CN117330650A - Construction method of vertical sequence pokeberry characteristic spectrum - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a vertical-order pokeberry characteristic map. The method comprises the steps of preparing a sample solution, adopting a specific gradient elution program, and measuring under chromatographic conditions to obtain a characteristic map of the pokeberry root. The feature spectrum of the vertical-sequence pokeberry provided by the invention has the advantages of better separation degree of each feature peak, clear material basis, capability of distinguishing the Liu Butong base sources, capability of separating main chemical components of the vertical-sequence pokeberry, more accurate and reliable result, good reproducibility and high sensitivity.

Description

Construction method of vertical sequence pokeberry characteristic spectrum

Technical Field

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a vertical-order pokeberry characteristic map.

Background

Phytolacca, phytolaccaceae, phytolacca perennial robusta herb, widely distributed in red soil low hilly area in the south of Yangtze river, and the varieties currently distributed in China mainly comprise Phytolacca (wild radish) and Phytolacca in vertical order (Phytolacca americana, and Phytolacca decastes). The Chinese medicinal materials are called amaranth, radish, pokeberry, rouge, etc. according to the form of the Chinese medicinal materials.

At present, the related reports on the feature patterns of the pokeberry root are relatively few, the related documents adopt an evaporative light scattering detector for analysis and research, the detection sensitivity is relatively low, the reproducibility is relatively poor, and the system comparison is not formed for different base sources. The prior art also carried out the assay in an isocratic elution procedure, which makes it difficult to isolate the major chemical components of pokeberry root.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defects of low detection sensitivity, poor reproducibility, difficulty in separating and obtaining main chemical components of the pokeberry features of the vertical sequence in the prior art when the pokeberry features of the vertical sequence are obtained, and the like, so that the construction method of the pokeberry features of the vertical sequence is provided.

For this purpose, the invention provides the following technical scheme.

The invention provides a construction method of a vertical sequence pokeberry characteristic map, which comprises the following steps:

preparation of test solution: preparing a sample solution from a sample;

and (3) measuring: the chromatographic conditions of the high performance liquid chromatography include: acetonitrile is taken as a mobile phase A, phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out;

the gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B; or alternatively, the first and second heat exchangers may be,

0-8min,1% mobile phase A,99% mobile phase B;8-18min, 1-8% mobile phase A, 99-92% mobile phase B;18-28min, 8-20% mobile phase A, 92-80% mobile phase B;28-35min, 20-30% mobile phase A, 80-70% mobile phase B;35-40min, 30-35% mobile phase A, 70-65% mobile phase B;40-50min,35% mobile phase A,65% mobile phase B; or alternatively, the first and second heat exchangers may be,

0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-40min, 30-35% mobile phase A, 70-65% mobile phase B;40-50min,35% mobile phase A,65% mobile phase B.

The chromatographic conditions of the high performance liquid chromatography further include: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, and the grain diameter is 4 mu m; and/or the column temperature is 25-35 ℃; and/or a wavelength of 210-254nm; and/or the sample injection amount is 5-15 mu L; and/or a flow rate of 0.8-1.2ml/min; and/or taking 0.03-0.05% phosphoric acid aqueous solution as mobile phase B.

The gradient elution procedure includes: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

The chromatographic conditions of the high performance liquid chromatography include: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is taken as a mobile phase A, and 0.04% phosphoric acid aqueous solution is taken as a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm.

The test sample is prepared from radix Phytolaccae granule, radix Phytolaccae standard decoction lyophilized powder, radix Phytolaccae medicinal material or radix Phytolaccae Liu Yinpian.

The preparation method of the sample solution comprises the following steps: extracting the sample with solvent, and filtering;

preferably, in preparing the sample solution, the extraction solvent is at least one of methanol and water;

preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%;

preferably, in preparing the sample solution, the extraction method is reflux extraction or ultrasonic extraction.

Further, when the test sample is freeze-dried powder or formulated granules, the mass of the extraction solvent corresponding to each 1ml of the test sample is 0.01g-0.08g;

when the sample is medicinal material or decoction piece, the mass of the water extract corresponding to each 1ml of the extraction solvent is 0.01g-0.08g.

The construction method also comprises the preparation of a reference substance solution of a reference medicinal material;

the preparation method of the reference substance solution of the reference medicinal material comprises the following steps: extracting reference materials with water to obtain water extract, adding extraction solvent into the water extract, mixing, and filtering;

preferably, in preparing the reference solution of the reference medicinal material, the extraction solvent is at least one of methanol and water;

preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%;

preferably, the volume of water corresponding to each 1g of medicinal material of the order-dropping quotient Liu Duizhao is 25-100ml;

preferably, the mass of the extraction solvent is 0.01g-0.08g per 1ml of the aqueous extract;

preferably, in obtaining the aqueous extract, the extraction method is reflux extraction or ultrasonic extraction.

In the present invention, about 0.2g to about 0.3g of the aqueous extract can be obtained per 1g of the medicinal material or decoction piece.

The construction method also comprises the preparation of a reference substance solution;

the reference substance is at least one of esculentoside A, erythrina base, adenosine, guanosine and L-tryptophan.

The characteristic map obtained by the construction method comprises 9 characteristic peaks;

peak 5 was used as reference peak 1, peak 1 had a relative retention time of 0.47,2, peak 3 had a relative retention time of 0.67, peak 4 had a relative retention time of 0.81, peak 6 had a relative retention time of 1.24, and peak 7 had a relative retention time of 1.64;

peak 8 as reference peak 2, peak 9 relative retention time 1.07;

the relative retention time should be within + -10% of the specified value.

The technical scheme of the invention has the following advantages:

1. the invention provides a construction method of a feature map of Phytolacca acinosa, which comprises the steps of preparing a sample solution, adopting a specific gradient elution program, and measuring under chromatographic conditions to obtain the feature map of Phytolacca acinosa. The feature spectrum of the vertical-sequence pokeberry provided by the invention has the advantages of better separation degree of each feature peak, clear material basis, capability of distinguishing the Liu Butong base sources, capability of separating main chemical components of the vertical-sequence pokeberry, more accurate and reliable result, good reproducibility and high sensitivity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a characteristic map of the pokeberry formulation granule and pokeberry control drug of example 1 of the present invention; FIG. 2 is a characteristic map of 3 batches of pokeberry formulation particles and 16 batches of pokeberry standard decoction lyophilized powder of the invention; FIG. 3 is a pokeberry root comparison signature of the present invention; FIG. 4 is a graph showing the comparison of characteristic patterns of pokeberry root formula particles with reference substances; FIG. 5 is a graph showing the result of the chromatographic condition 1 of section 1.2 in experimental example 1 of the present invention; FIG. 6 is a graph showing the condition 2 of section 1.2 of the present invention; FIG. 7 is a graph showing the result of the chromatographic condition 3 of section 1.2 in experimental example 1 of the present invention; FIG. 8 is a graph showing the condition 4 of section 1.2 of the present invention; FIG. 9 is a graph showing the condition 5 of section 1.2 of the present invention; FIG. 10 is a graph showing the condition 6 of section 1.2 of the present invention in Experimental example 1; FIG. 11 is a graph showing the result of the chromatographic condition 7 of section 1.2 in experimental example 1 of the present invention; FIG. 12 is a graph showing the condition 8 of section 1.2 of the present invention; FIG. 13 is a graph showing the condition 9 of section 1.2 of the present invention; FIG. 14 is a graph showing the condition 10 of section 1.2 of the present invention in Experimental example 1; FIG. 15 is a graph showing the condition 11 of section 1.2 of the present invention in Experimental example 1; FIG. 16 is a graph showing the condition 12 of section 1.2 of the present invention in Experimental example 1; FIG. 17 is a graph showing the condition 13 of section 1.2 of the present invention in Experimental example 1; FIG. 18 is a chromatogram obtained at 1.3 different wavelengths in Experimental example 1 of the present invention; FIG. 19 is a chromatogram obtained by examining the specificity of section 3.1 in Experimental example 3 of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention. The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Instrument for measuring and controlling the intensity of light

Reagent and reagent:

reagent	Grade/specification	Lot number	Manufacturer' s
				Esculentoside A	≥98％	14541	Shanghai Shidande Standard technical service Co., ltd
Acetic acid	Chromatographic purity	P1622914	THERMO FISHER SCIENTIFIC (CHINA) Co.,Ltd.
				Formic acid	Mass spectrum level	U9260048	Shanghai Anpu Experimental Technology Co.,Ltd.
Methanol	Chromatographic purity	0212230404	XINGKE HIGH PURITY SOLVENTS (SHANGHAI) Co.,Ltd.
				Methanol	Analytical grade	P2304265	SHANGHAI TITAN TECHNOLOGY Co.,Ltd.
Phosphoric acid	Chromatographic purity	F6130152	SHANGHAI TITAN TECHNOLOGY Co.,Ltd.
				Acetonitrile	Chromatographic purity	0114230403	XINGKE HIGH PURITY SOLVENTS (SHANGHAI) Co.,Ltd.
Phytolacca acinosa formula granule	1 g/bag	2208001-2208003	Huarun Sanjiu modern Chinese medicine Co.Ltd
				Freeze-dried powder of standard phytolacca acinosa decoction	/	2102001-2102016	Huarun Sanjiu modern Chinese medicine Co.Ltd

Example 1

The embodiment provides a construction method of a feature map of a pokeberry root formula particle, which comprises the following steps:

preparation of test solution: taking about 0.2g of a test sample of the pokeberry root formula particles in vertical order, precisely weighing, placing in a conical bottle with a plug, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30min, taking out, cooling, shaking uniformly, filtering, and taking out a subsequent filtrate to obtain a test sample solution.

Preparation of control drug reference solution: taking 1g of radix Phytolaccae (Phytolacca acinosa L. In order of vertical) reference medicine, placing into a conical flask with a plug, adding 50ml of water, heating and refluxing for 60min, centrifuging, taking supernatant, concentrating under reduced pressure to dryness to obtain water extract, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30min, cooling, shaking, filtering, and collecting the subsequent filtrate as reference substance solution of reference medicine.

Preparation of a control solution: taking a proper amount of a pokeberry saponin A reference substance and a erythrina base reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.2mg of pokeberry saponin A and 0.1mg of erythrina base per 1 ml.

And taking appropriate amounts of adenosine reference substance, guanosine reference substance and L-tryptophan reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.1mg of adenosine, 0.1mg of guanosine and 0.1mg of L-tryptophan per 1 ml.

And (3) measuring: the chromatographic conditions of the high performance liquid chromatography include: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is taken as a mobile phase A, and 0.04% phosphoric acid aqueous solution is taken as a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm. The gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

Respectively precisely sucking 10 μl of each of the sample solution and the reference solution of the reference material, and injecting into a liquid chromatograph for measurement. The characteristic spectrum of the pokeberry root formula granule is shown as 1a in figure 1, and the spectrum of the pokeberry root reference medicinal material is shown as 1b in figure 1.

Example 2

The embodiment provides a construction method of a vertical sequence pokeberry characteristic map, which comprises the following steps:

preparation of test solution: taking about 0.2g of a standard phytolacca acinosa decoction freeze-dried powder test sample, precisely weighing, placing into a conical flask with a plug, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (with the power of 300W and the frequency of 40 kHz) for 30min, taking out, cooling, shaking uniformly, filtering, and taking out a subsequent filtrate to obtain a test sample solution.

Preparation of control drug reference solution: taking 1g of radix Phytolaccae (Phytolacca acinosa L. In order of vertical) reference medicine, placing into a conical flask with a plug, adding 50ml of water, heating and refluxing for 60min, centrifuging, taking supernatant, concentrating under reduced pressure to dryness to obtain water extract, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30min, cooling, shaking, filtering, and collecting the subsequent filtrate as reference substance solution of reference medicine.

Preparation of a control solution: taking a proper amount of a pokeberry saponin A reference substance and a erythrina base reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.2mg of pokeberry saponin A and 0.1mg of erythrina base per 1 ml.

And taking appropriate amounts of adenosine reference substance, guanosine reference substance and L-tryptophan reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.1mg of adenosine, 0.1mg of guanosine and 0.1mg of L-tryptophan per 1 ml.

And (3) measuring: the chromatographic conditions of the high performance liquid chromatography include: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is taken as a mobile phase A, and 0.04% phosphoric acid aqueous solution is taken as a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm. The gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

Respectively precisely sucking 10 μl of each of the sample solution and the reference solution of the reference material, and injecting into a liquid chromatograph for measurement.

Example 3

The embodiment provides a construction method of a vertical sequence pokeberry characteristic map, which comprises the following steps:

preparation of test solution: taking about 1g of a test sample of radix Phytolaccae medical material in vertical order, placing the test sample in a conical flask with a plug, adding 50ml of water, heating and refluxing for 60min, centrifuging, taking supernatant, concentrating under reduced pressure to dryness to obtain about 0.2g-0.3g of water extract, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30min, cooling, shaking, filtering, and taking subsequent filtrate to obtain a test sample solution.

Preparation of control drug reference solution: taking 1g of radix Phytolaccae (Phytolacca acinosa L. In order of vertical) reference medicine, placing into a conical flask with a plug, adding 50ml of water, heating and refluxing for 60min, centrifuging, taking supernatant, concentrating under reduced pressure to dryness to obtain water extract, precisely adding 10ml of 60% methanol water solution, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30min, cooling, shaking, filtering, and collecting the subsequent filtrate as reference substance solution of reference medicine.

Preparation of a control solution: taking a proper amount of a pokeberry saponin A reference substance and a erythrina base reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.2mg of pokeberry saponin A and 0.1mg of erythrina base per 1 ml.

And taking appropriate amounts of adenosine reference substance, guanosine reference substance and L-tryptophan reference substance, precisely weighing, and adding 60% methanol water solution to prepare a mixed solution containing 0.1mg of adenosine, 0.1mg of guanosine and 0.1mg of L-tryptophan per 1 ml.

And (3) measuring: the chromatographic conditions of the high performance liquid chromatography include: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is taken as a mobile phase A, and 0.04% phosphoric acid aqueous solution is taken as a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm. The gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

Respectively precisely sucking 10 μl of each of the sample solution and the reference solution of the reference material, and injecting into a liquid chromatograph for measurement.

Identification of characteristic peaks

3 batches of pokeweed formula particles are measured according to example 1 to obtain a spectrum (see 2b in fig. 2), 16 batches of pokeweed standard decoction freeze-dried powder are measured according to example 2 to obtain a spectrum (see 2a in fig. 2), a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition in fingerprint similarity evaluation software compiled by pharmacopoeia committee is adopted, and a pattern of 'multi-point correction and Mark peak matching' is adopted to perform fitting to generate a pokeweed comparison characteristic spectrum, see fig. 3. The contrast characteristic spectrum has 9 common characteristic peaks, wherein the peak 5 is taken as a reference peak 1, the peak 1 has a relative retention time of 0.47,2, the peak 3 has a relative retention time of 0.67, the peak 4 has a relative retention time of 0.81, the peak 6 has a relative retention time of 1.24 and the peak 7 has a relative retention time of 1.64; peak 8 as reference peak 2, peak 9 relative retention time 1.07; the relative retention time should be within + -10% of the specified value. The feature patterns of the pokeberry root formula particles and the pokeberry root standard decoction freeze-dried powder are basically consistent, which shows that the pokeberry root formula particles and the pokeberry root formula particle extract have better transmissibility. The preparation method of the pokeberry root formula granule extract comprises the following steps: taking radix Phytolaccae medicinal material in order of radix Phytolaccae, removing impurities, and preparing into radix Phytolaccae Liu Yinpian; decocting Verticillium Liu Yinpian in water for 2 times, filtering with 200 mesh filter cloth, mixing filtrates, concentrating to fluid extract, and spray drying.

The characteristic peaks of 9 common chromatographic peaks in the HPLC characteristic spectrum of the vertical pokeberry root formula granule are identified and identified by combining HPLC and LC-MS/MS with a reference substance, and the results are shown in the following table. It was determined that peak 1 was adenosine, peak 2 was guanosine, peak 3 was L-tryptophan, peak 5 was erythrina base, and peak 8 was esculentoside A. Further, by combining the literature investigation analysis with the mass spectrometry analysis, the structures of the peak 4, the peak 6, the peak 7 and the peak 9 are analyzed, the peak 4 is presumed to be 4-aminotryptophan or an isomer thereof, the peak 6 is Saccharomyces molside C, the peak 7 is siberian polygalase A5, and the peak 9 is esculentoside B, and the above 4 substances are unknown in composition or difficult to obtain as a reference substance, and the alignment of the substances cannot be confirmed temporarily by adopting the reference substance. The results are shown in the following table and FIG. 4.

TABLE 1 LC-MS/MS analysis and detection results of pokeberry formulation particles

Note that: because mass spectrum is not compatible with phosphoric acid solution, when UPLC-Q-TOF-MS analysis is carried out, 0.1% formic acid solution is used for replacing the original mobile phase with 0.04% phosphoric acid solution for analysis, so that the retention time and the peak outlet sequence of each characteristic image peak are different from those of the original image, and the compound corresponding to each characteristic image peak is determined through comparison of a reference substance and analysis of a spectrogram.

Experimental example 1 determination of chromatographic conditions

1.1 gradient elution procedure

Taking the same batch of test products of pokeberry root formula particles, preparing 3 parts of test product solution according to the embodiment 1, and measuring to obtain a characteristic map, wherein the difference is that: the gradient elution procedure was different and is shown in table 2.

(1) Gradient elution procedure 1:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～5	1	99
5～15	1→8	99→92
			15～25	8→20	92→80
25～32	20→30	80→70
			32～37	30→35	70→65
37～50	35	65

(2) Gradient elution procedure 2:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～8	1	99
8～18	1→8	99→92
			18～28	8→20	92→80
28～35	20→30	80→70
			35～40	30→35	70→65
40～50	35	65

(3) Gradient elution procedure 3:

time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～5	1	99
5～15	1→8	99→92
			15～25	8→20	92→80
25～32	20→30	80→70
			32～40	30→35	70→65
40～50	35	65

TABLE 2 test results from different gradient elution procedures

From the above results, it can be seen that the gradient elution procedures 1-3 can all obtain satisfactory feature patterns. Further, the gradient elution procedure 1 is preferably a gradient elution procedure for constructing a feature map of pokeberry root in order, since each chromatographic peak in the chromatogram obtained by the gradient elution procedure 1 is uniformly distributed and the acquisition time is relatively short.

1.2 chromatographic conditions

The same batch of test articles of pokeberry root formula particles is prepared according to the example 1, and the test article solution is measured to obtain the characteristic spectrum, wherein the difference is that: the chromatographic conditions were different, and the respective chromatographic conditions were as follows.

(1) Chromatographic condition 1: using a Waters H-class instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,t3 (2.1X100 mm,1.6 μm) was used as a column, and gradient elution was performed as follows; the flow rate is 0.35ml/min; the column temperature is 30 ℃; the detection wavelength is 210nm and 254nm respectivelyThe sample volume was 2. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～10	1→8	99→92
10～20	8→30	92→70
			20～30	30→42	70→58

As can be seen from the results of FIG. 5, the chromatographic information at 210nm is richer than 254nm, and phytolacca methylsine, the main characteristic component, can be detected, so that the subsequent optimization is carried out at 210nm.

(2) Chromatographic condition 2: using a Waters H-class instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,t3 (2.1X100 mm,1.6 μm) was used as a column, and gradient elution was performed as specified in the following table; the flow rate is 0.35ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 2. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～5	1	99
5～13	1→8	99→92
			13～23	8→20	92→80
23～28	20→30	80→70
			28～40	30→42	70→58

As can be seen in fig. 6, increasing the aqueous phase elution time did not improve the separation effect of the first 5 minutes chromatographic peak and resulted in a poorer separation effect of the subsequent chromatographic peak.

(3) Chromatographic condition 3: using a Waters H-class instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,t3 (2.1X100 mm,1.6 μm) was used as a column, and gradient elution was performed as specified in the following table; the flow rate is 0.35ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 2. Mu.L.

As can be seen from FIG. 7, the chromatographic peaks with retention times of 8-12min need to be further optimized.

(4) Chromatographic condition 4: using a Waters H-class instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,t3 (2.1X100 mm,1.6 μm) was used as a column, and gradient elution was performed as specified in the following table; the flow rate is 0.35ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 2. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～8	1→6	99→94
8～15	6	94
			15～25	6→20	94→80
25～30	20→30	80→70
			30～40	30→42	70→58

As can be seen from FIG. 8, the chromatographic peaks with retention times of 9-12min need to be further optimized.

(5) Chromatographic condition 5: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～13	1→8	99→92
			13～23	8→20	92→80
23～33	20→23	80→77
			33～50	23→60	77→40

As can be seen from FIG. 9, the chromatographic information and separation effect of the chromatographic column are significantly better than those of the chromatographic columnT3 (2.1X100 mm,1.6 μm) was optimized on the basis of the column.

(6) Chromatographic condition 6: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.1% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～15	1→8	99→92
			15～25	8→20	92→80
25～35	20→30	80→70
			35～45	30→60	70→40

As is clear from FIG. 10, the separation effect of each of the other chromatographic peaks was relatively excellent except for the 7-9min chromatographic peak, and an attempt was made to change the concentration of phosphoric acid in the mobile phase.

(7) Chromatographic condition 7: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.03% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～15	1→8	99→92
			15～25	8→20	92→80
25～30	20→30	80→70
			30～45	30→60	70→40

As can be seen from FIG. 11, in the case of the condition 6, the separation of the chromatographic peak was still poor in the range of 7 to 9 minutes, and no chromatographic peak was observed in the range of 30 to 40 minutes, and the shortening of the analysis time was attempted.

(8) Chromatographic condition 8: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.03% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～15	1→8	99→92
			15～25	8→20	92→80
25～30	20→30	80→70
			30～40	30→60	70→40
40～45	60	40

As can be seen from FIG. 12, the separation effect of the chromatographic peak at 7-9min was not good, the specificity of the chromatographic peak at the first 10min was not good, and the chromatographic condition was discarded.

(9) Chromatographic condition 9: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.03% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L. The results are shown in FIG. 13.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	3	97
4～15	3→8	97→92
			15～25	8→20	92→80
25～30	20→30	80→70
			30～40	30→60	70→40
40～45	60	40

(10) Chromatographic condition 10: using a Waters e2695 instrument, using acetonitrile as a mobile phase A, using a 0.03% phosphoric acid solution as a mobile phase B,(4.6X250 mm,5 μm) as a column, gradient elution was performed as specified in the following table; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L. The results are shown in FIG. 14.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～8	2	98
8～15	2→8	98→92
			15～25	8→20	92→80
25～30	20→30	80→70
			30～40	30→60	70→40
40～45	60	40

As can be seen from chromatographic conditions 9 and 10, the first 7min chromatogram was still not ideal and an attempt was made to replace the column Porosill 120SB-Aq (4.6X105 mm,4 μm).

(11) Chromatographic condition 11: gradient elution was performed using an instrument Waters e2695, acetonitrile as mobile phase a,0.03% phosphoric acid solution as mobile phase B, poroshell 120SB-Aq (4.6x250 mm,4 μm) as chromatographic column, as specified in the table below; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength was 210nm and the sample volume was 10. Mu.L. The results are shown in FIG. 15.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～15	1→8	99→92
			15～25	8→20	92→80
25～30	20→30	80→70
			30～40	30→60	70→40
40～42	60	40

As shown in the result, each chromatographic peak in the chromatogram has uniform distribution and good separation effect, but the chromatographic peak (phytolacca acinosa) of about 32min is closer to the adjacent peak, so that the chromatographic peak can be further optimized.

(12) Chromatographic condition 12: gradient elution was performed using an instrument Waters e2695, acetonitrile as mobile phase a,0.03% phosphoric acid solution as mobile phase B, poroshell 120SB-Aq (4.6x250 mm,4 μm) as chromatographic column, as specified in the table below; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength is 210nm, the sample injection volume is 10 mu L, and the spectrum is shown in figure 16.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～4	1	99
4～15	1→8	99→92
			15～25	8→20	92→80
25～35	20→30	80→70
			35～50	30→40	70→60

(13) Chromatographic condition 13: gradient elution was performed using an instrument Waters e2695, acetonitrile as mobile phase a,0.03% phosphoric acid solution as mobile phase B, poroshell 120SB-Aq (4.6x250 mm,4 μm) as chromatographic column, as specified in the table below; the flow rate is 1ml per minute; the column temperature is 30 ℃; the detection wavelength is 210nm, and the sample injection volume is 10L. The results are shown in FIG. 17.

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0～5	1	99
5～15	1→8	99→92
			15～25	8→20	92→80
25～32	20→30	80→70
			32～37	30→35	70→65
37～50	35	65

As shown by the result, each chromatographic peak in the chromatogram is uniformly distributed, and the separation effect is good, so that the method is used as an initial condition for carrying out subsequent system optimization.

1.3 wavelength

According to the chromatographic condition 13 of 1.2 in experimental example 1, the sample solution of the pokeberry formulation particles with vertical order is injected into a chromatograph, and full-wave scanning is carried out at 210-400nm, as shown in figure 18, chromatograms at different absorption wavelengths of 210nm, 230nm, 254nm, 280nm, 300nm and 330nm are compared according to the information content of the chromatogram, so as to detect the number of chromatographic peaks, the absorption intensity corresponding to the retention time and the baseline stability condition as the evaluation indexes. The experimental result shows that the number of chromatographic peaks is obviously reduced under the detection wavelength of 280nm-330nm, and the number of the chromatographic peaks at 210nm, 230nm and 254nm is not obviously different, and the detection wavelength of the vertical sequence pokeberry characteristic spectrum is preferably 210nm.

1.4 flow Rate

Taking the same batch of pokeberry formulation particles, preparing 3 parts of test solution according to the embodiment 1, and measuring according to the embodiment 1 by taking the flow rate as a variable to obtain a map corresponding to different flow rates, wherein the result is as follows; wherein the flow rates are respectively 0.8ml/min, 1.0ml/min and 1.2ml/min.

Table 3 different flow rates investigation System applicability parameters

From the above results, it can be seen that the effect is optimal when the flow rate is 1.0 ml/min.

1.5 column temperature

Taking the same batch of pokeberry root formula particles, preparing 3 parts of test solution according to the embodiment 1, and measuring according to the embodiment 1 by taking column temperature as a variable to obtain maps corresponding to different column temperatures, wherein the results are as follows; wherein the column temperature is 25 ℃,30 ℃ and 35 ℃ respectively.

Table 4 different column temperature investigation system applicability parameters

From the above results, when the column temperature is 30 ℃, the degree of separation of each characteristic peak is better, and 30 ℃ is preferable as the column temperature.

1.6 chromatography column

Taking the same batch of pokeberry root formula particles, preparing 3 parts of test solution according to the embodiment 1, and measuring according to the method of the embodiment 1 by taking chromatographic columns of different batches as variables to obtain corresponding maps of different batches, namely batches 1-3 respectively, wherein the results are as follows;

TABLE 5 System applicability parameters of the chromatographic peaks obtained for different chromatographic columns

The results show that the spectra obtained by the chromatographic columns of different batches have no obvious difference, and the system applicability of each characteristic peak meets the measurement requirement.

1.7 Mobile phase B concentration

Taking the same batch of pokeberry formulation granule test products, preparing 4 parts of test product solution according to the method of example 1, and measuring according to the method of example 1 by taking different mobile phase B concentrations as variables to obtain a map; wherein, the concentration of the mobile phase B is respectively 0.02%, 0.03%, 0.04% and 0.05%.

TABLE 6 applicability parameters of different phosphoric acid concentration investigation systems

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From the above results, it can be seen that the applicability parameters of the respective chromatographic peak systems are good and have no significant difference when the mobile phase B concentration is 0.03 to 0.05%. Subsequent studies were performed to select a phosphoric acid concentration of 0.04% in order to improve the durability of the process.

In summary, chromatographic conditions and system suitability test preferred protocol: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is a mobile phase A, and a 0.04% phosphoric acid aqueous solution is a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm. The gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

Experimental example 2 investigation of sample solution preparation method

2.1 extraction solvent

Taking the same batch of pokeberry formulation particles, taking the extraction solvent as a variable, preparing a test solution according to the embodiment 1, and measuring to obtain a map; wherein the extraction solvents are water, 30% methanol, 60% methanol, 80% methanol and methanol, respectively.

TABLE 7 Phytolacca acinosa formula particle chromatogram parameters for different extraction solvent sagging sequences

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The above results show that when the extraction solvent is 60% methanol, 80% methanol, the chromatographic peak area and the system applicability do not significantly change, and 60% methanol is preferable as the extraction solvent for saving the amount of methanol.

2.2 extraction modes

Taking the same batch of pokeberry formulation particles, taking an extraction mode as a variable, preparing a test solution according to the embodiment 1, and measuring to obtain a map; wherein the extraction mode is ultrasonic treatment or heating reflux. Wherein, ultrasonic treatment: the power is 300W, the frequency is 40kHz, and the time is 30min; and (3) heating and refluxing: the time was 30min.

TABLE 8 Phytolacca acinosa formula particle chromatogram parameters for different extraction modes

The information quantity of chromatographic peaks and the system applicability parameters are used as main investigation indexes. From the above results, it can be seen that the extraction mode has no significant influence on the chromatographic information amount and the system applicability parameter, and ultrasound is preferred as the extraction mode for the sake of operational simplicity.

2.3 extraction time investigation

Taking the same batch of pokeberry formulation particles, taking the extraction time as a variable, preparing a test solution according to the embodiment 1, and measuring to obtain a map; wherein the extraction time is 20min, 30min, 40min.

TABLE 9 Phytolacca acinosa formula particle chromatogram parameters for different extraction times

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The chromatographic peak information and the system applicability parameters are used as main investigation indexes. From the above results, it can be seen that the extraction time has no significant effect on the chromatographic peak information amount and the system applicability parameter, and for complete extraction, ultrasound for 30min is preferred as the extraction time.

2.4 sample size

Sample solutions were prepared according to example 1 and measured to obtain maps corresponding to different sample amounts, with the sample amounts of the sample as variables, wherein the sample amounts of the sample were 0.1g, 0.2g, 0.4g, and 0.8g, respectively.

TABLE 10 Phytolacca acinosa formula particle chromatogram parameters for different sample volumes

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The information quantity of chromatographic peaks and the system applicability parameters are used as main investigation indexes. From the above results, it can be seen that the peak area of the chromatographic peak increases by a corresponding multiple with increasing sampling amount, indicating that the sampling amount is in the range of 0.1 to 0.8g, and the components in the vertical pokeberry formulation particles can be completely extracted, preferably 0.2g as the sampling amount in view of the response value and the baseline smoothness.

2.5 sample injection volume

Sample volumes were 5. Mu.L, 10. Mu.L and 15. Mu.L, respectively, as variables, sample solutions were prepared and measured as in example 1 to obtain maps corresponding to the different sample volumes.

TABLE 11 chromatographic peak System applicability parameters at different sample volumes

The information quantity of chromatographic peaks and the system applicability parameters are used as main investigation indexes. From the above results, it can be seen that the peak area of the chromatographic peak increases by a corresponding multiple with increasing sample injection amount. In view of peak shape, degree of separation of chromatographic peaks and baseline smoothness, 10. Mu.L is preferable as the sample amount.

In summary, the preparation method of the sample solution preferably comprises: taking a proper amount of a test sample, grinding, taking about 0.2g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 60% methanol, performing ultrasonic treatment (power 300W, frequency 40 kHz) for 30 minutes, taking out, cooling, shaking uniformly, filtering, and taking the subsequent filtrate.

Experimental example 3 methodology investigation

3.1 investigation of specificity

Test solution, reference solution and reference solution of the pokeberry root formula particles are prepared according to the method of example 1, chromatographic conditions and system applicability of the pokeberry root formula particles are examined according to the method of example 1, and whether blank auxiliary materials (maltodextrin) cause interference is examined, wherein the blank auxiliary material patterns are shown in fig. 19, and the results are as follows:

table 12 control line system applicability parameters

Chromatographic peak	Name of the name	Retention time	Peak area	Peak height	Degree of separation	Tailing factor	Number of theoretical plates
								1	Erythrina base	15.464	4844944	214748	11613	1.10	/
2	Esculentoside A	40.387	536585	581164	444328	1.24	/

Table 13 reference drug system applicability parameters

Chromatographic peak	Retention time	Peak area	Peak height	Degree of separation	Tailing factor	Number of theoretical plates
							1	7.256	734760	54516	\	1.60	6422
2	8.457	228720	14380	3.06	1.22	6503
							3	10.422	291258	14468	4.18	1.45	7388
4	12.424	339912	20617	4.28	0.87	13516
							5	15.503	581058	31390	6.71	1.32	15525
6	19.160	94516	8172	9.57	1.60	75678
							7	25.426	90823	12434	2.57	1.54	278058
8	40.372	403796	35645	58.19	1.56	318912
							9	43.143	98830	6502	7.56	1.12	215704

Table 14 test line system applicability parameters

Sequence number	Retention time	Peak area	Peak height	Degree of separation	Tailing factor	Number of theoretical plates
							1	7.155	335069	43301	3.29	1.28	19735
2	8.489	201479	15816	4.69	1.14	10198
							3	10.720	335642	28276	6.64	1.18	20200
4	12.649	211725	22721	6.93	1.31	41660
							5	15.624	617965	51474	10.58	1.45	40593
6	19.328	70167	8814	1.88	0.82	164439
							7	25.573	60013	11889	3.92	1.04	555281
8	40.516	151246	22284	21.1	1.15	832408
							9	43.344	31896	3738	8.90	0.93	566417

The results show that the test solution, the reference solution and the reference solution of the pokeberry root formula particles have good applicability; figure 19 illustrates that the blank auxiliary material has no interference to the characteristic spectrum of the test sample, and the chromatographic method has good system applicability and specificity.

3.2 precision

A sample solution of the vertical pokeberry root formula particles is prepared according to the example 1, and is continuously sampled for 6 times, the relative retention time and the relative peak area of each characteristic peak are calculated, and the result shows that the relative retention time of each characteristic peak has RSD less than 2% and the relative peak area has RSD less than 5%, which indicates that the instrument precision is good.

Table 15 results of the instrument precision versus retention time test

Table 16 results of instrument precision versus peak area test

3.3 method reproducibility

Taking the same batch of pokeberry formulation particles in vertical order, preparing 6 parts of test solution according to the embodiment 1, measuring the disease, obtaining a map, and calculating the relative retention time and the relative peak area of each characteristic peak. The results showed that the relative retention time of each characteristic peak was less than 2% and the relative peak area was less than 5% indicating good reproducibility of the process.

Table 17 repeatability test versus retention time test results

TABLE 18 results of repeatability versus peak area test

3.4 intermediate precision

(1) Intermediate precision of different personnel

According to the method, 3 different workers (A, B, C) respectively prepare 2 parts of test sample solutions according to the method in example 1, the test is carried out on the test sample solutions in the same instrument to obtain characteristic maps of pokeberry formulation granules with different vertical sequences, the relative retention time and the relative peak area of each characteristic peak are calculated, the result shows that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, and the method has good operation precision for different workers.

TABLE 19 results of intermediate precision versus retention time tests for different personnel

Table 20 results of intermediate precision versus peak area tests for different personnel

(2) Intermediate precision of different instruments

6 parts of sample solution of the pokeberry root formula particles are prepared according to the example 1, and are sampled and measured at different times and by different instruments to obtain a map, and the relative retention time and the relative peak area of each characteristic peak are calculated. The result shows that the relative retention time of each characteristic peak is less than 5% and the relative peak area is less than 10%, which indicates that the method has good intermediate precision in different instruments.

Table 21 results of intermediate precision versus retention time tests for different instruments

Table 22 results of intermediate precision versus peak area tests for different instruments

3.5 durability

Sample solutions of the vertical pokeberry root formula particles were prepared according to example 1, and sample injections were performed at 0h, 2h, 4h, 8h, 12h, 18h, 24h, 36h, and 48h, respectively, after the preparation of the sample solutions was completed, and the relative retention time and relative peak area of each characteristic peak were determined. The result shows that the relative retention time of each characteristic peak has RSD value less than 2% and the relative peak area of each characteristic peak has RSD value less than 5%, which indicates that the sample solution is stable within 48h and meets the measurement requirement.

Table 23 stability investigation relative retention time test results

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Table 24 stability investigation of the relative peak area test results

3.6 durability inspection of different flow rates

Taking the same batch of pokeberry formulation particles in vertical order, obtaining 3 parts of test solution according to the embodiment 1, measuring the same test solution by taking the flow rate as a variable, and calculating the relative retention time and the relative peak area of each characteristic peak; the flow rates were 0.9ml/min, 1.0ml/min, and 1.1ml/min, respectively. The results show that the relative retention time of each characteristic peak has an RSD value of less than 5% and the relative peak area of each characteristic peak has an RSD value of less than 10%, and the method has certain durability to the flow rate.

Table 25 comparison of different flow rates versus retention time results

Table 26 comparison of different flow rates versus peak area results

3.7 investigation of durability at different column temperatures

Taking the same batch of pokeberry formulation particles, preparing 3 parts of test solution according to the embodiment 1, measuring by taking column temperature as a variable, and calculating the relative retention time and the relative peak area of each characteristic peak; the column temperatures were 28 ℃,30 ℃ and 32 ℃ respectively. The results show that the relative retention time of each characteristic peak has an RSD value of less than 5% and the relative peak area of each characteristic peak has an RSD value of less than 10%, which indicates that the column has certain durability to column temperature.

Table 27 comparison of results of different column temperatures versus retention time

Table 28 comparison of Peak area results for different columns Wen Xiangdui

3.8 investigation of durability of different chromatography columns

Taking the same batch of pokeberry root formula particle test products, preparing 2 parts of test product solutions according to the embodiment 1, examining the influence of chromatographic columns of different batches on characteristic peaks, and the result shows that the RSD value of the relative retention time of each characteristic peak is less than 5%, and the RSD value of the relative peak area of each characteristic peak is less than 10%, which indicates that the method has good durability on the chromatographic columns. Wherein, the batch numbers of the chromatographic columns are B21336 and B21193 respectively; porosill 120SB-Aq, 4.6X105 mm,4 μm.

Table 29 comparison of the results of different chromatographic columns with respect to retention time

Table 30 comparison of the results of the relative peak areas of different chromatographic columns

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. The construction method of the vertical sequence pokeberry characteristic map is characterized by comprising the following steps of:

preparation of test solution: preparing a sample solution from a sample;

and (3) measuring: the chromatographic conditions of the high performance liquid chromatography include: acetonitrile is taken as a mobile phase A, phosphoric acid aqueous solution is taken as a mobile phase B, and gradient elution is carried out;

the gradient elution procedure included: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B; or alternatively, the first and second heat exchangers may be,

0-8min,1% mobile phase A,99% mobile phase B;8-18min, 1-8% mobile phase A, 99-92% mobile phase B;18-28min, 8-20% mobile phase A, 92-80% mobile phase B;28-35min, 20-30% mobile phase A, 80-70% mobile phase B;35-40min, 30-35% mobile phase A, 70-65% mobile phase B;40-50min,35% mobile phase A,65% mobile phase B; or alternatively, the first and second heat exchangers may be,

0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-40min, 30-35% mobile phase A, 70-65% mobile phase B;40-50min,35% mobile phase A,65% mobile phase B.

2. The method of claim 1, wherein the chromatographic conditions of the high performance liquid chromatography further comprise: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, and the grain diameter is 4 mu m; and/or the number of the groups of groups,

the column temperature is 25-35 ℃; and/or the number of the groups of groups,

the wavelength is 210-254nm; and/or the number of the groups of groups,

the sample injection amount is 5-15 mu L; and/or the number of the groups of groups,

the flow rate is 0.8-1.2ml/min; and/or the number of the groups of groups,

taking 0.03-0.05% phosphoric acid aqueous solution as mobile phase B.

3. The construction method according to claim 1 or 2, wherein the gradient elution procedure comprises: 0-5min,1% mobile phase A,99% mobile phase B;5-15min, 1-8% mobile phase A, 99-92% mobile phase B;15-25min, 8-20% mobile phase A, 92-80% mobile phase B;25-32min, 20-30% mobile phase A, 80-70% mobile phase B;32-37min, 30-35% mobile phase A, 70-65% mobile phase B;37-50min,35% mobile phase A,65% mobile phase B.

4. A method of constructing as claimed in any one of claims 1 to 3 wherein the chromatographic conditions of said high performance liquid chromatography comprise: the poroshall 120SB-Aq is used as a chromatographic column, and the specification of the chromatographic column is as follows: the inner diameter is 4.6mm, the column length is 250mm, the grain diameter is 4 mu m, acetonitrile is taken as a mobile phase A, and 0.04% phosphoric acid aqueous solution is taken as a mobile phase B; the flow rate is 1ml/min; the column temperature is 30 ℃; the detection wavelength was 210nm.

5. The method according to any one of claims 1 to 4, wherein the test sample is a pokeberry formulation granule, pokeberry standard decoction lyophilized powder, pokeberry medicinal material or pokeberry Liu Yinpian.

6. The method of constructing according to any one of claims 1 to 5, wherein the method of preparing the test solution comprises: extracting the sample with solvent, and filtering;

preferably, in preparing the sample solution, the extraction solvent is at least one of methanol and water;

preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%;

preferably, in preparing the sample solution, the extraction method is reflux extraction or ultrasonic extraction.

7. The method according to claim 6, wherein when the sample is lyophilized powder or formulated granule, the mass of the sample per 1ml of the extraction solvent is 0.01g to 0.08g;

when the sample is medicinal material or decoction piece, the mass of the water extract corresponding to each 1ml of the extraction solvent is 0.01g-0.08g.

8. The method of any one of claims 1-7, further comprising preparing a control drug reference solution;

the preparation method of the reference substance solution of the reference medicinal material comprises the following steps: extracting reference materials with water to obtain water extract, adding extraction solvent into the water extract, mixing, and filtering;

preferably, in preparing the reference solution of the reference medicinal material, the extraction solvent is at least one of methanol and water;

preferably, the extraction solvent is an aqueous methanol solution; preferably, the volume fraction of methanol in the aqueous methanol solution is not less than 60%;

preferably, the volume of water corresponding to each 1g of medicinal material of the order-dropping quotient Liu Duizhao is 25-100ml;

preferably, the mass of the extraction solvent is 0.01g-0.08g per 1ml of the aqueous extract;

preferably, in obtaining the aqueous extract, the extraction method is reflux extraction or ultrasonic extraction.

9. The method of any one of claims 1-8, further comprising preparing a control solution;

the reference substance is at least one of esculentoside A, erythrina base, adenosine, guanosine and L-tryptophan.

10. The method according to any one of claims 1 to 9, wherein the profile obtained by the method comprises 9 characteristic peaks;

peak 5 was used as reference peak 1, peak 1 had a relative retention time of 0.47,2, peak 3 had a relative retention time of 0.67, peak 4 had a relative retention time of 0.81, peak 6 had a relative retention time of 1.24, and peak 7 had a relative retention time of 1.64;

peak 8 as reference peak 2, peak 9 relative retention time 1.07;

the relative retention time should be within + -10% of the specified value.