Method for constructing characteristic spectrum of Eriocaulon buergerianum and its application

Abstract

The present invention provides a method for constructing a characteristic chromatogram of Eriocaulon buergerianum and its application. The construction method includes: detecting reference substances, reference medicinal materials and test samples by high performance liquid chromatography, calibrating the common peaks to obtain the characteristic chromatogram of Eriocaulon buergerianum; in the high performance liquid chromatography, acetonitrile is used as mobile phase A, and formic acid aqueous solution is used as mobile phase B; the mass percentage content of formic acid in the formic acid aqueous solution is 0.25-0.45%. In the present invention, the construction method has good resolution, can comprehensively reflect the characteristic peak information of the sample, has high precision, good reproducibility and good stability, and can efficiently realize the quality control of Eriocaulon buergerianum formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for constructing a characteristic fingerprint of Eriocaulon buergerianum and its application. Background Art

[0002] The medicinal material of Eriocaulon buergerianum is the dried capitulum with scape of the plant Eriocaulon buergerianum in the family Eriocaulaceae, also known as Dai Xing Cao, Wen Xing Cao, Meteor Grass, etc., and is an annual herb in the family Eriocaulaceae. Its functions and indications are: dispersing wind-heat, improving eyesight and removing nebula; used for diseases such as wind-heat red eyes, swelling and pain with photophobia, corneal nebula, wind-heat headache, etc. Modern research shows that Eriocaulon buergerianum has functions such as antibacterial, antioxidant, α-glucosidase inhibition, and protection against 6-OHDA-induced nerve damage. Moreover, Eriocaulon buergerianum can also be used as a compound component in combination with other traditional Chinese medicines and is widely used in ophthalmic diseases, such as viral keratitis, uveitis cataract, amaurotic optic neuritis, epidemic conjunctivitis, etc., and the treatment efficiency of the diseases can reach more than 90%. Therefore, its medicinal value has been increasingly emphasized by people. At present, the legal standard quantitatively analyzes the medicinal material of Eriocaulon buergerianum with vanillic acid as the component, and the identified components are few, which is not conducive to the quality control of the medicinal material.

[0003] Therefore, developing a method that can comprehensively reflect the characteristic peak information of the sample, has good resolution, stability, high precision, good reproducibility, and can efficiently detect the content of the effective components of Eriocaulon buergerianum is an urgent problem to be solved in this field. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for constructing a characteristic fingerprint of Eriocaulon buergerianum and its application. The construction method has good resolution, can relatively comprehensively reflect the characteristic peak information of the sample, high precision, good reproducibility, good stability, and can efficiently realize the quality control of the effective components of Eriocaulon buergerianum.

[0005] To achieve this purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum, and the construction method includes:

[0007] The reference substance, reference crude drug and test sample are detected by high performance liquid chromatography, and the common peaks are calibrated to obtain the characteristic chromatogram of the Eriocaulon buergerianum Miq.; in the high performance liquid chromatography, acetonitrile is used as mobile phase A, and formic acid aqueous solution is used as mobile phase B; the mass percentage content of formic acid in the formic acid aqueous solution is 0.25-0.45% (for example, it can be 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, etc.).

[0008] In the present invention, the mobile phase is selected as acetonitrile and formic acid aqueous solution with a specific mass concentration to elute the sample, and the separation degree is good. There are more characteristic peaks in the obtained chromatogram, which can more comprehensively reflect the information of the characteristic peaks of the sample; and the method has high precision, good reproducibility and good stability, and can efficiently realize the quality control of the effective components of Eriocaulon buergerianum Miq.

[0009] Preferably, the Eriocaulon buergerianum Miq. includes Eriocaulon buergerianum Miq. crude drug, Eriocaulon buergerianum Miq. formula granules or Eriocaulon buergerianum Miq. decoction pieces.

[0010] Preferably, in the high performance liquid chromatography, the sample is gradient eluted with the mobile phase.

[0011] Preferably, in the high performance liquid chromatography, the elution gradient is as follows:

[0012] 0→5 min: the volume percentage content of mobile phase A is 1%, and the volume percentage content of mobile phase B is 99%; 5→32 min: the volume percentage content of mobile phase A is 1-16%, and the volume percentage content of mobile phase B is 99-84%; 32→40 min: the volume percentage content of mobile phase A is 16-17%, and the volume percentage content of mobile phase B is 84-83%; 40→45 min: the volume percentage content of mobile phase A is 17-21%, and the volume percentage content of mobile phase B is 83-79%; 45→50 min: the volume percentage content of mobile phase A is 21-23%, and the volume percentage content of mobile phase B is 79-77%; 50→55 min: the volume percentage content of mobile phase A is 23-27%, and the volume percentage content of mobile phase B is 77-73%.

[0013] In the present invention, gradient elution of the sample is carried out with a specific elution gradient, and a test sample chromatogram with more characteristic peaks can be obtained, with high separation degree and high detection efficiency.

[0014] In the present invention, by way of example, the elution gradient is as follows: 5→32 min: the volume percentage of acetonitrile in the mobile phase is 1 to 16%, and the volume percentage of the aqueous formic acid solution is 99 to 84%, which means that at 32 min, the composition of the mobile phase becomes 16% acetonitrile and 84% aqueous formic acid solution.

[0015] Preferably, the flow rate of the mobile phase is 0.25 to 0.35 mL / min, for example, it can be 0.25 mL / min, 0.26 mL / min, 0.27 mL / min, 0.28 mL / min, 0.29 mL / min, 0.3 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, 0.35 mL / min, etc.

[0016] Preferably, in the high performance liquid chromatography, the column temperature is 30 to 40 °C, for example, it can be 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C, etc.

[0017] Preferably, in the high performance liquid chromatography, the stationary phase includes octadecylsilyl-bonded silica gel.

[0018] Preferably, the particle size of the stationary phase is 1.8 μm.

[0019] Preferably, the column length of the chromatographic column is 150 mm; the inner diameter of the chromatographic column is 2.1 mm.

[0020] Preferably, in the high performance liquid chromatography, the detection wavelength is 250 to 280 nm, for example, it can be 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 262 nm, 265 nm, 268 nm, 270 nm, 272 nm, 275 nm, 280 nm, etc.

[0021] Preferably, in the high performance liquid chromatography, the injection volume is 1 to 3 μL, for example, it can be 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, etc.

[0022] Preferably, the reference substance, the reference medicinal material and the test sample each independently exist in the form of a solution.

[0023] Preferably, the preparation methods of the reference medicinal material solution and the test sample solution each independently include:

[0024] Mix the reference medicinal material or the test sample with a solvent and ultrasonicate to obtain the reference medicinal material solution or the test sample solution.

[0025] Preferably, the solvent includes an aqueous methanol solution.

[0026] Preferably, the mass concentration of the aqueous methanol solution is 40-60%, for example, it can be 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, etc.

[0027] Preferably, the concentrations of the control crude drug solution and the test sample solution are each independently 0.02-0.06 g / mL, for example, it can be 0.02 g / mL, 0.025 g / mL, 0.03 g / mL, 0.035 g / mL, 0.04 g / mL, 0.045 g / mL, 0.05 g / mL, 0.055 g / mL, 0.06 g / mL, etc.

[0028] Preferably, the power of the ultrasonic wave is 200-300 W, for example, it can be 200 W, 220 W, 240 W, 260 W, 280 W, 300 W, etc.; the time of the ultrasonic wave is 20-40 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, etc.

[0029] In the present invention, the frequency of the ultrasonic wave is 30-50 kHz, for example, it can be 30 kHz, 35 kHz, 40 kHz, 45 kHz, 50 kHz, etc.

[0030] In the present invention, the control crude drug is the control crude drug of Eriocaulon buergerianum Koern.

[0031] In the present invention, the preparation method of the reference substance solution includes: mixing the reference substance with an aqueous methanol solution having a mass concentration of 40-60% to obtain the reference substance solution; the concentration of the reference substance solution is 10-30 μg / mL, for example, it can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, etc.

[0032] In the present invention, the reference substances include uridine, guanosine, adenosine, protocatechuic acid, vanillic acid, caffeic acid, ferulic acid, rutin, etc.

[0033] Preferably, the number of common characteristic peaks in the characteristic chromatogram of the test sample is ≥15.

[0034] Preferably, the characteristic peaks include the corresponding characteristic peaks of uridine, adenosine, guanosine, protocatechuic acid, vanillic acid, caffeic acid, ferulic acid, and rutin.

[0035] Preferably, the construction method includes the following steps:

[0036] Detecting the reference substance, the control crude drug, and the test sample by high performance liquid chromatography, calibrating the common peaks, and obtaining the characteristic chromatogram of Eriocaulon buergerianum Koern.

[0037] In the high performance liquid chromatography, the stationary phase includes octadecylsilyl bonded silica gel, acetonitrile is used as mobile phase A, and an aqueous formic acid solution with a mass percentage of 0.25-0.45% is used as mobile phase B. The sample is subjected to gradient elution, and the elution gradient is as follows: 0→5 min: the volume percentage of mobile phase A is 1%, and the volume percentage of mobile phase B is 99%; 5→32 min: the volume percentage of mobile phase A is 1-16%, and the volume percentage of mobile phase B is 99-84%; 32→40 min: the volume percentage of mobile phase A is 16-17%, and the volume percentage of mobile phase B is 84-83%; 40→45 min: the volume percentage of mobile phase A is 17-21%, and the volume percentage of mobile phase B is 83-79%; 45→50 min: the volume percentage of mobile phase A is 21-23%, and the volume percentage of mobile phase B is 79-77%; 50→55 min: the volume percentage of mobile phase A is 23-27%, and the volume percentage of mobile phase B is 77-73%; the flow rate of the mobile phase is 0.25-0.35 mL / min; the column temperature is 30-40 °C, the injection volume is 1-3 μL, and the detection wavelength is 250-280 nm.

[0038] In a second aspect, the present invention provides an application of the construction method as described in the first aspect in detecting the content of active ingredients in Eriocaulon buergerianum.

[0039] The numerical ranges described in the present invention not only include the above-listed point values, but also any point values between the above numerical ranges not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The construction method of the characteristic fingerprint of Eriocaulon buergerianum provided by the present invention uses acetonitrile and an aqueous formic acid solution with a specific mass concentration as the mobile phase to elute the sample, with good resolution, a large number of characteristic peaks in the obtained fingerprint, and can more comprehensively reflect the information of the characteristic peaks of the sample; and the method has high precision, good reproducibility and good stability, and can efficiently realize the quality control of the active ingredients in Eriocaulon buergerianum. Description of the Drawings

[0042] Figure 1 It is a control characteristic fingerprint obtained by software simulation;

[0043] Among them, peak 1 corresponds to uridine; peak 2 corresponds to adenosine; peak 3 corresponds to guanosine; peak 4 corresponds to protocatechuic acid; peak 5 (S) corresponds to vanillic acid; peak 6 corresponds to caffeic acid; peak 9 corresponds to ferulic acid; peak 11 corresponds to rutin;

[0044] Figure 2The characteristic chromatograms of Eriocaulon buergerianum formula granules of different batches obtained by using the construction method provided in Example 1 of the present invention;

[0045] Among them, R(15) is the control characteristic chromatogram; S1(15) is the characteristic chromatogram of Eriocaulon buergerianum formula granules with batch number K463CP01; S2(15) is the characteristic chromatogram of Eriocaulon buergerianum formula granules with batch number K463CP10; S3(15) is the characteristic chromatogram of Eriocaulon buergerianum formula granules with batch number K463CP12;

[0046] Figure 3 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 2 of the present invention;

[0047] Figure 4 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 3 of the present invention;

[0048] Figure 5 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 4 of the present invention;

[0049] Figure 6 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 5 of the present invention;

[0050] Figure 7 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 6 of the present invention;

[0051] Figure 8 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 7 of the present invention;

[0052] Figure 9 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 8 of the present invention;

[0053] Figure 10 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 9 of the present invention;

[0054] Figure 11 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 10 of the present invention;

[0055] Figure 12 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 11 of the present invention;

[0056] Figure 13 The characteristic chromatogram of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 12 of the present invention;

[0057] Figure 14 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 13 of the present invention;

[0058] Figure 15 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Example 14 of the present invention;

[0059] Figure 16 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 1 of the present invention;

[0060] Figure 17 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 2 of the present invention;

[0061] Figure 18 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 3 of the present invention;

[0062] Figure 19 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 4 of the present invention;

[0063] Figure 20 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 5 of the present invention;

[0064] Figure 21 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 6 of the present invention;

[0065] Figure 22 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 7 of the present invention;

[0066] Figure 23 It is the characteristic spectrum of Eriocaulon buergerianum formula granules obtained by using the construction method provided in Comparative Example 8 of the present invention. Detailed implementation manners

[0067] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0068] The instruments and materials used in the present invention are as follows:

[0069] Instruments: Analytical balance (Sartorius, SQP QUINTIX224-1CN, Germany), Waters high performance liquid chromatograph (equipped with TUV detector, temperature-controlled column oven, temperature-controlled auto sampler, USA), high-speed bench centrifuge (SIGMA, 1-14, Germany), ultrasonic cleaner (Tianjin Outsen Science & Technology Co., Ltd., AS20500BT, China).

[0070] Test drugs: Eriocaulon buergerianum formula granules (batch numbers K463CP01, K463CP10, K463CP12), prepared by Beijing Kangrentang Pharmaceutical Co., Ltd.;

[0071] Eriocaulon buergerianum cut crude drugs (batch numbers: K463YP01, K463YP10, K463YP12), provided by Beijing Kangrentang Pharmaceutical Co., Ltd.

[0072] Reference crude drug of Eriocaulon buergerianum (batch number: 121631-201903, National Institutes for Food and Drug Control).

[0073] Uridine (batch number: 110887-202104, National Institutes for Food and Drug Control).

[0074] Guanosine (batch number: 111977-201501, National Institutes for Food and Drug Control).

[0075] Adenosine (batch number: 110879-201703, National Institutes for Food and Drug Control).

[0076] Protocatechuic acid (batch number: 110809-201906, National Institutes for Food and Drug Control).

[0077] Vanillic acid (batch number: 110776-201503, National Institutes for Food and Drug Control).

[0078] Caffeic acid (batch number: 110885-201703, National Institutes for Food and Drug Control).

[0079] Ferulic acid (batch number: 110773-201915, National Institutes for Food and Drug Control).

[0080] Rutin (batch number: 100080-202012, National Institutes for Food and Drug Control).

[0081] Reagents: Acetonitrile (Tianjin Bohua Chemical Reagent Co., Ltd., 20210220, chromatographic grade), methanol (Tianjin Bohua Chemical Reagent Co., Ltd., 20210301, analytical grade), formic acid (Tokyo Chemical Industry Co., Ltd., 3C7ON-NJ, chromatographic grade).

[0082] Preparation Example 1

[0083] Reference substance solution: Mix reference substances uridine, guanosine, adenosine, protocatechuic acid, vanillic acid, and rutin with 50% methanol respectively to prepare a reference substance solution with a concentration of 20 μg / mL.

[0084] Control crude drug solution: Mix 1.0 g of the control crude drug of Eriocaulon buergerianum with 25 mL of 50% methanol aqueous solution, ultrasonically treat for 30 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, cool, weigh, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the control crude drug solution.

[0085] Test sample solution: Mix 1.0 g of the finely ground Eriocaulon buergerianum formula granules with 25 mL of 50% methanol aqueous solution, ultrasonically treat for 30 minutes under the conditions of a power of 250 W and a frequency of 40 kHz, cool, weigh, make up the lost weight with 50% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test sample solution.

[0086] Preparation Example 2

[0087] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, 30% methanol aqueous solution is used, and the others are the same as in Preparation Example 1.

[0088] Preparation Example 3

[0089] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, 70% methanol aqueous solution is used, and the others are the same as in Preparation Example 1.

[0090] Preparation Example 4

[0091] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, instead of ultrasonic treatment, heat reflux for 30 min is carried out, and the others are the same as in Preparation Example 1.

[0092] Preparation Example 5

[0093] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, the ultrasonic treatment time is 20 min, and the others are the same as in Preparation Example 1.

[0094] Preparation Example 6

[0095] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, the volume of the methanol aqueous solution is 15 mL, and the others are the same as in Preparation Example 1.

[0096] Preparation Example 7

[0097] The difference from Preparation Example 1 is only that in the preparation method of the test sample solution, the volume of the methanol aqueous solution is 50 mL, and the others are the same as in Preparation Example 1.

[0098] Preparation Example 8

[0099] The difference from Preparation Example 1 is only that in the preparation method of the test solution, the ultrasonic treatment time is 40 min, and the others are the same as those in Preparation Example 1.

[0100] Example 1

[0101] This example provides a method for constructing a characteristic chromatogram of Eriocaulon buergerianum formula granules, which specifically includes the following steps:

[0102] Respectively absorb 2 μL of the reference substance solution, the reference crude drug solution and the test solution (Preparation Example 1), inject them into an ultra-high performance liquid chromatograph for detection, calibrate the common peaks, and obtain the characteristic chromatogram of the Eriocaulon buergerianum formula granules; the conditions of the ultra-high performance liquid chromatography are as follows:

[0103] Stationary phase: octadecylsilane-bonded silica gel (particle size 1.8 μm);

[0104] Chromatographic column: ACQUITY HSS T3, with a length of 150 mm and an inner diameter of 2.1 mm; the column temperature is 33 °C;

[0105] Mobile phase: acetonitrile and formic acid aqueous solution with a mass concentration of 0.3%; the flow rate of the mobile phase is 0.3 mL / min;

[0106] Gradient elution, and the elution gradient is as follows:

[0107]

[0108]

[0109] The detection wavelength is 260 nm.

[0110] Example 2

[0111] This example provides a method for constructing a characteristic chromatogram of Eriocaulon buergerianum formula granules. The difference from Example 1 is only that the flow rate of the mobile phase is 0.28 mL / min, and the other conditions are the same as those in Example 1.

[0112] Example 3

[0113] This example provides a method for constructing a characteristic chromatogram of Eriocaulon buergerianum formula granules. The difference from Example 1 is only that the flow rate of the mobile phase is 0.32 mL / min, and the other conditions are the same as those in Example 1.

[0114] Example 4

[0115] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the column temperature is 31°C, and other conditions are the same as those in Example 1.

[0116] Example 5

[0117] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the column temperature is 35°C, and other conditions are the same as those in Example 1.

[0118] Example 6

[0119] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the detection wavelength is 250 nm, and other conditions are the same as those in Example 1.

[0120] Example 7

[0121] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided by Preparation Example 2, and other conditions are the same as those in Example 1.

[0122] Example 8

[0123] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided by Preparation Example 3, and other conditions are the same as those in Example 1.

[0124] Example 9

[0125] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided by Preparation Example 4, and other conditions are the same as those in Example 1.

[0126] Example 10

[0127] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided by Preparation Example 5, and other conditions are the same as those in Example 1.

[0128] Example 11

[0129] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided by Preparation Example 6, and other conditions are the same as those in Example 1.

[0130] Example 12

[0131] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided in Preparation Example 7, and other conditions are the same as those in Example 1.

[0132] Example 13

[0133] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the test solution is the test solution provided in Preparation Example 8, and other conditions are the same as those in Example 1.

[0134] Example 14

[0135] This example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the injection volume is 1 μL, and other conditions are the same as those in Example 1.

[0136] Comparative Example 1

[0137] This comparative example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the mass concentration of the formic acid aqueous solution is 0.1%, the column temperature is 30 °C, and the elution gradient is as follows:

[0138]

[0139] Other conditions are the same as those in Example 1.

[0140] Comparative Example 2

[0141] This comparative example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the mass concentration of the formic acid aqueous solution is 0.1%, the column temperature is 30 °C, and the elution gradient is as follows:

[0142]

[0143] Other conditions are the same as those in Example 1.

[0144] Comparative Example 3

[0145] This comparative example provides a method for constructing a characteristic fingerprint of Eriocaulon buergerianum formula granules, which is only different from Example 1 in that the mass concentration of the formic acid aqueous solution is 0.1%, the column temperature is 30 °C, and the elution gradient is as follows:

[0146]

[0147]

[0148] Other conditions are the same as those in Example 1.

[0149] Comparative Example 4

[0150] This comparative example provides a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula granules, which differs from Example 1 only in that the mass concentration of the formic acid aqueous solution is 0.1%, the column temperature is 30°C, and the elution gradient is as follows:

[0151]

[0152] Other conditions are the same as in Example 1.

[0153] Comparative Example 5

[0154] This comparative example provides a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula granules, which differs from Example 1 only in that the mass concentration of the formic acid aqueous solution is 0.1%, and other conditions are the same as those in Example 1.

[0155] Comparative Example 6

[0156] This comparative example provides a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula granules, which differs from Example 1 only in that the formic acid aqueous solution is replaced by an acetic acid aqueous solution with a mass concentration of 0.1%, and other conditions are the same as those in Example 1.

[0157] Comparative Example 7

[0158] This comparative example provides a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula granules, which differs from Example 1 only in that the mass concentration of the formic acid aqueous solution is 0.2%, and other conditions are the same as those in Example 1.

[0159] Comparative Example 8

[0160] This comparative example provides a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula particles, which specifically comprises the following steps:

[0161] 1 μL of the reference solution, the reference medicinal material solution and the test solution (Preparation Example 1) were respectively taken and injected into an ultra-high performance liquid chromatograph for detection and calibration of the common peaks to obtain the characteristic spectrum of the Herba Corydalis glutinosae formula granules; the conditions of the ultra-high performance liquid chromatography were:

[0162] Stationary phase: octadecylsilane bonded silica gel (particle size 1.8 μm);

[0163] Column: ACQUITY HSS T3, length 100 mm, inner diameter 2.1 mm; column temperature 30 °C;

[0164] Mobile phase: acetonitrile (mobile phase A) and 0.1% phosphoric acid solution (mobile phase B); mobile phase flow rate: 0.3 mL / min;

[0165] Gradient elution, the elution gradient is as follows:

[0166]

[0167] The detection wavelength is 260 nm.

[0168] Test Example 1

[0169] Establishment of characteristic fingerprint

[0170] The characteristic fingerprints of different batches of Eriocaulon buergerianum formula granules and Eriocaulon buergerianum control medicinal materials were obtained by using the construction method provided in Example 1, and the results are as Figure 2 (different batches of Eriocaulon buergerianum formula granules) shown;

[0171] Among them, Figure 1 is the control characteristic fingerprint simulated by software; among them, peak 1 corresponds to uridine; peak 2 corresponds to adenosine; peak 3 corresponds to guanosine; peak 4 corresponds to protocatechuic acid; peak 5 (S) corresponds to vanillic acid; peak 6 corresponds to caffeic acid; peak 9 corresponds to ferulic acid; peak 11 corresponds to rutin.

[0172] Taking the control characteristic fingerprint as the reference fingerprint, calculated by the median, the control fingerprint of Eriocaulon buergerianum granules was obtained, and the common peaks were identified. And for the convenience of comparison, the control characteristic fingerprint [R(15)] and the characteristic fingerprints of Eriocaulon buergerianum formula granules with batch numbers K463CP01 [S1(15)], K463CP10 [S2(15)], and K463CP12 [S3(15)] were presented in the same figure, and the results are as Figure 2 shown: The Eriocaulon buergerianum formula granules of different batches all showed 15 characteristic peaks, and the retention times of the 15 characteristic peaks corresponded to those in the control characteristic fingerprint. Among them, the retention times of 8 peaks corresponded to those of the corresponding reference substance peaks of the reference substances respectively. The peak corresponding to the vanillic acid reference substance peak is the S peak. Calculate the relative retention times of peaks 7, 8, 10, 12, 13, 14, and 15 with the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 1.15 (peak 7), 1.29 (peak 8), 1.53 (peak 10), 1.69 (peak 12), 2.16 (peak 13), 2.18 (peak 14), 2.26 (peak 15).

[0173] The characteristic fingerprints of Eriocaulon buergerianum formula granules (K463CP01) obtained by using the construction methods provided in Examples 2 to 14 and Comparative Examples 1 to 8 are as Figures 3 to 21 shown. Among them, it can be seen from Example 1 and the comparative examples that it is not the specific elution gradient and mobile phase composition that result in different characteristic fingerprints.

[0174] Test Example 2

[0175] Test of precision

[0176] Appropriately take the Eriocaulon buergerianum formula granules (batch number: K463CP12), and determine them according to the method provided in Example 1. Using the vanillic acid peak as the reference peak, calculate its relative retention time and relative peak area, and calculate the RSD value. The results are shown in Table 1.

[0177] Table 1 Precision of the characteristic chromatogram of Eriocaulon buergerianum formula granules - relative retention time

[0178]

[0179] As can be seen from Table 1, the RSDs of the relative retention time and relative peak area of the characteristic peaks are both less than 3%, indicating good precision.

[0180] Test Example 3

[0181] Intermediate precision

[0182] Appropriately take the Eriocaulon buergerianum formula granules (batch number: K463CP12), and determine them according to the method provided in Example 1. Using the vanillic acid peak as the reference peak, calculate its relative retention time and relative peak area, and calculate the RSD value. The results are shown in Table 2.

[0183] Table 2 Intermediate precision of the characteristic chromatogram of Eriocaulon buergerianum formula granules - relative retention time

[0184]

[0185] As can be seen from Table 2, the RSDs of the relative retention time and relative peak area of the characteristic peaks are both less than 3%, indicating good repeatability.

[0186] Test Example 4

[0187] Durability test

[0188] 4.1 Stability test

[0189] Take the test solution of Eriocaulon buergerianum formula granules (batch number: K463CP12), and inject samples for determination at 0, 3, 6, 9, 12, and 24 h respectively according to the method provided in Example 1. Record the retention time and peak area of the identified peaks. Using vanillic acid as the S peak, calculate the relative retention time of other characteristic peaks and the S peak. The results are shown in Table 3.

[0190] Table 3 Stability of the characteristic chromatogram of Eriocaulon buergerianum formula granules - relative retention time

[0191]

[0192]

[0193] The results show that the RSDs of the relative retention time and relative peak area of the characteristic peaks are both less than 1%, indicating good stability of the test sample within 24 h.

[0194] 4.2 Investigation of Different Column Temperatures

[0195] The test solution of Eriocaulon buergerianum granules (batch number: K463CP12) was determined by the method provided in Example 1 at different column temperatures (31 °C, 33 °C, and 35 °C) respectively to investigate the durability of the experimental method with respect to the column temperature. The results are shown in Table 4.

[0196] Table 4 Retention Time and Relative Retention Time of Eriocaulon buergerianum at Different Column Temperatures

[0197]

[0198] As can be seen from Table 4, the RSD of the relative retention time of the labeled peaks of the samples at different temperatures is in the range of 0.0% - 1.1%, indicating that the method provided by the present invention has good durability with respect to the column temperature.

[0199] 4.3 Investigation of Different Flow Rates

[0200] The test solution of Eriocaulon buergerianum granules (batch number: K463CP12) was used at different flow rates (0.28 mL / min, 0.30 mL / min, 0.32 mL / min) respectively, and other steps were the same as those in Example 1; the durability of the experimental method with respect to the flow rate was investigated; the results are shown in Table 5.

[0201] Table 5 Retention Time and Relative Retention Time of Eriocaulon buergerianum at Different Flow Rates

[0202]

[0203] As can be seen from Table 5: at different flow rates, the RSD of the relative retention time of the labeled peaks of the samples is in the range of 0.0% - 1.9%, indicating that the method provided by the present invention has good durability with respect to the flow rate.

[0204] In summary, from the results of the durability investigation of different column temperatures and different flow rates, the method provided by the present invention has good durability.

[0205] Test Example 5

[0206] According to the requirements of traditional Chinese medicine fingerprint technology, the characteristic fingerprints of 3 batches of Eriocaulon buergerianum granules were analyzed. Using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (version 2004.A), the similarities between the characteristic fingerprints of 3 batches of Eriocaulon buergerianum granules and the reference characteristic fingerprint were calculated, and the results are shown in Table 6.

[0207] Table 6

[0208]

[0209] As can be seen from Table 6, the similarity between the characteristic chromatograms of the three batches of Eriocaulon buergerianum granules and the reference characteristic chromatogram is above 0.995, indicating that there are basically no differences in the chromatograms.

[0210] In summary, the construction method provided by the present invention can comprehensively reflect the characteristic peak information of the sample, with good resolution. Moreover, the method is stable, has high precision and good reproducibility, and can well complete the quality control of Eriocaulon buergerianum formula granules.

[0211] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for constructing a characteristic chromatogram of Eriocaulon buergerianum Characterized in that The construction method includes: Detecting reference substances, reference medicinal materials and test samples by ultra-high performance liquid chromatography, calibrating the common peaks, and obtaining the characteristic chromatogram of Eriocaulon buergerianum; the Eriocaulon buergerianum includes Eriocaulon buergerianum medicinal materials, Eriocaulon buergerianum formula granules and Eriocaulon buergerianum decoction pieces; The reference substances, reference medicinal materials and test samples each independently exist in the form of a solution; the preparation methods of the reference medicinal material solution and the test sample solution each independently include: mixing the reference medicinal material or test sample with a solvent and ultrasonically treating to obtain the reference medicinal material solution or test sample solution; the solvent includes an aqueous methanol solution; In the ultra-high performance liquid chromatography, acetonitrile is used as mobile phase A, and an aqueous formic acid solution is used as mobile phase B; The mass percentage content of formic acid in the aqueous formic acid solution is 0.25 - 0.45%; In the ultra-high performance liquid chromatography, gradient elution of the sample is carried out with the mobile phase; In the ultra-high performance liquid chromatography, the elution gradient is: 0 - 5 min: the volume percentage content of mobile phase A is 1%, and the volume percentage content of mobile phase B is 99%; 5 - 32 min: the volume percentage content of mobile phase A is 1 - 16%, and the volume percentage content of mobile phase B is 99 - 84%; 32 - 40 min: the volume percentage content of mobile phase A is 16 - 17%, and the volume percentage content of mobile phase B is 84 - 83%; 40 - 45 min: the volume percentage content of mobile phase A is 17 - 21%, and the volume percentage content of mobile phase B is 83 - 79%; 45 - 50 min: the volume percentage content of mobile phase A is 21 - 23%, and the volume percentage content of mobile phase B is 79 - 77%; 50 - 55 min: the volume percentage content of mobile phase A is 23 - 27%, and the volume percentage content of mobile phase B is 77 - 73%; The chromatographic column is ACQUITY UPLC® HSS T3; the particle size of the chromatographic column is 1.8 μm; the column length of the chromatographic column is 150 mm, and the inner diameter of the chromatographic column is 2.1 mm; In the ultra-high performance liquid chromatography, the detection wavelength is 250 - 280 nm; The number of characteristic peaks in the characteristic chromatogram of the test sample is 15.

2. The construction method according to claim 1, Characterized in that The flow rate of the mobile phase is 0.25 - 0.35 mL / min.

3. The construction method according to claim 1, Characterized in that In the ultra-high performance liquid chromatography, the column temperature is 30 - 40°C.

4. The construction method according to claim 1, Characterized in that In the ultra-high performance liquid chromatography, the injection volume is 1 - 3 μL.

5. The construction method according to claim 1, Characterized in that The mass concentration of the aqueous methanol solution is 40 - 60%.

6. The construction method according to claim 1, Characterized in that The concentrations of the reference medicinal material solution and the test sample solution are each independently 0.02 - 0.06 g / mL.

7. The construction method according to claim 1, Characterized in that The power of the ultrasound is 200 - 300 W, and the time of the ultrasound is 20 - 40 min.

8. According to the construction method described in claim 1, characterized in that the characteristic peaks include the corresponding characteristic peaks of uridine, adenosine, guanosine, protocatechuic acid, vanillic acid, caffeic acid, ferulic acid, and rutin.

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