HPLC (High Performance Liquid Chromatography) quantitative determination and characteristic chromatogram construction method of Tibetan medicine twenty-five-ingredient pearl pills and application of HPLC quantitative determination and characteristic chromatogram construction method

Abstract

The invention relates to an HPLC (High Performance Liquid Chromatography) quantitative determination and characteristic chromatogram construction method of Tibetan medicine twenty-five-ingredient pearl pills and application. Belongs to the technical field of detection analysis. According to the method, a test solution is obtained by a simple and rapid pretreatment method, and a high performance liquid chromatograph and a gradient elution mode are adopted; the contents of bitter crocin (250 nm), agarotetrol (252 nm), cinnamaldehyde (290 nm), hydroxysafflor pigment A (403 nm) and crocin I and II (440 nm) in the Tibetan medicine twenty-five-ingredient pearl pills can be determined at the same time by switching different wavelengths under the same elution condition, and a characteristic spectrum (280 nm) is constructed. And the practical purposes of multi-component evaluation by one measurement, high efficiency, low consumption and easiness in popularization are realized. According to the polarity difference of the substances, the components can be separated and accurately detected by adopting a gradient elution procedure.

Description

Technical Field

[0001] The present invention relates to a one-test-multiple-evaluation HPLC quantitative determination and characteristic spectrum construction method and application of Tibetan medicine 25-flavor Pearl Pills. That is, by using a high performance liquid chromatograph and gradient elution, the contents of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II in Tibetan medicine 25-flavor Pearl Pills can be simultaneously determined and a characteristic spectrum can be constructed by switching different wavelengths under the same elution condition. It belongs to the field of detection and analysis technology. Background Art

[0002] The Twenty-five Ingredients Pearl Pill has the national medicine approval number Z54020085. It calms the mind and opens the orifices. It is used for stroke, hemiplegia, facial paralysis, coma, mental disorder, delirium, etc.

[0003] The prescription composition and preparation process are as follows:

[0004] Prescription: pearl, mother of pearl, nutmeg, travertine, safflower, grass fruit, clove, agarwood, cardamom, terminalia chebula, sandalwood, emblica, agarwood, cinnamon, terminalia chebula, crab, costus root, mallow fruit, piper longum, shidasazeng, gold ore, in vitro cultivated bezoar, fragrant celery, saffron, black grass seeds, artificial musk, buffalo horn concentrated powder.

[0005] Preparation process: Except for pearl, in vitro cultured bezoar and artificial musk, the remaining nutmeg and other ingredients are crushed into fine powder. Pearl, in vitro cultured bezoar and artificial musk are ground into fine powder respectively, mixed with the above powders, used for water-flooded pills, and dried to obtain the product.

[0006] At present, most of the research on Twenty-five Pearl Pills is pharmacological research, and there are few studies on quality and fingerprint. The 2020 edition of the "Chinese Pharmacopoeia" only includes crocin I in the content determination. This prescription has many medicinal flavors, and it is difficult to control the intrinsic quality of the preparation. After consulting many literatures, the content determination of eugenol is GC method, which is unstable; the baseline of the costus lactone and costus hydroxylactone content determination spectrum deviates, the peak shape is not beautiful, and the published method is outdated. Other content determinations are UV tandem ELSD, and the combined detection of crocin and bile acid has high requirements for the detection machine, is difficult, and has no general applicability.

[0007] In order to ensure the therapeutic effect of Twenty-five Pearl Pills with high quality and strictly control the quality of medicinal materials, it is planned to conduct a simultaneous quantitative study on crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II in the pills and construct a characteristic map.

[0008] According to the literature, the chemical structures of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II are as follows:

[0009]

[0010] From the above structural formula, we know that according to the polarity difference of each substance, a gradient elution program can be used to separate and accurately detect the above components. So far, there is no report on the simultaneous determination of these six medicinal ingredients (crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II) in Twenty-five Pearl Pills. Summary of the invention

[0011] The present invention is based on the above background, in order to improve detection efficiency, reduce detection cost and reduce environmental pollution, for the first time, obtains a test solution by a simple and fast pretreatment method, adopts a high performance liquid chromatograph (diode array detector), and uses A (0.2% phosphoric acid)-B (acetonitrile) as a mobile phase for gradient elution, and the elution procedure is as follows: 0-18min 3-8% B, 18-30min 8% B, 30-48min 8-13% B, 48-67min 13-19% B, 67-88min 19B%, 88-95min 19-32% B, 95-120min 32B%, 120-125min 32-13% B, 125-130min 3% B, 135min 3%. The same elution condition can achieve simultaneous determination of six index components and establishment of a characteristic spectrum by switching wavelengths. The detection wavelengths were 280 nm (for establishing characteristic spectra), 250 nm (crocin), 252 nm (agaretol), 290 nm (cinnamaldehyde), 403 nm (hydroxysaffron pigment A), and 440 nm (crocin I and II); the column temperature was 25 °C; the flow rate was 0.8 mL min -1 ; Injection volume 10μL. The theoretical plate number of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II peaks were calculated to be no less than 5000. The peak separation was good, achieving the purpose of one test for multiple evaluations, high efficiency, low consumption, and easy popularization. The simultaneous determination of six index components of precious medicinal materials was reported for the first time.

[0012] Through methodological investigation, the injection amount of crocin I was 22.70-227.00 ng, the injection amount of crocin II was 18.22-182.20 ng, the injection amount of crocin was 20.00-200.00 ng, the injection amount of cinnamaldehyde was 19.48-194.80 ng, the injection amount of agarwood tetraol was 18.96-189.60 ng, and the injection amount of hydroxysaffron pigment A was 20.82-208.20 ng, which showed a good linear relationship with the peak area (see Tables 1-6, Figures 1 to 6 ), the regression equations are: Y = 0.0001X + 0.321 for crocin I, R 2 =0.9999; Y of crocin II = 0.0001X-0.0476, R 2=0.9999; Crocin Y = 0.0005X + 1.6511, R 2 =0.9998; Y of cinnamaldehyde = 7E-05X + 0.2625, R 2 =0.9998; Y of agarwood tetraol = 0.0004X-2.9648, R 2 =0.9995; Y of hydroxysafflower color A = 0.0002X + 1.1294, R 2 =0.9997. The results of the sample recovery experiment showed that the average recovery rate of crocin I was 98.39% for 6 determinations, and the RSD was 1.54% (see Table 7); the average recovery rate of crocin II was 97.75% for 6 determinations, and the RSD was 2.02% (see Table 8); the average recovery rate of crocin was 99.28% for 6 determinations, and the RSD was 1.38% (see Table 9); the average recovery rate of cinnamaldehyde was 100.96% for 6 determinations, and the RSD was 1.74% (see Table 10); the average recovery rate of agarwood tetraol was 97.41% for 6 determinations, and the RSD was 1.37% (see Table 11); the average recovery rate of hydroxysaffron pigment A was 98.45% for 6 determinations, and the RSD was 1.32% (see Table 12). Precision (see Table 13), stability (see Table 14), repeatability (see Table 15), UV spectra of the determined components (see Figures 7 to 12 ), reference substance chromatogram (see Figures 13 to 18 ), sample chromatogram (see Figures 19 to 23 ), specificity (see Figures 24 to 27 ), 3 batches of sample determination (see Table 16), ultrasonic time investigation (see Table 17), the experimental method data are in line with the methodological requirements. Simple, accurate and efficient, it can be used for the simultaneous quantitative determination of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II in 25-flavor pearl pills, and at the same time establish the fingerprint of 25-flavor pearl pills (280nm) for better quality control (see Fig.28 ).

[0013] The technical solution adopted by the present invention to solve the technical problem is:

[0014] (1) A. Chromatographic conditions and system suitability test; 0.2% phosphoric acid water was used as mobile phase A and acetonitrile was used as mobile phase B; gradient elution, the elution program was as follows: 0-18 min 3-8% B, 18-30 min 8% B, 30-48 min 8-13% B, 48-67 min 13-19% B, 67-88 min 19B%, 88-95 min 19-32% B, 95-120 min 32B%, 120-125 min 32-13% B, 125-130 min 3% B, 135 min 3%. The detection wavelengths were 280 nm (for establishing characteristic spectra), 250 nm (crocin), 252 nm (agaretol), 290 nm (cinnamaldehyde), 403 nm (hydroxysaffron pigment A), and 440 nm (crocin I and II); the column temperature was 25 °C; the flow rate was 0.8 mL min -1 ; Injection volume 10μL. Theoretical plate number of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II peaks are not less than 5000;

[0015] B. Preparation of reference solution: Accurately weigh appropriate amount of crocin, agave tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II, accurately weigh, add methanol to make a solution containing 10 μg of each per 1 ml, shake well, and obtain;

[0016] C. Preparation of Test Solution

[0017] Take 10 pills of Twenty-five Flavor Pearl Pills, grind them into powder, weigh about 0.5 g, weigh accurately, put them in a stoppered conical flask, add 25 mL of 70% methanol, stopper it tightly, weigh the weight, ultrasonically treat it (500 W, frequency 40 Hz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol solution, shake it well, filter it, and take the filtrate to obtain the product.

[0018] D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the six components respectively.

[0019] The principle of the present invention is as follows:

[0020] Based on the principle of similar-miscible extraction and the difference in dosage forms of substances, gradient elution is used, with polarity from small to large, using the maximum absorption of each substance (see Figures 7 to 12 ), as the detection wavelength, not only makes the size and area of ​​the component peaks suitable, but also greatly reduces the interference peaks at this detection wavelength, presents a stable baseline of the liquid phase spectrum, and has good peak separation, laying the foundation for the simultaneous determination of multiple components. Then, based on the good linear relationship between the injection volume of the multiple components and their peak areas within a certain range, they are used for quantitative determination.

[0021] The innovative features and beneficial effects of the present invention are as follows:

[0022] (1) Gradient elution is adopted, with 0.2% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B; the same elution condition is used to simultaneously determine six index components and establish characteristic spectra by switching wavelengths. It enables several effective ingredients of traditional Chinese medicine with completely different properties and structures to be quantitatively determined once using the same mobile phase. The quantitative chromatograms of multiple components have a stable baseline and good peak separation. The method has no extraction, no concentration, no evaporation, is simple, fast, accurate, reproducible, easy to popularize and master, and effectively improves detection efficiency, reduces detection costs, and reduces environmental pollution.

[0023] (2) According to the literature review, most of the current research on Twenty-five Pearl Pills is pharmacological research, and there are few studies on quality and fingerprint. The 2020 edition of the Chinese Pharmacopoeia only includes crocin I in the content determination. This prescription has many medicinal ingredients, and it is difficult to control the intrinsic quality of the preparation. After reviewing many literatures, the content determination of eugenol is a gas phase method, which is unstable; the baseline of the costus lactone and costus hydroxylactone content determination spectrum deviates, the peak shape is not beautiful, and the published method is outdated. Other content determinations are ultraviolet tandem ELSD evaporative light detectors, and the combined detection of crocin and bile acid has high requirements for the detection machine, is difficult, and has no general applicability.

[0024] The method for simultaneously determining these six components (saffron crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II) is reported for the first time. In addition, the characteristic spectrum has a total of 40 peaks. In addition to the above-mentioned components, gallic acid and corilagin were also identified. The detection efficiency is greatly improved and the instrument cost is significantly reduced. Not only that, the more critical thing is to upgrade one test and one evaluation to one test and multiple evaluations, achieving twice the result with half the effort. Its practical value in saving resources and improving the detection phase rate and detection indicators is obvious to all.

[0025] (3) The advent of the method of the present invention not only provides a method for the simultaneous detection of precious medicinal materials in the Twenty-five Flavors Pearl Pills, but also provides a reference basis and research ideas for the simultaneous determination of these ingredients in other compound preparations, which has a demonstrative and exemplary role.

[0026] (4) The key technology of the present invention is to make use of the characteristic of acetonitrile in the mobile phase that the peak retention time of various chemical components is the premise, and the combination with phosphoric acid solution can make the acidic components exist as molecules and eliminate the ion tailing performance. Through the study of the gradient ratio between acetonitrile and phosphoric acid of various concentrations for an unlimited number of times, an elution condition is explored that can make a variety of chemical components with large polarity differences appear in the same mobile phase, and the peak retention time is appropriate, the baseline is stable, and the reproducibility is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Linear relationship diagram of crocin I;

[0028] Figure 2 Linear relationship diagram of crocin II;

[0029] Figure 3 Linear relationship diagram of crocin;

[0030] Figure 4 Linear relationship graph of cinnamaldehyde;

[0031] Figure 5 Linear relationship diagram of agarwood tetraol;

[0032] Figure 6 Linear relationship diagram of hydroxysafflorchrome A;

[0033] Figure 7 Ultraviolet absorption spectrum of crocin Ⅰ;

[0034] Figure 8 Ultraviolet absorption spectrum of crocin II;

[0035] Fig. 9 The UV absorption spectrum of crocin;

[0036] Fig.10 UV absorption spectrum of cinnamaldehyde;

[0037] Fig.11 UV absorption spectrum of agarwood tetraol;

[0038] Fig.12 UV absorption spectrum of hydroxysafflorchrome A;

[0039] Fig.13 HPLC chromatogram of crocin I;

[0040] Fig.14 HPLC chromatogram of crocin II;

[0041] Fig.15 HPLC chromatogram of picrocrocin;

[0042] Fig.16 HPLC chromatogram of cinnamaldehyde;

[0043] Fig.17 HPLC chromatogram of agarwood tetraol;

[0044] Fig.18 HPLC chromatogram of hydroxysafflorchrome A;

[0045] Fig.19 HPLC chromatogram of 25-flavor Pearl Pill sample (crocin I, II 440nm);

[0046] Fig. 20 HPLC chromatogram of 25-flavor Pearl Pill sample (bitter crocin 250nm);

[0047] Fig.21 HPLC chromatogram of 25-flavor Pearl Pill sample (cinnamaldehyde 290nm);

[0048] Fig. 22 HPLC chromatogram of 25-flavor Pearl Pill sample (Agarwood Tetraol 252nm);

[0049] Fig.23 HPLC chromatogram of 25-flavor Pearl Pill sample (hydroxysafflower pigment A403nm);

[0050] Fig.24 is the HPLC chromatogram of a blank sample without saffron;

[0051] Fig.25 is the HPLC chromatogram of a blank sample without cinnamon;

[0052] Fig.26 This is the HPLC chromatogram of a blank sample without agarwood;

[0053] Fig. 27 is the HPLC chromatogram of a blank sample without safflower;

[0054] Fig.28 Characteristic spectrum of 25-flavor Pearl Pills;

[0055] Figures 1 to 5 In the figure, the ordinate is the peak area; the abscissa is the injection amount (ng);

[0056] Figures 6 to 12 In the figure, the ordinate is the absorption intensity (mAU); the abscissa is the absorption wavelength (nm);

[0057] Figures 13 to 27 In the figure, the horizontal axis is the retention time (minutes), and the vertical axis is the response value (mAU);

[0058] Figures 13 to 23 In the figure, the horizontal axis is the retention time (minutes), the vertical axis is the response value (mAU), and the labels 1 is crocin I, 2 is crocin II, 3 is picrocrocin, 4 is cinnamaldehyde, 5 is agaretatetraol, and 6 is hydroxysaffron pigment A;

[0059] Fig.28 In the figure, the horizontal axis is the retention time (minutes), the vertical axis is the response value (mAU), and the labels 1 is gallic acid, 2 is hydroxysaffron pigment A, 3 is picrocrocin, 4 is corilagin, 5 is agaretatetraol, 6 is crocin I, 7 is crocin II, and 8 is cinnamaldehyde. DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0061] (1) A. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; 0.2% phosphoric acid water was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution, the elution program was as follows: 0-18 min 3-8% B, 18-30 min 8% B, 30-48 min 8-13% B, 48-67 min 13-19% B, 67-88 min 19B%, 88-95 min 19-32% B, 95-120 min 32B%, 120-125 min 32-13% B, 125-130 min 3% B, 135 min 3%. The detection wavelengths were 280 nm (for establishing characteristic spectra), 250 nm (crocin), 252 nm (agaretol), 290 nm (cinnamaldehyde), 403 nm (hydroxysaffron pigment A), and 440 nm (crocin I and II); the column temperature was 25 °C; the flow rate was 0.8 mL min -1 ; Injection volume 10μL. Theoretical plate number of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II peaks are not less than 5000;

[0062] B. Preparation of reference solution: Accurately weigh appropriate amount of crocin, agave tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II, accurately weigh, add methanol to make a solution containing 10 μg of each per 1 ml, shake well, and obtain;

[0063] C. Preparation of test solution Take 10 Twenty-five-flavor Pearl Pills, grind them into powder, weigh about 0.5 g, accurately weigh, put in a stoppered conical flask, add 25 mL of 70% methanol, stopper, weigh, ultrasonically treat (500 W, frequency 40 Hz), take out, cool, weigh again, make up the lost weight with 70% methanol solution, shake well, filter, and take the filtrate to obtain the test solution.

[0064] D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the six components respectively.

[0065] Table 1 Injection amount and peak area of ​​crocin I

[0066]

[0067] Table 2 Injection amount and peak area of ​​crocin Ⅱ

[0068]

[0069] Table 3 Injection amount and peak area of ​​crocin

[0070]

[0071] Table 4 Injection amount and peak area of ​​cinnamaldehyde

[0072]

[0073] Table 5 Agarwood tetraol injection amount and peak area

[0074]

[0075] Table 6 Hydroxysafflor A injection amount and peak area

[0076]

[0077] Table 7 Results of the test on the recovery of crocin I in samples

[0078]

[0079] Table 8 Results of the test on the recovery of saffron glycoside II in samples

[0080]

[0081] Table 9 Results of the test on the recovery rate of crocin in samples

[0082]

[0083] Table 10 Cinnamaldehyde recovery test results in samples

[0084]

[0085] Table 11 Agarwood tetraol recovery test results in samples

[0086]

[0087] Table 12 Results of the recovery test of hydroxysafflorchrome A in samples

[0088]

[0089] Table 13 Precision test results

[0090]

[0091] Table 14 Stability test results

[0092]

[0093] Table 15 Repeatability test results (mg / g)

[0094]

[0095] Table 16 Determination results of the contents of 6 ingredients in 3 batches of Ershiwuwei Shanhu Wan (mg / g, n=2)

[0096]

[0097] Table 17 Ultrasonic time investigation (mg / g)

[0098]

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A one-test-multiple-evaluation HPLC quantitative determination and characteristic spectrum construction method of Tibetan medicine 205-flavor Pearl Pills, characterized by: The following steps are involved: A. Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; 0.2% phosphoric acid water was used as mobile phase A, and acetonitrile was used as mobile phase B; the detection wavelengths were 280nm, 250nm, 252nm, 290nm, 403nm, and 440nm; the column temperature was 25°C; the flow rate was 0.8mL·min -1 ; Sample injection volume: 10 μL; Theoretical plate number of crocin, agarwood tetraol, gallic acid, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II peaks should not be less than 5000; B. Preparation of reference solution: Accurately weigh appropriate amount of crocin, agave tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II, accurately weigh, add methanol to make a solution containing 10 μg of each per 1 ml, shake well, and obtain; C. Preparation of test solution Take 10 pills of 25-flavor Pearl Pills, grind them into powder, weigh about 0.5 g, accurately weigh, put into a stoppered conical flask, add 25 mL of 70% methanol, stopper tightly, weigh the weight, ultrasonically treat at 500 W and 40 Hz, take out, cool, weigh again, make up the lost weight with 70% methanol solution, shake well, filter, and take the filtrate to obtain; D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the six components respectively.

2. The one-test-multiple-evaluation HPLC quantitative determination and characteristic spectrum construction method of Tibetan medicine Ershiwuwei Zhenzhu Wan according to claim 1, characterized in that: The one-measurement-multiple-evaluations method means that the contents of crocin, agarwood tetraol, cinnamaldehyde, hydroxysaffron pigment A, crocin I and II in the Tibetan medicine Twenty-five Flavors Pearl Pills can be simultaneously determined by switching different wavelengths under the same elution condition and constructing a characteristic spectrum.

3. The one-test-multiple-evaluation HPLC quantitative determination and characteristic spectrum construction method of Tibetan medicine Ershiwuwei Zhenzhu Wan according to claim 1, characterized in that: 280nm is used to establish the characteristic spectrum, 250nm is used to detect crocin, 252nm is used to detect agarwood tetraol, 290nm is used to detect cinnamaldehyde, 403nm is used to detect hydroxysaffron pigment A, and 440nm is used to detect crocin I and II.

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