes: taking 10 μL of a mixed reference substance of normal alkanes C7-C40, adding ethyl acetate to 1 mL to obtain a 1% mixed reference substance solution of normal alkanes; The preparation of the test solution includes: precisely weighing 1 g of Guben Yanling Pills, placing it in a conical flask, adding 3 mL of water, soaking and stirring until it becomes a suspension, adding 10 mL of ethyl acetate, ultrasonically extracting for 30 min, then cooling to room temperature, filtering with filter paper, separating the filtrate through a separating funnel, taking the upper ethyl acetate extract, transferring it to a 10 mL volumetric flask, making up the volume to the mark with ethyl acetate, shaking well, and filtering through a 0.22 μm filter membrane to obtain the test solution.

2. The method for establishing the GC-MSMS fingerprint of Guben Yanling Pills according to claim 1, wherein: The preparation of the instrument includes a GCMS-TQ8040 gas chromatograph-mass spectrometer, a SH-Rxi-5SilMS capillary chromatographic column, an electronic analytical balance, and a KQ-250B ultrasonic cleaner.

3. The method for establishing the GC-MSMS fingerprint of Guben Yanling Pills according to claim 1, wherein: The preparation of the reagents includes n-hexane, ethyl acetate, n-butanol, a mixed reference substance of C7-C40 normal alkanes, and distilled water, and the n-hexane, ethyl acetate, and n-butanol are all of analytical grade.

4. The method for establishing the GC-MSMS fingerprint of Guben Yanling Pills according to claim 1, characterized in that: In the EI ionization, the data acquisition mode is the full scan mode.

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