CN112816597B - Standard fingerprint spectrum and quality consistency evaluation method of standard preparation mode of gardenia golden flower pills - Google Patents

Standard fingerprint spectrum and quality consistency evaluation method of standard preparation mode of gardenia golden flower pills Download PDF

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CN112816597B
CN112816597B CN202110204451.7A CN202110204451A CN112816597B CN 112816597 B CN112816597 B CN 112816597B CN 202110204451 A CN202110204451 A CN 202110204451A CN 112816597 B CN112816597 B CN 112816597B
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孙国祥
兰丽丽
孙万阳
张晶
巩丹丹
陈金玉
王刚
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Shenyang Pharmaceutical University
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Abstract

The invention relates to the field of traditional Chinese medicine analysis, and discloses a standard fingerprint and quality consistency evaluation method of a standard preparation mode of gardenia golden flower pills. The invention realizes the overall quality control mode and method of the gardenia golden flower pills, establishes the standard fingerprint control mode of the standard preparation of the gardenia golden flower pills, is the standard fingerprint of the standard preparation formed on the basis of the quantitative fingerprint research of 32 batches of gardenia golden flower pill preparations of 14 manufacturers, and can be used for controlling the overall quality of the gardenia golden flower pills; the invention is different from any multi-index quantitative quality control method and Chinese pharmacopoeia fingerprint spectrum inspection method, and uses macro quantitative similarity to control the amplitude range and quality consistency of the total effective substances of the gardenia golden flower pill preparation.

Description

Standard fingerprint spectrum and quality consistency evaluation method of standard preparation mode of gardenia golden flower pills
Technical Field
The invention relates to the field of traditional Chinese medicine analysis, in particular to a standard fingerprint spectrum and quality consistency evaluation method of a standard preparation mode of gardenia golden flower pills.
Background
The traditional Chinese medicine is a treasure of thousands of years of culture of Chinese nationalities, has a long history, and through thousands of years of medical practice, the safety and the effectiveness of the traditional Chinese medicine have been verified, but the traditional Chinese medicine has not been universally accepted internationally, and the key problem of quality control must be solved in order to lead the traditional Chinese medicine to the world. However, the traditional Chinese medicine standard system is still imperfect at present, and the quality control must be carried out from the overall macroscopic perspective and the multidimensional quantitative perspective to improve the quality standard of the traditional Chinese medicine in order to develop the traditional Chinese medicine into one of the main bodies of the worldwide medicinal culture. Under the existing conditions, a single chemical component analysis method is not suitable for a traditional Chinese medicine system with complex components, and an effective means for solving the problem is to establish a new quality control method and a standard fingerprint control mode of a standard preparation and strengthen the scientization and standardization of traditional Chinese medicine quality evaluation. At present, the fingerprint spectrum technology established on the basis of the research of Chinese medicine ingredient systems is an effective means for evaluating the quality consistency of Chinese medicines by applying the analysis means of modern instruments. The quality of the medicinal materials is controlled according to the characteristics of the quality of the medicinal materials, such as species, form, producing area, harvesting period and the like of the medicinal materials, and then the quality of the preparation can be controlled well.
The gardenia golden flower pill relates to 8 raw medicinal materials, cannot identify the components of each medicinal material one by one, but can intensively and quantitatively control index components capable of reflecting the quality of the medicinal materials, and can establish a quantitative fingerprint control method of a standard preparation of the medicinal materials. The precision of instruments, the repeatability of methods and the stability of samples are examined to ensure the reliability of the fingerprint spectrum establishment method and the quality characteristic attribute of the gardenia golden pills, and finally the established fingerprint spectrum can effectively and comprehensively reflect the quality and the drug effect of the traditional Chinese medicine preparation, so that a great number of technical problems need to be overcome.
Because the traditional Chinese medicine has the characteristic of complexity, the inventor develops a standard fingerprint spectrum and a quality consistency evaluation method of a standard preparation of the gardenia golden flower pill after overcoming the related technical problems.
Disclosure of Invention
Based on the problems, the invention provides a standard fingerprint and quality consistency evaluation method of a standard preparation mode of a gardenia golden flower pill, the invention uses macro quantitative similarity to control the amplitude range and quality consistency of total effective substances of the gardenia golden flower pill preparation, the invention realizes the integral quality control mode and method of the gardenia golden flower pill, establishes the standard fingerprint control mode of the standard preparation of the gardenia golden flower pill, and can be used for controlling the overall quality of the gardenia golden flower pill.
In order to solve the technical problems, the invention provides a standard fingerprint spectrum and quality consistency evaluation method of a standard preparation mode of gardenia golden flower pills, which comprises the following steps:
s1: preparing test solution
Weighing 1-5g of ground gardenia golden flower pill powder, putting the gardenia golden flower pill powder into a 25ml measuring bottle, and respectively carrying out the following treatment: placing the mixture into a 25ml measuring flask, precisely adding 25ml of an extraction solvent, wherein the extraction solvent is 80% methanol aqueous solution containing 0.5% phosphoric acid, precisely weighing, carrying out ultrasonic treatment at 45 ℃ for 10-40min, the ultrasonic treatment power is 240W, the frequency is 40KHz, standing to room temperature, precisely weighing, and supplementing the loss weight by using the extraction solvent; secondly, refluxing and extracting the gardenia golden flower pill powder for 2 times by using 25ml of the extraction solvent, wherein the refluxing and extracting time for two times is 1 hour and 40 minutes respectively, then merging the filtrates obtained by the refluxing and extracting for two times, and fixing the volume to 50 ml;
shaking up the first and the second to be tested respectively, filtering the mixture by a 0.45 mu m filter membrane, and taking the subsequent filtrate to obtain first and second test solution which are obtained by different extraction modes;
s2: preparation of Dual reference solutions
Preparing a mixed reference solution with the baicalin reference substance concentration of 300-;
s3: preparation of Mixed control solutions
Preparing a mixed reference solution, wherein the mixed reference solution comprises the following components in concentration: baicalein 650 mu g/ml, wogonin 160 mu g/ml, baicalin 1200 mu g/ml, wogonoside 300 mu g/ml, geniposide 450 mu g/ml, isochlorogenic acid A50 mu g/ml, berberine 260 mu g/ml, emodin 30 mu g/ml, chrysophanol 100 mu g/ml, rhein 80 mu g/ml, physcion 270 mu g/ml, aloe-emodin 30 mu g/ml;
s4: chromatographic conditions
A chromatographic column: COSMOSIL 5C18-MS-II, with column length 250mm, inner diameter 4.6mm, particle diameter 5 μm, and filler being octadecylsilane chemically bonded silica;
column temperature: 25-45 ℃; the sample amount is 1-50 mul;
mobile phase: an aqueous phase A: 0.2% phosphoric acid solution containing 0.005mol/L sodium heptanesulfonate; and (3) organic phase B: acetonitrile-methanol mixed solution with the volume ratio of 9: 1; gradient elution, elution procedure was as follows:
Figure GDA0003751021010000021
Figure GDA0003751021010000031
detection wavelength: setting detection wavelengths to be 220nm, 238nm, 254nm, 278nm and 326nm, and simultaneously collecting a 190-400 nm three-dimensional chromatogram by using the DAD;
s5: fingerprint spectrum checking method
The number of common fingerprint peaks of the test sample under the detection wavelengths of 220nm, 238nm, 254nm, 278nm and 326nm is respectively 30, 30 and 24, wherein baicalin is used as a reference peak for identification and recognition of the common fingerprint peaks, and the baicalin peak and the baicalein peak are used as double-reference quantitative measurement correction peaks, which are respectively calculated according to the peak area and retention time of the common fingerprint peaks, the macro qualitative similarity of the fingerprint spectrum of the test sample is not less than 0.90, and the macro quantitative similarity is 80-120%; wherein the peak area ratios of No. 15 fingerprint peak at two wavelengths of 220nm and 254nm, 238nm and 278nm, and 278nm and 254nm are respectively 4.0-5.0, 4.68-5.72 and 5.4-6.6;
s6: index component quantitative method
The method can be used for quantitatively measuring 12 compound components in the gardenia golden flower pills by using a standard curve method, and the linearity, range, detection limit and quantitative limit of the 12 compound components in the gardenia golden flower pills are as follows:
Figure GDA0003751021010000032
the method can also be used for quantitatively measuring 12 compound components in the gardenia golden flower pill by utilizing a one-line multiple evaluation method, the S compound is any one of baicalin, berberine, baicalein, wogonin and chrysophanol, the concentrations of other 11 compounds can be obtained by measuring the peak area of the S compound with known concentration during analysis, and the calculation formula is as follows:
Figure GDA0003751021010000033
wherein A is s Is a measurement of the peak area of the compound, C s To determine the concentration of the compound, A i Peak area of compound to be evaluated, C i The concentrations of other 11 compounds to be tested which are remained after selecting one of the five compounds are determined; relative correction factor values f for five of the compounds si The following were used:
peak number S compound No. P7 P12 P15 P19 P20 P22 P23 P24 P26 P28 P29 P30
Peak 15 Baicalin f si 1.44 0.91 1.00 0.80 0.43 0.68 0.30 0.51 0.53 0.24 0.29 0.53
Peak 20 Berberine f si 3.34 2.11 2.32 1.85 1.00 1.58 0.70 1.20 1.22 0.56 0.67 1.23
Peak 22 Baicalein f si 1.45 0.92 1.01 0.80 0.43 1.00 0.30 0.52 0.53 0.24 0.29 0.53
Peak 24 Wogonin f si 2.73 1.73 1.90 1.51 0.82 1.29 0.57 1.00 1.00 0.46 0.55 1.00
Peak 29 Chrysophanol f si 5.00 3.16 3.48 2.77 1.50 2.37 1.05 1.79 1.83 0.84 1.00 1.83
Further, the detection wavelengths in step S5 include, but are not limited to, 220nm, 238nm, 254nm, 278nm and 326nm, and include any wavelength within 190-400 nm of DAD acquisition for fingerprint detection.
Further, the number of common fingerprint peaks in step S5 includes, but is not limited to, 30, and 24, and fingerprint peaks whose signal is more than 5 times noise can be regarded as valid fingerprint peaks.
Compared with the prior art, the invention has the beneficial effects that: the invention realizes the whole quality control mode and method of the gardenia golden flower pill (watered pill), establishes the standard fingerprint control mode of the standard preparation of the gardenia golden flower pill, is the standard fingerprint of the standard preparation formed on the basis of the quantitative fingerprint research of 32 batches of gardenia golden flower pill preparations of 14 manufacturers, and can be used for controlling the overall quality of the gardenia golden flower pill; the invention is different from any multi-index quantitative quality control method and Chinese pharmacopoeia fingerprint spectrum inspection method, and uses macro quantitative similarity to control the amplitude range and quality consistency of the total effective substances of the gardenia golden flower pill preparation.
Drawings
FIG. 1 is an HPLC standard fingerprint (obtained by detecting 80 mg/ml of test solution) of a standard preparation of a gardenia golden flower pill (watered pill) at 220nm according to an embodiment of the invention;
FIG. 2 is a standard fingerprint of a 238nm detection standard preparation (obtained by detecting 80 mg/ml of a test solution) in an embodiment of the present invention;
FIG. 3 is a standard fingerprint of a standard preparation measured at 254nm (obtained by measuring 80 mg/ml of test solution) according to an embodiment of the present invention;
FIG. 4 is a standard fingerprint of a 278nm assay standard formulation (obtained by assaying test solution at 80 mg/ml) according to an embodiment of the present invention;
FIG. 5 is a standard fingerprint of a standard preparation measured at 326nm (obtained by measuring 80 mg/ml of test solution) according to an embodiment of the present invention;
FIG. 6 is a graph of the first four wavelengths of a fingerprint detected at five wavelengths according to an embodiment of the present invention having 30 fingerprint peaks in common;
FIG. 7 is a fingerprint of a standard preparation measured at a wavelength of 326nm according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b):
the embodiment provides a standard fingerprint spectrum and quality consistency evaluation method of a standard preparation mode of gardenia golden flower pills (water pills), which comprises the following steps:
s1: preparing test solution
Taking 5-30g of gardenia golden flower pills, grinding, weighing 1-5g of ground gardenia golden flower pill powder, putting the gardenia golden flower pill powder into a 25ml measuring bottle, and respectively carrying out the following treatment: placing the mixture into a 25ml measuring flask, precisely adding 25ml of extraction solvent, wherein the extraction solvent is 80% (ml/ml) methanol aqueous solution containing 0.5% (ml/ml) phosphoric acid, precisely weighing, carrying out ultrasonic treatment at 45 ℃ for 10-40min, the ultrasonic treatment power is 240W, the frequency is 40KHz, standing to room temperature, precisely weighing, and supplementing the loss weight by using the extraction solvent; secondly, refluxing and extracting the gardenia golden flower pill powder for 2 times by using 25ml of the extraction solvent, wherein the refluxing and extracting time for two times is 1 hour and 40 minutes respectively, then merging the filtrates obtained by the refluxing and extracting for two times, and fixing the volume to 50 ml;
shaking up the first and the second to filter with 0.45 μm filter membrane, and collecting the subsequent filtrate to obtain the first and the second test solution obtained by different extraction methods;
s2: preparation of Dual reference solutions
Preparing a mixed reference solution with the baicalin reference substance concentration of 300-;
s3: preparation of Mixed control solutions
Preparing a mixed reference solution, wherein the mixed reference solution comprises the following components in concentration: 650 mu g/ml of baicalein, 160 mu g/ml of wogonin, 1200 mu g/ml of baicalin, 300 mu g/ml of wogonoside, 450 mu g/ml of geniposide, 50 mu g/ml of isochlorogenic acid A, 260 mu g/ml of berberine, 30 mu g/ml of emodin, 100 mu g/ml of chrysophanol, 80 mu g/ml of rhein, 270 mu g/ml of physcion and 30 mu g/ml of aloe-emodin;
s4: chromatographic conditions
A chromatographic column: COSMOSIL 5C18-MS-II, with column length 250mm, inner diameter 4.6mm, particle diameter 5 μm, and filler being octadecylsilane chemically bonded silica;
column temperature: 25-45 ℃; the sample amount is 1-50 μ l (the sample amount is determined with the maximum chromatographic peak absorbance not more than 1);
mobile phase: an aqueous phase A: 0.2% phosphoric acid solution containing 0.005mol/L sodium heptanesulfonate; and (3) organic phase B: acetonitrile-methanol mixed solution with the volume ratio of 9: 1; gradient elution, elution procedure was as follows:
time (min) Mobile phase A (%) Mobile phase B (%)
0～10 94→79 6→21
10～30 79→65 21→35
30～55 65→45 35→55
55～65 45→18 55→82
65～70 18→15 82→85
70～75 15 85
75～80 15→94 85→6
Detection wavelength: setting detection wavelengths to be 220nm, 238nm, 254nm, 278nm and 326nm, and simultaneously collecting a 190-400 nm three-dimensional chromatogram by using the DAD;
s5: fingerprint spectrum checking method
Referring to the attached drawings 1, 2, 3, 4 and 5, the number of common fingerprint peaks of the test sample under the detection wavelengths of 220nm, 238nm, 254nm, 278nm and 326nm is respectively 30, 30 and 24, wherein baicalin is used as a reference peak for identification and identification of the common fingerprint peaks, a baicalin peak and a baicalein peak are used as double-reference quantitative measurement correction peaks, and are respectively calculated according to the peak area and retention time of the common fingerprint peaks, the macro qualitative similarity of the fingerprint of the test sample is not lower than 0.90, and the macro quantitative similarity is between 80% and 120%; wherein the peak area ratios of No. 15 fingerprint peak at two wavelengths of 220nm and 254nm, 238nm and 278nm, and 278nm and 254nm are respectively 4.0-5.0, 4.68-5.72 and 5.4-6.6; the detection wavelength of the embodiment includes, but is not limited to, 220nm, 238nm, 254nm, 278nm and 326nm, and any wavelength within 190-400 nm of DAD acquisition is used for fingerprint detection; the number of common fingerprint peaks in the present embodiment includes, but is not limited to, 30, and 24, and fingerprint peaks whose signal is more than 5 times of noise can be regarded as valid fingerprint peaks;
the fingerprint data (obtained by measuring 80 mg/ml of test solution) of the standard preparation measured at 220nm in this example are as follows:
Figure GDA0003751021010000061
Figure GDA0003751021010000071
the fingerprint data (obtained by measuring the test solution at 80 mg/ml) of the standard preparation at 238nm are as follows:
peak number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Retention time/min 3.0 5.0 5.4 9.5 9.8 10.4 11.6 12.1 13.6 15.6 16.8 18.3 20.0 22.5 23.7
Peak area 316.5 151.2 251.3 329.6 1073.5 900.1 4323.6 630.4 1183.9 768.5 336.7 732.6 744.4 330.6 10736.5
Relative retention time 0.13 0.21 0.23 0.40 0.41 0.44 0.49 0.51 0.57 0.66 0.71 0.77 0.84 0.95 1.00
Relative peak area 0.03 0.01 0.02 0.03 0.10 0.08 0.40 0.06 0.11 0.07 0.03 0.07 0.07 0.03 1.00
Peak number 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Retention time/min 26.6 28.1 29.4 29.9 32.1 34.5 35.5 41.4 44.1 44.9 45.8 46.3 57.0 63.6 66.7
Peak area 586.3 1483.2 581.9 2042.3 4970.1 222.9 5351.7 196.1 1294.4 546.9 592.1 473.4 264.1 1031.3 230.8
Relative retention time 1.12 1.19 1.24 1.26 1.35 1.46 1.50 1.74 1.86 1.89 1.93 1.95 2.40 2.68 2.81
Relative peak area 0.05 0.14 0.05 0.19 0.46 0.02 0.50 0.02 0.12 0.05 0.06 0.04 0.02 0.10 0.02
The standard formulation fingerprint data (obtained by measuring 80 mg/ml of test solution) measured at 254nm is as follows:
peak number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Retention time/min 3.0 5.0 5.4 9.5 9.8 10.4 11.6 12.1 13.6 15.6 16.8 18.3 20.0 22.5 23.7
Peak area 376 139.4 521.9 350.2 813 772.2 2756.7 725.4 616 945.4 392.6 441.6 409.8 443.3 11112.3
Relative retention time 0.13 0.21 0.23 0.40 0.41 0.44 0.49 0.51 0.57 0.66 0.71 0.77 0.84 0.95 1.00
Relative peak area 0.03 0.01 0.05 0.03 0.07 0.07 0.25 0.07 0.06 0.09 0.04 0.04 0.04 0.04 1.00
Peak number 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Retention time/min 26.6 28.1 29.4 29.9 32.1 34.5 35.5 41.3 44.1 44.9 45.8 46.3 57.0 63.6 66.7
Peak area 717.9 1896.3 822.4 2970.6 3904.7 273.2 6793.3 328.6 1563.5 614 463.7 749.1 377.7 1724 283.8
Relative retention time 1.12 1.19 1.24 1.26 1.35 1.46 1.50 1.74 1.86 1.89 1.93 1.95 2.40 2.68 2.81
Relative peak area 0.06 0.17 0.07 0.27 0.35 0.02 0.61 0.03 0.14 0.06 0.04 0.07 0.03 0.16 0.03
The standard formulation fingerprint data (obtained by measuring 80 mg of test solution per ml of test solution) measured at 278nm is as follows:
Figure GDA0003751021010000072
Figure GDA0003751021010000081
referring to FIG. 6, when the above detection is performed at 220nm, 238nm, 254nm and 278nm, the total fingerprint peaks are 30 (326nm), and the identification peaks are 12 fingerprint peaks, wherein: 7. geniposide; 12. isochlorogenic acid A; 15. baicalin; 19. wogonoside; 20. berberine; 22. baicalein; 23. aloe-emodin; 24. wogonin; 26. rhein; 28. emodin; 29. chrysophanol; 30. physcion;
the standard formulation fingerprint data (obtained by measuring 80 mg/ml of test solution) measured at 326nm is as follows:
peak number 1 2 3 4 5 6 7 8 9 10 11 12
Retention time/min 5.4 9.5 10.4 12.1 13.6 15.6 16.9 18.3 20.0 23.7 26.6 28.1
Peak area 292.7 185.1 1800.1 392.7 1673.7 587.1 379.4 1289.7 920.7 16436.4 321.4 1873.4
Relative retention time 0.23 0.40 0.44 0.51 0.57 0.66 0.71 0.77 0.84 1.00 1.12 1.19
Relative peak area 0.02 0.01 0.11 0.02 0.10 0.04 0.02 0.08 0.06 1.00 0.02 0.11
Peak number 13 14 15 16 17 18 19 20 21 22 23 24
Retention time/min 29 30 32 35 35 44 45 46 46 57 64 67
Peak area 195.2 1712.8 3171.5 230.7 8625.6 1015.9 384.3 88.4 833 30.3 82.9 26
Relative retention time 1.24 1.26 1.35 1.45 1.50 1.86 1.89 1.93 1.95 2.40 2.68 2.81
Relative peak area 0.01 0.10 0.19 0.01 0.52 0.06 0.02 0.01 0.05 0.00 0.01 0.00
See fig. 7, which shows 24 common fingerprint peaks, three dotted lines represent the partition lines of fingerprint peaks, and when the number of common fingerprint peaks is 24 (326nm), 10 fingerprint peaks are identified, wherein: 8. isochlorogenic acid A; 10. baicalin; 14. wogonoside; 15. berberine; 17. baicalein; 18. wogonin; 20. rhein; 22. emodin; 23. chrysophanol; 24. 10 fingerprint peaks such as physcion;
s6: index component quantitative method
The method can be used for quantitatively measuring 12 compound components in the gardenia golden flower pills by using a standard curve method, and the linearity, range, detection limit and quantitative limit of the 12 compound components in the gardenia golden flower pills are as follows:
Figure GDA0003751021010000082
Figure GDA0003751021010000091
the method can also be used for quantitatively measuring 12 compound components in the gardenia golden flower pill by utilizing a one-line multiple evaluation method, the S compound is any one of baicalin, berberine, baicalein, wogonin and chrysophanol, the concentrations of other 11 compounds can be obtained by measuring the peak area of the S compound with known concentration during analysis, and the calculation formula is as follows:
Figure GDA0003751021010000092
wherein A is s Is measured as the peak area of the compound, C s To determine the concentration of the compound, A i Peak area of compound to be evaluated, C i The concentrations of other 11 compounds to be tested which are remained after selecting one of the five compounds are determined; relative correction factor values f for five of the compounds si The following were used:
peak number S compound No. P7 P12 P15 P19 P20 P22 P23 P24 P26 P28 P29 P30
Peak
15 Baicalin f si 1.44 0.91 1.00 0.80 0.43 0.68 0.30 0.51 0.53 0.24 0.29 0.53
Peak 20 Berberine f si 3.34 2.11 2.32 1.85 1.00 1.58 0.70 1.20 1.22 0.56 0.67 1.23
Peak 22 Baicalein f si 1.45 0.92 1.01 0.80 0.43 1.00 0.30 0.52 0.53 0.24 0.29 0.53
Peak 24 Wogonin f si 2.73 1.73 1.90 1.51 0.82 1.29 0.57 1.00 1.00 0.46 0.55 1.00
Peak 29 Chrysophanol f si 5.00 3.16 3.48 2.77 1.50 2.37 1.05 1.79 1.83 0.84 1.00 1.83
The above is an embodiment of the present invention. The embodiments and specific parameters thereof are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and all equivalent structural changes made by applying the contents of the specification and the drawings shall be embraced by the scope of the invention.

Claims (4)

1. The standard fingerprint spectrum and quality consistency evaluation method of the standard preparation mode of the gardenia golden flower pills is characterized by comprising the following steps of:
s1: preparing test solution
Weighing 1-5g of ground gardenia golden flower pill powder, putting the gardenia golden flower pill powder into a 25ml measuring flask, and respectively carrying out the following treatment: placing the mixture into a 25ml measuring flask, precisely adding 25ml of an extraction solvent, wherein the extraction solvent is 80% methanol aqueous solution containing 0.5% phosphoric acid, precisely weighing, carrying out ultrasonic treatment at 45 ℃ for 10-40min, the ultrasonic treatment power is 240W, the frequency is 40KHz, standing to room temperature, precisely weighing, and supplementing the loss weight by using the extraction solvent; secondly, refluxing and extracting the gardenia golden flower pill powder for 2 times by using 25ml of the extraction solvent, wherein the refluxing and extracting time for two times is 1 hour and 40 minutes respectively, then merging the filtrates obtained by the refluxing and extracting for two times, and fixing the volume to 50 ml;
shaking up the first and the second to be tested respectively, filtering the mixture by a 0.45 mu m filter membrane, and taking the subsequent filtrate to obtain first and second test solution which are obtained by different extraction modes;
s2: preparation of Dual reference solutions
Preparing a mixed control solution with the baicalin control solution concentration of 300-2000 mu g/ml and the baicalein control solution concentration of 300-1200 mu g/ml, and shaking up to be used as a double-reference solution;
s3: preparation of Mixed control solutions
Preparing a mixed reference solution, wherein the mixed reference solution comprises the following components in concentration: 650 mu g/ml of baicalein, 160 mu g/ml of wogonin, 1200 mu g/ml of baicalin, 300 mu g/ml of wogonoside, 450 mu g/ml of geniposide, 50 mu g/ml of isochlorogenic acid A, 260 mu g/ml of berberine, 30 mu g/ml of emodin, 100 mu g/ml of chrysophanol, 80 mu g/ml of rhein, 270 mu g/ml of physcion and 30 mu g/ml of aloe-emodin;
s4: chromatographic conditions
A chromatographic column: COSMOSIL 5C18-MS-II, with column length 250mm, inner diameter 4.6mm, particle diameter 5 μm, and filler being octadecylsilane chemically bonded silica;
column temperature: 25-45 ℃; the sample injection amount is 1-50 mu l;
mobile phase: an aqueous phase A: 0.2% phosphoric acid solution containing 0.005mol/L sodium heptanesulfonate; and (3) organic phase B: acetonitrile-methanol mixed solution with the volume ratio of 9: 1; gradient elution, elution procedure was as follows:
Figure FDA0003785849870000011
Figure FDA0003785849870000021
detection wavelength: setting detection wavelengths to be 220nm, 238nm, 254nm, 278nm and 326nm, and simultaneously collecting a 190-400 nm three-dimensional chromatogram by using the DAD;
s5: fingerprint spectrum checking method
The number of common fingerprint peaks of the test sample under the detection wavelengths of 220nm, 238nm, 254nm, 278nm and 326nm is respectively 30, 30 and 24, wherein baicalin is used as a reference peak for identification and recognition of the common fingerprint peaks, and the baicalin peak and the baicalein peak are used as double-reference quantitative measurement correction peaks, which are respectively calculated according to the peak area and retention time of the common fingerprint peaks, the macro qualitative similarity of the fingerprint spectrum of the test sample is not less than 0.90, and the macro quantitative similarity is 80-120%; wherein the peak area ratios of No. 15 fingerprint peak at two wavelengths of 220nm and 254nm, 238nm and 278nm, and 278nm and 254nm are respectively 4.0-5.0, 4.68-5.72 and 5.4-6.6;
s6: index component quantitative method
The method can be used for quantitatively measuring 12 compound components in the gardenia golden flower pills by using a standard curve method, and the linearity, range, detection limit and quantitative limit of the 12 compound components in the gardenia golden flower pills are as follows:
Figure FDA0003785849870000022
or a multi-evaluation method can be used for quantitatively measuring 12 compound components in the gardenia golden flower pill, the S compound is any one of baicalin, berberine, baicalein, wogonin and chrysophanol, the concentrations of other 11 compounds can be obtained by measuring the peak area of the S compound with known concentration during analysis, and the calculation formula is as follows:
Figure FDA0003785849870000023
wherein A is s Is measured as the peak area of the compound, C s To determine the concentration of the compound, A i Peak area of compound to be evaluated, C i The concentrations of other 11 compounds to be tested which are remained after selecting one of the five compounds are determined; relative correction factor values f for five of the compounds si The following were used:
peak number S compound No. P7 P12 P15 P19 P20 P22 P23 P24 P26 P28 P29 P30 Peak 15 Baicalin f si 1.44 0.91 1.00 0.80 0.43 0.68 0.30 0.51 0.53 0.24 0.29 0.53 Peak 20 Berberine f si 3.34 2.11 2.32 1.85 1.00 1.58 0.70 1.20 1.22 0.56 0.67 1.23 Peak 22 Baicalein f si 1.45 0.92 1.01 0.80 0.43 1.00 0.30 0.52 0.53 0.24 0.29 0.53 Peak 24 Wogonin f si 2.73 1.73 1.90 1.51 0.82 1.29 0.57 1.00 1.00 0.46 0.55 1.00 Peak 29 Chrysophanol f si 5.00 3.16 3.48 2.77 1.50 2.37 1.05 1.79 1.83 0.84 1.00 1.83
。
2. The standard fingerprint spectrum and quality consistency evaluation method of the standard preparation mode of gardenia golden flower pills as claimed in claim 1, wherein the detection wavelengths in the step S5 include, but are not limited to, 220nm, 238nm, 254nm, 278nm and 326 nm.
3. The method for evaluating the standard fingerprint spectrum and the quality consistency of the standard preparation mode of the gardenia golden flower pills according to claim 1, wherein the detection wavelength in the step S5 comprises any wavelength within 190-400 nm acquired by DAD for fingerprint spectrum detection.
4. The method for evaluating the standard fingerprint spectrum and quality consistency of the standard preparation mode of gardenia golden flower pills according to claim 1, wherein the number of common fingerprint peaks in step S5 includes 30, 30 and 24 but is not limited to the above number of fingerprint peaks, and fingerprint peaks whose signals are more than 5 times of noise can be regarded as effective fingerprint peaks.
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