A HPLC-UV fingerprint spectrum determination method for Fufang Shenqi oral liquid and the measured fingerprint spectrum

Abstract

The present invention proposes a method for determining the HPLC-UV fingerprint spectrum of Fufang Shenqi oral liquid and the measured fingerprint spectrum, which belongs to the field of analytical detection technology. The fingerprint spectrum of Fufang Shenqi oral liquid was determined by HPLC-UV, and a total of 12 common characteristic peaks were generated, which were numbered from left to right. Peak No. 6 was calycosin isoflavone glucoside. The present invention uses the HPLC-UV method to establish a method for determining the qualitative fingerprint spectrum of Fufang Shenqi oral liquid, providing a new experimental basis for the quality control of Fufang Shenqi oral liquid.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for determining a HPLC-UV fingerprint spectrum of a Fufang Shenqi oral liquid and a measured fingerprint spectrum. Background Art

[0002] Fufang Shenqi oral liquid is a prescription given by Professor Huang Yingru, a famous traditional Chinese medicine doctor of Guangxi University of Chinese Medicine. It is an OTC Chinese patent medicine exclusively developed and produced by the pharmaceutical factory of Guangxi University of Chinese Medicine. It has been included in the Chinese Pharmacopoeia for three consecutive editions. It has the effects of replenishing qi and blood, strengthening the spleen and nourishing the heart. It is clinically used to treat symptoms such as fatigue, lack of words, dizziness, thirst and insomnia caused by insufficient qi and blood, and deficiency of both heart and spleen. The preparation is a compound preparation made from three medicinal materials: astragalus, fufang tsao and ginseng. At present, the quality control method of Fufang Shenqi oral liquid is mainly to determine the content of saponins in the preparation by HPLC-ELSD method, except for the thin layer identification of ginseng and fufang tsao in the original standard and the content determination of astragaloside IV by thin layer chromatography scanning. There are few studies and reports on other components in astragalus and fufang tsao in the preparation. The composition of the compound preparation is complex, and the method of controlling the quality of the preparation only by determining the saponins in the preparation does not achieve the purpose of preparation quality control. Fufang Shenqi Oral Liquid has low toxicity and side effects and is highly safe. Over the past 30 years, the product has been widely used in clinical practice due to its mature production process, stable quality, definite efficacy, and very few adverse reactions. It is necessary to more comprehensively control the quality of Fufang Shenqi Oral Liquid.

[0003] Therefore, the present invention uses HPLC-UV fingerprint method to study Fufang Shenqi oral liquid, and provides more comprehensive information for its quality control. Summary of the invention

[0004] The purpose of the present invention is to propose a HPLC-UV fingerprint spectrum determination method for Fufang Shenqi oral liquid and the measured fingerprint spectrum, and to establish a method for determining the qualitative fingerprint spectrum of Fufang Shenqi oral liquid by HPLC-UV method, so as to provide a new experimental basis for the quality control of Fufang Shenqi oral liquid.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The invention provides a HPLC-UV fingerprint spectrum determination method for Fufang Shenqi oral liquid. HPLC-UV is used to determine the fingerprint spectrum of Fufang Shenqi oral liquid, and 12 common characteristic peaks are generated, which are numbered from left to right. Peak No. 6 is calycosin isoflavone glucoside.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. Preparation of test solution: accurately measure Fufang Shenqi oral liquid, place it in a separatory funnel, add water-saturated n-butanol, shake to extract, combine the water-saturated n-butanol solution and transfer it completely to another conical flask, add ammonia reagent and ultrasonicate, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, transfer to a volumetric flask, add methanol to the scale, shake well, centrifuge, filter, and take the filtrate to obtain;

[0009] S2. Chromatographic conditions: Chromatographic column; mobile phase A is acetonitrile, phase B is 0.1-0.3% phosphoric acid solution, gradient elution is used, detection wavelength: 250-270nm; injection volume 2-5μL; column temperature 20-30℃; flow rate 0.5-1.5mL / min;

[0010] S3. Accurately pipette the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure them according to the chromatographic conditions described in step S2.

[0011] As a further improvement of the present invention, the number of extractions in step S1 is 3 times.

[0012] As a further improvement of the present invention, the volume ratio of the Fufang Shenqi oral liquid to the three extractions of water-saturated n-butanol is 15:15-25:15-25:5-15.

[0013] As a further improvement of the present invention, the ammonia reagent in step S1 is a concentrated ammonia solution with a concentration of 25-28 wt%.

[0014] As a further improvement of the present invention, the specification of the chromatographic column in step S2 is 250 mm×4.6 mm, and the particle size is 5 μm.

[0015] As a further improvement of the present invention, the chromatographic conditions in step S2 are: Chromatographic column; mobile phase A is acetonitrile, phase B is 0.2% phosphoric acid solution, detection wavelength: 254nm; injection volume: 5μL; column temperature: 25℃; flow rate: 1mL / min.

[0016] As a further improvement of the present invention, the gradient elution condition in step S2 is:

[0017] Time / min Acetonitrile / % 0.2% Phosphoric acid / % 0 15 85 10 15 85 20 18 82 30 30 70 50 50 50 .

[0018] The present invention further protects a HPLC-UV fingerprint spectrum of Fufang Shenqi oral liquid measured by the above-mentioned HPLC-UV fingerprint spectrum determination method of Fufang Shenqi oral liquid.

[0019] As a further improvement of the present invention, 12 common characteristic peaks are generated, which are numbered from left to right, and peak No. 6 is calycosin isoflavone glucoside.

[0020] The invention has the following beneficial effects: the invention takes Fufang Shenqi oral liquid as the research object, adopts HPLC-UV method to conduct qualitative fingerprint spectrum research on Fufang Shenqi oral liquid, provides method and basis for comprehensive quality control of Fufang Shenqi oral liquid, adopts mobile phase: acetonitrile-phosphoric acid (0.2%); adopts gradient elution method; column temperature: 25°C; wavelength: 254nm; analysis time: 50min; SunFireC18 chromatographic column (250mm×4.6mm, 5μm); injection volume: 5μL, flow rate: 1min / mL.

[0021] By applying the HPLC-UV method to determine the fingerprints of 11 batches of Fufang Shenqi oral liquid, the chromatographic peak of calycosin isoflavone glucoside was used as the reference peak, and the common pattern of the fingerprints of Fufang Shenqi oral liquid was established. By analyzing the fingerprints of 11 batches of Fufang Shenqi oral liquid, a total of 12 common peaks were generated; the spectrum of each medicinal material and the sample spectrum were compared to determine the attribution of each common peak. The data of the fingerprints of 11 batches of Fufang Shenqi oral liquid were processed and analyzed, and the results showed that the similarity of the fingerprints of the 11 batches of samples was above 0.9. The present invention uses the HPLC-UV method to establish a method for determining the qualitative fingerprint of Fufang Shenqi oral liquid, providing a new experimental basis for the quality control of Fufang Shenqi oral liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 It is the HPLC chromatogram of water-saturated n-butanol extraction;

[0024] Figure 2 This is the HPLC chromatogram of ethyl acetate extraction;

[0025] Figure 3 HPLC chromatogram of water-saturated n-butanol extraction once;

[0026] Figure 4 HPLC chromatograms of 2 extractions with water-saturated n-butanol;

[0027] Figure 5 HPLC chromatogram of 3 extractions with water-saturated n-butanol;

[0028] Figure 6 It is a HPLC chromatogram with a mobile phase system of methanol-water;

[0029] Figure 7 It is a HPLC chromatogram with a mobile phase system of methanol-0.1% phosphoric acid aqueous solution;

[0030] Figure 8 It is a HPLC chromatogram with a mobile phase system of methanol-0.2% phosphoric acid aqueous solution;

[0031] Fig. 9 It is a HPLC chromatogram with the mobile phase system being acetonitrile-water system;

[0032] Fig.10 It is a HPLC chromatogram with a mobile phase system of acetonitrile-0.1% phosphoric acid aqueous solution;

[0033] Fig.11 It is a HPLC chromatogram with the mobile phase system being acetonitrile-0.2% phosphoric acid aqueous solution system;

[0034] Fig.12 This is the chromatogram at a column temperature of 25°C;

[0035] Fig.13 This is the chromatogram at a column temperature of 30°C;

[0036] Fig.14 This is the chromatogram at a column temperature of 35°C;

[0037] Fig.15 It is a ZORBAX SB-Aq (4.6×250 mm 5-Micron) column HPLC chromatogram;

[0038] Fig.16 It is a ZORBAX SB-C18 (4.6×250mm 5-Micron) column HPLC chromatogram;

[0039] Fig.17 for (4.6×250mm 5μm) column HPLC chromatogram;

[0040] Fig.18 This is the chromatogram at a flow rate of 0.8 mL / min;

[0041] Fig.19 This is the chromatogram at a flow rate of 1 mL / min;

[0042] Fig. 20 This is the chromatogram at a flow rate of 1.2 mL / min;

[0043] Fig.21 This is the 3D atlas of Fufang Shenqi oral liquid;

[0044] Fig. 22It is HPLC chromatogram at 254nm wavelength;

[0045] Fig.23 It is HPLC chromatogram at 254nm wavelength;

[0046] Fig.24 It is HPLC chromatogram with wavelength of 280nm;

[0047] Fig.25 It is HPLC chromatogram with wavelength of 310nm;

[0048] Fig.26 It is HPLC chromatogram with wavelength of 360nm;

[0049] Fig. 27 This is the HPLC chromatogram of Fufang Shenqi oral liquid;

[0050] Fig.28 This is the HPLC chromatogram of precision No. 1;

[0051] Fig.29 It is the HPLC chromatogram of precision No. 2;

[0052] Fig.30 This is the HPLC chromatogram of precision No. 3;

[0053] Fig.31 It is the precision No. 4 HPLC chromatogram;

[0054] Fig.32 It is the HPLC chromatogram of precision No. 5;

[0055] Fig.33 It is the precision No. 6 HPLC chromatogram;

[0056] Fig.34 is the stability 0h HPLC chromatogram;

[0057] Fig.35 is the stability 2h HPLC chromatogram;

[0058] Fig.36 is the stability 4h HPLC chromatogram;

[0059] Fig.37 is the stability 8h HPLC chromatogram;

[0060] Fig.38 is the stability 12h HPLC chromatogram;

[0061] Fig.39 is the stability 24h HPLC chromatogram;

[0062] Fig.40 is the reproducibility HPLC chromatogram No. 1;

[0063] Fig.41 is the reproducible HPLC chromatogram No. 2;

[0064] Fig.42 is the reproducible HPLC chromatogram No. 3;

[0065] Fig.43 is the reproducible HPLC chromatogram No. 4;

[0066] Fig.44 is the reproducible HPLC chromatogram No. 5;

[0067] Fig.45 is the reproducible HPLC chromatogram No. 6;

[0068] Fig.46 This is the superposition of HPLC fingerprints of 11 batches of Fufang Shenqi oral liquid;

[0069] Fig.47 This is the common pattern of the HPLC fingerprint of Fufang Shenqi oral liquid;

[0070] Fig.48 is the blank solvent HPLC fingerprint;

[0071] Fig.49 is the chromatogram of calycosin glucoside;

[0072] Fig.50 This is the chromatogram of Fufang Shenqi oral liquid;

[0073] Fig.51 Overlapping graph of sample and reference substance, calycosin glucoside;

[0074] Fig.52 This is the HPLC chromatogram of the Fufang Shenqi oral liquid test sample;

[0075] Fig.53 This is the HPLC chromatogram of the test sample of Euonymus fortunei.

[0076] Fig.54 This is the HPLC chromatogram of the Astragalus membranaceus medicinal material test sample;

[0077] Fig.55 This is the HPLC chromatogram of the ginseng medicinal sample. DETAILED DESCRIPTION

[0078] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] The instruments used in the embodiments of the present invention are shown in Table 1:

[0080] Table 1

[0081]

[0082]

[0083] The materials used in the embodiments of the present invention are as follows:

[0084] Ginseng is the dried root of the cultivated product of Panax ginseng CAM ey. of Araliaceae; Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of Leguminosae; Euonymus fortunei is the dried stem and leaf of Euonymus fortunei (Tu rcz.) Hand-M azz. of Celastraceae, all of which were provided by Guangxi University of Traditional Chinese Medicine Pharmaceutical Factory. The finished products of each batch of Fufang Shenqi oral liquid were provided by Guangxi University of Traditional Chinese Medicine Pharmaceutical Factory, see Table 2.

[0085] Table 2

[0086]

[0087] Example 1

[0088] This embodiment provides a method for determining the HPLC-UV fingerprint spectrum of Fufang Shenqi oral liquid, comprising the following steps:

[0089] S1. Preparation of test solution: accurately measure 15 mL of Fufang Shenqi oral liquid, place it in a separatory funnel, add 20 mL, 20 mL, and 10 mL of water-saturated n-butanol, respectively, shake and extract 3 times, combine the water-saturated n-butanol solutions and transfer them completely to another conical flask, add 1 mL of ammonia reagent and ultrasonicate for 5 min, evaporate to dryness in a water bath, add an appropriate amount of methanol to dissolve the residue, transfer it to a 5 mL volumetric flask, add methanol to the scale, shake well, centrifuge, filter with a 0.45 μm microporous filter membrane, and take the filtrate to obtain;

[0090] S2. Chromatographic conditions: Chromatographic column (250 mm × 4.6 mm, particle size 5 μm); mobile phase A is acetonitrile, phase B is 0.2% phosphoric acid solution, detection wavelength: 254 nm; injection volume 5 μL; column temperature 25 ° C; flow rate 1 mL / min;

[0091] The gradient elution conditions are:

[0092] Time / min Acetonitrile / % 0.2% Phosphoric acid / % 0 15 85 10 15 85 20 18 82 30 30 70 50 50 50

[0093] S3. Accurately pipette the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure them according to the chromatographic conditions described in step S2.

[0094] Example 2: Investigation of extraction method

[0095] Accurately measure 3 portions of 15mL of Fufang Shenqi oral liquid (batch number 20190701), add 50mL of water-saturated n-butanol, and extract by extraction, ultrasound, and reflux, respectively, for 30 minutes. Add 1mL of ammonia test solution and ultrasound for 5 minutes, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, transfer to a 5mL volumetric flask, add methanol to the scale, shake well, centrifuge, filter with a 0.45μm microporous filter membrane, and take the filtrate.

[0096] Accurately pipette 5 μL of three test sample solutions respectively, inject them into high performance liquid chromatography for determination, and record the peak area.

[0097] The experimental results show that the test sample is an oral liquid with a high sugar content, and the ultrasound and reflux methods cannot accurately quantify it, so the present invention adopts the extraction method.

[0098] Example 3: Investigation of extraction solvents

[0099] Accurately measure 2 portions of 15mL of Fufang Shenqi oral liquid (batch number 20190701), place them in a separatory funnel, add 50mL of ethyl acetate and water-saturated n-butanol for extraction, transfer the upper solution completely to another conical flask, add 1mL of ammonia test solution and ultrasonicate for 5min, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, centrifuge, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain.

[0100] Accurately pipette 5 μL of each test solution, inject into high performance liquid chromatography, and record the peak area. Figure 1 , Figure 2 .

[0101] Depend on Figure 1 , Figure 2 The results showed that when the extraction solvent was water-saturated n-butanol, the Fufang Shenqi oral liquid had more peaks and higher chromatographic peak heights, so water-saturated n-butanol was selected for extraction.

[0102] Example 4: Investigation of extraction times

[0103] Accurately measure 3 portions of 15mL of Fufang Shenqi oral liquid (batch number 20190701), place the first portion in a separatory funnel, add 50mL of water-saturated n-butanol to extract once; add 25mL and 25mL of water-saturated n-butanol to the second portion, respectively, and extract twice in a separatory funnel; add 20mL, 20mL, and 10mL of water-saturated n-butanol to the third portion, respectively, and extract three times in a separatory funnel, transfer the upper solution completely to another conical flask, add 1mL of ammonia test solution to each of the three samples, ultrasonicate for 5min, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve it, transfer it to a 5mL volumetric flask, add methanol to the scale, shake well, centrifuge, filter with a 0.45μm microporous filter membrane, and take the filtrate to obtain.

[0104] Accurately pipette 5 μL of the test solution, inject it into the high performance liquid chromatograph for determination, and record its peak area. Figure 3 , Figure 4 , Figure 5 , Table 3.

[0105] Table 3

[0106]

[0107] The experimental results show that when the number of water-saturated n-butanol extractions is 3 times, the peak area of ​​each chromatographic peak is higher than that when the number of extractions is 1 time or 2 times, so the number of water-saturated n-butanol extractions is determined to be 3 times.

[0108] Example 5 Determination of Chromatographic Conditions

[0109] 1. Mobile phase selection

[0110] The present invention investigates the methanol-water system, the methanol-phosphoric acid aqueous solution (0.1%, 0.2%) system, the acetonitrile-water system, and the acetonitrile-phosphoric acid aqueous solution (0.1%, 0.2%) system. The results show that the acetonitrile-0.2% phosphoric acid aqueous solution system can achieve better separation of the target components and the obtained chromatogram peak shape is better, so the acetonitrile-0.2% phosphoric acid aqueous solution system is selected for analysis. Figure 6-11 .

[0111] 2. Investigation of column temperature

[0112] The present invention investigated the chromatograms at different column temperatures of 25°C, 30°C, and 35°C. The results showed that the target chromatographic peak separation was better and the peak shape was sharp at 25°C, so the column temperature was selected to be 25°C. Figure 12-14 .

[0113] 3. Selection of chromatographic column

[0114] The present invention investigates the separation effects of different chromatographic columns under the same chromatographic conditions. The chromatographic columns selected are (1) ZORBAX SB-Aq (4.6×250mm 5-Micron), (2) ZORBAX SB-C18 (4.6×250mm 5-Micron), (3) (4.6×250mm 5μm), the same test solution was analyzed under the same chromatographic conditions. The separation and peak shape of each component of the (4.6×250mm 5μm) column are good, so it is selected The results are shown in Figure 15-17 .

[0115] 4. Investigation of different flow rates

[0116] The present invention investigated the chromatograms at different flow rates of 0.8mL / min, 1.0mL / min and 1.2mL / min. The results showed that the chromatographic peak separation and peak shape at a flow rate of 1.0mL / min were better, so the flow rate was selected to be 1.0mL / min. Figure 18-20 .

[0117] 5. Investigation of wavelength

[0118] The present invention adopts HPLC-UV fingerprint method to study the non-saponin components in Fufang Shenqi oral liquid, which are mainly derived from Fufang truncatum medicinal materials. The 3D spectrum of Fufang Shenqi oral liquid is obtained by full wavelength scanning using a DAD detector. Fig.21 , and 5 wavelengths were selected for further detailed comparison, and the chromatograms are shown in Figure 22-26 Comparison of chromatograms at different wavelengths showed that at 254nm, the components corresponding to the Fufangteng medicinal material in the Fufangshenqi oral liquid had more peaks and better ultraviolet absorption, so the wavelength was determined to be 254nm.

[0119] In summary, the chromatographic conditions of the present invention are determined as follows: Chromatographic column (250 mm × 4.6 mm, 5 μm); mobile phase acetonitrile (A)-0.2% phosphoric acid (B), detection wavelength: 254 nm; injection volume 5 μL; column temperature 25 ° C; flow rate 1 mL / min. In order to improve the separation and peak shape of the fingerprint spectrum, the mobile phase gradient was adjusted, and the final elution gradient was determined as shown in Table 4.

[0120] Table 4 Gradient elution mobile phase ratio

[0121] Time / min Acetonitrile / % 0.2% Phosphoric acid / % 0 15 85 10 15 85 20 18 82 30 30 70 50 50 50

[0122] Example 6 Precision Test

[0123] Accurately measure Fufang Shenqi oral liquid (batch number 20190701), prepare the test solution according to the method of step S1 in Example 1, and continuously inject 6 times under the chromatographic conditions in step S2, measure the HPLC chromatogram, and record the precision 1-6 respectively. Take the retention time and peak area of ​​peak No. 6 (calycosin isoflavone glucoside) as a reference, record the retention time and peak area of ​​the common peaks, and calculate that the relative retention time RSD of the 11 common peaks except for calycosin isoflavone glucoside is between 0.08%-0.11%, and the relative peak area RSD is between 0.13%-1.21%. The results show that the instrument has good precision and meets the relevant requirements and regulations for fingerprint determination. Results are shown in Figure 28-33 , Table 5, Table 6.

[0124] Table 5 Common peak precision experiment-relative retention time (n=6)

[0125] Peak number 1 2 3 4 5 6 RSD% 1 0.2665 0.2661 0.2662 0.2667 0.2668 0.2668 0.11 2 0.3599 0.3594 0.3593 0.3601 0.3602 0.3602 0.11 3 0.4091 0.4087 0.4082 0.4090 0.4090 0.4089 0.08 4 0.5575 0.5569 0.5567 0.5578 0.5580 0.5578 0.09 5 0.9707 0.9692 0.9691 0.9710 0.9707 0.9705 0.09 6 1.0000 0.9983 0.9983 1.0003 1.0000 1.0000 0.09 7 1.1462 1.1447 1.1451 1.1475 1.1474 1.1471 0.11 8 1.2861 1.2846 1.2847 1.2876 1.2876 1.2869 0.10 9 1.4139 1.4120 1.4122 1.4154 1.4154 1.4150 0.11 10 1.5501 1.5482 1.5485 1.5518 1.5521 1.5517 0.11 11 1.5802 1.5784 1.5785 1.5820 1.5821 1.5817 0.11 12 1.9895 1.9870 1.9870 1.9917 1.9915 1.9910 0.11

[0126] Table 6 Common peak precision experiment - relative peak area (n = 6)

[0127]

[0128]

[0129] Example 7 Stability Test

[0130] Accurately measure 15 mL of Fufang Shenqi oral liquid (batch number 20190701), prepare the test solution according to the method of step S1 in Example 1, and measure at 0h, 2h, 4h, 8h, 12h, and 24h under the chromatographic conditions in step S2, and record the stability 1-6 respectively. Take the retention time and chromatographic peak area of ​​peak No. 9 (calycosin isoflavone glucoside) as a reference, record the retention time and peak area of ​​the common peaks, and calculate that the relative retention time RSD of the 11 common peaks except for calycosin isoflavone glucoside is between 0.36%-1.91%, and the relative peak area RSD is between 0.52%-2.97%. The results show that Fufang Shenqi oral liquid is stable within 24h and meets the relevant requirements and regulations for fingerprint determination. See Figure 34-39 , Tables 7-8.

[0131] Table 7 Common peak stability experiment-relative retention time (n=6)

[0132] Peak number 0h 2h 4h 8h 12h 24h RSD% 1 0.2656 0.2658 0.2557 0.2660 0.2654 0.2663 1.56 2 0.3573 0.3580 0.3447 0.3588 0.3582 0.3550 1.51 3 0.4087 0.4085 0.3931 0.4090 0.4084 0.4091 1.58 4 0.5547 0.5553 0.5344 0.5563 0.5559 0.5567 1.59 5 0.9681 0.9685 0.9776 0.9699 0.9701 0.9704 0.36 6 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 7 1.1455 1.1450 1.1111 1.1457 1.1456 1.1452 1.23 8 1.2866 1.2857 1.2444 1.2857 1.2850 1.2849 1.32 9 1.4144 1.4133 1.3589 1.4281 1.4129 1.4124 1.72 10 1.5496 1.5484 1.4876 1.5573 1.5745 1.5483 1.91 11 1.5814 1.5799 1.5189 1.5945 1.6020 1.5788 1.87 12 1.9909 1.9890 1.9120 2.0556 2.0122 1.9875 2.34

[0133] Table 8 Common peak stability test - relative peak area (n = 6)

[0134] Peak number 0h 2h 4h 8h 12h 24h RSD% 1 0.0530 0.0548 0.0549 0.0554 0.0553 0.0549 1.61 2 0.2407 0.2413 0.2420 0.2402 0.2393 0.2386 0.52 3 0.0787 0.0790 0.0810 0.0798 0.0793 0.0788 1.11 4 0.3306 0.3355 0.3375 0.3317 0.3308 0.3317 0.85 5 0.2954 0.2967 0.2941 0.2923 0.2980 0.2955 0.66 6 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 7 1.1614 1.1578 1.1932 1.1578 1.1577 1.1571 1.23 8 1.6199 1.6312 1.7298 1.6618 1.7000 1.6707 2.48 9 0.3414 0.3391 0.3420 0.3298 0.3356 0.3348 1.37 10 0.1006 0.1004 0.1003 0.0948 0.0993 0.0995 2.22 11 0.3977 0.3977 0.3961 0.3833 0.3902 0.3938 1.42 12 0.1108 0.1129 0.1102 0.1043 0.1057 0.1083 2.97

[0135] Example 8 Repeatability Test

[0136] Accurately measure 6 portions of Fufang Shenqi oral liquid (batch number 20190701), each about 15 mL, and prepare the test solution according to the method of step S1 in Example 1. Under the chromatographic conditions in step S2, take the retention time and chromatographic peak area of ​​Peak 6 (calycosin isoflavone glucoside) as a reference, record the retention time and peak area of ​​the common peaks, and calculate that the relative retention time RSD of the 11 common peaks except clycosin isoflavone glucoside is between 0.01%-0.07%, and the relative peak area RSD is between 0.05%-1.10%. The results show that this method has good repeatability and meets the requirements of the fingerprint spectrum. Figure 40-45 , Tables 9-10.

[0137] Table 9 Common peak repeatability experiment-relative retention time (n=6)

[0138]

[0139]

[0140] Table 10 Common peak repeatability experiment-relative peak area (n=6)

[0141] Peak number 1 2 3 4 5 6 RSD% 1 0.0548 0.0546 0.0545 0.0544 0.0540 0.0550 0.65 2 0.2543 0.2538 0.2517 0.2515 0.2509 0.2513 0.57 3 0.0753 0.0752 0.0749 0.0744 0.0747 0.0753 0.51 4 0.3321 0.3321 0.3333 0.3335 0.3340 0.3309 0.34 5 0.3094 0.3073 0.3068 0.3038 0.3019 0.3102 1.04 6 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 7 1.1580 1.1583 1.1570 1.1571 1.1571 1.1578 0.05 8 1.7121 1.7124 1.7101 1.7100 1.7053 1.7051 0.19 9 0.3326 0.3322 0.3318 0.3344 0.3303 0.3349 0.51 10 0.0998 0.0996 0.0995 0.0992 0.0992 0.0998 0.26 11 0.3944 0.3943 0.3933 0.3927 0.3923 0.3940 0.22 12 0.1043 0.1037 0.1036 0.1036 0.1031 0.1063 1.10

[0142] Example 9

[0143] 1. Determination of fingerprint spectrum of Fufang Shenqi oral liquid

[0144] Accurately measure 15 mL of each of 11 batches of Fufang Shenqi oral liquid, prepare the test solution according to the method of step S1 in Example 1, and record the chromatogram of each sample under the chromatographic conditions in step S2.

[0145] 2. Establishment of fingerprint of Fufang Shenqi oral liquid

[0146] Based on the HPLC chromatograms of 11 batches of Fufang Shenqi oral liquid samples, the fingerprints of each sample were analyzed using the "Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012.130723 Edition). The results are shown in Fig.46 The HPLC fingerprint common mode of Fufang Shenqi oral liquid was established, and 12 common characteristic peaks were generated. Fig.47 Among them, peak 6 has better separation, higher chromatographic response value and suitable retention time, so it is selected as the reference peak.

[0147] 3. Blank test

[0148] Under the chromatographic conditions in step S2 of Example 1, 5 μL of methanol was accurately measured and injected into the HPLC. The results showed that the reagent had no impurity interference with the sample. (See Fig.48 ).

[0149] 4. HPLC spectrum determination of reference substance and characteristic peak attribution analysis

[0150] The solution of the reference substance of calycosin isoflavone glucoside was accurately measured, and the chromatogram was measured and analyzed under the chromatographic conditions in step S2 of Example 1 and recorded, and compared with the common peaks in the sample fingerprint spectrum. The results showed that the retention time of the characteristic peak No. 6 in the common mode of the fingerprint spectrum was consistent with the retention time of the reference substance of calycosin isoflavone glucoside, so it was confirmed that the peak No. 6 in the spectrum of Fufang Shenqi oral liquid was calycosin isoflavone glucoside. The results are shown in Figure 49-51 .

[0151] 5. HPLC chromatogram determination of main medicinal materials and attribution of characteristic peaks

[0152] Take appropriate amount of Astragalus and Folium Fangzi respectively, extract them according to the process, prepare the test solution according to the method of step S1 in Example 1, and record the chromatogram of each sample. Compare the fingerprint of the main medicinal materials with the reference spectrum, and attribute the common peaks in the fingerprint of Folium Fangzi oral liquid to the medicinal materials. Confirm that peaks 1, 4, 6, 9, 11, and 12 of the fingerprint are the medicinal material components of Astragalus, peaks 2, 3, 4, 5, 7, and 10 are the medicinal material components of Folium Fangzi, and peaks 4 and 11 are the medicinal material components of Ginseng. Results are shown in Figure 52-55 .

[0153] 6. Common peak analysis

[0154] By analyzing the chromatograms of 11 batches of samples, chromatographic peaks with good repeatability and obvious characteristics were selected as common peaks, and a total of 12 common peaks were marked. Peak 6 (calycosin isoflavone glucoside) with a relatively large peak area, a medium retention time and good stability was selected as the reference peak in the sample spectrum, and the relative peak areas and relative retention times of the remaining 11 peaks except for the calycosin isoflavone glucoside were calculated. The RSD of the relative retention time of the 11 batches of samples was less than 0.77%, which was in line with relevant regulations. The RSD of the relative peak area was 23.87% at the minimum and 62.92% at the maximum, which may be related to the different origins of the raw medicinal materials of the preparations or the differences in the preparation production process. Detailed results are shown in Tables 11-12.

[0155] Table 11 Relative peak area

[0156] batch number 1 2 3 4 5 6 7 8 9 10 11 12 1 0.116 0.413 0.073 0.343 0.342 1.000 1.355 0.941 0.386 0.126 0.601 0.149 2 0.094 0.356 0.073 0.398 0.335 1.000 1.237 1.928 0.351 0.120 0.628 0.144 3 0.124 0.282 0.071 0.405 0.361 1.000 1.297 1.569 0.242 0.096 0.599 0.128 4 0.148 0.320 0.065 0.430 0.305 1.000 1.078 1.770 0.297 0.113 0.566 0.148 5 0.215 0.272 0.093 0.658 0.220 1.000 0.720 1.586 0.305 0.109 0.689 0.162 6 0.218 0.334 0.116 0.687 0.246 1.000 0.783 0.924 0.281 0.117 0.654 0.150 7 0.216 0.296 0.138 0.624 0.375 1.000 1.289 2.329 0.269 0.154 0.847 0.192 8 0.317 0.931 0.253 0.813 0.948 1.000 4.132 2.261 0.161 0.399 1.896 0.403 9 0.235 0.877 0.187 0.668 0.827 1.000 2.716 4.864 0.189 0.267 1.545 0.318 10 0.154 0.449 0.124 0.558 0.560 1.000 1.676 1.625 0.252 0.171 1.022 0.234 11 0.056 0.247 0.078 0.337 0.302 1.000 1.157 1.689 0.326 0.104 0.394 0.110 RSD 43.78 55.28 50.95 30.20 54.79 0.000 62.92 54.39 23.87 57.22 53.81 46.50

[0157] Table 12 Relative retention time

[0158] serial number 1 2 3 4 5 6 7 8 9 10 11 12 1 0.265 0.355 0.405 0.547 0.970 1.000 1.140 1.277 1.401 1.535 1.566 1.971 2 0.267 0.359 0.408 0.556 0.971 1.000 1.147 1.286 1.414 1.550 1.580 1.990 3 0.269 0.361 0.411 0.558 0.970 1.000 1.152 1.292 1.445 1.582 1.611 2.002 4 0.266 0.357 0.407 0.564 0.967 1.000 1.152 1.295 1.427 1.564 1.596 2.011 5 0.266 0.358 0.408 0.556 0.969 1.000 1.146 1.287 1.415 1.550 1.582 1.991 6 0.268 0.360 0.409 0.556 0.970 1.000 1.145 1.286 1.412 1.547 1.578 1.987 7 0.267 0.358 0.408 0.555 0.971 1.000 1.147 1.286 1.415 1.550 1.581 1.991 8 0.268 0.359 0.409 0.557 0.972 1.000 1.148 1.288 1.415 1.556 1.585 1.992 9 0.267 0.359 0.409 0.557 0.972 1.000 1.147 1.287 1.416 1.551 1.582 1.993 10 0.268 0.360 0.409 0.556 0.973 1.000 1.152 1.291 1.418 1.554 1.585 1.995 11 0.266 0.359 0.409 0.557 0.968 1.000 1.147 1.288 1.416 1.552 1.583 1.994 RSD 0.44 0.45 0.37 0.70 0.18 0.00 0.31 0.35 0.77 0.75 0.71 0.49

[0159] 7. Similarity analysis

[0160] According to the "Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012.130723 Edition), the fingerprints of 11 batches of Fufang Shenqi Oral Liquid were processed and calculated in the test data. The results showed that the similarity of the 11 batches of Fufang Shenqi Oral Liquid was above 0.9, with good similarity, which met the analysis requirements of the fingerprint. The results are shown in Tables 13-14.

[0161] Table 13 Original data table of similarity calculation of Fufang Shenqi oral liquid

[0162]

[0163]

[0164] Table 14 Similarity evaluation results of 11 batches of Fufang Shenqi oral liquid

[0165] sample Similarity sample Similarity 20170901 0.954 20180104 0.989 20170904 0.995 20180105 0.913 20171001 0.997 20190201 0.962 20171103 0.994 20190303 0.986 20180101 0.971 20190701 0.989 20180102 0.947

[0166] The present invention selects the most suitable extraction method by examining the extraction method (ultrasound method, reflux method, extraction method), extraction solvent (ethyl acetate, water-saturated n-butanol), extraction times (1, 2, 3), etc. It is finally determined that the best extraction method is the extraction method, the extraction solvent is water-saturated n-butanol, and the extraction times are 3 times. By testing different mobile phase systems (methanol-water system, methanol-phosphoric acid aqueous solution (0.1%, 0.2%) system, acetonitrile-water system, acetonitrile-phosphoric acid aqueous solution (0.1%, 0.2%) system); different column temperatures (25°C, 30°C, 35°C); different chromatographic columns ((1) ZORBAX SB-Aq (4.6×250mm 5-Micron), (2) ZORBAX SB-C18 (4.6×250mm 5-Micron), (3) (4.6×250mm 5μm)); different flow rates (0.8mL / min, 1.0mL / min, 1.2mL / min), etc., and finally the chromatographic conditions were determined to be acetonitrile-0.2% phosphoric acid aqueous solution system, column temperature 25°C, Chromatographic column, flow rate 1mL / min. After scanning with DAD detector, the components corresponding to Fufangteng medicinal materials in Fufangshenqi oral liquid have more peaks at 254nm and better ultraviolet absorption, so the wavelength is selected as 254nm.

[0167] The methodological investigation and verification results of the present invention show that the precision, repeatability and stability are good, and all meet the relevant requirements and regulations for fingerprint determination. Finally, the common mode spectrum of the HPLC fingerprint spectrum of Fufang Shenqi oral liquid was established. Through reference substances and related medicinal materials, 12 common peaks were determined, among which peaks 1, 4, 6, 9, 11, and 12 are components of Astragalus medicinal materials, peaks 2, 3, 4, 5, 7, and 10 are components of Fufang Thunbergia medicinal materials, and peaks 4 and 11 are components of ginseng medicinal materials. However, at present, only the chemical composition of peak 6 is determined to be calycosin isoflavone glucoside, and the chemical composition of other common peaks needs further study. The relative retention time of the 12 common peaks of 11 batches of Fufang Shenqi oral liquid is relatively stable, but the relative peak area difference of different batches is too large, which may be related to the different origins of the medicinal materials in the preparations or the differences caused during the processing and production process.

[0168] The research on the quality control of Fufang Shenqi oral liquid mainly focuses on saponin components, which mainly come from the two medicinal materials of Astragalus and Ginseng in the prescription; while there are few reports on the characteristic components of Fufang Shenqi medicinal materials in Fufang Shenqi oral liquid. The present invention attempts to use HPLC-UV fingerprint spectrum method to study the non-saponin components in Fufang Shenqi oral liquid. 11 different batches of Fufang Shenqi oral liquid were selected to determine 12 common peaks, of which 6 characteristic peaks belong to Fufang Shenqi medicinal materials. The similarity of the established fingerprint spectrum is higher than 0.9. The method is accurate and reliable, and can provide a reference for establishing a more comprehensive quality control method for Fufang Shenqi oral liquid. However, due to limited time, the present invention only established a fingerprint spectrum determination method for Fufang Shenqi oral liquid, and determined the medicinal materials to which the common peaks belonged by comparing with the medicinal material spectrum, but only the chemical composition of one common peak has been determined, and the chemical composition of other common peaks needs further study.

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the HPLC-UV fingerprint spectrum of Fufang Shenqi oral liquid, characterized in that: The fingerprint of Fufang Shenqi oral liquid was determined by HPLC-UV, and 12 common characteristic peaks were generated, which were numbered from left to right. Peak 6 was calycosin isoflavone glucoside; The following steps are involved: S1. Preparation of test solution: accurately measure Fufang Shenqi oral liquid, place it in a separatory funnel, add water-saturated n-butanol, shake to extract, combine the water-saturated n-butanol solution and transfer it completely to another conical flask, add ammonia reagent and ultrasonicate, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, transfer to a volumetric flask, add methanol to the scale, shake well, centrifuge, filter, and take the filtrate to obtain; S2. Chromatographic conditions: C18 chromatographic column; mobile phase A is acetonitrile, phase B is 0.1-0.3% phosphoric acid solution, gradient elution is used, detection wavelength is 250-270nm; injection volume is 2-5μL; column temperature is 20-30℃; flow rate is 0.5-1.5mL / min; the specification of the chromatographic column is 250mm×4.6mm, and the particle size is 5μm; S3. Precision draw reference solution and test solution, respectively, injected into a high performance liquid chromatograph, according to the chromatographic conditions of step S2, to obtain; The gradient elution conditions were: 。 2. The HPLC-UV fingerprint analysis method of Fufang Shenqi oral liquid according to claim 1, characterized in that: The number of extractions in step S1 is 3 times.

3. The HPLC-UV fingerprint analysis method of Fufang Shenqi oral liquid according to claim 1, characterized in that: The volume ratio of the Fufang Shenqi oral liquid to the three extractions of water-saturated n-butanol is 15:15-25:15-25:5-15.

4. The HPLC-UV fingerprint analysis method of Fufang Shenqi oral liquid according to claim 2, characterized in that: The ammonia reagent in step S1 is a concentrated ammonia solution with a concentration of 25-28 wt%.

5. The HPLC-UV fingerprint analysis method of Fufang Shenqi oral liquid according to claim 2, characterized in that: The chromatographic conditions in step S2 are: C18 chromatographic column; mobile phase A is acetonitrile, phase B is 0.2% phosphoric acid solution, detection wavelength: 254nm; injection volume 5μL; column temperature 25℃; flow rate 1mL / min.

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